Keywords: preimmune repertoire, CD8 T cell dysfunction, TCR signaling, viral immunology, chronic inflammation, tissue immunity

The CD8 preimmune repertoire is defined as the set of circulating antigen-specific T CD8 lymphocytes that have not been activated yet by their cognate antigen. Because those cells are very rare, they have not been evaluated in humans. We developed a tetramer-based enrichment protocol that allowed for the first time direct detection and enumeration of those rare naive antigen-specific CD8 T cells in healthys. We then used this tool to characterize the CD8 preimmune repertoire in patients with chronic hepatitis C viral infection. We found that their naive CD8 T cells are dysregulated, being hypersensitive to TCR signals, and with increased proportions of memoryphenotype (MP) cells in inexperienced populations. These perturbations are reversible after viral clearance, highlighting the added benefit of early antiviral treatment. Finally, using a transgenic model (OTI), we observed high proportions of MP inexperienced T cells in the blood of cxcr3-deficient unimmunized mice. This suggests that CXCR3-dependent lymphocyte trafficking could account for some preimmune repertoire alterations. Altogether, our work demonstrates that inexperienced T cells can lose their naiveté in several pathological situations. The impact of these findings will need to be considered when designing future immunotherapeutic strategies -especially when « inflammatory » patients are being targeted. Additionally, we highlight the challenge of interpreting T-cell immunophenotyping studies without getting knowledge into antigen-specific populations.

Résumé

Le répertoire T CD8 préimmun correspond aux lymphocytes T spécifiques d'antigène circulant en périphérie, et n'ayant pas encore été activés. Ces cellules sont très rares, et de ce fait, n'ont jusqu'ici pas pu être étudiées de façon approfondie. Nous avons dans un premier temps développé un protocole d'enrichissement basé sur la technologie des tétramères. Nous avons pu détecter et énumérer des lymphocytes T CD8 naïfs spécifiques d'antigènes dans le sang de sujets sains. Nous avons ensuite utilisé cet outil pour évaluer le répertoire T préimmun de patients chroniquement infectés par le virus de l'hépatite C (cHCV). Nous avons démontré que celui-ci est significativement perturbé, avec des cellules hypersensibles à l'activation TCR et une proportion importante de lymphocytes T de phénotype mémoire alors qu'ils n'ont pourtant pas rencontré leur antigène cible. Ces anomalies disparaissent après résolution de l'infection, soulignant l'intérêt d'instaurer précocément un traitement antiviral chez ces patients. Enfin, nous avons observé dans un modèle de souris transgéniques (OTI) une proportion importante de T inexpérimentés de phénotype mémoire chez les animaux nonimmunisés déficients en cxcr3. Nos travaux démontrent que les lymphocytes T inexpérimentés peuvent perdre leur naïveté dans différentes situations pathologiques. Ces résultats devraient être pris en considération pour l'optimisation de futures stratégies d'immunothérapie, notamment lorsque des patients dits "inflammatoires" sont la cible de vaccinations. Enfin, nos résultats soulignent la difficulté d'interpréter les données d'immunophénotypage en l'absence d'information sur la spécificité antigénique.

Mots-clés : répertoire préimmun; lymphocyte T CD8; signal TCR; immunologie anti-virale; inflammation chronique; immunité tissulaire ............................................................................................................................ 57! (b)! Virus genome .............................................................................................................................. 58! (c)! Tropism and entry receptors ...................................................................................................... 59! (d) ! From acute to chronic infection ................................................................................................. 61! (e) ! Extrahepatic manifestations ....................................................................................................... 64! (f) ! Treatment and clearance benefits ............................................................................................... 65! (g)! Antiviral immune responses ....................................................................................................... François Lemoine, Andrès Alcover and Ludovic Deriano for their participation to the Jury. I would like to thank also Lars Rogge, Fernando Arenzana-Seisdos, and Sébastien André, for nurturing excellent discussions about science, and for their support during the entire course of my PhD.

List of figures

List of tables

Classical, but so obvious, I will start by thanking my mentor, Matthew. For such a great time in your lab. For being on my side since the very begining. For your tender acceptation (and self control) after I announced my first second third pregnancy. For your active support for women in science -which includes an exceptional capacity to answer a mean of 5 emails during late evenings and weekends. For your permanent optimism, your laughters, and your contagious joie de vivre. For your amazing ability to sense (and teach) non-spoken things. For being so unpredictable… and so open to unpredicted projects or discoveries. And because you also are a daily challenge to work with… but in such a good way ☺! Difficult to list here all the family members implicated in this crazy project. My love thank goes to my grandmother, Marie-Rose: your generosity and intelligence are an infinite source of inspiration (I imagine your face when you will hear that I wrote that in English…!). The golden medal is then attributed to my husband, Alexandre. For being still there after three babys and four years of PhD -you should be awarded. For your (almost) infinite tolerance and love. For the paupiettes -I will never forget. For your amazing gestion of the home team, at the same time you build your own brilliant career. You should be proud. Thanks to my mother, Jeanne-Marie, for the friday nursery and other concrete supports, and for being an example of working-mother; to my father, Christian, for your permanent and strong support, for relaxing Sunday evenings, and for actively preparing my progeny for Roland Garros tournament (soon?); to Patrizia, for offering an inspirative example of private life/work organization, and for your woman-to-woman support and advices; to my brother Nicolas, for being the first one (and unique one actually) proposing to build a CD5-agonist (or antagonist?) trial in his intensive care unit patients, for never revealing the incredible secret about the t…., and for being always behind me; to my brother Davide, for great help with the kids, for taking care of our lunchs, and for simply being you; to my sister Carole, for her love and support -my only regret is that you did not initiate Nader (and me) to the pole-dance art; and to my brother Lucas, for incredible help with the kids over last spring. Thanks also to Florence, for very nice discussions -your turn now ☺, to Ariel, Marc, Marie-Laure, all the cousins and "related": we are a great family team. Last but not least, I would like to thank my family-in-law, Elisabeth, Georges, and Edouard, as well as all the Alanio/Detton members: you are my second and so warm and supportive family. A special thank to Emile and Martin who literally saved my life in the last month of my PhD.

a big thank to my children Malo, Titouan and Ambre, for your patience. I will never have made all this work without you. You learned me that doing 100 things at the same time is completely possible, that sleeping is a detail, and that the important thing in life is to be equilibrated. You are an inexhaustible source of giggles and happiness. I love you. And yes, now we can go to Disneyland ☺.

Crucial thank to Aurélie. Not necessarily because of very productive discussions on the data themselves (hum), but more for all your support: this started with Radio Pipette and Radio Diva, and later evolved into more private conversations (in the middle of the corridor), where I think we shared almost all of the spicy details of our hectic mother/wife/worker life. Our trip in San Francisco was the "apothéose" of a strong friendship. And yes, I still think that the immunophenotyping is dead, and that you have so much to learn from reading carefully the protocols ☺.

I am very happy to count you, Milena, as a close friend. We shared very nice moment inside and outside the lab. I appreciate your honesty and integrity. I am very grateful that you never installed video cameras into the P2 + -CIH to respect the privacy of (my) late evening musically inspired sorts… Well… I just discovered that you indeed installed a video camera… without advertising me? Since when? Do you keep the records?

He is also my friend and brother (because you are born in 1989!!), and so smart and charming… Nader, thank you for your support over these past years (and for ordering the coffee!). This was an honor -and a challengeto have you as co-PhD. Your maturity is impressive. You will do an amazing career. Looking forward to build Alanio-Yatim (or Yatim-Alanio!?) projects together in the near future.

a special thought for the CIH members who never blamed me after my disrespectful move to the second floor… for all my P2 + friends… and for Lars and Elisabetta, as well as all the members of their lab, that Few more lines to thank namely members of ICD andCIH (mostly 2007-2015) families (apologize for the very few recent additions, or those that I might have forgotten). Thanks to: Armanda, for your strong help to settle in the lab in the first year(s) of my PhD; Biljana -always a pleasure to spend time with you; Brieuc (or Pierre?), because each time I see you I feel happy and it makes me laugh. I wish you all the best for the years to comeyou will be awesome; Caroline, I wish you all the best in life and science; Claire, your work during PhD was very inspirative: thanks for your support even after leaving the lab -and the template ;-); Clémence, for your cheerfulness and your spontaneity; Darragh, for your careful relectures of the thousands of versions of the HCV paper; Dilay, for your "joie de vivre"; Estelle, for you amazing work and your nice attentions with the home team; Gaby, for your support and nice words; Helen L., for your help in starting the project; Hélène S., for saving my life several times by removing the dead flies on my desk with a lot of courage, and also for your mature and helpful advices with the kids; Isabelle, for being a faithful member of the pulldown team, and for you strong support since I arrived in the lab; Jérémie -it is a real pleasure to have you as close neighbor, your "insatiable curiosité" is fantastic -thanks for your advices for the paper as well as the writing of this manuscript; Marie-Laure, for being international expert in orchidées's breeding (what is your secret finally?) and for a communicative happiness; Marine, for your work while I was not there; Matt, I think I never laughed as much as during our few days in Amsterdam; Melissa, because each time I go to the FACS I imagine you knitting in front of your data and that is a very relaxing picture (how do you pass the tubes??); Molly, because I admire you, and feel proud and inspired to be by your side -even if you sometimes stress me by being more organized than me (is it possible?) -looking forward to build 25-combicolor panels with you ;-)!; Philippe, for sharing my excitation about phospho experiments -even when it was not at all exciting, and for all our fun discussions; Pierre-Emmanuel, for being devoted to eat all what I don't like during official lunchs and dinners, and for carrying the heavy secret of the "coquilles saint Jacques" with a lot of discretion; Rosa, for being such a positive person, for learning me everything about the mice -including how to speak to them "à la portuguaise" and some breeding secrets, and because nobody else would have runned to exchange that spleen kidney…; Stéphanie, because you were extremely supportive from the very beginning (do you remember the "charges publiques/privées" ratio?) and all over the course of my career. It is always a great pleasure to discuss and work with you -a shot in the arm when facing difficulties on the regular project(s). You should be very proud of what you are accomplishing in the Labex project; Tracy, for taking care of my children almost as carefully as of my mice; Valentina, for your smile and life energy; Vincent B., for your deep commitment to work in general, and tetramer technology in particular -looking forward to celebrating the 10mg production!; Vincent R., for all your unbiased analysis and your help with R -always a great pleasure to interact with you (except for society games…!).

Foreword

Life is about learning and growing. When I joined Matthew Albert's lab, I thought applying for a PhD would mean being part of a scientific project, focusing on a specific topic for years, hopefully making some interesting discoveries, and at the end, becoming an expert of the subject. What took me a while is to realize that PhD is far more than that. It is a life experience. What it learns you is far beyond science, but this requires to be well surrounded. And I could not dream of better mentor than Matthew.

From the beginning, he offered me freedom, and trust. The first year, I was so free that I was surprised, stressed by the hurry to start getting results, and frustrated and angry. He accepted that, heared my complaints, but never changed his way of doing. I realized a bit later that this was the best gift he could offer me. This learned me curiosity, this learned me to talk to people in the lab as well as outside, to build a project from the very beginning -balancing my own interests with feasibility, and to realize that every scientific experience -even far from my initial interests, is extremely enriching. I am now convinced that there is no better learning than floating, touching to different subjects, testing different techniques, and acquiring a big picture before going deeper into one direction. Behind that, it was also essential to my own growth. I am proud to measure the progress made, and I really hope to be able one day to drive myself students in such a good way.

My interest to immunology started when I learned that we all have garbage cells, capable of purifying our body from the inside. I still have the poster that my SVT teacher offered me after realizing my fascination about them. My interest to naïve T cells came a bit later. Naïve T cells are like young PhD students: well educated, more or less mature, full of good intentions, but with no concrete idea of what is expected from them. Similar to young PhD students, everything is still possible for them, they have a lot of potential, but their maturation will depend on their environment and on the encounters they will have. Like our children, they are our future. And their diversity is an incredible richness and the basis of a harmonious and tolerant (homeostatic) society. We are responsible for preserving and encouraging that diversity.

By this late-evening disgression, I hope to illustrate my profound commitment to my project, as well as how much fun I had during these four years of PhD and the writing of this manuscript

Research Plan

The preimmune T cell repertoire is defined as the set of α/β T cell-receptor (TCR)expressing lymphocytes that have completed thymic development and entered the secondary lymphoid organs, but have not yet been activated by high-affinity TCR binding to a foreign peptide-major histocompatibility complex (pMHC) [START_REF] Jenkins | On the Composition of the Preimmune Repertoire of T Cells Specific for Peptide-Major Histocompatibility Complex Ligands[END_REF]. These mature inexperienced T cells continuously circulate from bloodstream to lymphoid organs and back into the blood, and are maintained by low-affinity contacts with non-cognate ligands in the periphery, as well as survival cytokines. Antigen (Ag)-specific CD8 + T cells get activated when their TCR bind to class I pMHC at the surface of an antigen-presenting cell (APC). They undergo robust proliferation, with up to 50 000 fold expansion possible within a week [START_REF] Blattman | Estimating the precursor frequency of naive antigen-specific CD8 T cells[END_REF], and differentiate into effector T cells that disseminate throughout the body to combat infection. CD8 + T cells are one of the major components of adaptative immunity. They play a critical role in both antiviral and antitumor immunity. To adequately function, the immune system has to be able to mount exquisitely specific responses to a vast repertoire of possible antigens. To establish the basis for such a diverse response to potential foreign antigens, the genes coding for the α and β chains of the TCR undergo somatic recombination and nucleotide insertion, allowing for the potential generation of ~10 15 different specificities. The loss of over 95% of thymocytes during thymic development reflects the stringent selection processes that shapes the T cell repertoire [START_REF] Kyewski | A central role for central tolerance[END_REF]. Remarkably, only 1 to 3% of double positive thymocytes survive thymic selection.

While this might suggest that the repertoire size is in the order of 10 13 unique clones, there is another limiting factor -the capacity of the human body. Estimates have suggested that the total αβ TCR diversity in the blood is 2.5x10 7 [START_REF] Arstila | A direct estimate of the human alphabeta T cell receptor diversity[END_REF]. Many questions remain about the ~10 6 fold gap between the theoretical and observed numbers of different TCR in the peripheral blood of human. One thing is clear, even for identical twins possessing the same genetic information, their T cell repertoires are distinct and in some cases may account for discordance in the development of diseases such as type I diabetes or multiple sclerosis. Given the number of CD8 + T cells present in an adult human (5x10 9 ), the estimated number of cells per specificity is in the range of 100 -1000 cells, i.e. a precursor frequency of 10 -6 -10 -7 (= 1 cell in 1 to 10 millions of CD8). These very low numbers explain that thus far, the question of the frequency, natural diversity, and migration of naive CD8 T cells in normal or pathological situations, as well as their impact on primary and memory immune responses has not been evaluated in humans. Results for these questions hold important implications for understanding the dynamics of an immune response, in particular in relation to vaccine design. To address them, we aimed to:

I. DEVELOP TOOLS FOR SCREENING RARE ANTIGEN-SPECIFIC RESPONSES IN HUMANS

Recent studies in the mouse model just reported an elegant method combining pMHC tetramer staining and magnetic bead enrichment, which allows the tracking in vivo, under physiological conditions, of Ag-specific CD4 + and CD8 + T cells from the naive preimmune repertoire through the entire stages of an immune response. Specifically, it permitted the first time detection and characterization of the CD4 + and CD8 + preimmune repertoire in these animals [START_REF] Moon | Naive CD4+ T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude[END_REF][START_REF] Dalloul | CD5: A safeguard against autoimmunity and a shield for cancer cells[END_REF][START_REF] Obar | Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection[END_REF]. Based on these technical improvments, as well as our own experience in the lab, we developed and standardized a similar tetramer-associated magnetic enrichment protocol (TAME) for detecting rare Ag-specific CD8 + T cell responses in human samples [START_REF] Alanio | Tracking antigen-specific CD8⁷ T cells using MHC class I multimers[END_REF].

We applied it to healthy donors, in order to explore the preimmune repertoire in physiological conditions. This allowed for the first time direct detection, enumeration and characterization of rare naive Ag-specific T cells in healthy humans [START_REF] Alanio | Enumeration of human antigenspecific naive CD8+ T cells reveals conserved precursor frequencies[END_REF]. Interestingly, we detected higher than 100-fold differences in precursor frequency across these epitopes (from 0.6x10 -6 to 1.3x10 -4 ), but conserved frequencies among individuals. These results were published in Blood in 2010 (Manuscript 1) [START_REF] Alanio | Enumeration of human antigenspecific naive CD8+ T cells reveals conserved precursor frequencies[END_REF]. The detailed method of enrichment in both mouse and human was separately published in Methods in Molecular Biology in 2013 (Manuscript 2) [START_REF] Alanio | Tracking antigen-specific CD8⁷ T cells using MHC class I multimers[END_REF].

II. EVALUATE THE IMPACT OF CHRONIC INFECTION

ON THE PREIMMUNE REPERTOIRE

Chronic inflammation is known to perturb immune homeostasis and negatively impact the T cell repertoire. Functional impairments of CD8 + T cells have been characterized in several persistent viral infections, including HIV and hepatitis C virus (HCV) infection in humans, SIV infection in macaques, and lymphocytic choriomeningitis virus (LCMV) infection in mice [START_REF] Ahmed | Immunological memory[END_REF]. Such impairments have been shown at the level of differentiated memory/effector viral-specific CD8 + T cells [START_REF] Stelekati | Chronic Bystander Infections and Immunity to Unrelated Antigens[END_REF]. Recently, chronic infections were shown to also impair the development of unrelated immune responses in mouse models, as well as in humans chronically infected with HCV [START_REF] Stelekati | Chronic Bystander Infections and Immunity to Unrelated Antigens[END_REF]. We investigated whether this effect could be the reflect of a bystander effect of HCV chronic viral infection on the CD8 + T cell preimmune repertoire.

We performed a cross-sectional study of patients with chronic hepatitis C viral infection (cHCV), assessed during chronic phase or post-virologic clearance (Manuscript 3, Submitted). Using TAME and functional assays, we studied Ag-inexperienced CD8 + T cells (tumor or viral antigen-specific populations in tumor-free and CMV neg patients). cHCV patients showed increased differentiation towards memory-phenotype (MP) cells, which correlated with lower surface expression of CD5, a negative regulator of TCR signaling. Accordingly, we demonstrated increased TCR-induced ERK phosphorylation and high susceptibility to cell death. Our study provides the first evidence that naive CD8 + T cells are dysregulated during the course of a chronic viral infection, and suggest that bystander alteration of the preimmune repertoire might participate to the negative impact of chronic infections on the development of CD8 + T cell memory.

III. STUDY THE IMPACT OF CXCR3 RECEPTOR ON INEXPERIENCED T CELLS

Our laboratory recently provided the first evidence for dipeptidylpeptidase 4 (DPP4) mediated chemokine antagonism in chronic HCV pathogenesis. DPP4 (also known as CD26) is a member of the family of X-prolyl dipeptidylpeptidases, and is capable of enzymatically removing the N-terminal two amino acids of proteins. Specifically, we detected N-terminal truncation of the chemokine interferoninduced protein 10 (IP-10 or CXCL10) in cHCV patients, which correlated with higher DPP4 plasma activity, reduced trafficking of CXCR3-expressing lymphocytes and poor response to treatment.

As both CXCL10 and its receptor CXCR3 have been occasionally reported as implicated in intrathymic retention of CD8SP T cells, we hypothesized that disruption of chemokine gradients might perturb the development of mature and fully functional naive T cells. Utilizing animal models, we investigated the impact of Cxcr3 deletion on CD8 + inexperienced T cells development and behavior in periphery. In the thymus, we observed a subset of CD8 + T cells that is Cxcr3-dependent, and could correspond to rehoming memory T cells. In the periphery, we observed an increase in proportions of MP CD8 + T cells. To be able to track naive Ag-specific populations in the context of chemokine gradient disruption, we crossed OTI transgenic (i.e. mice expressing only CD8 + T cells specific for the ovalbumin antigen) with CXCR3KO mice. At steady state, we found that OTI T cell populations harvested from the spleen and blood of OTI CXCR3KO animals contain an increased proportion of MP cells as compared to WT animals. This correlated with a distinct pattern of activation of those cells after stimulation by the Ova peptide in vitro. In parallel, we found homeostatic MP CD69 + CD103 + OTI cells in the skin of WT but not CXCR3KO animals, resembling resident memory T cells [START_REF] Ariotti | Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert[END_REF]. Whether it is tempting to speculate that high proportions of MP cells are circulating into the blood as a result of trafficking defect, additional results on mixed bone marrow chimeras favor the hypothesis of a direct effect of Cxcr3-deficiency.

Ultimately, our work aims at identify if shaping the preimmune repertoire towards higher or lower reactivity is possible, and could be used as a strategy for improving vaccine efficacy.

Chapter 1. General Introduction

CD8 + T cells are one of the major components of adaptive immunity [START_REF] Rocha | CD8 T cell memory[END_REF]. They play a critical role in both antiviral and antitumor immunity. Mature circulating CD8 + T cells that have not yet encountered their specific antigens are known as naive T cells. Naive T cells continuously circulate from bloodstream to lymphoid organs and back to the blood, making contact with thousands of antigen presenting cells (APCs) within the lymphoid tissue every day. They get activated when their TCR bind to peptide-MHC Class I complexes (pMHCI) at the surface of an APC.

Activated antigen (Ag)-specific CD8 + T cells undergo robust proliferation, with up to 50 000 fold expansion possible within one week, at the same time differentiating into effector T cells that can disseminate throughout the body to combat infection [START_REF] Blattman | Estimating the precursor frequency of naive antigen-specific CD8 T cells[END_REF]. The expansion phase is followed by contraction, marked by high levels of cell death, resulting in a small population of memory CD8 + T cells, generally 5-10% of the peak cell number. The memory pool is thought to persist throughout one's life, a level 100-fold higher than the initial frequency [START_REF] Badovinac | Initial T Cell Receptor Transgenic Cell Precursor Frequency Dictates Critical Aspects of the CD8+ T Cell Response to Infection[END_REF], and further subsets into "effector memory" (T EM ) and "central memory" (T CM ) depending on their ability to traffic to lymphoid tissues [START_REF] Sallusto | Central Memory and Effector Memory T Cell Subsets: Function, Generation, and Maintenance[END_REF]. Upon secondary encounter with antigen, memory cells undergo a faster expansion, thus providing a more rapid response to subsequent infection.

During all these steps, CD8 + T cells undergo characteristic changes in surface expression of various molecules, as presented in Table 1. In humans, naive T cells are characterized by basal expression of CD45RA, homing surface molecules (CD62L), and lymph node chemokine receptors (CCR7), together with co-stimulation molecules (CD28, CD27). They do not display activation markers (CD25, CD69, CD44, HLA-DR) and typically express certain adhesion molecules (CD57) but not others (CD11a). In mouse, naive and memory T cell subsets are identified by their pattern of expression of the homing surface molecules CD62L and the activation marker CD44. In both mouse and humans, while none of these molecules alone may specify a naive T cell, their combination allows precise identification [START_REF] De Rosa | 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity[END_REF].

Table 1: Naive and memory phenotype of CD8 T cells in humans and mice.

To adequately function, the immune system has to be able at any moment to mount exquisitely specific responses to a vast repertoire of possible antigens. To establish the basis for such a diverse response to potential foreign antigens, the genes coding for the α and β chains of the TCR undergo somatic recombination and nucleotide insertion, allowing for the potential generation of ~10 15 different specificities. This diversity is stringently regulated as evidenced by the loss of over 95% of thymocytes during thymic development, further shaping the T cell repertoire [START_REF] Kyewski | A central role for central tolerance[END_REF]. First, positive selection provides survival signals for those double-positive thymocytes bearing a TCR capable of interacting with self-pMHC. Coincidently, negative selection induces apoptosis in cells bearing a TCR that reacts with self-pMHC with a too high affinity. Remarkably only 1% of double positive thymocytes survive thymic selection. While this might suggest that the repertoire size is in the order of 10 13 unique clones, there is another limiting factor -the capacity of the human body.

Only a small fraction of this potential repertoire is used during an individual's lifetime. Estimates have suggested that the total αβ TCR diversity in the human blood is 2.5x10 7 [START_REF] Arstila | A direct estimate of the human alphabeta T cell receptor diversity[END_REF]. Given the number of CD8 + T cells present in an adult human (5x10 9 ), the estimated number of cells per specific population is in the range of 100 -1000 cells, giving a precursor frequency of 10 -6 -10 -7 (= 1 naive Ag-specific T cell into 10 6 -10 7 CD8 + T cells). In fact, it is not only the diversity but also the high degree of cross-reactivity built in the T cell repertoire that ensures recognition of virtually any presentable peptide [START_REF] Zippelius | Thymic selection generates a large T cell pool recognizing a self-peptide in humans[END_REF].

I. PREIMMUNE REPERTOIRE

My work has focused on the preimmune repertoire in physiological and pathological conditions. In this section, I introduce thymus anatomy and function, in order to provide a context for understanding naive T cell homeostasis.

A. Thymic development

1) Thymus anatomy and function

The thymus is a gland in the upper part of the chest, behind the sternum and the lungs (Figure 1A) [START_REF] Chidgey | The Thymic Niche and Thymopoiesis (Encyclopedia of Life Sciences)[END_REF]. It starts developing before birth. In newborns and young children, it is large as compared to the size of the body (Figure 1B). It grows during childhood, reaching its largest size (about 40 grams) at puberty. Then, the thymus slowly involutes, and the proportion of fatty tissue increases during adulthood. The thymus is composed of 2 irregularly shaped lobes, each of which has 3 layers (Figure 1C):

• The capsule is the connective tissue covering the outside of the thymus;

• The cortex is the middle layer;

• The medulla is the inner layer.

The cortex of the thymus is supplied by extensive networks of capillaries that are connected to postcapillary venules and arterioles at the corticomedullary junction (CMJ).

The thymus plays a unique role in the adaptive immune system by supporting T cell development.

Intrathymic cells can be divided into 4 categories depending on their origin:

• T lymphocytes in development (thymocytes);

• Cells derived from hematopoietic stem cells : dendritic cells (DCs) -including CD8α + SIRPα - cDCs and pDCs, as well as small populations of B cells, macrophages, and natural killer (NK) cells;

• Resident stromal cells that are derived from non-hematopoietic lineage : this includes epithelial cells that reside either in the medulla or the cortex, mesenchymal cells, and endothelial cells;

• CD8α -SIRPα + cDCs migrating from the peripheral blood [START_REF] Wu | Heterogeneity of thymic dendritic cells[END_REF], which promote central tolerance by bringing blood-borne antigens to single-positive (SP) thymocytes [START_REF] Hadeiba | Plasmacytoid Dendritic Cells Transport Peripheral Antigens to the Thymus to Promote Central Tolerance[END_REF];

• Mature CD4 + and CD8 + T lymphocytes that rehome into the thymus after having been released in the circulation [START_REF] Hale | Back to the thymus: peripheral T cells come home[END_REF] : this was first demonstrated in unmanipulated mice transgenic for green fluorescent protein (GFP) under the control of the RAG2 promoter (Rag2p-GFP) [START_REF] Yu | Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5' of RAG2[END_REF]. Comparative quantification of GFP + and GFP -mature T cells within the thymus of these mice evaluated the proportion of recirculating mature CD4 + and CD8 + T cells around 10-20%, with an interesting positive correlation with age [START_REF] Hale | Back to the thymus: peripheral T cells come home[END_REF]. Most of these cells express high levels of CD44, and locate to the medulla. Based on their long term persistence, they are now identified as intra-thymic T resident memory (T RM ) cells [START_REF] Mackay | Tissue-resident memory T cells: local guards of the thymus[END_REF]. As compared to T CM and T EM memory subsets, T RM have specific non-circulating properties [START_REF] Shin | Tissue-resident memory T cells[END_REF]. They have been identified in parabiosis and transplant experiments as resting tissue memory CD8 + T cells that are maintained independently of the circulating memory population [START_REF] Clark | Resident memory T cells in human health and disease[END_REF]. They are conserved across species (mouse, rat, pigs), and found in multiple tissues such as the gastrointestinal tract, lung, skin and reproductive tract. In the thymus, T RM have been shown to provide potent local immunity, preserving thymic integrity and normal T-cell development in the face of infection with thymus-invading pathogens [START_REF] Hofmann | Thymus-resident memory CD8 +T cells mediate local immunity[END_REF]. Whether they also play a role into thymic selection processes is a matter of debate [START_REF] Hale | Back to the thymus: peripheral T cells come home[END_REF].

The CD3 molecule is detectable (i) intracellularly in all thymocytes, and (ii) at the surface of positively selected double-positive (DP) thymocytes and in all later stages of T cell development [START_REF] Brodeur | Expression of fully assembled TCR-CD3 complex on double positive thymocytes: synergistic role for the PRS and ER retention motifs in the intra-cytoplasmic tail of CD3[END_REF]. As such, it is a useful immunohistochemical marker for T-cells in paraffinembedded tissue sections (Figure 1D). It permits distinction between (i) the cortex -composed of a network of cortical thymic epithelial cells (cTECs) and DCs surrounding densely packed immature thymocytes; and (ii) the medulla -containing relatively fewer mature thymocytes supported by medullary thymic epithelial cells (mTECs) and DCs.

Involution of the thymus happens with age [START_REF] Boehm | Thymus involution and regeneration: two sides of the same coin?[END_REF]. It corresponds to changes in the macroscopic architecture as well as decreased function [START_REF] Ahmed | Clonal expansions and loss of receptor diversity in the naive CD8 T cell repertoire of aged mice[END_REF]. It has been evidenced by the decrease of the number of signal-joint T cell receptor delta excision circles (sjTRECs) detected by RT-PCR in T lymphocytes isolated from age-stratified healthy donors [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF].

sjTRECs are episomal DNA circles generated by the excision of the TCRδ locus during the recombination of the α chain of the TCR. Importantly, they localize to the cytoplasm of T cells and are not replicated. As deletion of the TCRδ locus usually occurs in both alleles, it is assumed that cells that have successfully rearranged their α chain contain 2 copies of sjTREC. sjTREC copy numbers are usually normalized (i) to albumin gene copy number -two copies are also present per cell, and (ii) per 150 000 cells. Two biological parameters complicate the interpretation of sjTREC as a measure of thymic function: longevity of naive T cells, and sjTREC dilution by cell division. Naive T cells are long lived (up to 7 years in humans), and data from thymectomized individuals have reported that sjTREC-containing cells do not necessarily reflect recently-produced cells [START_REF] Sauce | Lymphopenia-driven homeostatic regulation of naive T cells in elderly and thymectomized young adults[END_REF].

Additionally, naive T cell division -even without phenotypic changes, can occur [START_REF] Martin | Self-gratification yields not-so-naive T cells[END_REF][START_REF] Tough | Turnover of naive-and memory-phenotype T cells[END_REF], and as sjTRECs do not replicate, any T cell division dilutes sjTREC content by 2 (Hazenberg et al., 2000b). Estimating thymic function by the amount of sjTREC in the periphery therefore is a powerful but indirect method that needs to be interpreted with caution [START_REF] Hazenberg | Thymic output: a bad TREC record[END_REF]. Recent studies on thymic tissue from age-stratified humans have confirmed involution of the thymus with aging, with the CD8 + T cell compartment being more affected than its CD4 + counterpart, but there is considerable variation among individuals. Importantly, although macroscopically rearranged, the thymus was demonstrated to be functional in individuals over the age of 70 [START_REF] Ferrando-Martínez | Thymopoiesis in elderly human is associated with systemic inflammatory status[END_REF], and higher thymic function correlated with reduction of allcause mortality in the elderly [START_REF] Ferrando-Martínez | Thymic function failure and C-reactive protein levels are independent predictors of all-cause mortality in healthy elderly humans[END_REF].

2) Early thymocyte development

Within the bone marrow, haematopoietic stem cells differentiate into multipotent progenitors (Figure 2). A subset of them expressing high levels of FLT3 initiates transcription of recombination activating gene 1 (RAG1) and RAG2. These cells are termed lymphoid-primed multipotent progenitors (LMPPs) or early lymphoid progenitors, and develop into common lymphoid progenitors (CLPs). T lymphocytes are the unique hematopoietic cells that mature outside of the bone marrow. Their generation requires CLPs to be released from the bone marrow into the blood, and to enter the thymus through blood vessels located at the corticomedullary junction (CMJ) [START_REF] Ciofani | A survival guide to early T cell development[END_REF]. These thymus-settling progenitors undergo massive proliferation, and their progeny of early thymocyte precursors seed into the cortex region of the thymus, where they constitute the more immature subset of the CD4 -CD8 -double negative (DN1) compartment. ETPs are phenotypically identified as CD117 + CD44 + CD25 -. They further divide into three sequential subsets: DN2 (CD117 + CD44 + CD25 + ), DN3 (CD117 lo/-CD44 -CD25 + ), and DN4 (CD117 -CD44 -CD25 -) [START_REF] Godfrey | A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8-triple-negative adult mouse thymocytes defined by CD44 and CD25 expression[END_REF]. DN subsets are all restricted to the cortex, and support critical events in T cell maturation: rearrangement of the TCRβ chain in DN2 cells, expression of pre-TCR (β chain and invariant α chain) and β-selection in DN3 cells, and rearrangement of the TCRα chain in DN4 cells [START_REF] Starr | Positive and negative selection of T cells[END_REF].

These processes are critically regulated by the activity of RAG1 and 2, as illustrated by mice deficient in RAG1, RAG2, or any component of the pre-TCR complex, presenting a blockage in αβ T cell development at the DN3 stage. Of note, at this stage, DN precursors can also rearrange TCRγ and TCRδ loci, and give rise to TCRγδ T cells.

3) Thymic selection of the MHC-restricted preimmune repertoire Still in the cortex, DN4 cells differentiate into double positive (DP: CD4 + CD8 + ) thymocytes, and undergo positive and negative selection. Only 1% of them survive these processes [START_REF] Bouneaud | Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion[END_REF]. Briefly, the latest event of cortical maturation is the positive selection process, mediated by cTECs (cortical TECs), where early DP cells with TCR conformations that are non-responsive to self-pMHC ligands die by neglect, and those that are overly reactive to self-pMHC undergo programmed cell death [START_REF] Klein | Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see)[END_REF]. It ensures that the preimmune repertoire is MHC-restricted and composed of low affinity thymocytes. Positive selection is also the step where (i) a thymocyte becomes a CD4 + (if recognizing self-pMHCII complexes) or a CD8 + (if recognizing self-pMHCI complexes) T cell [START_REF] Tubo | TCR signal quantity and quality in CD4[END_REF], and (ii) T cells undergo a process of TCR desensitization [START_REF] Grossman | Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function[END_REF]. Positively selected late DP cells and SP CD8 + or CD4 + spend about 4-5 days in the medulla. There, TCR engagement by autoreactive immature thymocytes induces their apoptosis. This is negative selection, which is mediated by mTECs that have the unique ability to express the transcription factor Aire, and promote the intrathymic expression of tissue-specific antigens (TSAs). It ensures that the post-selection repertoire exported from the thymus comprises T cells that are largely non-responsive to self, yet capable of responding with remarkable specificity to foreign peptides.

Interestingly, there is a concerted regulation of thymocyte migration at several critical steps of their maturation [START_REF] Dzhagalov | How to find your way through the thymus: a practical guide for aspiring T cells[END_REF]. This is mediated by (i) tonic TCR signals in the cortex, and (ii) differential expression of chemokines (Figure 2) [START_REF] Halkias | Opposing chemokine gradients control human thymocyte migration in situ[END_REF]. Chemokines are a group of low-molecular-weight, structurally related molecules that provide attractive or repulsive signals to target cells through interactions with seven-transmembrane, G protein-coupled receptors [START_REF] Thelen | How chemokines invite leukocytes to dance[END_REF]. They induce leukocyte movement (chemokinesis) and support directed migration (chemotaxis), either as soluble gradients or as anchored forms on the extracellular matrix [START_REF] Sarris | Inflammatory Chemokines Direct and Restrict Leukocyte Migration within Live Tissues as Glycan-Bound Gradients[END_REF].

Schematically, intrathymic chemokines regulate:

• The seeding of LMPP and CLP progenitors into the thymus, which involves CC-chemokine receptor 9 (CCR9; which is expressed by subsets of LMPPs and CLPs), CCR7 (which is expressed by some LMPPs and highly expressed by CLPs), and PSGL1 (P-selectin glycoprotein ligand-1);

• The retention of DN4 and early DP thymocytes into the cortex, which involves interaction between CXCL12 (or SDF-1) and its receptor CXCR4 [START_REF] Halkias | Opposing chemokine gradients control human thymocyte migration in situ[END_REF];

• The attraction of late DP and mature SP thymocytes into the medulla, involving interaction between the chemokines CCL19 and CCL21 and their receptor CCR7.

Switch of expression from one receptor to another, for example from CXCR4 to CCR7 as a consequence of successful positive selection, is part of the maturation process. Only thymocytes with sufficient maturity are able to migrate towards a new subpart of the thymus -and thus the next step of maturation [START_REF] Dzhagalov | How to find your way through the thymus: a practical guide for aspiring T cells[END_REF][START_REF] Halkias | Opposing chemokine gradients control human thymocyte migration in situ[END_REF].

4) Maturation and emigration of mature SP thymocytes

During their estimated 4-5 days into the medulla, in parallel with negative selection, SP thymocytes undergo an ordered terminal differentiation program, transitioning from a semi-mature CD69 hi CD62L lo CD24 hi phenotype to a mature CD69 lo CD62L hi CD24 lo phenotype. They also progressively acquire resistance to apoptosis. Most of the medullary SP thymocytes express high levels of CCR7, and restricted subsets also express CCR8, CCR9 and CXCR4. However, these data arise from studies on mouse CD4SP maturation, and very little is known about the relevance of this finding for the CD8SP compartment. Recent studies focusing on human CD8SP documented that 20-40% of the CD8SP subset expresses CXCR3 -and not CCR7 [START_REF] Annunziato | CXCR3 and E 7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intraepithelial lymphocytes[END_REF]. These cells, as well as TCRγδ and NK cells that also express CXCR3 in the thymus, were successfully attracted by CXCR3 ligands in transwell experiments, although CXCL11 (I-TAC) appeared to be less active than CXCL10 (IP-10) and CXCL9 (MIG). All three chemokines are actively produced in the thymic medulla [START_REF] Annunziato | Chemokines and lymphopoiesis in human thymus[END_REF], and thus likely play a role in retention of these specific subsets of cells in the medulla.

After completing their developmental program, mature SP thymocytes leave the thymus and join the population of peripheral circulating lymphocytes. The majority of thymocytes emigrate via perivascular spaces at the CMJ [START_REF] Zachariah | Neural Crest-Derived Pericytes Promote Egress of Mature Thymocytes at the Corticomedullary Junction[END_REF]. Similar to intrathymic migration, thymic export is not a passive, but rather an active process, where sphingosine-1-phosphate (S1P) receptor 1 (S1P 1 ) has to be selectively turned on. Schematically, when semi-mature SP thymocytes are receiving signals through engagement of TCR with self-ligands in the context of negative selection screens, FOXO1 is phosphorylated by AKT, and moves away from its transcriptional activity (Figure 3). This leads to the downregulation of Krüppel-like factor 2 (Klf2) expression, which is required for the transcription of CCR7, Sell (which encodes CD62L) and S1PR1 (which encodes S1P 1 ). In addition, TCR signaling induces CD69 expression, which downregulates surface expression of S1P 1 . When the cell is mature, negative selection and TCR signaling cease. This releases FOXO1, which translocates to the nucleus, and promotes the transcription of several genes, including S1PR1. Additionally, downregulation of CD69 increases S1P 1 surface expression. These events render SP thymocytes responsive to S1P present in high concentrations into the blood, and induce their export from the thymus [START_REF] Garris | Sphingosine-1-phosphate receptor 1 signalling in T cells: trafficking and beyond[END_REF].

B. Peripheral naive T cells 1) Recent Thymic Emigrants

Recent thymic emigrants (RTEs) constitute the population of T cells that just exited the thymus [START_REF] Fink | The biology of recent thymic emigrants[END_REF]. RTE numbers decrease in parallel to global thymic function [START_REF] Appay | Naive T cells: the crux of cellular immune aging?[END_REF]. They are phenotypically and functionally immature, and it takes about 3 weeks before they acquire the features of standard « mature naive T-cells » [START_REF] Dong | Homeostatic Properties and Phenotypic Maturation of Murine CD4+ Pre-Thymic Emigrants in the Thymus[END_REF][START_REF] Fink | The biology of recent thymic emigrants[END_REF]. RTEs were identified in the mouse by direct injection of fluorescein isothiocyanate (FITC) into the thymus -and retrieval of FITC + peripheral T cells later on, or analysis of mice surgically implanted with thymic lobes from congenic donors. More recently, they were tracked using RAG1-GFP (recombination activating genegreen fluorescent protein) knockin and RAG2p-GFP transgenic (Tg) mice in which the RAG1 or RAG2 promoters drive GFP expression. In the RAG2p-GFP, the GFP signal remains detectable even after RAG gene expression is over, and GFP + peripheral T cells are considered as RTEs. In all of these models, RTEs were found to account for a large fraction of the peripheral naive T cells in neonates, but less than 10% in adults. Most but not all RTEs are Qa2 lo CD24 hi . Post-thymic maturation includes:

• downregulation of CD24 and CD3/TCR and upregulation of Qa2, CD28, CD45RB, IL-7R!, and Ly6C for CD4 + RTEs;

• downregulation of !4!7, !E integrins (LPAM-1 and CD103 respectively), and CCR9 for CD8 + RTEs [START_REF] Staton | CD8+ recent thymic emigrants home to and efficiently repopulate the small intestine epithelium[END_REF].

If stimulated by foreign pMHC during this short maturation period, RTEs proliferate poorly, and produce fewer cytokines and fewer effector and memory cells as compared to « mature » naive T cells [START_REF] Boursalian | Continued maturation of thymic emigrants in the periphery[END_REF]. Their progeny typically display decreased levels of Ly6C at their cell surface. This might contribute to the relative immunodeficiency observed in newborns. TCRs expressed by RTEs contain longer complementarity determining region 3 (CDR3) regions, suggesting the existence of more autoreactive cells [START_REF] Kyewski | A central role for central tolerance[END_REF][START_REF] Matsutani | Comparison of CDR3 length among thymocyte subpopulations: Impacts of MHC and BV segment on the CDR3 shortening[END_REF]. The expression of inhibitory receptors such as CTLA-4 and PD-1 may also help prevent autoreactive RTEs from harmful tissue damage [START_REF] Fink | Post-thymic maturation: young T cells assert their individuality[END_REF]. Additionally, a subset of RTEs highly express !4!7, !E integrin, and CCR9, facilitating their homing to gut-associated lymphoid tissues where they likely play a role in the tolerance to self-and harmless food-antigens [START_REF] Staton | CD8+ recent thymic emigrants home to and efficiently repopulate the small intestine epithelium[END_REF].

How much these findings translate to humans is largely unknown, as there are no clear markers of RTEs in humans. sjTRECs have long been used as markers of recent thymic emigrants, but sjTREC data must be interpreted cautiously, as discussed above [START_REF] Hazenberg | Thymic output: a bad TREC record[END_REF]. While some human CD4 + RTEs have been shown to express CD31 and PTK7, no markers are available to track CD8 + RTEs, although they are likely enriched in the CD103 + subset of naive peripheral CD8 + T cells [START_REF] Mcfarland | Identification of a human recent thymic emigrant phenotype[END_REF]. As such, very little is known in humans about their existence, their functionality, their repertoire, as well as variations in numbers during physiological or pathological situations, which could significantly impact the adaptive immune responses.

2) Population size and TCR repertoire How many T cells do we have in our body? This question is seemingly a simple one, but is still unresolved [START_REF] Ganusov | Do most lymphocytes in humans really reside in the gut?[END_REF]. Mouse have about 9x10 7 αβT cells (60% are CD4 + T cells and 40% are CD8 + ), and at steady-state, 70% of them have a naive phenotype (Table 2) [START_REF] Jenkins | On the Composition of the Preimmune Repertoire of T Cells Specific for Peptide-Major Histocompatibility Complex Ligands[END_REF]. 95% of those naive T cells are located into secondary lymphoid organs (SLOs) through interactions of their surface receptors (CD62L, CCR7, α4β7 integrin) to specific ligands (PNAd, CCL21, MadCAM) [START_REF] Andrian | T-cell function and migration. Two sides of the same coin[END_REF]. In humans, these numbers are extrapolated from animal studies, and normalized by the body surface area. Thus, it is estimated that we have 10 10 T cells in the blood, 40x10 10 cells in lymphoid tissues, 3x10 10 cells in the gut, 3x10 10 cells in the lungs, and 10 10 cells in other organs. This is a total of about 5x10 11 αβT cells in our body [START_REF] Ganusov | Do most lymphocytes in humans really reside in the gut?[END_REF][START_REF] Thome | Spatial Map of Human T Cell Compartmentalization and Maintenance over Decades of Life[END_REF]. 70% of them are CD4 + T cells and 30% are CD8 + T cells. Within both circulating subsets, 30% display a naive phenotype. Both naive CD4 + and CD8 + T cell numbers decrease with age, but less dramatically than thymocyte numbers. This reflects peripheral compensation of T cell loss by increased proliferation of naive T cells [START_REF] Braber | Maintenance of Peripheral Naive T Cells Is Sustained by Thymus Output in Mice but Not Humans[END_REF][START_REF] Ferrando-Martínez | Age-related deregulation of naive T cell homeostasis in elderly humans[END_REF] -which does not necessarily translate into loss of their naive phenotype [START_REF] Tough | Turnover of naive-and memory-phenotype T cells[END_REF]. However, if this phenomenon partially compensates for the number of circulating naive T cells, it does not prevent the loss of diversity of the peripheral TCR repertoire, and ultimately leads to naive T cell oligoclonality, likely influencing immune responses in the elderly [START_REF] Qi | Diversity and clonal selection in the human T-cell repertoire[END_REF].

Table 2. Mouse and human adults preimmune repertoire numbers.

Grey area indicates results from estimations, whereas white areas indicate experimental measurements.

T cell specificity relies on the somatic TCR gene rearrangements during T cell development, and relates to the capacity of a given T cell to specifically recognize its cognate epitope when presented in the context of a pMHC complex. Given that the number of potential TCR amino acid sequences potentially produced by V(D)J recombination (> 10 15 ) is greater than the total number of naive cells, it is theoretically possible that each cell carries a unique TCR and responds to a different foreign pMHC.

However, estimates of T cell repertoire composition have revealed that the average T cell clone exists in 30 copies in mice and 10 000 copies in humans. This must arise by cell division after TCRα locus rearrangement, either in the thymus or during homeostatic proliferation in periphery. Many questions remain about the ~10 6 fold gap between the theoretical and observed numbers of different TCRs in the peripheral blood of humans.

In generating a « useful » repertoire, there are some major constraints [START_REF] Mason | A very high level of crossreactivity is an essential feature of the T-cell receptor[END_REF]):

• The repertoire must be able to react to a very large number of individual peptides presented by the MHC class I and II antigens, to ensure that pathogenic peptides do not go unrecognized. It is estimated that 1 peptide is generated every 30 aminoacids (aa), and « only » 10 10 different peptides can be presented by one given MHC molecule;

• it must be highly specific to allow responses to foreign but not to self-peptides;

• the frequency of inexperienced T cells that can recognize a given foreign peptide must be sufficiently large to ensure a rapid response to a foreign antigen containing that peptide, but at the same time sufficiently low to fit into a size-limited animal.

As there are far more potential foreign peptides in the environment than the organism has T cells, a very high level of crossreactivity -i.e., the ability for an individual T cell to recognize multiple peptides -is now recognized as an essential feature of the T-cell receptor [START_REF] Nelson | T cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity[END_REF]Su and Davis, 2013;Su et al., 2013). One given naive T cell is estimated to have the capacity to bind 10 6 different peptides. Inversely, one given pMHC complex can be recognized by at least 500 different clones [START_REF] Kedzierska | Quantification of repertoire diversity of influenza-specific epitopes with predominant public or private TCR usage[END_REF]. Those clones can be restricted by the same or different haplotypes, and it is not excluded that CD4 + T cells participate in the complexity [START_REF] Legoux | Impact of TCR Reactivity and HLA Phenotype on Naive CD8 T Cell Frequency in Humans[END_REF]. With the number of foreign nonamer peptides estimated to be ~10 10 for a given MHC molecule, there is theoretically less than one chance in 10 4 that a T cell reacts with one chosen at random. This makes the T cell repertoire appear artificially highly antigen specific. As such, the composition of the foreign peptide-specific T cell repertoire is shaped by the degree of TCR cross-reactivity between similar self and foreign peptides [START_REF] Nelson | T cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity[END_REF]. However, since a T cell must see at least 100 immunogenic pMHC complexes to get activated, a self-antigen that is under this level of expression in the periphery will be ignored by the immune system -the majority of those that exceed this threshold being deleted into the thymus. Importantly, when a T cell is primed, this leads to a reduction in the heterogeneity of TCR gene usage, with a degree of affinity selection that ensures that only T cells that respond to low concentrations of peptide give rise to memory cells [START_REF] Mcheyzer-Williams | Antigen-specific development of primary and memory T cells in vivo[END_REF]. As a consequence, the repertoire of memory cells is less diverse, and as a consequence less crossreactive than naive cells.

The generation of MHC class I tetrameric or tetrameric complexes (commonly referred as MHC tetramers) represents a major technical advance for the study of T cell repertoires [START_REF] Altman | Phenotypic analysis of antigen-specific T lymphocytes[END_REF][START_REF] Altman | MHC-peptide tetramers to visualize antigen-specific T cells[END_REF] (Figure 5). MHC class I tetramers are reagents that carry multiple pMHC complexes, and thus have the ability to interact with multiple TCRs on a single CD8 + T cell.

Fluorescent-labeling of MHC tetramers permits identification of Ag-specific T lymphocytes based on the avidity of their TCR, independent of their functional or differentiation state [START_REF] Davis | Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis[END_REF].

Using MHC tetramers, it is possible to directly track and quantitate Ag-specific T cells (i.e. all T cell clones that are able to recognize a given pMHC complex) during the course of immunization [START_REF] Klenerman | Tracking T cells with tetramers: new tales from new tools[END_REF]. And by co-staining with antibodies directed against phenotypic cell surface proteins, one can define subsets of cells of interest based on their activation or differentiation state, or chemokine receptor expression [START_REF] Romero | Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma[END_REF]. MHC tetramer technology has been successfully coupled to conventional functional assays (e.g., CFSE dilution, intracellular cytokine staining -ICS), and specific T cells can be sorted for elispot, cytotoxicity assays, gene expression studies, or for generating long-term cultures [START_REF] Baitsch | Exhaustion of tumor-specific CD8+ T cells in metastases from melanoma patients[END_REF]. Tetramers are now widely used in the immune monitoring of T cell responses following therapeutic or prophylactic vaccination. Finally, the availability of good manufacturing practice (GMP)-grade tetramers is enabling the ex vivo expansion of T cells for immunotherapy [START_REF] Kanodia | PR1specific T cells are associated with unmaintained cytogenetic remission of chronic myelogenous leukemia after interferon withdrawal[END_REF].

However, the limit of detection of multimers, of 10 -5 , does not allow exploration of the preimmune repertoire prior to peptide-induced expansion. To solve that issue, in 2007, MHC tetramers were coupled to magnetic bead enrichment in mice [START_REF] Moon | Naive CD4+ T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude[END_REF][START_REF] Obar | Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection[END_REF]. This permitted for the first time, a direct evaluation of Ag-specific CD4 + -and later CD8 + -T cell precursor frequencies (i.e., frequencies of T cells specific for peptides that have never been seen by the immune system). They were found in the range of 10 -6 to 10 -4 (i.e. 1 cell in 10,000 to 1,000,000 T cells).

Following from these studies, we and others developed a similar enrichment protocol for human CD8 + T cells (see Chapter 2) [START_REF] Alanio | Enumeration of human antigenspecific naive CD8+ T cells reveals conserved precursor frequencies[END_REF][START_REF] Legoux | Impact of TCR Reactivity and HLA Phenotype on Naive CD8 T Cell Frequency in Humans[END_REF]. With that tool, we found precursor frequencies also ranging from 10 -6 to 10 -4 . Similar ranges were later observed for the human CD4 + T cell preimmune repertoire [START_REF] Kwok | Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire[END_REF]Su et al., 2013).

3) Homeostasis and homeostatic proliferation

Mature naive phenotype T cells are relatively long-lived, with a half-life of 7-11 weeks in mice and 4-7 years in humans [START_REF] Braber | Maintenance of Peripheral Naive T Cells Is Sustained by Thymus Output in Mice but Not Humans[END_REF][START_REF] Jenkins | On the Composition of the Preimmune Repertoire of T Cells Specific for Peptide-Major Histocompatibility Complex Ligands[END_REF][START_REF] Sprent | Normal T cell homeostasis: the conversion of naive cells into memoryphenotype cells[END_REF]. They have low basal metabolic activity, and actively maintain quiescence while sustaining their naive differentiation state [START_REF] Hamilton | CD8 T cell quiescence revisited[END_REF][START_REF] Tough | Turnover of naive-and memory-phenotype T cells[END_REF]. Quiescence is a complex process requiring the interplay of a number of different regulatory mechanisms that prevent T cell activation, including:

• repression of NFkB activity via the continuous synthesis of IkB (inhibitor of NFkB) induced by two members of the forkhead family of transcription factors, FOXO3 and FOXJ1;

• continuous inactivation of NFAT by inhibition of its dephosphorylation that avoid its translocation to the nucleus;

• KLF2-, ELF4-, and KLF4-dependent downregulation of the expression of genes essential for cell cycle progression such as Cdk2.

Two homeostatic signals are crucials to maintain naive T cells alive (Figure 6) [START_REF] Hamilton | CD8 T cell quiescence revisited[END_REF][START_REF] Jameson | Not all naive CD8 T cells are created equal[END_REF]):

• IL-7. This cytokine is synthesized by CD45-negative stromal cells, and is primarily produced in the thymus, lymph nodes and bone marrow. In IL-7 -/-and IL-7R(CD127) -/-mice, both CD4 + and CD8 + naive cells die within 1-2 weeks after their transfer. IL-7 binding to IL-7R, via JAK1-3/Stat5 signaling, promotes survival by tilting the balanced expression of anti/pro apoptotic molecules such as Bcl-2 and Bim towards the former [START_REF] Schluns | Cytokine control of memory T-cell development and survival[END_REF]. IL-7 responsiveness is controlled by modulation of expression of IL-7R via (i) an autocrine loop, and (ii) the transcription factor Foxo1;

• TCR-self-pMHC interactions. Both tonic TCR signaling of T cells, and MHC contacts maintain cell viability [START_REF] Sprent | Normal T cell homeostasis: the conversion of naive cells into memoryphenotype cells[END_REF]. In their absence, T cells die within several weeks. This interaction is subtly controlled by TCR tuning during T cell development. Indeed, although intrathymic DP and SP T cells are more sensitive to activation by antigen than mature T cells, they undergo a process of TCR desensitization (or tuning) in the late step of maturation [START_REF] Grossman | Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function[END_REF]. This sets the threshold for TCR activation just above the level required for T cell maintenance in the periphery, thereby keeping the cells in a selftolerant state.

Contact with these survival factors occurs in secondary lymphoid organs, such as lymph nodes (LNs), where professional APCs and local stromal cells provide a key source of MHC molecules and IL-7 respectively. In line with this model, the numbers of naive T cells are significantly reduced under conditions where homing to lymph nodes is hampered [START_REF] Boyman | Homeostatic maintenance of T cells and natural killer cells[END_REF]. This is further supported by the common transcriptional regulation of key elements of homing and survival such as CD62L (Lselectin), CCR7, and IL-7R of naive T cells [START_REF] Kerdiles | Foxo1 links homing and survival of naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor[END_REF].

Homeostatic proliferation (HP) occurs naturally in the neonatal period, but is most prominent in hosts with acute lymphopenia. In the latter case, it is driven by sudden high concentrations of cytokines, such as IL-7, IL-15 and IL-2. This is a result of low consumption by dramatically decreased numbers of lymphocytes. HP corresponds to the expansion, but also in some cases differentiation and conversion to memory-phenotype (MP) cells, of inexperienced T cells occurring independently of antigenic stimulation. CD8 + T cells are more sensitive to HP than CD4 + T cells [START_REF] Sprent | Normal T cell homeostasis: the conversion of naive cells into memoryphenotype cells[END_REF]. In humans, HP-derived T cells are chronically activated (CD28 -CD57 + ), highly proliferative (Ki67 + ), oligoclonal, memory-like (usually CCR7 -), and capable of producing proinflammatory cytokines. In mice, HP cells typically have a central-memory phenotype, and are functionally indistinguishable from Ag-driven memory cells [START_REF] Sallusto | Central Memory and Effector Memory T Cell Subsets: Function, Generation, and Maintenance[END_REF]. However, they display low levels of α4-integrin (CD49d) [START_REF] Cheung | Memory-Like CD8+ T Cells Generated during Homeostatic Proliferation Defer to Antigen-Experienced Memory Cells[END_REF]. The reasons why half of CD8 + naive T cells express this marker in non-lymphopenic adult animals are unclear, but could reflect under-estimated homeostatic proliferation rates. Importantly, HP-derived cells have been identified as a driving force for lymphopenia-associated autoimmunity in both mice [START_REF] King | Homeostatic expansion of T cells during immune insufficiency generates autoimmunity[END_REF][START_REF] Saout | Memory-like CD8+ and CD4+ T cells cooperate to break peripheral tolerance under lymphopenic conditions[END_REF] and humans [START_REF] Jones | Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation[END_REF].

Recent studies using tetramer-enrichment tools demonstrated that MP cells represent up to 30% of inexperienced Ag-specific T cells in both mice and humans [START_REF] Haluszczak | The antigen-specific CD8+ T cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion[END_REF][START_REF] Legoux | Impact of TCR Reactivity and HLA Phenotype on Naive CD8 T Cell Frequency in Humans[END_REF]Su and Davis, 2013). These cells may reflect cross-reactivity, and/or more likely, derive from HP, as: (i) large numbers of specificities are concerned, (ii) they are found in germ-free animals [START_REF] Haluszczak | The antigen-specific CD8+ T cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion[END_REF], and (iii) they increase in numbers in the context of acute lymphopenia [START_REF] Goldrath | Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation[END_REF]. Notably, in the latter case in mice, Ag-specific inexperienced T cell populations were found to reverse to a naive phenotype within about a month after homeostatic conditions were restored. This parallels the observation of restoration of bulk naive T cell polyclonality within two years in humans [START_REF] Jones | Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation[END_REF]. The mechanisms underlying these observations are unclear.

4) CD5 expression and dynamic tuning of lymphocytes

One of the key molecules in TCR tuning is CD5 (Figure 7) [START_REF] Grossman | Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function[END_REF][START_REF] Soldevila | The immunomodulatory properties of the CD5 lymphocyte receptor in health and disease[END_REF]. CD5 is a type I glycoprotein of 67-kDa, constitutively expressed at the cell surface of all thymocytes and mature T cells, as well as a subset of B cells. It belongs to the type B superfamily of scavenger receptor cysteine-rich, and also exists in a soluble form, which acts as a decoy receptor. It is composed of three extracellular regions and a highly conserved cytoplasmic domain containing multiple Ser/Thr and Tyr phosphorylation sites, including two potential immunotyrosine-based inhibition motifs [START_REF] Raman | CD5, an important regulator of lymphocyte selection and immune tolerance[END_REF].

Both soluble and membrane-bound human CD5 molecules bind and sense pathogen-associated molecular patterns (PAMPS) of fungal origin (β-glucans) via the extracellular portion of the molecule [START_REF] Fenutría | Transgenic Expression of Soluble Human CD5 Enhances Experimentally-Induced Autoimmune and Anti-Tumoral Immune Responses[END_REF]. CD5 has also been reported to interact with several surface molecules present on APCs including CD5L, CD72, gp40-80, gp150, and IgV(H) framework region sequences, but whether these are true ligands remains unclear [START_REF] Soldevila | The immunomodulatory properties of the CD5 lymphocyte receptor in health and disease[END_REF].

Via its intracytoplasmic portion, CD5 negatively regulates TCR signaling. This effect was first observed in CD5 -/-mice, where T cells are hyperresponsive to TCR stimulation, with increased proliferation, and enhanced phosphorylation of TCR signaling effectors such as LAT, phospholipase Cγ1, or Vav [START_REF] Tarakhovsky | A role for CD5 in TCR-mediated signal transduction and thymocyte selection[END_REF]. The mechanism of action has now been clarified [START_REF] Grossman | Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function[END_REF]. Upon TCR ligation, CD5 is rapidly engaged in the immunological synapse, where it physically associates with the CD3 molecule, and recruits inhibitory molecules of TCR signal such as SHP-1, RasGFP and Cbl.

The level of CD5 expressed at the cell surface of antigen-specific naive T cells is set on thymocytes at the time of positive selection, and positively correlates with the strength of encounter with self-pMHC ligands. CD5 hi cells are thymocytes with the highest affinity for self-ligands -just under the level triggering intrathymic deletion -and thus those for which the threshold for TCR activation needs to be significantly lowered in the periphery. CD5 and IL-7R expression are positively associated, providing a mechanistic basis for crosstalk between TCR and cytokine signals during homeostasis. In the context of inflammation, CD5 hi naive CD8 + T cells show superior use of inflammatory cytokines in vitro and in vivo, as well as improved clonal recruitment and expansion in response to foreign pMHC. This highlights that (i) predetermined heterogeneity dictates the capacity of naive T cells to respond to foreign antigens, and that (ii) T cells with the strongest reactivity to intrathymic self-peptides dominate the response to foreign pMHC. However, in more complex systems, peripheral modulation of CD5 level does exist. It decreases when cells are deprived in pMHC contact [START_REF] Smith | Sensory adaptation in naive peripheral CD4 T cells[END_REF], or IL-7 and IL-21 cytokines [START_REF] Soldevila | The immunomodulatory properties of the CD5 lymphocyte receptor in health and disease[END_REF]. Inversely, it increases when cells are chronically exposed to self-antigen in the periphery [START_REF] Hawiger | Immunological unresponsiveness characterized by increased expression of CD5 on peripheral T cells induced by dendritic cells in vivo[END_REF]. Levels of CD5 are negatively regulated at the transcriptional level by E47 and GATA-3 [START_REF] Arman | Transcriptional regulation of human CD5: important role of Ets transcription factors in CD5 expression in T cells[END_REF][START_REF] Ling | GATA3 controls the expression of CD5 and the T cell receptor during CD4 T cell lineage development[END_REF], and positively by ETS1 [START_REF] Tung | The regulation of CD5 expression in murine T cells[END_REF].

Peripheral down-and up-regulation of CD5 levels translates into increased and decreased responsiveness of CD8 + T cells to antigenic stimulation respectively. It is a way to tune (i) tumorinfiltrating lymphocytes to promote increased antitumor activity [START_REF] Tabbekh | T-cell modulatory properties of CD5 and its role in antitumor immune responses[END_REF], or (ii) autoreactive cells to diminish responsiveness to chronically exposed self-antigens [START_REF] Hawiger | Immunological unresponsiveness characterized by increased expression of CD5 on peripheral T cells induced by dendritic cells in vivo[END_REF][START_REF] Stamou | Chronic exposure to low levels of antigen in the periphery causes reversible functional impairment correlating with changes in CD5 levels in monoclonal CD8 T cells[END_REF].

5) Activation, differentiation and activation-induced cell death

Engagement of the TCR occurs when it binds to its agonist pMHC complex with sufficient avidity to overcome the threshold for activation established during T cell development [START_REF] Poltorak | TCR activation kinetics and feedback regulation in primary human T cells[END_REF]. It results in the sequential recruitment and phosphorylation of the src-family kinase Lck, the TCRassociated γ, δ, and ζ chains, the kinase ZAP-70, the coreceptor-associated linker for activation of T cells (LAT), and the mitogen-activated protein (MAP) kinase ERK. At the single cell level, the ERK response is digital: 70-80% of maximum signal reached within few minutes after activation with different agonists concentrations, versus no significant response with antagonists [START_REF] Grossman | Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function[END_REF]. At the population level, increase in ligand concentration results in an increased number of activated cells with a maximal ERK response, likely reflecting the pre-existing variations among individual cells. These proximal events translate into transcriptional promotion of the expression of genes involved in T cell differentiation and proliferation. Schematically, important events of T cell activation include (Figure 8):

• At hour (H)6, upregulation of the T cell activation marker CD69, a calcium-dependent type II transmembrane receptor of the lectin superfamily. Its expression peaks at 24 hours, and is down-regulated after 48 hours;

• At H12, upregulation of CD25, the α-chain of the IL-2 receptor. Its expression peaks at 48 hours, and diminishes after 72-96 hours;

• From day (D)2 to D4, modification of the surface phenotype, with acquisition of memory markers. Of note, CD45RA and CD45RO are two different isoforms, and the transition in mRNA expression is detectable as soon as 24 hours after activation. Late persistence of the CD45RA molecule result from slow turnover of the molecule at the cell surface;

• At D2, blastogenesis with limited division. It is transient, lasting only one day;

• At D3, proliferation and rapid expansion (up to 50 000 fold expansion within one week) as illustrated by high levels of 3H-thymidin incorporation [START_REF] Blattman | Estimating the precursor frequency of naive antigen-specific CD8 T cells[END_REF].

Ultimately, the activated T cells differentiate into effector cells that disseminate throughout the body to combat infection.

A properly functioning immune system is dependent on programmed cell death at every stage of lymphocyte development. Following T-cell activation and proliferation, activation-induced cell death (AICD) controls the expansion of activated T lymphocytes and ensures tolerance and homeostasis. It is an apoptotic pathway triggered by (i) the death receptor CD95 (APO-1, Fas) -a subset of the tumor necrosis factor receptor (TNFR) family, and its natural ligand (CD95L, FasL), engagement of which leads to caspase activation [START_REF] Green | Activation-induced cell death in T cells[END_REF]; and (ii) TNF, TRAIL, and Bim molecules. AICD regulation happens predominantly at the transcriptional level, with regulation of FasL, TRAF1 and TRAF2, c-IAPs, and c-FLIP gene expression -the latter being a negative regulator of Fas and TRAIL receptor signaling.

Besides its action on regulating T cell activation, CD5 has emerged as an important regulator of T-cell survival -low levels of CD5 correlate with increased susceptibility to cell death (Figure 7). This is the mechanism accounting for the paradoxical observation of delayed onset and decreased severity of EAE in CD5 -/-mice [START_REF] Axtell | CD5-CK2 binding/activation-deficient mice are resistant to experimental autoimmune encephalomyelitis: protection is associated with diminished populations of IL-17-expressing T cells in the central nervous system[END_REF]. Ag-reactive T cells from those mice are hypersensitive to activation, but unable to persist after the proliferation phase. Similarly, low levels of CD5 on human TILs increases their susceptibility to AICD [START_REF] Tabbekh | Rescue of Tumor-Infiltrating Lymphocytes from Activation-Induced Cell Death Enhances the Antitumor CTL Response in CD5-Deficient Mice[END_REF]. CD5-mediated T-cell survival involves different pathways than those implicated in the regulation of TCR activation. This is evidenced by bone marrow chimera (BMC) experiments, where reconstitution of CD5 /-mice with CK2-binding deficient mutant CD5 restores resistance to AICD, but not sensitivity to activation [START_REF] Axtell | CD5-CK2 binding/activation-deficient mice are resistant to experimental autoimmune encephalomyelitis: protection is associated with diminished populations of IL-17-expressing T cells in the central nervous system[END_REF]. CD5 expression promotes lymphocyte survival through activation of CK2, which induces the expression of pro-survival molecules such as Bcl-2 and Bcl-xl, and inhibits proteins associated with apoptotic pathways, such as FasL, caspases and Bid.

6) Phenotypic and functional diversity

Naive T cells have long been seen as a homogeneous population [START_REF] Stemberger | Stem cell-like plasticity of naïve and distinct memory CD8(+) T cell subsets[END_REF]. However, over the last decade, it became evident that the preimmune repertoire contains a considerable degree of phenotypic and functional heterogeneity.

Schematically, in homeostatic conditions, the preimmune repertoire is a mixture of:

• 10% RTEs (cf Chapter IB1);

• 70% mature naive-phenotype cells, further subdivided into: o CD49d hi or CD49d lo cells -in the mouse CD8 + compartment (cf Chapter IB3); o CD5 hi and CD5 lo populations (cf Chapter IB4);

• and 20% memory-phenotype cells, which is a mixture of: o cells arising from HP (cf Chapter IB3); o crossreactive cells (cf Chapter IB2); o innate memory cells, or virtual memory (VM). These cells appear soon after birth, and increase in frequency during aging [START_REF] Jameson | Innate Memory T cells[END_REF]. They are present in germ-free mice. They are phenotypically similar to cells arising from homeostatic proliferation (CD49d lo ). However, they have unique functional properties: poor TCR-induced IFNγ synthesis, and preferential differentiation into central memory cells [START_REF] Lee | Virtual memory CD8 T cells display unique functional properties[END_REF]. VM cells lead to enhanced pathogen control in mouse models, and might be involved in preimmune resistance to infection [START_REF] Rudd | Nonrandom attrition of the naive CD8+ T-cell pool with aging governed by T-cell receptor: pMHC interactions[END_REF].

Obviously, any change in the proportion of one or multiple of these subpopulations will tune the preimmune repertoire from poor to high reactivity. Table 3 shows the characteristics of each of these subsets, and their expected contribution to the global response of the Ag-specific population. Table 3. Diversity of the CD8 T cell preimmune repertoire.

7) Impact of precursor frequency and diversity

Direct measurements of Ag-specific populations in mice have shown that the size of Ag-specific inexperienced populations vary in a predictable fashion, with significant differences among distinct epitopes, but highly conserved frequencies among individuals that express at least one similar MHC allele. The impact of such variations on later immune responses is key, and has been investigated in mice. Early (<1 week) after peptide immunization, whatever the dose of peptide utilized, there is a positive correlation between pre-and post-immunization CD4 + T cell frequencies (Figure 9A).

Similar observations were reported for CD8 + T cells, but when precursor frequencies are significantly different (>5-fold) [START_REF] Kotturi | Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance[END_REF][START_REF] Obar | Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection[END_REF]. Besides magnitude, T cell precursor frequency was also demonstrated in mice as a key factor for early kinetics (an increased precursor frequency resulting in a more rapid response), and immunodominance hierarchy [START_REF] Kotturi | Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance[END_REF][START_REF] Lo | T cell immunodominance is dictated by the positively selecting self-peptide[END_REF][START_REF] Obar | Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection[END_REF]. It also predicts qualitative aspects of the memory T cell response, with increased precursor frequencies translating into rapid acquisition of a CD62L hi central-memory phenotype [START_REF] Obar | Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection[END_REF]. Finally, precursor frequencies is a mode of virus escape in LCMV model of infection [START_REF] Kotturi | Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance[END_REF]. In humans, the same impact of precursor frequency on the magnitude and immunodominance patterns of responses were found in studies of influenza-infected humanized HLA-transgenic mice [START_REF] Tan | Precursor Frequency and Competition Dictate the HLA-A2-Restricted CD8+ T Cell Responses to Influenza A Infection and Vaccination in HLA-A2.1 Transgenic Mice[END_REF], HCV-specific peripheral T cells [START_REF] Schmidt | Immunodominance of HLA-A2-Restricted Hepatitis C Virus-Specific CD8+ T Cell Responses Is Linked to Naive-Precursor Frequency[END_REF], and B.anthracis-vaccinated individuals [START_REF] Kwok | Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire[END_REF].

It is important to consider that some studies on mice infected with influenza [START_REF] Cukalac | The Influenza Virus-Specific CTL Immunodominance Hierarchy in Mice Is Determined by the Relative Frequency of High-Avidity T Cells[END_REF][START_REF] Gruta | Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion[END_REF], or Respiratory Syncytial Virus (RSV) [START_REF] Ruckwardt | Neonatal CD8 T-cell Hierarchy Is Distinct from Adults and Is Influenced by Intrinsic T cell Properties in Respiratory Syncytial Virus Infected Mice[END_REF] did not confirm the « precursor frequency to magnitude » correlation. One possible explanation for this discrepancy is that the impact of precursor frequency will only manifest in situations where the other parameters that influence immunodominance are controlled and comparable. This includes efficiency of antigen processing and presentation, MHC binding properties, epitope abundance at the surface of presenting APCs, viral load and kinetics of viral protein expressions. Alternatively, qualitative properties of the initial Ag-specific population, such as the proportion of high-affinity T cells, might influence its behavior after immunization [START_REF] Cukalac | The Influenza Virus-Specific CTL Immunodominance Hierarchy in Mice Is Determined by the Relative Frequency of High-Avidity T Cells[END_REF]. In other words, the work presented above has now established precursor frequency as a crucial factor in shaping adaptive immune responses, but qualitative parameters must also be taken into account when trying to predict the effective epitopespecific response after antigenic challenge [START_REF] Jenkins | The Role of Naive T Cell Precursor Frequency and Recruitment in Dictating Immune Response Magnitude[END_REF]. In particular, each of the subsets identified as part of the T cell inexperienced populations (as reported in Table 3) has a specific threshold for TCR activation and sensitivity to cytokine signals, and a specific pattern of function.

RTEs and CD5 hi NP cells have increased threshold for TCR activation [START_REF] Berkley | Recent Thymic Emigrants and Mature Naive T Cells Exhibit Differential DNA Methylation at Key Cytokine Loci[END_REF][START_REF] Dalloul | CD5: A safeguard against autoimmunity and a shield for cancer cells[END_REF]. Inversely, MP cells are hyperresponsive when encountering their cognate antigen [START_REF] Haanen | Selective expansion of crossreactive CD8(+) memory T cells by viral variants[END_REF][START_REF] Lee | Virtual memory CD8 T cells display unique functional properties[END_REF][START_REF] Younes | Memory Phenotype CD4 T Cells Undergoing Rapid, Nonburst-Like, Cytokine-Driven Proliferation Can Be Distinguished from Antigen-Experienced Memory Cells[END_REF]. However, how much the proportion of each of these subsets inside a given Ag-specific population influences the global behavior of that population during homeostasis and after immunization is unknown (Figure 9B).

Interestingly, in both CD4 + and CD8 + naive T cells in mice, early in the course of a primary response (i.e. 4-6 days after peptide immunization), TCR diversity of the expanded population is similar to the one present before immunization [START_REF] Moon | Naive CD4+ T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude[END_REF] [START_REF] Obar | Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection[END_REF]. This apparently conflicts with the idea that priming of cells results in a drastic reduction in the heterogeneity of TCR usage as early as day 6 after immunization [START_REF] Mcheyzer-Williams | Antigen-specific development of primary and memory T cells in vivo[END_REF]. However, clonal selection of high-affinity clones happens predominantly after day 4 [START_REF] Malherbe | Clonal selection of helper T cells is determined by an affinity threshold with no further skewing of TCR binding properties[END_REF]. Additionally, whereas the two conflicting studies examine the repertoire at the level of Vα and/or Vβ expression profile, the latter explore the diversity at the level of CDR3 sequences. More recent studies indicate that the diversity of the naive population is more at the level of the CDR3 diversity than at the level of Vβ chain selection [START_REF] Neller | Naive CD8 T-cell precursors display structured TCR repertoires and composite antigen-driven selection dynamics[END_REF]. This means that individual Vα and Vβ families are likely to have the same ability to be recruited during the course of an infection, but that does not exclude the fact that within each of those families, there are predominant CDR3-divergent clones that are selected. The preimmune repertoire is composed of mature naive-phenotype cells (NP, dark blue), recent thymic emigrants (RTEs, light blue), and memory-phenotype cells (MP, red) that can arise by homeostatic proliferation (HP), cross-reactivity (CR, one black point), or be virtual memory cells (VM, two black points). A. Precursor frequency positively correlates with the magnitude of subsequent responses. In this simple situation, considering other parameters influencing immunodominance are controlled, epitope 2 will drive bigger expansion than epitope 1. B. However, the composition of a given antigen-specific population is likely to be more complex, and based on specific functional features of each of these subsets, likely influence the behavior of the population during and after expansion.

II. CHRONIC INFLAMMATION

In this section, I introduce general notions about inflammation. I present the hepatitis C virus (HCV) and characteristics of related chronic infection, and detail the reasons why we chose HCV as a model of chronic inflammation.

1) General introduction

Inflammation is the immune response of tissues to injury. Classical signs of acute inflammation include pain, heat, and redness. It is usually acute and localized, in response to trauma or infection.

However, if the causal agent persists, inflammation can become chronic and/or sytemic. Circulating monocytes are first attracted to the site of inflammation, where they differentiate into long-lived tissue-homing macrophages. Locally, they get activated by macrophage activating factors such as IFN-γ and MCP-1, while they are actively retained by migration inhibition factors, such as GM-CSF and IFN-γ [START_REF] Feghali | Cytokines in acute and chronic inflammation[END_REF]. Activated macrophages contribute to the inflammatory process by chronically elaborating low levels of IL-1 and TNF. This induces a cascade of recruitment/activation of immune cells that secrete a complicated network of multiple cytokines.

Schematically, we can differentiate:

• cytokines participating in humoral inflammation: IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-13, and transforming growth factor-β (TGF-β);

• cytokines contributing to cellular inflammation: IL-1, IL-2, IL-3, IL-4, IL-7, IL-9, IL-10, IL-12, interferons (IFNs), IFN-γ inducing factor, TGF-β, and tumor necrosis factor (TNF)-α andβ.

The Labex Milieu Interieur [START_REF] Thomas | The Milieu Intérieur study -An integrative approach for study of human immunological variance[END_REF] is a large-scale study ongoing in the lab, with the aim to assess the determinants underlying human immunological variance within the healthy population. On 1,000 healthy donor samples, it illustrated that the global pattern of secretion rather than the level of each individual cytokine defines a very specific and conserved proteomic signature to a given stimulus (Figure 10) [START_REF] Duffy | Functional Analysis via Standardized Whole-Blood Stimulation Systems Defines the Boundariesof a Healthy Immune Response to Complex Stimuli[END_REF]. Deconvoluting pathogen complex responses into a sum of specific signatures now appears as a reasonable strategy, and will hopefully result in identification of critical pathways that could be targeted with therapies.

Macrophages also release ROS and proteases, leading to important tissue damages -a hallmark of chronic inflammation. Some tissues have only little ability to regenerate (e.g. skin, neurons, cardiac cells), and are more affected than others. Aberrant repairing is frequent, and triggers fibrosis and neoangiogenesis, leading to restructurated and often less functional tissues.

Chronic inflammation is a feature of many human diseases including:

• allergy or food intolerance: asthma, celiac disease;

• inflammatory bowel diseases: Crohn's disease;

• rheumatologic disorders: rheumatoid arthritis, spondyloarthritis;

• persistent infection with mycobacteria, parasites, or fungi;

• autoimmune disorders: systemic lupus erythematosus, multiple sclerosis, psoriasis;

• atherosclerosis, obesity, diabetes, cancer, and Alzheimer's disease;

• and chronic viral infections: HIV, HCV, HBV, CMV, as well as other latent viruses such as EBV, HSV1/2, or HTLV1.

2) HCV as a model of chronic inflammation

Hepatitis literally means inflammation of the liver. It can be acute or chronic, symptomatic or not, and relate to many different causes. Viral hepatitis is the leading cause worldwide, and refers to infection with one of the five hepatotropic viruses A, B, C, D and E. Major characteristics are summarized in Table 4.

Table 4. Hepatotropic viruses.

Virological and clinical characteristics of HAV, HBV, HCV, HDV and HEV infections. From Park et al, Immunity 2014.

Our laboratory focuses on chronic infection with the hepatitis C virus (HCV), because it is a major public health problem, and a leading cause of primary liver cancer. HCV naturally trigger an immunemediated inflammatory response (hepatitis) that is either:

• acute, self-limited, and beneficial -leading to clearance of the infection in about one-third of the patients;

• chronic, systemic and deleterious -slowly destroying the liver and possibly leading to extrahepatic manifestations, including extra-hepatic carcinogenesis.

(a) Epidemiology

Hepatitis C virus (HCV) was identified in 1988, and cloned in 1997. Chronic infection by the HCV virus concerns 2-3% of population worldwide, i.e. 130-170 million people [START_REF] Mohd Hanafiah | Global epidemiology of hepatitis C virus infection: New estimates of age-specific antibody to HCV seroprevalence[END_REF]. Prevalence vary from one country to another, from <1% to >10%. Higher attack rate concerns Africa and Middle East, with a maximum of 14% in Egypt. The latter is a unique situation, where high prevalence of HCV infection is related to the usage of collective material for massively treating schistosomia in the 70's [START_REF] Frank | The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt[END_REF]. In France, prevalence of positive serology -defined as the detection in blood of anti-HCV IgG antibodies, is estimated 1%, i.e. ~350 000 individuals. Twothird are chronically infected, i.e. with detectable viral RNA in their blood. It is important to note that humans are the only known natural host for this virus; however, it can be transmitted to chimpanzeesand recently to genetically humanized mice -for experimental purposes [START_REF] Dorner | Completion of the entire hepatitis C virus life cycle ingenetically humanized mice[END_REF].

HCV virus circulates as 7 different genotypes, classified from 1 to 7, and further subdivided in subtypes identified by an alphabetical letter (1a, 1b,...). These subtypes correspond to nucleotidic variations from 20 to 30% [START_REF] Simmonds | Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes[END_REF]. Different genotypes and subtypes of HCV virus comes from its high nucleosidic mutation rate of 1.5×10 -3 /protein/year, which result from error prone replication by the RNA-dependent RNA viral polymerase. As such, different isolates -or quasispecies, also coexist within subtypes, and their evolution is largely dependent on the selective pressure of targeting CD8 + T cells [START_REF] Bailey | Constraints on Viral Evolution during Chronic Hepatitis C Virus Infection Arising from a Common-Source Exposure[END_REF]. In France, genotype 1 is predominant (60%)with 30% of subtypes 1b and 25% of genotype 1a, then genotype 3 (20%), genotype 2 (9%), genotype 4 (9%), genotype 5 (2%) and genotype 6 (<1%) [START_REF] Hajarizadeh | Epidemiology and natural history of HCV infection[END_REF] (Figure 11).

HCV virus is mainly parenterally transmitted, by transfusion or transplantation of infected samples, or usage of drugs intravenously (IV). The latest is now the main contamination mode in France (4,000 new cases, mainly in the two first years of usage), whereas transfusion of infected blood samples account for 40% of infection worldwide. Short exposure, trauma with infected needle, or mucosal exposure are less infectious. Percutaneous transmission is unlikely, which is very different from the hepatitis B virus (HBV) [START_REF] Park | Immune Responses to HCV and Other Hepatitis Viruses[END_REF]. Of note, reinfection after treatment and clearance of the virus is possible if risk factors are not controlled. [START_REF] Shimizu | Lipoprotein component associated with hepatitis C virus is essential for virus infectivity[END_REF]. These lipoviro-particles (LVP) have a diameter >100 nm, and contain the apolipoprotein B (ApoB). It has been proposed that LVP protect the virus from the host immune surveillance and/or increase interaction with lipoprotein receptors, which could act as a HCV receptor.

(c) Tropism and entry receptors

HCV liver tropism is mediated by expression of entry receptors at the cell surface of hepatocytes [START_REF] Penin | Structural biology of hepatitis C virus[END_REF][START_REF] Sattentau | Avoiding the void: cell-to-cell spread of human viruses[END_REF] (Figure 13). By circulating within blood, HCV is in direct contact with the basolateral surface of hepatocytes where it ligates surface receptors allowing its entry within the cells. Early step of viral attachment is a low-affinity interaction between the LPV and the LDL receptor and/or glycosaminoglycanes, both largely expressed on the target cell membrane. Then, HCV can use numerous higher affinity receptors to enter hepatocytes in a coordinated multistep process.

This includes the scavenger receptor class B type I, the tetraspanin CD81, the tight junction proteins claudin 1 and occludin (OCLN), the receptor tyrosine kinases epidermal growth factor receptor and ephrin receptor A2, and the cholesterol uptake receptor Niemann-Pick C1 like 1. Of these, CD81 and OCLN comprise the minimal human factors needed for HCV uptake into rodent cells [START_REF] Dorner | Completion of the entire hepatitis C virus life cycle ingenetically humanized mice[END_REF].

Other sites of replication have been reported, including biliary cells, salivary glands, epithelial cells, pancreas, thyroidis, bone marrow, spleen, lymph nodes and brain [START_REF] Zignego | Extrahepatic manifestations of HCV infection: facts and controversies[END_REF]. The more debated concerns the ability of HCV to infect lymphocytes. Whereas some studies where biased by identifying the virus within cells possibly contaminated by viral particles (viremic patients at the time of collection), others clearly demonstrate replication in the absence of detection of the virus into the blood [START_REF] Kondo | Direct effects of hepatitis C virus on the lymphoid cells[END_REF]. In a recent study, despite comparable expression levels of CD81, only T cell lines displaying moderate to high levels of CD5 protein were found to be susceptible to HCV infection [START_REF] Sarhan | Hepatitis C Virus Infection of Human T Lymphocytes Is Mediated by CD5[END_REF]. Both anti-CD5 and anti-CD81 MAbs inhibited infection of permissive T cells, indicating that both are required for HCV entry into T cells.

Considering the pan-T cell expression of CD5, it suggests that while CD81 may contribute to broad recognition of cells by HCV, CD5 facilitates HCV tropism specifically toward lymphocytes. As degree of infection usually correlates with levels of expression of entry receptors, it is possible that naive T cells, which express the highest levels of CD5, are the main target lymphocyte population for HCV, and a previously ignored reservoir for the virus.

(d) From acute to chronic infection

Acute infection by HCV leads to acute hepatitis -i.e. that started less than 6 months ago. The incubation period averages 6 to 10 weeks. 80% of HCV-induced hepatitis are asymptomatic, with cases being detected by serology. Others present clinical symptoms such as asthenia, fever, jaundice, acholic stools, and dark urines. After exposure, it takes (Figure 14):

• 2-14 days for the virus to be detectable in the blood, in parallel with an increase in transaminases;

• 8-12 weeks to cellular immunity to take place, and thus to anti-HCV antibodies to develop and be detectable by serology [START_REF] Halliday | Vaccination for hepatitis C virus: closing in on an evasive target[END_REF] ;

• 20 weeks to the virus to be either spontaneously cleared (30%), or persistent (70%). Chronic infection by HCV is defined as persistence of the virus for more than 6 months. It associates with hepatic injury such as necrosis, inflammation and fibrosis. HCV is not cytopathic, except in rare immunocompromised situations. It rather leads to immune-mediated liver injury. Histology testing distinguishes persisting chronic hepatitis -considered as benign, and active chronic hepatitis. Chronic HCV infection may evolve to cirrhosis in 10-20% of patients, depending on the presence or absence of cofactors [START_REF] Wiese | Evaluation of liver disease progression in the German hepatitis C virus (1b)contaminated anti-D cohort at 35 years after infection[END_REF]. Cirrhosis can complicate with liver-related complications (ascites, hepatic encephalopathy, icterus, variceal bleeding, spontaneous bacterial peritonitis), or lead to hepatocellular carcinoma (HCC). Incidence for disease progression is 2-4%/year [START_REF] Kew | Hepatocellular carcinoma in developing countries: Prevention, diagnosis and treatment[END_REF]

(e) Extrahepatic manifestations

Clinical manifestations of chronic HCV are not restricted to the liver [START_REF] Zampino | Chronic HCV infection and inflammation: Clinical impact on hepatic and extra-hepatic manifestations[END_REF]. Several extra-hepatic manifestations are occasionally observed in those patients (Figure 15):

• 50% present with mixed cryoglobulinemia (MC), a benign B-cell lymphoproliferative disease.

Its definition is biological, and corresponds to the detection into the serum of immunoglobulins (Igs) capable to reversibly precipitate at low temperatures. HCV-related MC are typically type II (the precipitate is composed of both monoclonal and polyclonal Igs), or type III (the precipitate is only formed by polyclonal Igs). Viral antigens and HCV-RNA are consistently isolated from the precipitate, suggesting a direct role of the virus in MC pathogenesis. Only 15% of patients with MC are symptomatic, associating with a poor prognosis related to development of B-cell malignancies in 10% of them. Clinical symptoms of MC are those of vasculitis due to precipitation of Igs complexes in small-and mediumsized blood vessels. Livedo, purpura, ulcers, arthralgia, and arthritis are related to thrombosis of small vessels and systemic inflammation. Vasculitis can also affect the kidneys, the gastrointestinal tract, the peripheral nervous system, and/or other body compartments;

• 50% present neurological symptoms including cognitive disorders, depression, and asthenia, with perturbed quality of life;

• 30-50% present steatosis, insulinoresistance, and/or metabolic syndrome;

• some develop cardiovascular diseases, including atherosclerosis and coronaropathy, independently of the precited metabolic loss of balance, which translates into increased mortality [START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a community-based long-term prospective study[END_REF] ;

• some develop extra-hepatic cancers, which are not restricted to a particular site, suggesting disruption of a common mechanism for carcinogenesis. Esophagus, prostate, and thyroid cancers, as well as non-Hodgkin B cell lymphomas are more frequently reported [START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a community-based long-term prospective study[END_REF].

Non-cryoglobulinemic extrahepatic manifestations are thought to reflect the activation of proinflammatory immune responses rather than to be related to the virus itself.

(f) Treatment and clearance benefits

HCV is to date the only chronic viral infection that can be cured. Curing is achieved by induction of a Sustained Virologic Response (SVR) by appropriate treatment. Sustained virologic responders (SVRs)

-i.e. patients achieving SVR, are individuals with no detectable virus in the serum 3-6 months after the end of the treatment. It probably correspond to complete eradication of the virus from the blood, the liver, and any potential other reservoir. Indeed, after SVR, reappearance of the virus never happens, even when patients later undergo severe immunosuppression (e.g. chemotherapy or organ transplantation).

In the 80's, patients were initially treated with subcutaneous injections (SC) of alpha-interferon (IFN α). It acts both directly on the virus, and indirectly by immuno-modulating the cellular immune response, reinforcing the endogenous interferon naturally present in response to HCV. It cured only 6-12% of patients after 6 months. Extension to 12 months increased prolonged response to 16-20%. In the 90's, a nucleosidic analogue -ribavirine (RBV), was proposed in association with IFNα. Also it had no effect in monotherapy, it permitted to double virological efficacy when associated to IFNα With improved knowledge on the cell cycle of HCV, direct acting antiviral agents (DAA) have been recently developed [START_REF] Pol | Oral antiviral therapies for chronic hepatitis C infection[END_REF] (Figure 12). First molecules in 2011 were inhibitors of NS3/NS4A protease (telaprevir and boceprevir). Association of these molecules to peg-IFNα and RBV permitted to increased SVR to 75 % in genotype 1 naive patients, and is the recommended treatment for genotype 1 patients, especially those at risk of non responding to classical bitherapy.

However, protease inhibitors can lead to serious secondary events such as cutaneous eruptions, or severe anemia in 49% of treated patients. Also, these treatments are expensive -35 000 euros for one tritherapy, and thus not affordable worldwide. Since then, other direct molecules have been developed: second generation of proteases inhibitors, nucleos(t)idic or non nucleos(t)idic polymerase NS5B inhibitors, and inhibitors of the NS5A replication complex. The current interferon-free oral combinations allow more than 90% of cure for all patients. The main limitation remains their cost, estimated between 40 000 to 75 000 euros per individual.

On the liver, viral clearance leads to fibrosis -and even cirrhosis -regression, and significantly reduces liver-related morbi-mortality. The estimated 10-years mortality rate is 1.9% on SVR patients, in comparison with 27.4% on non-responders (van der [START_REF] Van Der Meer | Is there sufficient evidence to recommend antiviral therapy in hepatitis C?[END_REF]. Sustained virologic response also leads to normalisation of transaminases, and improvement of extrahepatic manifestations, both MC-related or not [START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a community-based long-term prospective study[END_REF].

(g) Antiviral immune responses

Viral infection leads to several intracellular events that permit to initiate the host immune response, both inside the infected cells and within the micro environment [START_REF] Gale | Evasion of intracellular host defence by hepatitis C virus[END_REF]. HCV viral RNA is sensed inside the hepatocytes by pathogen-associated molecular pattern molecules (Figure 15). Beside hepatocytes, HCV virus can also be sensed by:

Several

• Macrophages after phagocytosis;

• Plasmacytoid dendritic cells (pDCs) after take up HCV RNA via exosomes (demonstrated in vitro, but not yet in vivo).

Inside those cells, the virus triggers TLR7, and initiates an inflammatory IL-1β response, as well as IFNα production [START_REF] Park | Immune Responses to HCV and Other Hepatitis Viruses[END_REF]. With no inhibition by viral particles, they could thus be the source of ISG-inducing type I IFNs.

High levels of IFNα in the early steps of infection contribute to NK cells activation. After recognition and elimination of infected cells, they produce proinflammatory cytokines (IFNγ) and participate to polarization of T cell responses toward cytotoxicity. NK cells with the greater capacity to produce IFNγ translates into more efficient clearance of HCV [START_REF] Albert | Plasmacytoid dendritic cells move down on the list of suspects: In search of the immune pathogenesis of chronic hepatitis C[END_REF].

Inflammatory chemokines such as RANTES, MIP-1α, and MIP-1β quickly recruit circulating immature DC (imDC) to the site of inflammation (e.g. the HCV-infected liver), where they can take up viral antigens. This is mediated by their binding to their corresponding chemokine receptors (CCR1, CCR2, and CCR5 respectively) at the surface of imDC. This is followed by functional maturation of DC, in parallel with modifications of the pattern of expression of chemokine receptors.

Mature DC typically express high levels of CCR7, which leads them to quit the tissue, and home into CCL19 and CCL21-expressing T cell areas of lymphoid tissues. This migration step is required for appropriate priming of HCV-specific T cells. As HCV-specific T cell responses play a key role in viral clearance, and are often found as defective in the context of chronic infection, the functionality of cDCs in the context of cHCV has been regularly questioned, and led to controversial conclusions.

Initial reports have shown impaired maturation and allostimulation of monocyte-in vitro-derived DCs from patients with chronic HCV infection. However, more recent analysis using direct strategies to identify and characterize circulating DCs reported no phenotypic or functional differences between cHCV patients and healthy donors [START_REF] Longman | Normal functional capacity in circulating myeloid and plasmacytoid dendritic cells in patients with chronic hepatitis C[END_REF].

Despite rapid innate responses, HCV-specific adaptive immune responses are not detectable before 8-12 weeks after infection [START_REF] Park | Immune Responses to HCV and Other Hepatitis Viruses[END_REF]. This is observed irrespectively of clinical outcome. Importantly, the appearance of HCV-specific CD8 T cells in the blood precisely coincides with the CD8 T-cell infiltration to the liver, as assessed by CD8β mRNA levels. This delay is very unique to HCV. It has been proposed that secretion of high levels of chemokines by the HCV-infected liver at early time points could sequester unprimed CD8 T cells expressing the corresponding receptors. After this delay, one can observe:

• HCV-specific humoral responses: they are mainly targeting the highly variable region 1 (HVR1) of the E2 glycoprotein [START_REF] Halliday | Vaccination for hepatitis C virus: closing in on an evasive target[END_REF]. Their appearance is variable between individuals, and always significantly delayed after the infection. They are restricted in their isotype profile, found at low levels only, and not maintained for life. In sum, HCV-specific antibodies happening during the course of chronic infection do not confer protection: reinfection can occur after clearance, and clearance can occur without detection of specific antibodies.

• HCV-specific cellular immune responses [START_REF] Klenerman | T cell responses in hepatitis C: the good, the bad and the unconventional[END_REF] : HCV-specific T cells likely play a key role in the elimination of the virus as (i) there is a temporal correlation between increased expression of CD8β and IFNγ mRNA into the liver, viral clearance and the appearance of HCV-specific CD8 + T cell responses in both blood and the liver of acutely infected humans and chimpanzees [START_REF] Thimme | Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease[END_REF] ; (ii) depletion of either CD4 + or CD8 + T cells prevents HCV clearance in the chimpanzee model [START_REF] Shoukry | Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection[END_REF] ; and

(iii) there is a strong correlation between protective class I HLA alleles such as HLA-B27, HLA-B57, and HLA-A3 and spontaneous viral clearance [START_REF] Fitzmaurice | Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection[END_REF].

Specifically:

" patients who later clear the virus typically mount broad CD4 + T cell responses, with high proliferation and cytokine production capacities [START_REF] Missale | Lack of full CD8 functional restoration after antiviral treatment for acute and chronic hepatitis C virus infection[END_REF][START_REF] Shoukry | Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection[END_REF]. In contrast, HCV-specific CD4 + T cell responses are absent or weak in those who subsequently develop chronic infection. In vivo depletion of CD4 + T cells from HCV-recovered chimpanzees abrogates protective CD8 + T cell-mediated immunity upon rechallenge. This highlights that CD4 + T cell help is required for the generation and maintenance of protective CD8 + T cells, but do not seem to mediate direct antiviral effects [START_REF] Shoukry | Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection[END_REF] ;

" in contrast, whatever the clinical outcome, all patients typically present ''stunned'' HCV-specific CD8 + T cells, with impaired proliferation, IFNγ production, and cytotoxicity, as well as transient surface expression of PD-1 and CD38. Antiviral therapy in this phase of the infection results in a rapid decay of CD8 T cell responses, suggesting that most of these HCV-specific CD8 + T cells are short-lived, antigen-dependent effector cells. If HCV infection is self-limited, dysfunction progressively resolves, HCV-specific CD8 + T cell responses are stronger, broader, higher-affinity, and express high levels of Bcl2 and CD127 [START_REF] Neveu | Selection of high-avidity CD8 T cells correlates with control of hepatitis C virus infection[END_REF]. If not, they remain weak, restricted, and low-affinity, and the infection evolves toward chronicity (Figure 17).

Schematically, perturbations of CD8 T cell responses in cHCV patients concern:

• their number: HCV-specific responses are weak and hardly detectable even by cell-based (tetramer) assays [START_REF] Klenerman | T cell responses in hepatitis C: the good, the bad and the unconventional[END_REF];

• their affinity: cell lines derived from cHCV patients are typically low-avidity, and express low levels of CD3. Importantly, this phenomenon is reversible as emergence of high-avidity clones correlates with the control of the infection [START_REF] Neveu | Selection of high-avidity CD8 T cells correlates with control of hepatitis C virus infection[END_REF];

• their phenotype: HCV-specific responses are classically blocked into an early central-memory phenotype [START_REF] Appay | Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections[END_REF].

Several mechanims have been identified that participate to this failure:

• a lack of CD4 help. As mentioned above, CD4 + T-cell help is required for the induction and maintenance of sustained CD8 + effector T-cell functions [START_REF] Shoukry | Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection[END_REF];

• chronic antigenic stimulation: this has been demonstrated in mice with lymphocytic choriomeningitis virus (LCMV) infection. High levels of persisting viral antigen result in sequential loss of T cell function: the capacity to produce IL-2 is lost first, followed by cytotoxicity, TNF-α, and ultimately IFN-γ production [START_REF] Park | Immune Responses to HCV and Other Hepatitis Viruses[END_REF]) ;

• T-cell dysfunction: antiviral T cells typically express high levels of pro-apoptotic and activation molecules such as Bim and PD-1 respectively [START_REF] Larrubia | Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1+/CD127-HCV-specific CD8+ cells targeting the virus in chronic hepatitis C virus infection[END_REF], and low levels of Tbet [START_REF] Kurktschiev | Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction[END_REF]. They have reduced effector functions, with a poor capacity of proliferating and secreting IFN-γ after in vitro stimulation. Restoration of HCV-specific CD8 + T cells can be achieved in vitro by blocking both CTLA4 and PD-1/PD-L1 interactions [START_REF] Fuller | Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1)[END_REF];

• increased numbers of Treg cells and inhibitory cytokines (e.g., IL-10 and TGF-β) [START_REF] Larrubia | Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1+/CD127-HCV-specific CD8+ cells targeting the virus in chronic hepatitis C virus infection[END_REF] : the liver is a naturally tolerogenic environment. One of the mechanisms is that Kuppfer cells, as well as intrahepatic regulatory T cells, constitutively express IL- 3) HBV as a comparative model of chronic hepatitis

The hepatitis B virus is a DNA virus composed of a nucleocapsid core (HBc Ag) surrounded by an outer lipoprotein coat (envelope) containing the hepatitis B surface antigen (HBs Ag) (Table 4). It replicates via reverse transcription of a pregenomic RNA (pgRNA). Similar to HCV, HBV virus is hepatotropic, non cytopathogenic, and a major public health problem. Indeed, 350 millions persons are currently infected with HBV worldwide, with high risk of developing severe liver disease -chronicity, cirrhosis, liver failure and hepatocellular carcinoma (HCC), like in chronic HCV infection.

After acute infection, 95% of infected adults clear spontaneously the HBV virus -but only 1% in the case of vertical transmission from mother to neonate. This is a major difference with HCV infection, where spontaneous clearance happens in 1/3 of patients whatever their age. Schematically, chronic HBV infection evolves through three immune phases (Figure 18):

• immune tolerant (IT): HBe Ag + , HBV DNA high , ALT normal ;

• immune clearance, or immunoactive (IA): HBe Ag + , HBV DNA high , ALT elevated, clinical symptoms ;

• inactive chronic carrier (ICC): HBe Ag -, HBV DNA low , ALT normal.

HBe Ag is a viral protein that is secreted by the HBV-infected cells. It is associated with chronic HBV infections. HBe Ag positivity is a marker of active viral disease and infectiousness. However, rare cases of e-Ag negative virus mutations can lead to false negativity of HBe Ag, and to misinterpretations.

The phases of chronic HBV infection are not considered static and patients who initially develop the IT phase usually progress to the IA phase, and later on to the ICC state. Progression in the reverse direction can also happen, either spontaneously, or after withdrawal of antiviral or immunosuppresive therapies (corticosteroids, TNFα blockers, cancer chemotherapy). The HBV genome form a covalently closed circular DNA (cccDNA) microchromosome that persists in the nucleus of virusinfected hepatocytes, serving as a template for viral mRNA synthesis [START_REF] Zhang | Regulation of hepatitis B virus replication by epigenetic mechanisms and References microRNAs[END_REF]. It also integrate into the host genome (Jiang et al., 2012b). As such, in contrast to HCV, current antiviral agents control but do not eliminate HBV. Integrative events and very low levels of cccDNA persist indefinitely into hepatocytes, and are key events of hepatocarcinogenesis. This explains lifelong (i) risk of HCC emergence, and (ii) maintenance of anti-HBV responses, despite clinical resolution of the infection.

Like HCV, acute HBV is cleared by a strong adaptive immune response that confers lifelong protective immunity. However, due to its special replication strategy, HBV behaves as a stealth virus, and do not induce ISG expression, even if infecting almost 100% of hepatocytes. This is a major difference between the two viruses. HBV rather induces a type III interferon response, that leads to increased expression of CXCL10, OAS2, and RSAD2 in the human liver [START_REF] Sato | The RNA Sensor RIG-I Dually Functions as an Innate Sensor and Direct Antiviral Factor for Hepatitis B Virus[END_REF]. This does not impair the induction of a vigorous HBV-specific CD4 + T cell response, that appear much earlier than in HCV infection. Similar to HCV, vigorous multi-epitope-specific CD4 + and CD8 + T-cell responses are essential for viral clearance, and as illustrated by the negative effect of depletion of either CD8 + or CD4 + T cells. When evolving towards chronicity, there is an inverse correlation between virus levels, and detection of HBV-specific T cells in the blood. CD8 + T cell failure is mainly due to T cell dysfunction, with similar characteristics as during HCV infection (high sensitivity to cell death, high expression of PD-1, low levels of Tbet, and poor effector functions). This exhaustion phenotype is efficiently reversed by PD1/PDL1 blockade.

Comparison between HBV and HCV physiopathogenesis raise the hypothesis that the absence rather than presence of a strong type I IFN response is critical for the induction of successful CD4 + and CD8 + T cell responses. Mechanisms accounting for this observation are unknown.

4) Viro-induced chronic inflammation

Inflammation is a physiological pathway in response to exogenous stimuli. However, a chronic inflammatory state or an excessive inflammatory response can produce deleterious effects. Liver cells, including hepatocytes, hepatic stellate cells (HSCs), Kuppfer cells (KCs), bile duct epithelial cells and sinusoidal endothelial cells, are implicated in the synthesis and secretion of a large number of proinflammatory stimuli (proinflammatory cytokines, chemokines, growth factors and inflammatory enzymes), and thus plays a central role in the initiation and the regulating this process [START_REF] Zampino | Chronic HCV infection and inflammation: Clinical impact on hepatic and extra-hepatic manifestations[END_REF].

HCV causes major liver inflammation by:

• Direct pathways:

" NS5A promotes inappropriate upregulation of cyclooxygenase-2 (COX-2), which is an inducible COX isozyme able to contribute to chronic inflammation and fibrosis through production of various prostaglandins [START_REF] Nunez | Increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2, and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis C virus infection: role of viral core and NS5A proteins[END_REF];

" Non-structural proteins (NS3, NS4, NS5) lead to TNF-α and IL-1β production by KCs;

• Indirect pathways: after its recognition by hepatocyte and innate cells, it triggers recruitment and activation of inflammatory cells into the liver. Interleukin-1β (IL-1β) and related inflammasome play a central role in orchestration of this inflammatory response [START_REF] Petrilli | The inflammasome: a danger sensing complex triggering innate immunity[END_REF], inducing local production of several pro-inflammatory cytokines and chemokines, such as IL-6, IL-8, and macrophages inflammatory proteins (MIP-1α and MIP-1β), by hepatocytes ;

• Mediating the recruitment of both virus-and non-virus-specific T cells into the liver [START_REF] Maini | The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection[END_REF], leading to overreaction with liver antigens, and liver damages.

Outside of the liver, cHCV also triggers systemic inflammation, as illustrated by increased levels of pro-inflammatory cytokines in their serum, activated blood monocytes, and increased local and systemic oxidative stress [START_REF] Mencin | Toll-like receptors as targets in chronic liver diseases[END_REF]. Recent studies highlighting the frequency of extrahepatic HCV-related diseases brought new interests into systemic inflammation [START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a community-based long-term prospective study[END_REF]. In particular, increased ratios of proinflammatory/anti-inflammatory cytokines (TNF-α/IL10

and IL-6/IL-10) correlates with an increased risk of cardiac failure and death in cHCV patients [START_REF] Oliveira | Effects of Hepatitis C virus on cardiovascular risk in infected patients: a comparative study[END_REF]. Additionally, increased oxidative stress has been proposed as a strong inducer of damaged DNA, potentially promoting the development and progression of hepatocellular carcinoma (HCC) as well as extrahepatic cancers [START_REF] Maki | Predictive Power of Biomarkers of Oxidative Stress and Inflammation in Patients with Hepatitis C Virus-Associated Hepatocellular Carcinoma[END_REF]. Several groups are now dissecting HCV-related systemic inflammation with the aim to identify targets for anti-inflammatory therapies that could help prevent or treat related complications, as well as pathological pathways involved into carcinogenesis or diabetes development.

HBV also induces strong liver inflammation in parallel of viral replication. However, the exact mechanisms are unknown. It was recently demonstrated that RIG-I can sense some pregenomic RNAs within the infected cells, triggering a specific type III/not type I interferon response [START_REF] Sato | The RNA Sensor RIG-I Dually Functions as an Innate Sensor and Direct Antiviral Factor for Hepatitis B Virus[END_REF].

Clinically, it is less frequently associated with extrahepatic manifestations, and those are different from those induced by HCV in both their localization and pathogenesis (vessels in polyarteritis nodosa, kidney in glomerulonephritis).

5) T cells and inflammation

Naive CD8 + T cells need three concomittant signals: (i) TCR, (ii) CD28 ligation, and (iii) inflammatory cytokines [START_REF] Richer | Pathogen-Specific Inflammatory Milieux Tune the Antigen References Sensitivity of CD8+ T Cells by Enhancing T Cell Receptor Signaling[END_REF][START_REF] Wilson | Inflammation Makes T Cells Sensitive[END_REF] to get activated, and differentiate into a spectrum of effector populations possessing unique capacities for survival, proliferation, cytokine expression, cytotoxicity, and memory formation. Currently, there is great interest in understanding how distinct inflammatory cytokines direct and tune CD8 + T cell responses.

Indeed, while there is a detailed literature on CD8 + memory T cells generation and function in response to specific pathogens, it is less clear how these response behave in the context of concurrent infection. This becomes an important distinction since approximately one third of the world's population harbors a persistent infection -mostly HIV, malaria, and tuberculosis [START_REF] Krishnamurty | Inflammatory interference of memory formation[END_REF]. If non-pathogenic microbes are considered (such as herpes viruses), this rate is clearly 100% [START_REF] Virgin | The Virome in Mammalian Physiology and Disease[END_REF], and we currently know very few about how they shape the immune system.

To date, only two bystander effect of chronic infection on unrelated T cell responses have been extensively documented:

• inflammatory cytokines tune the affinity of unrelated memory T cells for a faster and more efficient effector response to cognate antigens [START_REF] Richer | Pathogen-Specific Inflammatory Milieux Tune the Antigen References Sensitivity of CD8+ T Cells by Enhancing T Cell Receptor Signaling[END_REF]. This has been well demonstrated in the model of listeria monocytogenes (LM) and lymphocytic choriomeningitis virus (LCMV) infections. This is mediated by IL-12 and IFN-I cytokines [START_REF] Raué | Cytokine-Mediated Programmed Proliferation of Virus-Specific CD8+ Memory T Cells[END_REF], that enhance the downstream TCR signaling ;

• the inflammatory environment associated with a persistent infection divert memory cells from a long-lived fate to a more terminally differentiated effector phenotype, with a conserved inflammation-driven gene expression program (Figure 19).

Importantly, those two effects are reversible when infection is resolved, meaning that they are not hard-wired, but rather an active phenomenon. The possibility that similar effects affect the other T cell compartments has not been explored yet.

III. T CELL TRAFFICKING

1) Short introduction about chemokines

Chemokines are 8-to 12-kDa heparin-binding molecules with the ability to drive leukocyte trafficking and positioning within tissues. Chemokine-mediated trafficking of leukocytes allows these immune players to be directed to the places where they are needed. Therefore these processes play a critical role in regulating not only immune responses and inflammation but also homeostatic cell positioning.

To allow cell migration, multiple chemokines and chemokine receptors can be involved, ensuring flexibility and specificity of leukocyte recruitment [START_REF] Oo | The Role of Chemokines in the Recruitment of Lymphocytes to the Liver[END_REF].

In the human system, there are more than 50 chemokines and 20 chemokine receptors that can be divided into structural subsets based on the presence of NH2-terminal cysteine motifs (Figure 20 and Table 5). The large CC chemokine family consists of chemokines in which the first two cysteine residues are adjacent, whereas in CXC chemokines, they are separated by a single amino acid residue.

Fractalkine (CX3CL1) is the only member of the CX3C chemokine family in which three amino acid residues separate the first two cysteines. Finally, two related chemokines, XCL1 and XCL2, both of which bind the XCR1 receptor, lack two adjacent cysteine residues. Chemokines exert their chemotactic functions by binding to cell-surface G protein-coupled receptors (GPCRs), resulting in mobilization of intracellular Ca 2+ , which results in receptor internalization and the initiation of signaling pathways that facilitate chemotaxis. Consistent with Gi association, the majority of chemokine responses are inhibited by treatment with pertussis toxin, which blocks the global G protein receptor. Chemokines are generally classified into two functional groups, 'inflammatory' and 'homeostatic/constitutive', based on whether they are induced by inflammation or constitutively expressed and involved in homeostatic immune regulation. However, this distinction is somehow erroneous, as classical homeostatic chemokines such as CCL19 and CCL21 (CCR7 ligands) and CXCL13 (CXCR5 ligand) are also induced at sites of chronic inflammation.

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers [START_REF] Wei | A stochastic view of lymphocyte motility and trafficking within the lymph node[END_REF]. It consists ligand recognition, cell polarization, and directed cell migration, and requires integration by the cell of numerous signals that will help it decide in which direction to move.

Chemotaxis starts occuring at relatively low concentrations of chemokines, and is dose-dependent.

However, at high concentrations, cells are paralyzed and no longer migrate.

Lymphocytes chemokinesis is the cell mobilization through cytoskeletal rearrangements [START_REF] Wei | A stochastic view of lymphocyte motility and trafficking within the lymph node[END_REF]. T cell progress by a series of repetitive lunges, repeatedly balling-up and then extending. This cycle appears to be driven by an intrinsic rhythmicity, with a period of about 2 minutes. During each cycle, the cell progresses an average distance of about 20 micrometers, i.e. T cells have a mean velocity of 10 micrometers per minute. However, following each pause, there is a high probability that the cell will take off in another direction. Thus, tracks of T cells display a random walk pattern when followed over long times, although motions at short time are more linear.

2) Naive T cell trafficking

To be able to engage an immune response, naive T cells permanently patrol SLOs, therefore optimizing the chance to encounter an APC harboring a cognate antigen. This processed is largely aided by CCR7-mediated migration, induced by CCL19 and CCL21 -expressed at high levels in high endothelial venules in SLOs. Similar as thymic export, egress from the lymph node is an active process, and requires both gradual desensitization to CCR7 signals, and re-expression of S1P 1 . The balance between « stay » and « go » signals through CCR7 and S1P 1 is a point of regulation of duration of naive T cells stay into the lymph nodes [START_REF] Takada | Naive T cell homeostasis: from awareness of space to a sense of place[END_REF]. Naïve T cells also express CXCR4. CXCR4 on naive T cells also promotes lymph node entry by binding to CXCL12, which is produced by the follicular reticular cells and presented on the high endothelial venules lumen. This is a minor signal compared with CCR7, as mice deficient in CXCR4 exhibit defects in naive T cell homing only in the absence of CCR7 signaling [START_REF] Okada | Chemokine Requirements for B Cell Entry to Lymph Nodes and Peyer's Patches[END_REF].

In the context of infection, CCR7 and its ligands CCL19 and CCL21 recruit naive T cells and mature APCs to the lymph nodes, and promote their mobility to optimize rare antigen-specific naive CD8 + T cell and cognate antigen-loaded DC encounters. During these early transient encounters, unknown signals upregulate the expression of various chemokine receptors, including CCR5, CCR4, and CXCR3, and downregulate CCR7 on the CD8 + T cells. CCR5 and CCR4 ligands are then produced by DCs that have come into contact with antigen-specific or activated CD4 + T cells, CD8 + T cells, and/or NKT cells, resulting in additional recruitment of naive CD8 + T cells [START_REF] Griffith | Chemokines and Chemokine Receptors: Positioning Cells References for Host Defense and Immunity[END_REF].

3) Effector and resident memory T cells trafficking

After activation, effector CD8 + T cells downregulate LN-homing CD62L and CCR7 and start expressing chemokine receptors such as CXCR3, CCR5, CXCR6, CCR6, CXCR1, and CX3CR1.

These induced chemokine receptors mediate effector CD8 + T cell migration to sites of inflammation.

Importantly, the site of priming appears to imprint effector T cells with the ability to traffic to particular organs [START_REF] Agace | Tissue-tropic effector T cells: generation and targeting opportunities[END_REF]. In the gut, CD103 + DCs and stromal cells from Peyer's patches and mesenteric LNs produce retinoic acid that triggers expression of α4β7 and CCR9 on T cells.

Interactions with their cognate ligands -endothelial-derived MAdCAM and epithelial-derived CCL25

-directs activated T cells to the small intestine. Similarly, T cells primed within the skin-draining inguinal LN preferentially migrate to the skin. This is mediated by cutaneous lymphocyte antigen (CLA), the mouse E/P-selectins, and the chemokine receptors CCR4, CCR8, and CCR10. CCR10 expression is induced by the active form of vitamin D3, locally synthesized by skin DCs [START_REF] Sigmundsdottir | DCs metabolize sunlight-induced vitamin D3 to "program" T cell attraction to the epidermal chemokine CCL27[END_REF].

T CM cells are defined by the coexpression of LN-homing CD62L and CCR7, and their recirculation through the blood and SLOs. Unlike naive cells, however, they express inflammatory chemokine receptors such as CXCR3 and CXCR4 that may serve as a CCR7-independent LN entry [START_REF] Scimone | CXCL12 Mediates CCR7-independent Homing of Central Memory Cells, But Not Naive T Cells, in Peripheral Lymph Nodes[END_REF]. Recently, the expression of CXCR3 on T CM has been found to have a critical role for their function [START_REF] Sung | Chemokine Guidance of Central Memory T Cells Is Critical for AntiviralRecall Responses in Lymph Nodes[END_REF]. The CD8 + T CM response was directly compared with naive CD8 + T cells after injection of antigen-pulsed DCs or infection with LCMV. Surprisingly, the response to antigenpulsed DCs was similar in naive T and TCM cells except for an enhanced IFN-γ response by the TCM. However, when the same comparisons were made during LCMV infection, T CM had higher CD69 expression, proliferation, and IFN-γ production compared with the naive cells. This was related to the specific recruitment of CXCR3 + T CM into specific T cell zones of the LN with a predominance of LCMV-infected macrophages. CXCR3 ligands chemokines then recruit additional CXCR3 + T CM to the periphery of the LN and sites of active LCMV infection in a feed-forward mechanism that resulted in their quick activation and expansion in response to infection. Thus, CCR7-mediated LN homing coupled with CXCR3 expression on T CM positions these cells to quickly control the spread of virus within the lymphatic system upon rechallenge.

Resident-memory T cells (T RM ) have been defined by their preferential tissue residence. These cells have been definitively identified in the skin, vaginal mucosa, central nervous system, salivary gland, lung, and intestine and are characterized by the increased expression of CD103 and CD69 and a lack of CD62L and CCR7 [START_REF] Mueller | Memory T Cell Subsets, Migration Patterns, and Tissue Residence[END_REF]. Chemokines are important in both the development and maintenance of CD8 + T RM . Into the skin, during HSV infection, CD8 + T RM development is dependent upon localization of CD8 + effector precursor cells to the epidermis by CXCR3-CXCL10 interactions, strong enough to counteract a CCR7-dependent tissue exit signal (Mackay et al., 2013). CD8 + T RM are further maintained in the tissue by downregulation of S1P 1 [START_REF] Skon | Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8[END_REF]. T RM trigger a state of tissue-wide pathogen alert, and provide protection from reinfection or reactivation of latent skin viruses [START_REF] Ariotti | Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert[END_REF][START_REF] Gebhardt | Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus[END_REF]Jiang et al., 2012a). CD8 + T RM quickly produce IFN-γ in response to cognate antigen. This results in robust local production of chemokines, such as CXCL9, which recruits circulating memory CD8 + T cells independent of their antigen specificity [START_REF] Ariotti | Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert[END_REF].

Finally, mucosal-associated invariant T (MAIT) cells are a conserved sub-population of T cells, characterized by their expression of the semi-invariant Vα7.2-Jα33 chain of the TCR, and restricted to the non-polymorphic MHC class I-related protein 1, MR1 [START_REF] Kurioka | MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets[END_REF]. Those cells are broadly reactive to microbes, and in particular critical in the control of Bacillus Calmette-Guérin (BCG), Klebsiella pneumonia, and Francisella tularensis infections in mice. MAIT cells are enriched in the intestine and the liver, and represent up to 20% of CD8 + T cells in the periphery in humans.

They have been described to be CD8ab, CD8aa, or double-negative (DN), although a differential role for these subsets has not been explored [START_REF] Walker | Human MAIT and CD8 cells develop from a pool of type-17 precommitted CD8+ T cells[END_REF]. In adult blood, MAIT cells are the dominant population among CD8+ T cells expressing the C-type lectin-like receptor CD161 at high levels. These CD161hi CD8+ T cells are polyclonal, and functionally and transcriptionally distinct from other CD8+ T cell populations, with the ability to be activated by Il-12 and Il-18 in a TCRindependent manner. Different from NKT, those cells are naive and low numbers in the newborn thymus and cord blood, and later evolve to abundant and effector-memory phenotype cells in adults.

They share similarities with Th17 cells, and are activated and enriched non-specifically at inflammatory sites such as liver and joints, where they have the ability to kill target cells via important cytotoxicity.

4) Role of CXCR3 in T cell biology

CXCR3 is a seven-transmembrane domain receptor (G protein-coupled receptor) that has the ability to bind three chemokines: CXCL9 (also known as MIG, monokine induced by gamma-interferon), CXCL10 (or IP-10, interferon-induced protein of 10kDa), and CXCL11 (or I-TAC, interferon inducible T cell alpha chemoattractant) [START_REF] Groom | CXCR3 in T cell function[END_REF]. In human T cells, three isoforms of CXCR3 exist as a result of alternative splicing: CXCR3-A -the classical and more abundant form of the receptor, CXCR3-B -which not only binds CXCL9, CXCL10 and CXCL11, but also PF4/CXCL4, and CXCR3-alt -which binds only CXCL11 [START_REF] Ehlert | Identification and partial characterization of a variant of human CXCR3 generated by posttranscriptional exon skipping[END_REF] for the activation of T cells (LAT), and phospholipase-C-g1 [START_REF] Smit | CXCR3-mediated chemotaxis of human T cells is regulated by a Gi-and phospholipase Cdependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase[END_REF]. Interestingly, recent data suggest that chemokine receptors are recruited to the immunological synapse, and that there is a crosstalk between T-cell receptor and CXCR3-signalling pathways and that this participates in the fine tuning of immune responses [START_REF] Dar | CXCR3-mediated T-cell chemotaxis involves ZAP-70 and is regulated by signalling through the T-cell receptor[END_REF].

The expression of CXCR3 is regulated at the transcription level by the transcription factor Tbet (T-box expressed in T cells), and permits the circulation of memory cells towards tissues that do express its specific ligands during the process of inflammation. CXCR3 and its ligands were shown to be implicated in the recruitment of:

• activated CD4 + and CD8 + T cells, and innate lymphocytes such as NK and NKT, into inflamed tissues;

• pDCs and subsets of B cells in the peripheral lymph nodes;

• and tissue-resident memory (T RM ) T cells into nonlymphoid tissues [START_REF] Clark | Resident memory T cells in human health and disease[END_REF] (Figure 21).

Of note, T RM T cells have been identified in several organs such as small intestine, vaginal mucosa, brain, lung, salivary glands, and thymus [START_REF] Mackay | Tissue-resident memory T cells: local guards of the thymus[END_REF][START_REF] Mueller | Tissue-resident T cells: dynamic players in skin immunity[END_REF], and are characterized by high expression levels of the αE integrin (CD103) and the marker CD69 [START_REF] Mueller | Tissue-resident T cells: dynamic players in skin immunity[END_REF]. These T cells are fully functional, quickly producing interferon-γ upon local peptide stimulation, and triggering a state of tissue-wide pathogen alert [START_REF] Ariotti | Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert[END_REF]. responses [START_REF] Groom | CXCR3 Chemokine Receptor-Ligand Interactions in the Lymph Node Optimize CD4+ T Helper 1 Cell Differentiation[END_REF]. In addition, CXCR3-engagemente in SLOs also results in T cell costimulation and activation, as evidenced by spatial association between CXCR3 and CD3 intracellular signaling [START_REF] Friedman | Surface-bound chemokines capture and prime T cells for synapse formation[END_REF]Newton et al., 2009); and T cell differentiation choice towards short-lived effector rather than memory cells [START_REF] Kurachi | Chemokine receptor CXCR3 facilitates CD8+ T cell differentiation into short-lived effector cells leading to memory degeneration[END_REF].

5) Focus on CXCL10

CXCL10 influences effector cells behavior into nonlymphoid tissues. This has been evidenced in mice chronically infected with Toxoplasma gondii in the brain, where treatment with anti-CXCL10

antibodies result in decreased numbers of local CD8 + T cells, and a subsequent increase in parasite burden [START_REF] Khan | IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection[END_REF]. This effect relates to a significant decrease in the average cell velocity within the tissue, and is reproducible by pertussis toxin (PTX) injection.

CXCL10 can be expressed by many different types of cells, including non-hematopoietic (endothelial, keratinocytes, fibroblasts, mesangial cells, astrocytes, hepatocytes), and hematopoietic (monocytes, neutrophils, DCs) cells. It is highly expressed in many Th1-type inflammatory diseases, often correlating with infiltration of T cells in the inflamed tissue. As many other molecules, CXCL10 can undergo post-translational modifications which might alter its function (Figure 22). It is believed that these modifications constitute a general mechanism to control leukocyte trafficking and inflammation [START_REF] Rot | Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells[END_REF]. Post-translational modifications of CXCL10 include:

• C-terminal cleavage by matrix metalloproteinase 9 (MMP9);

• Citrullination by peptidylarginine deiminase (PAD);

• and N-terminal cleavage of 2 amino acids by members of the X-prolyl dipeptidyl peptidase (DPP) family -the most characterized being dipeptidyl peptidase IV (DPP4 or CD26).

Whereas the two first modifications do not abrogate the chemotactic activity of CXCL10, DPP4mediated NH 2 truncation seems to generate a dominant negative form of the protein, capable of binding CXCR3 but not inducing signaling. DPP4-truncated CXCL10 acts, therefore, as an antagonist of the bioactive chemokine. Our lab provided in vivo evidence of a role for CXCL10 antagonism in chronic HCV disease pathogenesis (Casrouge et al., 2011b). Indeed, patients chronically infected by HCV have high levels of the antagonist form of IP10 in their blood, explaining the paradoxical observation that this correlates with poor control of the disease. It is curently unknown if similar perturbations can induce impairments of trafficking in other infection such as HIV and malaria.

Besides CXCL10, many other chemokines were shown to be altered by post-translational modifications, resulting in the formation of antagonist molecules. Pertinent in-vivo examples include the degradation of monocyte chemoattractant protein-3 (MCP3) by MMP2, reported to be relevant to the pathogenesis of spondyloarthritis; and the cleavage of stromal cell-derived factor-1 (SDF1 or CXCL12) in HIV-associated neurodegeneration, also mediated by MMP2 [START_REF] Moelants | In vivo regulation of chemokine activity by post-translational modification[END_REF].

Nevertheless, these studies are particularly challenging due to (i) the inability to discriminate the different forms of the analytes using sensitive immune-based assays, and (ii) the necessity to inhibit the activity of proteolytic plasma enzymes that may continue to act even after collection of the biological samples. Regarding the first challenge, specific assays that permit discrimination of the truncated (short, 3-77) from the untruncated (long, 1-77) form of CXCL10 have now been successfully developed, and permit to monitor these two molecules in biological samples (Casrouge et al., 2011a).

DPP4 is a transmembrane glycoprotein expressed by hepatocytes, kidney epithelium and various tissue endothelium [START_REF] Gorrell | Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders[END_REF]. DPP4 (CD26) is also expressed at the cell surface of B, T and NK lymphocytes. Its expression can be increased on T cells after TCR engagement [START_REF] Dang | Comitogenic effect of solidphase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathways[END_REF]. Its enzymatic activity is directed toward luminal or extracellular spaces, and its substrates include soluble and stromal factors (e.g., hormones and chemokines). DPP4 functions as a homodimer [START_REF] Chiravuri | Homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell proline dipeptidase[END_REF], and in addition to its dipeptidylpeptidase activity, it may have endopeptidase activity [START_REF] Bermpohl | Rat dipeptidyl peptidase IV (DPP IV) exhibits endopeptidase activity with specificity for denatured fibrillar collagens[END_REF]. Under steady state conditions, DPP4 may be shed (or possibly secreted), and enzymatically active soluble DPP4 (sDPP4) can be found in plasma, urine and semen [START_REF] Durinx | Molecular characterization of dipeptidyl peptidase activity in serum: soluble CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides[END_REF][START_REF] Wilson | Prostate specific origin of dipeptidylpeptidase IV (CD-26) in human seminal plasma[END_REF]. This phenomenon can be modulated during inflammation -for example during HCV chronic infection (Casrouge et al., 2011b). In addition to its enzymatic activity, DPP4

possess other biological functions that may be dictated by heterodimeric interactions with FAPα, plasminogen 2, ADA, CD45 or extracellular matrix proteins [START_REF] Gorrell | CD26: a multifunctional integral membrane and secreted protein of activated lymphocytes[END_REF]. Oral DPP4 Chapter 2. Develop tools for screening rare antigenspecific responses

I. INTRODUCTION

Standard quantitative assays for measuring specific CD8 + T cell responses, such as pMHCI tetramers, Intracellular Cytokine Staining (ICS) and Enzyme Linked Immunospot (Elispot), with a limit of detection of ~10 -4 (=1 cell in 10 4 CD8) and 5x10 -5 respectively, do not allow to study naive Agspecific CD8 + T cells [START_REF] Jenkins | On the Composition of the Preimmune Repertoire of T Cells Specific for Peptide-Major Histocompatibility Complex Ligands[END_REF]. Thus, most of our current knowledge about precursor frequencies prior to immunization and during first steps of activation relies on:

• Limiting Dilution Assays (LDA). In humans, it has long been the only way to evaluate precursor frequency. LDA are in-vitro assays in which 24-replicates cultures are set up at multiple dilutions of cells and stimulated in an Ag-specific manner (e.g. irradiated APCs, peptide and growth factors) [START_REF] Taswell | Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis[END_REF]. After one to two weeks, cultures are combined with functional detection of specific cells via Elispot or ICS, and negative wells are scored for each dilution. According to Poisson statistics, when 37% of the tested wells are negative, there is a 95% confidence that specific cells in the positive wells derived from a single precursor.

Even with recent improvments and standardization [START_REF] Geiger | Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells[END_REF], limits of this assay persist: (i) this requires exogenous stimulation, introducing potential bias and significant interassay variability; and (ii) it relies on one of many possible T-cell functions, and thus fail to detect epitope-specific T cells that lack that function.

• In vivo experimental models in mouse, utilizing adoptive transfer of monoclonal TCR transgenic (Tg) T cells into histocompatible mice secondarily exposed to their relevant antigen [START_REF] Moon | Tracking epitope-specific T cells[END_REF]. Their main advantage is to identify epitope-specific T cells purely on the basis of TCR expression. However, only a limited number of antigenic specificities are available, these cells are monoclonal, and transfer efficiencies for each are unknown (ranging from 1 -20%), making it difficult to estimate a broad panel of precursor frequencies. Also, these studies present some technical challenges. Indeed, high frequency transfer (1 to 5 x10 6 cells) generate populations 1,000 times larger than the endogenous naive epitope-specific populations, creating unphysiological situations where Ag-induced activation is inefficient due to increased competition. In contrast, transferring low number (1x10 4 cells) of TCR transgenic cells that approximates the endogenous naive repertoire is possible, but the detection of transferred T cells then necessitates a cell enrichment step, and more sophisticated flow cytometry techniques.

These limitations indicated the need to directly study polyclonal naive T cells [START_REF] Badovinac | Initial T Cell Receptor Transgenic Cell Precursor Frequency Dictates Critical Aspects of the CD8+ T Cell Response to Infection[END_REF][START_REF] Ford | Antigen-specific precursor frequency impacts T cell proliferation, differentiation, and requirement for costimulation[END_REF][START_REF] Marzo | Initial T cell frequency dictates memory CD8+ T cell lineage commitment[END_REF]. To this end, an elegant method combining pMHC tetramer staining and magnetic bead enrichment was recently developed [START_REF] Moon | Naive CD4+ T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude[END_REF][START_REF] Obar | Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection[END_REF].

It allowed for the first time direct detection, enumeration and characterization of rare naive Agspecific T cells in mice. With this tool, it was further demonstrated that precursor frequency is as a critical factor for the magnitude of primary and memory responses after immunization, immunodominance hierarchy, kinetic of responses, and differentiation patterns of memory. In humans, tetramers and magnetic beads enrichment have already been coupled to purify memory CMV-specific CD8 + T cells for clinical purposes [START_REF] Luxembourg | Biomagnetic isolation of antigen-specific CD8+ T cells usable in immunotherapy[END_REF], but never optimized for naive T cell detection. Thus, the question of the frequency of naive CD8 + T cells specific for a given Ag in humans, their repertoire, and their impact on primary and memory immune responses in physiological or pathological situations have not been yet evaluated.

In this context, we developed and validated a new assay for tracking rare epitope-specific naive CD8 + T cell from human peripheral blood. This assay, based on pMHCI tetramer staining and magnetic beads enrichment, permitted detection, enumeration and phenotyping of previously undetectable naive populations specific for HIV-1 Gag p17 77-85 , HCV NS3 1073-1081 , HCV Core 132-140 , NY-ESO-1 157-165 , and CMV pp65 495-503 in peripheral blood of seronegative healthy donors (HDs).

Interestingly, we detected higher than 100-fold differences in precursor frequency across these epitopes (from 0.6x10 6 to 1.3x10

II. TETRAMER ASSOCIATED MAGNETIC ENRICHMENT (TAME)

A. Preparation of pMHCI tetramers MHC tetramers are reagents that carry multiple peptide-MHC Class I (pMHCI) complexes, and thus have the ability to interact with multiple TCRs on a single CD8 + T cell [START_REF] Altman | MHC-peptide tetramers to visualize antigen-specific T cells[END_REF].

Using MHC tetramers, it is possible to directly track and quantitate Ag-specific T cells during the course of immunization, independently of their function or differentiation state [START_REF] Klenerman | Tracking T cells with tetramers: new tales from new tools[END_REF]. In our studies, we use manually prepared tetramers. Global scheme of their preparation is presented in Figure 23A (from [START_REF] Bousso | Generation of MHC-peptide tetramers: a new opportunity for dissecting T-cell immune responses[END_REF]). Detailed protocol is available in the Protocols section. Briefly, inclusion bodies of heavy chain of HLA molecules -with BirA substrate peptide fused to the extracellular portion -and β2-microglobuline protein (β2m) are prepared in E. Coli, purified and stored at -80°C (Figure 23B). At Day 0, we achieve the HLA-peptide refolding by diluting each inclusion bodies subunit in a large volume of buffer containing the antigenic peptide, and we maintain the reaction for 4 days at 4°C. To improve the yield, we perform two supplementary heavy chain injections at Day 1 and 2. At Day 4, we concentrate the folding reaction using a stirred cell with a 30kDa cutoff membrane, then perform biotinylation by incubation with recombinant BirA enzyme (Avidity, Denver, CO) overnight. At Day 5, we purify the trimolecular complexes MHC/β2m/peptide on a size exclusion chomatography column (Figure 23C). We collect fractions containing the biotinylated MHC peptide complexes (pMHC), pool them, and concentrate on a 30kDa cutoff membrane. The extent of biotinylation reaction is visualized by SDS-Page electrophoresis by incubating samples with an excess of avidin (Figure 23D). Biotinylated monomers are stored at -80°C. When needed, we add fluorescently labelled streptavidin (e.g., PE, APC, Qdots) to the biotinylated pMHCI complexes to generate fluorescent tetramers that can be stored for several weeks in the dark at 4°C (Figure 23E). PE streptavidin is often the first choice because of its brightness and stability. We regularly validate the quality of our production by comparing stainings obtained with commercially versus manually prepared tetramers.

B. Preparation of photocleavable pMHCI tetramers

The production of a single pMHC-complex as presented above is time-consuming and labor-intensive, and thus has limited application for high-throughput screening formats, or when low amounts of a large number of different specificities is needed. To address this issue, the group of Ton Schumacher developed conditional HLA ligands that authorize the generation of monomers in a highly adaptable manner [START_REF] Bakker | Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7[END_REF]Hadrup et al., 2009b;[START_REF] Toebes | Design and use of conditional MHC class I ligands[END_REF]. During the first months of my PhD, I had the great opportunity to be trained by Ton Schumacher's group on this technology. In brief, the generation of pMHCI photocleavable monomers follows the same steps as in the above section [START_REF] Bousso | Generation of MHC-peptide tetramers: a new opportunity for dissecting T-cell immune responses[END_REF] (Figure 24A). The UV peptide permits stabilization of the monomer, and thus production of large numbers of photocleavable stocks. Then, when needed, UV-mediated cleavage is performed, and the complex is disintegrated except if there is an efficient binding peptide in the medium that rescues it from degradation. Main advantages of this technology is (i) to permit generation of tetramers with up to 200 different specificities at the same time -thus authorizing extensive analysis of the repertoire, and (ii) to produce small amounts of each -which dramatically reduces costs and time when tetramers with different specificities are needed, or for testing on small numbers of samples. We now routinely produce and use in our lab the HLA-A 0201 , HLA-A 0101, HLA-A 0301, HLA-B 0801, HLA-A 1101, H2-D b and K b photocleavable tetramers, as well as CD8 null HLA-A 0201 , which allows avidity studies.

In addition to the use of UV-induced peptide exchange for the generation of pMHC reagents for T cell staining, this technology is also used to test in vitro the affinity of in silico predicted MHC-ligands (Hadrup et al., 2009b). This application relies on the demonstration that stabilization of the MHC molecule after peptide exchange is correlated to the binding capacities of a peptide (Figure 24B). We evaluate ligands affinity by a semi-quantitative ELISA standardized with known low, middle, and high-affinity binders. This assay can be performed in 96-and 384-well formats, allowing the screening of hundreds or thousands of candidate peptides (Figure 24C).

C. Development of TAME for human samples

Conventional pMHC tetramer stainings are of unsufficient sensitivity to study rare naive antigenspecific CD8 + T cells. Based on recent technological improvments in mouse models, we worked on the development of a pMHCI tetramer-based enrichment protocol for human studies. We used unqualified buffy coats collected on healthy donors in blood banks. Buffy coat represent the thin layer between the upper clear fluid (plasma) and the bottom red fluid (red blood cells) that is obtained after centrifugation of an anticoagulated blood sample, and contains most of the white blood cells and platelets (Figure 25A). We isolated PBMCs using standard procedures, and performed HLA-A2 immunophenotyping (Figure 25B). As expected based on frequencies in the general population, we found about half of these samples to be HLA-A2 positive. Importantly, FACS-based HLA-A2 typing identifies the serotype allele, but does not inform on the 8 possible A2 subtypes. In Caucasian populations, the assumption that HLA-A2 positive individuals are of HLA*A 0201 subtype is acceptable, and correct in a large majority of cases (100% in a pilot study of 20 samples collected in our laboratory). However, this is not true for non-caucasian populations, where HLA subtypes vary greatly, and where high-resolution molecular HLA definition is thus clearly needed. Indeed, inappropriate matching between the HLA alleles of the biological sample and the pMHC reagents used for monitoring can lead to dramatic changes in our ability to detect T cells and/or predict immunogenic peptides [START_REF] Van Buuren | HLA Micropolymorphisms Strongly Affect Peptide-MHC Multimer-Based Monitoring of Antigen-Specific CD8+ T Cell Responses[END_REF]. In future clinical studies, this issue will need to be addressed by (i) the use of high-resolution PCR-based haplotyping [START_REF] Erlich | HLA DNA typing: past, present, and future[END_REF], and (ii) the engineering and use of HLA subtype tools [START_REF] Van Buuren | HLA Micropolymorphisms Strongly Affect Peptide-MHC Multimer-Based Monitoring of Antigen-Specific CD8+ T Cell Responses[END_REF].

Our enrichment protocol starts with the staining of PBMCs with PE-labeled pMHCI tetramers, where we typically put aside a small aliquot for flow cytometry analysis, corresponding to the «Before» fraction (Figure 25C). We incubate stained samples with anti-PE antibodies linked to magnetic particles, allowing enrichment of bound-cells by magnetic-activated cell sorting (MACS) column. We then recover, count and phenotype bound (« Enriched ») and unbound (« Depleted ») fractions with multicolor flow cytometry. Detailed protocol is available in the Methods section [START_REF] Alanio | Tracking antigen-specific CD8⁷ T cells using MHC class I multimers[END_REF].

In order to optimize detection and enumeration of rare tetramer positive events, our protocol includes multiple levels of inclusion and exclusion gating strategies (Figure 25D). This permits to minimize background and focus on genuine T cell-events. First, a side-scatter area versus width plot is used to identify cell aggregates, which are excluded from the analysis. The remaining single-cell events are then analyzed on a plot of non-T cell lineage (dump) markers versus CD3. Non-T cell antibodies all being conjugated with the same fluorochrome, dump-positive events correspond to all mononuclear blood cells we are not interested in, comprising monocytes (CD14 + and CD16 + ), B lymphocytes (CD19 + ), NK cells (CD56 + ), and dead cells because of inclusion of a viability dye with similar fluorescence characteristics. Of note, CD56 has to be removed when considering enrichment of activated T cells. Gating on CD3 + Dump -events thus allows for putative T-cell events to be gated away from most other cell type, including dead cells and highly autofluorescent cells that potentially reside along the center diagonal of the plot. These CD3 + T-cell events are then plotted for CD8 versus tetramer stainings, which allows for the selection of CD8 + Tetramer + subpopulation. Of note, in some experiments, CD3 staining is not included in order to free a channel on the cytometer, and Dump - CD8 + T cells are directly gated, both strategies being demonstrated as equivalent (Hadrup et al., 2009a).

D. TAME increases detection of Ag-specific T cells by up to 100-fold

In order to validate our protocol, and to define the output of the assay, we first focused on three HLA-A 0201 -restricted epitopes targeted by specific T cell populations that can be routinely detected by conventional pMHCI tetramer staining in healthy donors:

• Matrix-1 (M1 58-66 ), which is an immunodominant epitope from the Influenza A virus (Flu) sequence. Most if not all of the adults will have encounter this antigen in their life, with a frequency of memory tetramer-positive cells in blood expected to be around 1.2x10 -3 of CD8 + T cells;

• CMVpp65 495-503 , which is an immunodominant peptide from the Cytomegalovirus (CMV)

sequence. About half of the French population is expected to be seropositive for the virus, with extremely high frequencies of memory tetramer-positive cells in blood (estimated to be 2x10 -2 ). The other half will be naive for this antigen, permitting eventual detection of inexperienced T cell populations in these individuals;

• Melanoma-associated Antigen recognized by T cells, MART1 26-35 (also known as MelanA), which is a skin self-restricted epitope targeted by very high numbers of naive CD8 + T cells in peripheral blood of healthy individuals, ~6x10 -4 of CD8 + T cells [START_REF] Pittet | High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals[END_REF].

We enriched cells for each of these specificities, and analysed «Before», «Enriched» and «Depleted» subsets by flow cytometry (Figure 26A andB). After enrichment, Ag-specific populations were clearly defined with few cells lost (usually 10-30%) in the depleted fraction. In other words, capture on the column is quite efficient and given that the majority of populations of interest are less abundant than CMV, we extrapolate that there is less than 10% loss in the depleted fraction. As limit of detection of conventional tetramer staining is only 10 -4 , we confirmed that there are no or few remaining specific cells in the « Depleted » fraction by a more sensitive assay, the IFNγ-ELISPOT (Figure 26C). We observed reactivity for Flu-M1 58-66 peptide from « Before » fraction, as well as from the fraction depleted with another pMHCI tetramer (MART1 [START_REF]Each time, pull each in Falcon15 tubes , add 2mL of PB to the upper fraction of the column, push the piston, and discard the colum. You obtain the Enriched Fraction. 27[END_REF][START_REF]Stain them and analyse on FACS machine using standard procedures[END_REF][28][29][30][31][32][33][34][35] ), but not from the fraction depleted with Flu-M1 58-66 tetramer, confirming that reactive cells were specifically retained within the magnetic column. Concerning MART1 26-35 , we did not detect any reactivity both before and after depletion, which is in accordance with specific cells reactive for this epitope being of naive phenotype, and not able to be activated by a short period of activation. By comparing frequencies before and after enrichment, we evaluated TAME's yield being 550x, 35x, and 260x for Flu, CMV, and MART1 populations, respectively -and classically up to 100-fold in multiple experiments. This is in agreement with mouse studies, where the enrichment procedure typically achieves 100-fold enrichment. The starting number of cells utilized in the assay determines both precursor frequency calculation, and in most instances the limit of sensitivity for the assay. For example, a population around 10 -6 (i.e. 1 cell in 10 6 CD8) will only be detectable with a minimum of 10 7 starting cells.

To quantify the size of the epitope-specific populations within each sample, we use a calculation described in following blue box adapted from [START_REF] Moon | Tracking epitope-specific T cells[END_REF]. The number of epitope-specific T cell-numbers is based on the absolute number of tetramer-positive cells within the « Enriched » fraction. We systematically analyze in each experiment the « Depleted » fraction, and if necessary, we add the number of remaining tetramer-positive cells to the number calculated in the « Enriched » to give the total number of specific cells for the entire sample. To validate the accuracy of our enumeration strategy, we compared enumeration of MART1 26-35 -specific CD8 + T cell before and after enrichment in 6 healthy donors. Mean precursor frequencies were 4x10 -4 (1.2 to 6.6x10 -4 ), and 1.5x10 - 4 (9x10 -5 to 2.1x10 -4 ) respectively. We find the observed 2.8-fold difference acceptable with the benefits far outweighing the loss: better definition of specific population with decrease in non-specific background, and the possibility of enumerating cells with precursor frequencies under 10 -4 . As an external validation, a recent study on library method -a recently developed high-throughput limiting dilution assay, reports a strong positive correlation between precursor frequencies determined by the two methods [START_REF] Campion | Proteome-wide analysis of HIV-specific naive and memory CD4+ T cells in unexposed blood donors[END_REF].

Table 5. Calculation of epitope-specific T cell numbers, and validation.

Box indicates the strategy for calculating epitope-specific T cell numbers (adapted from Moon et al., Nat Protocols 2009).

!

With potential clinical application in mind, we evaluated the impact of freezing cell procedures on our protocol (Figure 26D). From one single donor, fresh versus frozen cells were enriched using Flu-M1 58-66 , CMVpp65 495-503 and MART1 26-35 PE-labeled HLA-A0201 tetramers. Cell phenotypes and precursor frequencies were found to be similar in both conditions: 4.7x10 -3 versus 3.9x10 -3 for Flu-M1 58-66 , 3.5x10 -2 versus 3x10 -2 for CMVpp65 495-503 , 1.7x10 -4 versus 1.5x10 -4 for MART1 [START_REF]Each time, pull each in Falcon15 tubes , add 2mL of PB to the upper fraction of the column, push the piston, and discard the colum. You obtain the Enriched Fraction. 27[END_REF][START_REF]Stain them and analyse on FACS machine using standard procedures[END_REF][28][29][30][31][32][33][34][35] , on frozen and fresh samples, respectively.

Finally, we used remaining free FACS channels for naive/memory phenotypic T-cell markers. There is no way to functionally assert that a cell is naive -only that a cell lacks memory functions. However, the lack of effector function can also mean that it does not respond to the cognate antigen, and we cannot easily distinguish these two options. Naiveness of a T cell is thus a phenotypic definition.

Based on literature, we first identified naive T cells using antibodies targeting CD45RA, CD27, CD11a, and CCR7. CD45RO was excluded as it is the exact inverse picture of CD45RA, and thus redundant. CD62L was excluded because it is known to be lost during freeze/thaw procedures, and thus not suitable for clinical studies. To test the efficiency of our panel to discriminate naive versus experienced populations, we stained the three pre-cited enriched specific populations (Figure 26B).

Consistent with a memory phenotype, 99% of the cells specific for Flu-M1 58-66 were found CD45RA -, CD11a + , CD27 -. 99% of cells specific for CMVpp65 495-503 were also found CD11a + and CD27 -, but CD45RA -/+ , consistent with a predominantly late memory phenotype, as expected from literature [START_REF]Each time, pull each in Falcon15 tubes , add 2mL of PB to the upper fraction of the column, push the piston, and discard the colum. You obtain the Enriched Fraction. 27[END_REF].

This differed from MART1 26-35 -specific populations, where 98% of the cells were CD45RA + , CD11a dim/lo and CD27 + , consistent with their naive status. The Labex Milieu Interieur, driven by Matthew Albert and Luis Quitana-Murci, is a large-scale study ongoing in the lab, which aim at assessing the determinants underlying human immunological variance within the healthy population.

Eight-color antibody panels and procedures for staining and lysis of whole blood samples were standardized and automated, and permitted extensive and reproducible immunophenotyping of 1 000 healthy individuals [START_REF] Hasan | Semi-automated and standardized cytometric procedures for multi-panel and multi-parametric whole blood immunophenotyping[END_REF]. As such, it represents solid foundations for defining reference values in healthy donors. In the T cell panel, naive and memory T cell subsets were defined by their expression of CD45RA and CD27 [START_REF] Bretschneider | Discrimination of T-cell subsets and T-cell receptor repertoire distribution[END_REF][START_REF] Hamann | Phenotypic and functional separation of memory and effector human CD8+ T cells[END_REF], and the expression of CCR7 and HLA-DR were also evaluated (Figure 27A). A strong correlation between percentages of naive cells defined by both CD45RA/CCR7, and CD45RA/CD27 combinations confirmed the redundancy of those markers for naive T cell identification -which does not rule out the need of CCR7 in defining specific memory T cell subsets (Figure 27B). In order to provide additional validation, we:

• sorted from a healthy donor naive, central memory, effector memory and late effector memory CD8 + T cell subsets based on their CD45RA/CD27 expression patterns, and quantified sjTRECs as a measure of « naiveness » of the T cell population [START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF]) (Figure 27C). As expected, we found the highest levels of sjTRECs in CD45RA + CD27 + T cell population;

• tested intracellular release of calcium after ionomycin stimulation, and found distinct kinetics in naive T cells as compared to memory [START_REF] Roederer | Heterogeneous calcium flux in peripheral T cell subsets revealed by five-color flow cytometry using log-ratio circuitry[END_REF]) (Figure 27D);

• performed transcriptomic analysis on some selected autophagy and cell death related genes, and found lower Bcl2 and cIAP gene expression in naive T cells as compared to memory [START_REF] Ahmed | Immunological memory[END_REF]) (Figure 27E).

We now routinely define naive T cells as co-expressing high levels of CD45RA and CD27.

E. Detection of naive CD8 T cells specific for HIV Gag p17 77-85

To address the issue of naive Ag-specific T cells, we further applied our tetramer enrichment protocol to the detection of cells specific for an epitope unknown from the healthy immune system. We decided to focus on the HIV Gag p17 77-85 HLA-A0201 epitope, as our HLA-typed healthy individuals receive serologic testing from the blood bank to confirm the absence of HIV infection. Clinical relevance and tetramer availability were also important criteria in our choice.

Using TAME, we were able to detect naive CD8 + T cells for HIV Gag p17 77-85 epitope in a HIVnegative individual, with a measured physiological precursor frequency being 0.3x10 -6 (Figure 28A).

These cells were not detectable before the enrichment step, but defined a relevant population after enrichment. Further phenotyping found 95% of specific T cells sharing a naive phenotype (CD45RA + , CD27 + , CD11a dim/lo ) (Figure 28B). As internal control from the same donor, 99% of MART1 [START_REF]Each time, pull each in Falcon15 tubes , add 2mL of PB to the upper fraction of the column, push the piston, and discard the colum. You obtain the Enriched Fraction. 27[END_REF][START_REF]Stain them and analyse on FACS machine using standard procedures[END_REF][28][29][30][31][32][33][34][35] specific cells were of naive phenotype, and precursor frequency was 1.8x10 -4 , which is comparable to other experiments. To confirm specificity of our HIV Gag p17 77-85 tetramer staining, we stained a LYMPHOCYTE clone specific for this epitope, and found 100% of these CD8 + specific T cells correctly labeled. Further phenotyping confirmed an antigen-experienced status of these cells (CD45RA -, CD11a + , CCR7 -), which were derived from a HIV1-positive patient.

The lymphocyte clone specific for HIV Gag p17 77-85 also allowed us to better define the limit of detection of our assay (Figure 28C). We spiked titrated amounts of the clonal CD8 + T cells within known amounts of healthy PBMCs to achieve a precursor frequency of the T-cell clone of 0, 10 -7 , 10 -6 , 10 -5 , and 10 -4 , and tested our ability to detect these cells before and after enrichment. For example, to achieve a precursor frequency of 10 -5 , we spiked 200 clonal lymphocytes within 100 million PBMCs (of which 20 millions were CD8 + T cells). TAME was performed and pre-enriched vs. enriched tetramer-positive cells were compared. In the absence of tetramer enrichment, the limit of detection was between 10 -4 and 10 -5 , consistent with prior evaluation of the tetramer technology. Using TAME, we were able to detect as few as 2 cells spiked into 100 million PBMCs, corresponding to a limit of detection of 10 -7 . Given the ability to control for nonspecific staining with this protocol, the true limit is the number of PBMCs used in the protocol. As such, the theoretic limit may be less than 10 -7 , but for routine clinical monitoring we calculate a working limit of detection to be approximately 10 -6 , corresponding to observation of 10 to 20 cells in approximately 10x10 6 of CD8 + T cells contained in 50 mL of blood.

To confirm the specificity of tetramer-reactive cells, we enriched CD8 + T cells from PBMCs, and stimulated them for 7 days with autologous mature dendritic cells (DCs) pulsed with MART1 [START_REF]Each time, pull each in Falcon15 tubes , add 2mL of PB to the upper fraction of the column, push the piston, and discard the colum. You obtain the Enriched Fraction. 27[END_REF][START_REF]Stain them and analyse on FACS machine using standard procedures[END_REF][28][29][30][31][32][33][34][35] peptide (Figure 28D). As described, the MART1 26-35 -enriched population was naive before stimulation. After the 7-days coculture, the entire population down-regulated CD45RA and expressed CD11a, whereas the HIV Gag p17 77-85 -specific cells in the same culture well remained naive. These data suggest that the naive T cells measured in our assays are indeed capable of responding to Agspecific stimulation.

Finally, in order to maximize the number of informations obtained from a single tube, and reduce the amount of blood needed for several precursor frequency determinations, we developed multitetramer enrichment strategies. For this, we prepare HLA-A0201 tetramers with streptavidin-PE, plus same tetramers labeled with one specific color (Figure 28E). Enrichment is performed with anti-PE microbeads to simultaneously enrich all specific populations. We discriminate each specific T cell population by reading the second color on CD8 + PE + events. This strategy leads to comparable percentages, output, and precursor frequencies of epitope-specific T cells as compared to single labeled populations. As simultaneous anti-PE/anti-APC enrichment with anti-APC microbeads works fine, we can with this technique multiplex up to 12 simultaneous tests within a single enrichment assay. We did not had the chance to test it, but we are confident in its possible achievement.

F. Enrichment and enumeration of multiple specificities

After these steps of development, we applied our tool to the evaluation of precursor frequencies for multiple epitopes on 5 to 17 HDs (Figure 29A). We could detect T cells for all specificities. The calculated precursor frequency for MART1 26-35 was consistent with prior reports, with a median of 1.3x10 -4 (n=13) [START_REF] Pittet | High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals[END_REF]. Undetectable by conventional assays, it was also possible to establish a range for the frequency of NY-ESO-1, HIV-, HCV-, and CMV-specific T cells in cancerfree and seronegative persons. Median frequencies were 3.6x10 -6 for the NY-ESO1 157-165 self-antigen (n=5); 1.9x10 -6 for HIV Gag p17 77-85 (n=13); 0.6x10 -6 for HCV Core 132-140 antigen (n=5); 5.3x 10 -6 for HCV NS3 1406-1415 antigen (n=5); and 0.6x10 -6 specific T cells for CMVpp65 in seronegative patients (CMVpp65(sn); n=6). Although we found significant variations when comparing one epitope to another, quite surprisingly, precursor frequency for a single epitope persons was conserved on different individuals. These results were obtained by staining 100x10 6 of PBMC, and correspond to absolute cell numbers ranging from 6 to 202 (Median 50). Of note, although most of the cells belonging to these detected populations were of naive phenotype, a subset of of memory-phenotype cells -usually less than 30%, was often detected (Figure 29B).

III. DISCUSSION

A. TAME permits enumeration and characterization of rare Ag-specific T cells from human peripheral blood

Two recent studies in mice reported a method for the direct detection of naive antigen-specific CD4 + or CD8 + T cells [START_REF] Moon | Naive CD4+ T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude[END_REF][START_REF] Obar | Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection[END_REF]. We have now successfully modified this protocol and our study represents the first direct detection of naive antigen-specific CD8 + T cells, prior to immunization, on human peripheral blood. Of the 6 specificities tested in mice, observed frequencies of specific CD8 + T cells ranged from 6x10 5 to 3x10 4 , corresponding to 80 to 200 cells per animal. Here, excluding MART1 [START_REF]Each time, pull each in Falcon15 tubes , add 2mL of PB to the upper fraction of the column, push the piston, and discard the colum. You obtain the Enriched Fraction. 27[END_REF][START_REF]Stain them and analyse on FACS machine using standard procedures[END_REF][28][29][30][31][32][33][34][35] , which is known to be an exception [START_REF] Pittet | High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals[END_REF], we determined CD8 + T-cell precursor frequencies that ranged from 6.0x10 7 to 5.3x10 6 . With an estimated total number of CD8 + cells of approximately 10 11 [START_REF] Arstila | A direct estimate of the human alphabeta T cell receptor diversity[END_REF], measured frequencies correspond to 1 700 to 20 000 cells, a value that is in fact quite close to the estimations made by indirect methods [START_REF] Coulie | Cytolytic T-cell responses of cancer patients vaccinated with a MAGE antigen[END_REF]. However, our technique offers 2 important advantages:

• it permits direct detection of T cells by mean of their TCR and not by their functional capacity;

• it allows to track polyclonal specific T cell under physiological conditions;

• it does not require in vitro culture, thus removing the intrinsic biases and variations inherent to limiting dilution assays. Indeed, those assays require to stimulate T cells with cytokines, and thus assume that their sensitivity to these stimuli is identical, i.e. that no cell will be favored by this artificial environment. Recent papers demonstrate that this usually holds true under homeostatic conditions, with a good correlation between frequencies obtained with libraries generation and tetramer enrichments [START_REF] Geiger | Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells[END_REF]. However, heterogeneity of the T cell preimmune repertoire -especially under pathogenic conditions, makes it less clear that this can be a general assumption.

Although TAME is a promising tool for fundamental and clinical studies, it is important to recognize the limitations of this approach:

• There may not be an exact correlation between the capacity of a T cell to bind a pMHCI tetramer, and its capacity to become activated when confronted with the respective pMHCI on an APC. Specifically, T cells detected by tetramer staining may not be capable of responding to the specific stimulus because of unproductive cross-reactivity. Such T cells would not per se be anergic, but more likely responsive to an alternative (possibly related) peptide;

• Tetramer-based assays are restricted to a given HLA, and even to a certain subtype of HLA.

They thus require extensive HLA-phenotyping of the patients, as well as development of tools for every possible HLA subunit. Important efforts are actually made by the community to permit these achievements and already, our collection of five photocleavable HLA Class I tetramers (A0201, A0101, A0301, A1101, B801) permits a coverage of >90% of individuals of Western European populations;

• In comparison with mouse studies -where spleen and/or lymph nodes are usually examined, starting material in human studies is often peripheral blood. Our expectation that our assay accurately reflect naive precursor frequency is based on the assumption that naive T cells continuously circulate, and do not stop into specific tissues [START_REF] Andrian | T-cell function and migration. Two sides of the same coin[END_REF].

B. Epitopes have conserved precursor frequencies among Individuals

We applied tetramer enrichment for detection of populations for which, except for MART1 26-35 , precursor frequencies were unknown. For all of them, we were able to detect naive Ag-specific T cells from the peripheral blood of HDs. Perhaps the most surprising result is that precursor frequencies for Ag-specific inexperienced populations are very conserved among individuals. While similar observations were made in mice, the use of inbred strains resulted in the different animals sharing 6 of 6 histocompatibility molecules, as compared to the use of non-haploidentical human donors in our study. These results suggest that some extrinsic factor determines the number of cells per pMHC specificity. This has not been previously considered and may indicate that (i) positive selection is a highly conserved process in between individuals, or that (ii) peripheral self-pMHC complexes are involved in the determination of the diversity and pool size for the repertoire [START_REF] Bouneaud | Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion[END_REF][START_REF] Ziegler | Low-affinity peptides and T-cell selection[END_REF]. The latter assumption is in line with recent observations that crossreactivity between self and foreign peptides influences naive T cell population size and autoimmunity [START_REF] Nelson | T cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity[END_REF].

Our data also indicate significant differences in precursor frequencies from one epitope to another:

• MART1, also called MelanA, is a self-protein of unknown function that is expressed by melanocytes and the majority of malignant melanoma cells. Previous studies have found a large thymic output of naive CD8 T lymphocyte specific for this antigen in the peripheral blood of HDs [START_REF] Pittet | High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals[END_REF]. Here we confirm that precursor frequency is exceptionally high, and that targeting lymphocytes are for the most part of naive phenotype;

• HCV NS3 1073-1081 epitope is one of the most frequently targeted epitopes during chronic infection with the hepatitis C virus (HCV). NS3 1073-1081 -specific T cell responses being associated with clearance of acute HCV, this epitope is an interesting candidate for a peptide vaccine. We found a relatively high precursor frequency for this population. This is in agreement with the observation that HCV NS3 1073-1081 -but not CMVpp65 or HIV Gag p17 77-85

-specific T cell lines can be readily generated by in vitro priming from HDs [START_REF] Neveu | Selection of high-avidity CD8 T cells correlates with control of hepatitis C virus infection[END_REF]) ;

• T cells specific for HIV Gag p17 77-85 are 16-fold less frequent than those targeting NS3 1073-1081 .

Gag p17 77-85 was one of the first identified epitope of HIV. Despite its dominance in patients with chronic infection, this epitope is paradoxically considered as a poor immunogen because of its rare reactivity in acute infection [START_REF] Kan-Mitchell | Degeneracy and repertoire of the human HIV-1 Gag p17(77-85) CTL response[END_REF]. Future studies will be needed to determine if the poor precursor frequency can account for these observations;

• CMV is a ubiquitous herpes virus, which persists lifelong after primary infection and be pathogenic in immunocompromised individuals. About half of healthy donors are seropositive for this virus. Precursor frequency on seronegative individuals is 20-fold lower than for NS3 1073-1081 . Considering mouse studies which have shown correlation between precursor frequency and magnitude of the response, it may be surprising that this epitope, for which we confirm very abundant specific populations after immunization, has such a low precursor frequency. We interpret these data as a result of CMV being a chronic infection with continuous restimulation of CMV-specific CD8 + T cells.

With the exception of MART1, we observed up to 30% of memory-phenotype (MP) cells into Agspecific inexperienced populations. In the CD4 preimmune repertoire, those cells were found more abundant, with 20-90% of memory-phenotype cells in Ag-specific anti-viral T cells of unexposed individuals (Su and Davis, 2013;Su et al., 2013). Interestingly, same populations were found naïvephenotype when harvested from cord blood samples. In this study, crossreactivity with environmental antigens is identified as having a critical role in shaping the adult healthy CD4 preimmune repertoire.

Examples of CD8 + T cell crossreactivity have been occasionally reported, for example in between HCV NS3 1073-1081 or HIV Gag p17 77-85 and influenza A virus determinants [START_REF] Acierno | Cross-reactivity between HLA-A2-restricted Flu-M1:58-66 and HIV p17 Gag:77-85 epitopes in HIV-infected and uninfected individuals[END_REF][START_REF] Kennedy | The influence of T cell cross-reactivity on HCV-peptide specific human T cell response[END_REF]. Lower levels of memory-phenotype cells in the CD8 preimmune repertoire as compared to CD4 can be explained by the later being restricted to extracellular antigens, and thus intrinsically more sensitive to changes in their environment. Inversely, CD8 T cells are more sensitive to homeostatic signals, and the contribution of HP in that population is expected to be high [START_REF] Sprent | Normal T cell homeostasis: the conversion of naive cells into memoryphenotype cells[END_REF].

Overall, our data indicates the potential for up to 30-fold differences in precursor frequencies across different specificities. Using these tools, recent studies demonstrated that precursor frequency account for related immunogenicity in humanized mouse [START_REF] Tan | Precursor Frequency and Competition Dictate the HLA-A2-Restricted CD8+ T Cell Responses to Influenza A Infection and Vaccination in HLA-A2.1 Transgenic Mice[END_REF] and humans [START_REF] Schmidt | Immunodominance of HLA-A2-Restricted Hepatitis C Virus-Specific CD8+ T Cell Responses Is Linked to Naive-Precursor Frequency[END_REF]. Identifying epitopes with the highest precursor frequency is of great interest for the design of future vaccine strategies, as it will hopefully correlate with stronger CD8 + T cell responses, and a better clinical outcome of vaccination in healthy, or in viral and tumoral diseases.

IV. PROSPECTIVES

Tetramer-based enrichment assays are now standardized for both mouse and humans. However, some technical improvements can still be hoped in order to increase their use by the scientific communityand thus our knowledge about Ag-specific populations. Our experience in tetramer production makes us believe that there is only few space for optimization in the generation of the tetramer molecules itself. However, improvement of the enrichment technology is more feasible. First, the screening of a larger number of specificities on a minimal amount of sample can be achieved by more complex combicolor coding strategies, where each Ag-specific T cell is concomitantly labeled with a defined combination of two flurorochrome-labeled tetramers (Hadrup et al., 2009a). Although we developed these assays in our lab, we had difficulties in getting clean-enough datas to screen rare T cell populations. We are optimistic that the use of last-generation spectral analyzers such as the SP6800 from Sony -by removing the need of compensation, will permit to improve its sensitivity. Second, we would ultimately like to build sort of Luminex-beads tetramer enrichment assays, permitting highthroughput exploration of the Ag-specific repertoire -including cells with rare frequencies. This would require the use of a component that would at the same time label the cell for specificity, and have a specific physicochemical property that can be exploited for enrichment. This could be magnetism, like in the StrepTag technology, but others might also be possible. Alternatively, we plan to work with companies to standardize enrichment procedures and adapt them to constraints of clinical laboratories. Finally, an important point is also to improve detection of Ag-specific T cells within tissues slides. At the moment, coupling tetramer staining to classical immunhistochemical protocols is impossible. Here again, exploiting components with original physico-chemical properties would be of great help.

Beside technical aspects, we believe our enrichment protocol will permit to address a number of unresolved questions:

• Are precursor frequencies stable over time and/or over decades of life? Measuring precursor frequencies in a cohort of healthy donors several times sequentially will inform us on the distribution of inexperienced T cells in the body and the stability of the T cell preimmune repertoire;

• Are MP cells proportions in inexperienced populations stable over time and/or over decades of life? Are MP proportions fixed for a given specificity similarly as precursor frequency? Are there detectable ranges of physiological variations? What is the participation of HP to MP generation? Same analysis as mentioned before, coupled to naive/memory phenotyping, will help answering these questions. Comparing the middle-age adult preimmune repertoire to Agspecific T cells isolated from cord blood, infants, or elderly will also be of great interest. Cord blood samples being supposedly germ-free (though arguably, not totally free of stimulation by specific pathogens), this will offer precious information on the origin of MP cells;

• Are recent thymic emigrants (RTEs) part of inexperienced Ag-specific populations, and what is their impact on T cell responses? It is known that RTEs are part of the naive repertoire, but still unclear whether they have a specific antigenic repertoire. Using single-cell technologies and sjTRECs analysis on neonate thymi and age-stratified infants cohorts, we hope it is possible to identify phenotypic markers for those cells, and track them in different situations, for example during childhood vaccinations;

• Are circulating naive T cells representative of the global naive population, or are they a specific subset that inform us only little about the entire body situation?

• Is the preimmune repertoire similarly shaped in other (non-A2) haplotype?

• Finally, do quality matters? The impact of precursor frequency is established in both mice and humans [START_REF] Moon | Naive CD4+ T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude[END_REF][START_REF] Schmidt | Immunodominance of HLA-A2-Restricted Hepatitis C Virus-Specific CD8+ T Cell Responses Is Linked to Naive-Precursor Frequency[END_REF]. We know from mice studies that each subpopulation found into the preimmune repertoire has a particular behavior after immunization. But it is unclear how much these parameters impact the Ag-specific response when considered all together.

Answers to these questions will require large application of TAME to clinical trials. This tool is sensitive enough to monitor Ag-specific specific CD8+ T cell with a precursor frequency above 0.5x10-6 from 50-100mL of blood, an acceptable amount to obtain from patients during routine analysis. Some clinical settings would be of particular interest:

• Viral clearance, as described in Chapter 3 in the context of chronic HCV;

• Preventive and therapeutic vaccination -childhood vaccination, HBV-vaccine non-responders, and cancer vaccines;

• Susceptibility to autoimmunity;

• Organ and stem cell transplantation, with the hypothesis that immunosuppression could (i) lead to extinction of the smallest inexperienced populations when T cell number decreases, and (ii) favor the proliferation of MP cells, which may account for some specific clinical features. A. Buffy coat is defined as a thin central layer after centrifugation of heparinized human plasma. B. HLA-A2 typing was performed on whole blood on each buffy coat. Histograms present representative A2-positive (left) and A2-negative (right) results gated on lymphocytes. C. Schematic representation of TAME. D. Gating strategy to reduce background. 100x10 6 PBMC from healthy individuals were analyzed by eight-color flow cytometry following staining and TAME with a Flu-Matrix1 HLA-A0201 tetramer labelled with PE. Flu-specific CD8 + T cells from the enriched (bound) fraction were identified as side-scatter low dump -CD3 + CD8 + Tet + events. The tetramerpositive gate was set using the CD8 -gated events as a negative control. Represented results show 10 6 collected events.

I. INTRODUCTION

Functional impairments of CD8 + T cells have been characterized in several persistent viral infections, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections in humans, simian immunodeficiency virus (SIV) infection in macaques, and lymphocytic choriomeningitis virus (LCMV) infection in mice [START_REF] Ahmed | Immunological memory[END_REF]. In particular, it has been shown that chronic infection skews memory/effector CD8 + T cells differentiation [START_REF] Stelekati | Chronic Bystander Infections and Immunity to Unrelated Antigens[END_REF], and drives virus-specific CD8 + T cells towards an « exhausted » phenotypic state, as marked by high expression of the programmed cell death-1 (PD-1) molecule [START_REF] Kim | Features of responding T cells in cancer and chronic infection[END_REF]. Chronic infections have also been reported to impair immune responses to unrelated infectious microbes in mouse models [START_REF] Richer | Pathogen-Specific Inflammatory Milieux Tune the Antigen References Sensitivity of CD8+ T Cells by Enhancing T Cell Receptor Signaling[END_REF][START_REF] Stelekati | Chronic Bystander Infections and Immunity to Unrelated Antigens[END_REF], as well as in humans infected with HCV [START_REF] Park | Immune Responses to HCV and Other Hepatitis Viruses[END_REF]. This phenomenon correlates with a interferon (IFN) stimulated gene (ISG) transcriptional signature, suggesting an indirect effect of systemic type I IFN secondary to innate immune activation [START_REF] Stelekati | Bystander Chronic Infection Negatively Impacts Development of CD8[END_REF]. Following from these observations, we hypothesized that chronic infection may alter the T cell preimmune repertoire, and by this way significantly impair our ability to mount appropriate responses after vaccination. Employing TAME, we evaluated this prediction in patients with chronic HCV viral infection of the liver. These findings will be relevant for the optimization of future vaccination strategies, where therapeutic vaccines are delivered to patients usually suffering from disease-related chronic inflammation.

We studied these questions on patients infected with the hepatitis C virus, for the following reasons:

• it is a model of chronic infection that is currently studied in our laboratory;

• it can be cured, allowing comparison of chronic and resolved patients. Outcome of infection is strongly determined by the host immune response, and in part explained by genetic polymorphisms [START_REF] Klenerman | T cell responses in hepatitis C: the good, the bad and the unconventional[END_REF][START_REF] Park | Immune Responses to HCV and Other Hepatitis Viruses[END_REF][START_REF] Thimme | Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease[END_REF] ;

• T cells have a strong impact on the clinical outcome, with clearance of HCV during the acute phase of infection being dependent on efficient induction of sustained specific CD4 + and CD8 + T-cell responses [START_REF] Park | Immune Responses to HCV and Other Hepatitis Viruses[END_REF] ;

• therapeutic vaccination is actively tested in this pathology, and the ability to mount strong CD8 + T cell responses is a biomarker of clinical efficacy. Alteration of the preimmune compartment could then lead to defective antigen-specific responses, and would need to be taken into account for improving responses to the vaccine;

• HCV patients typically have a poor response to unrelated vaccinations [START_REF] Buxton | Hepatitis A and hepatitis B vaccination responses in persons with chronic hepatitis C infections: A review of the evidence and current recommendations[END_REF][START_REF] Moorman | Impaired hepatitis B vaccine responses during chronic hepatitis C infection: Involvement of the PD-1 pathway in regulating CD4+ T cell responses[END_REF];

• HCV infection often associates with systemic manifestations -also referred as "extra-hepatic manifestations" [START_REF] Ko | Morphologic Features of Extrahepatic Manifestations of Hepatitis C Virus Infection[END_REF][START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a community-based long-term prospective study[END_REF];

• It is a clinical situation where it is ethically acceptable to propose leukapheresis collection from the patients for the benefit of research -permitting the access to high numbers of cells for the development of the project.

Schematically, the actual model for T-cell mediated clearance of the virus is represented in Figure 30.

It requires (1) local antigen uptake and DC maturation and migration to draining lymph nodes; (2) immunodominant epitopes to be presented; (3) activated cells to traffic back to the liver and ( 4) lyse target cells into the tissue. However, this model does not address the question of the availability and quality of precursor T cells -that needs to be in place and fully functional to be efficiently activated.

We hypothesized that the study of naive T cells in patients chronically infected by HCV may provide new insights into dysregulated effector responses, and could account for both failure to control viral infection, and inflammation-related extrahepatic manifestations [START_REF] Zampino | Chronic HCV infection and inflammation: Clinical impact on hepatic and extra-hepatic manifestations[END_REF]. We also tested this hypothesis in the context of chronic infection with the hepatitis B virus (cHBV). Although HBV and HCV differ in their genomic structures, replication strategies and life cycles, both viruses are hepatotropic and can lead to development of liver cirrhosis and hepatocellular carcinoma [START_REF] Rehermann | Pathogenesis of chronic viral hepatitis:differential roles of T cells and NK cells[END_REF][START_REF] Schmidt | T-cell responses in hepatitis B and C virus infection: similarities and differences[END_REF]. However, HBV classically induces less inflammation, and is less associated with extra-hepatic manifestations.

II. EVALUATION OF THE PREIMMUNE REPERTOIRE DURING CHRONIC HCV A. Gating strategy

We mentioned in previous chapters the challenge of finding phenotypic markers for the accurate identification of naive/memory CD8 + T cell compartments. An additional complexity concerns the applicability of those markers identified on healthy donors to pathological conditions. It is important to realize that the antibodies we use in immunomonitoring studies are not only markers, but do target molecules that are associated to one or multiple functions. Briefly, CD45RA is implicated in the TCR signal downregulation; CD62L (L-selectin), CD11a (β2 integrin) and CCR7 (chemokine receptor) in the trafficking of T cells; CD28 and CD27 (member of the TNF superfamily) in the costimulation of T cells during priming [START_REF] Kern | Phenotype and function of human T lymphocyte subsets: Consensus and issues[END_REF]. All of these molecules can eventually be modulated directly or indirectly by the pathology, and this can lead to major biases in the evaluation of naive/memory compartments. When starting the project, using 5 phenotypic markers for naive or memory T cells, we confirmed that co-expression of high levels of CD45RA and CD27 identified naive T cells as efficiently in cHCV patients as in HD (Figure 31). Of note, although lower surface expression of CD27 after TCR stimulation have been reported on CD4 + T cells in the context of chronic HCV [START_REF] Yonkers | Reduced Naive CD4 T Cell Numbers and Impaired Induction of CD27 in Response to T Cell Receptor Stimulation Reflect a State of Immune Activation in Chronic Hepatitis C Virus Infection[END_REF], similar observations have not been reported for naive CD8 + T cells, and we did not see evidence for this in our studies. However, we believe that this would not impact our ability to detect naive T cells with the CD45RA/CD27 gating strategy as we use a sufficiently large gate to include both CD27 +hi and CD27 +lo subpopulations.

B. Bulk level

To test the hypothesis that chronic viral infection perturbs immune homeostasis, we evaluated the impact of cHCV and cHBV infections on the phenotype of circulating CD8 + T cells. 28 cHCV, 35 patients achieving SVR after treatment for cHCV, and 18 cHBV patients were included in the study (Table 6).

Table 6. Description of the patients included in the Tetra cohort.

Perturbed naive CD8 + T cell repertoire during chronic infection

Total lymphocyte numbers were within normal range for all tested patients (median 2.2+/-0.6G/l).

Within the CD3 + lymphocyte population, we observed similar percentages of circulating CD8 + T cells (Figure 32A,B) and CD4 + T cells (data not shown). However, absolute numbers of CD3 + were significantly increased in our cohort of cHCV (KW p<0.0001), translating into increased absolute numbers of CD8 + and CD4 + T cells in cHCV patients (KW p<0.0001) (Figure 32C). We further

! cHCV SVR cHBV p(value n=28 n=35 n=18
Male,&n&(%) 16&( 57) 20&( 57) 14&( 78 subsetted the CD8 + T cells according to their surface expression of CD45RA and CD27. Decreased percentages of naive CD8 + T cells have previously been reported in cHCV [START_REF] Shen | PD-1 expression on peripheral CD8+ TEM/TEMRA subsets closely correlated with HCV viral load in chronic hepatitis C patients[END_REF]. Here, we confirmed these findings in age-and CMV-matched chronically infected patients, and extend this observation to HBV-infected individuals (KW p=0.0006, Figure 32D,E). Interestingly, due to the higher CD8 + T cell numbers in cHCV patients, we found a the absolute numbers of naive CD8 + and CD4 + T cells to be in the normal range as compared to healthy donors (Figure 32F). We therefore interpreted the lower proportion of naive T cells to simply be a result of an expansion of the memory cell compartment.

To directly test this prediction, we isolated CD8 + T cells and measured the frequency of signal joint TCR excision circles (sjTREC), by-products of TCR rearrangement, and previously validated as a measure of thymic production [START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Rehermann | Immunology of hepatitis B virus and hepatitis C virus infection[END_REF]. Confirming previous studies, we found a significant decrease in sjTREC content of cHCV CD8 + and CD4 + T cells (MW p=0.009, Figure 33A,B). To address the bias due to differential naive T cell number, we isolated CD45RA + /CD27 + naive CD8 + T cells and assessed sjTREC frequencies. Surprisingly, we also observed within the naive compartment a significantly lower sjTREC content in cHCV patients as compared to HD (MW p=0.02, Figure 33C). To further characterize this phenotype, we assessed the Vβ distribution within the naive repertoire of cHCV patients. cHCV patients showed a biased repertoire with increased representation of selected Vβ families. A representative example of Vβ usage plotted as % over the 24 tested families, and ordered by increasing size from one cHCV patient and one HD is shown (Figure 33D). To compare distributions, Lorenz curves and Gini coefficients were calculated for cHCV and HD (n=7 per group), and compared by Mann Whitney (MW) test (p=0.03) (Figure 33E,F). These data support an overall perturbed naive CD8 + T cell repertoire in cHCV patients, with increased peripheral expansion of selected populations.

Altogether, these data support a perturbed naive CD8 + T cell repertoire in cHCV patients that results from peripheral expansion of selected populations.

C. Antigen-specific level

Mart1-specific CD8 + T cell MP during chronic infection may be reversed by viral clearance

To evaluate the impact of these perturbations on Ag-specific populations, we utilized TAME to evaluate Mart1-specific T cell populations. While similar absolute numbers of Mart1-specific CD8 + T cells were observed in our respective study groups (Figure 34A,B), SVR and cHCV patients showed a more differentiated phenotype (Figure 34C), defined by fewer CD45RA + /CD27 + (KW p<0.0001, Figure 34D) and increased proportions of memory-phenotype (MP) cells. Of note, when considering only naive-phenotype Mart1-specific cells, precursor frequencies were still comparable across the different study groups (data not shown).

We next compared cHCV patients to those who achieved viral clearance. Strikingly, sequential samples (available from a single patient who achieved cure) suggested that immune restoration of the naive compartment is possible (Figure 34E). Testing this observation in our cross-sectional cohort, we validated our findings and show a statistically significant increase in the frequency of naive-phenotype antigen-specific CD8 + T cells that return as a function of time (KW p=0.04; Figure 34F). In more detail in a single SVR patient, we clearly distinguish two distinct co-circulating Mart1-specific populations (Figure 34G). One expresses high levels of CD5, a negative regulator of TCR signaling, and is of naïve-phenotype. The other express lower levels of CD5, and present an activated phenotype.

Although we only have one time point available for analysis from this patient, based on the results presented before, we believe this is reflecting progressive reversion to a healthy phenotype after clearance of the HCV infection. Altogether, these results indicate that the MP cells within the preimmune repertoire of cHCV patients are a reflection of active infection, and unlikely to result from cross-reactivity or true memory T cell differentiation.

In preliminary experiments, we evaluated our capacity to enumerate sjTRECs and study repertoire of small quantities of sorted naive cells. Also sjTRECs and Vβ usage were accurately estimated from as low as 10 5 naive CD8+ T cells (Figure 35A,B), CDR3 profiles were no longer polyclonal when cell numbers were below 10 6 , reflecting biased analysis (Figure 35C). From leukapheresis, we were able to purify sufficient numbers of Mart1-specific naive-and memory-phenotype CD8 + T cells from one HCV patient to compare immunoscope analysis on the Vβ chain usage of these two populations (Figure 35D). In line with our data in bulk T cells, we observed a restricted repertoire of Mart1 naive T cells, with evidence of an expanded Vβ clonotype in memory cells. These data argue in favor of MP cells being the expanded progeny of a perturbed naive T cell repertoire.

We next extended our observations to other antigen specificities by using two additional tetramers (hTERT1 572-580 , and human CMV pp65 495-503 ) that are expected to detect inexperienced self-and virusspecific CD8 + T cell populations in tumor-free, CMV-seronegative individuals. Here again, we found lower percentages of CD45RA + /CD27 + cells and increased MP cells in the antigen-inexperienced populations of cHCV patients (representative plots are shown in Figure 36A andB; and combined results from 2-4 individuals per group in Figure 36C; 2-way Anova p=0.003). In accordance with their MP, after 7h of peptide stimulation, the Mart1-, hTERT-and CMV-specific CD8 + T cells present in cHCV patients were able to secrete higher amounts of IFNγ (2-way Anova p=0.01; Figure 36D). It is interested to note that experienced populations -as specific for EBV BMLF1 in immunized patient, that typically contains a small proportion of naive-phenotype cells, are here again found more differentiated in cHCV patients (Figure 36E).

Altogether, our data highlights an overall perturbation of antigen-specific responses within the preimmune CD8 + T cell compartment.

D. Naive T cell homeostasis during chronic HCV

Decreased expression of CD5 on naive CD8 + T cells associates with TCR hypersensitivity in cHCV patients

To establish a mechanistic understanding of our findings we considered the key homeostatic factors for maintenance of naive CD8 + T cells (Hazenberg et al., 2000a;[START_REF] Ho | Cardiovascular manifestations of HIV infection[END_REF][START_REF] Jenkins | On the Composition of the Preimmune Repertoire of T Cells Specific for Peptide-Major Histocompatibility Complex Ligands[END_REF]. We hypothesized that perturbations of the TCR activation threshold of naive T cells could explain increased differentiation of inexperienced populations. Among them, cell surface expression of CD5 expression has been shown to correlate with the threshold of activation in mice [START_REF] Grossman | Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function[END_REF]. CD5 expression increases during thymic differentiation (Figure 37A) and is the highest on SP T cells. In periphery, it diminishes as a function of T cell differentiation (Figure 37B).

Interestingly, inside bulk naive cells, Ag-specific populations display different levels of CD5 (Mart1 > HCV NS3 1073 ) (Figure 37C). When evaluating CD5 expression under inflammatory conditions, we observed abnormal profiles in cHCV patients, with a single narrowed peak in both CD8 + and CD4 + T cells (Figure 37D andE). This was not observed in spondylarthritis patients (SPA), tested here as another inflammatory condition (Figure 37D). When MFI of CD5 was plotted for 3 different patients, we noticed that the main difference was a lower expression on naive CD8 + T cells (Figure 37E andF). When studied across the entire cohort, we confirmed decreased abnormal CD5 levels that were highly significant for CD45RA + /CD27 + naive CD8 + T cells (KW p<0.0001, Figure 38A andB) but not CD4 + T cells -although there could be a trend also in this population (Figure 38C). Importantly, these phenotypic changes were also observed when focusing on Mart1-specific naive-phenotype cells (MW p=0.04, Figure 38D,E).

Based on its role in regulating TCR signaling, we predicted that lower CD5 expression on naive T cells would result in their being hyperactivated upon stimulation. Indeed, under homeostatic conditions, CD5 negative regulation ensures TCR signaling cascade happens only in the case of highaffinity TCR/pMHC interactions (Figure 39A). If the CD5-induced brake is released, TCR signaling will happen for low affinity interactions (Figure 39B). This was tested functionally by evaluating TCR signaling in naive CD8 + T cells, stimulating cells with low doses of plate-bound anti-CD3 and anti-CD28 Abs. In preliminary experiments, we (i) compared stimulation with soluble antibodies, beads-coated antibodies, and plate-bound antibodies, (ii) compared different doses of CD3 and CD28 antibodies, and (iii) evaluated induced kinetics of activation. Although not detailed here, these experiments lead us to define optimal conditions as (i) plate-bound stimulation (soluble is too weak, beads are too strong), (ii) 1ug/ml OKT3 + 0.5ug/ml CD28 (low as compared to the 10ug/ml usually used in literature), and (iii) t=5mins to give the optimal p-ERK and p-P38 signals in both HD and cHCV. Indeed, while only weak induction of phosphorylated ERK (p-ERK) could be observed in HD during the first hour of stimulation, TCR stimulation induced a strong p-ERK and pP38 signals in both naive and memory cells from cHCV patients (Figure 40A). Differences were more appearent for naive and EMRA subsets, and concerned both percentages and MFI (Figure 40B). A typical profile for naive T cell activation is shown in Figure 40C. When studied on the entire cohort, seven of twelve cHCV patients tested showed significantly higher fold change at 5 minutes (MW p=0.02, Figure 40D).

Using the same stimulation protocol, we investigated the activation of the cells at later time points (24-120 hours). From FSC/SSC plots, we observed some differences, with presence of blastics cells at baseline -and further blasting after 24 and 48 hours of activation (Figure 41A). Blasting cells were mostly CD8 + T cells, and part of the CD25 + subset after stimulation (Figure 41B). When studying the kinetic of CD25 expression on naive CD8 + T cells over a 24h period of activation, we noticed significantly higher levels at 24 hours for most of the cHCV patients (Figure 41C). Same effect was not appearant in the context of cHBV (Figure 41D). When studied on the entire cohort, we found significantly higher levels of CD25 after TCR stimulation in cHCV patients (KW p=0.006, Figure 41E).

Using similar experimental designs, we evaluated if the blastic cells were in the process of dying (Figure 42A). Those cells were both CD4 + and CD8 + T cells, but mostly of the CD45RA + /CD27 + phenotype (blue), and were expressing high levels of the activation marker CD25, as well as high levels of CD95 and proapoptotic Bcl2. This evaluation was done at H10 in order to precede activationinduced cell death (AICD) events. Activated caspase 3 was detectable at 24 hours, and significantly increased in naive T cell subsets from cHCV patients (Figure 42B), as well as total numbers of cells at 48 hours (Figure 42C). All of these findings highlighted an increased sensitivity to cell death after TCR activation in the context of cHCV.

CD5 blockade in HD recapitulates the hypersensitivity phenotype

In preliminary experiments, we observed that activation and AICD events at 24 hours were increased when T cells were receiving stronger TCR signals (Figure 43A). To establish a mechanistic link between CD5 expression and hyperactivation of naive T cells, we evaluated the effect of blocking CD5 signaling. HD PBMCs were exposed to blocking anti-CD5 Abs (αCD5), followed by TCR stimulation. We observed increased levels of p-ERK after 5 minutes of TCR stimulation when HD cells were pre-incubated with αCD5 (representative donor shown in Figure 43B; MW p=0.04, Figure 43C). Similarly, CD25 expression at 24 hours was higher when αCD5 was used prior to TCR stimulation (representative donor shown in Figure 43D; MW p=0.04, Figure 43E), as well as the percentage of dying naive CD8 T cells as assessed by active caspase 3 staining (2-way Anova p=0.04) (Figure 43F). These data support a model where low expression of CD5 on naive T cells in cHCV patients results in dysregulation of the homeostatic TCR threshold. Low-affinity contacts may in turn result in T cell differentiation, providing an explaining for the increased frequency of memoryphenotype Ag-inexperienced cells.

As hypersensitivity to TCR activation can be induced by cytokine microenvironment (especially mediated by high levels of IL-7 and IL-15), we investigated the pattern of cytokine expression evaluated in a comparable cohort of HD, SVR and cHCV patients (Figure 44). We found no significant differences in IL-7 levels in the plasma of patients, but a trend for more IL-15. Notable differences were higher levels of IP10 and TNFα, and lower levels of Eotaxin and MCP1.

Finally, we used in vitro priming assays to evaluate the impact of naive dysregulation on the ability of those cells to be primed, recruited and expanded in a given Ag-specific response. We evaluated if we could grow Mart1-specific cells from either naïve or memory CD8 + T cell subsets. We separated both using magnetic-enrichment kits targeting CD45RO cells. An example of the naive/memory phenotype of cells obtained in the enriched (CD45RO + ) and in the depleted (CD45RO -) fractions of one HD and one cHCV is provided in Figure 45A. We primed enriched T cells with autologous mature DCs pulsed with Mart1 peptide. A cocktail of cytokines (TNFα, IL-1β, PGE2, and IL-7) was added at the time of the priming. Cultures were harvested at Day 10, and stained with Mart1-and unrelated PGTtetramers. From the HD, after tetramer enrichment, we observed an expansion of Mart1-but not PGTspecific cells predominantly from the RO -fraction. In comparison, we did not detect any Mart1specific T cells expanded from the cHCV patient (Figure 45B). This is to be interpreted in the context of lower numbers of total cells recovered from the cHCV patients. In a second independent assay, we tested on 3 cHCV patients two doses of peptides (low 10 -8 , and high 10 -6 ), and two concomitant staining with either conventional or CD8 null Mart1-specific tetramers (Figure 45C). The latter is a modified construct that was shared by P.Romero and M.Bonneville, and stains only high-affinity CD8 + T cells [START_REF] Neveu | Impact of CD8-MHC class I interaction in detection and sorting efficiencies of antigenspecific T cells using MHC class I/peptide multimers: contribution of pMHC valency[END_REF]. With the low dose of peptide, we could detect a population of expanded Mart1 cells at Day 7 that further increased at Day 11. About half of the Day 7 population also stained with the CD8 null tetramer -thus representing high affinity cells. As compared to Day 7, Day 11 cells showed fewer CD25 and CD5 expression, indicating their evolution towards a resting MP (Figure 45D). With the high dose of peptide, we detected a more abundant Mart1-specific population at Day 7, but similar proportions of high affinity cells. Interestingly, those cells were not detectable at Day 11, in the context of notable cell death in the well. Those datas were similar in the 3 tested cHCV individuals. However, for unclear reasons, expansions were highly variable from the HD, and our difficulties in getting robust controls lead us to consider alternative protocols that will be detailed in Prospectives.

III. DISCUSSION

Our study offers the first evidence for chronic viral infection as a cause of CD8 + T cell preimmune repertoire dysregulation. Specifically, we demonstrated that naive CD8 + T cells are dysregulated in the context of cHCV, marked by (i) decreased sjTRECs levels, (ii) a restricted Vβ repertoire, and (iii) a lower threshold for TCR engagement that correlates with lower surface expression of CD5.

Prior examples suggestive of preimmune repertoire perturbations have been documented in humans. Increased threshold for TCR activation in naive CD4 + T cells in elderly persons has been proposed as participating in the diminished response to vaccination that occurs with increasing age [START_REF] Li | Decline in miR-181a expression with age impairs T cell receptor sensitivity by increasing DUSP6 activity[END_REF]. Conversely, a decreased threshold for TCR activation, secondary to sustained cytokine production, leads to diverse autoimmune manifestations in rheumatoid arthritis patients [START_REF] Deshpande | IL-7-and IL-15-mediated TCR sensitization enables T cell responses to self-antigens[END_REF][START_REF] Singh | ERK-Dependent T Cell Receptor Threshold Calibration in Rheumatoid Arthritis[END_REF]. With respect to chronic infection, functional defects in the naive T cell compartment have also been documented in HIV-infected individuals, with non-cognate activation of T cells correlating with disease progression [START_REF] Favre | HIV disease progression correlates with the generation of dysfunctional naive CD8low T cells[END_REF]. These studies have all addressed global dysregulation of the naive T cell repertoire.

Due to their low frequency, little is known about the perturbations of antigen-specific populations in the context of chronic infection or inflammation. Due to the possibility of studying viremic vs. cured patients, we chose to investigate this question in cHCV patients. Analyzing rare (i.e., frequency = 10 -7 -10 -5 ) Ag-specific inexperienced CD8 + T cells populations, we demonstrate increased proportions of memory-phenotype cells in cHCV patients. This is in line with previous data showing that 34% of cHCV patients without detectable tumor secrete IFNγ when their harvested T cells are stimulated in vitro with tumor-specific peptides, as compared to 0% in comparable healthy donors [START_REF] Mizukoshi | Comparative analysis of various tumor-associated antigen-specific t-cell responses in patients with hepatocellular carcinoma[END_REF]. Additionally, we showed the naive T cells to be hyperreactive to TCR signaling. Despite these altered phenotypes, the absolute number of Ag-specific cells was comparable to healthy donors. This may be explained by the higher sensitivity to cell death, as indicated by ex vivo functional studies. Of note, cHCV patients are not thought to experience altered thymic output. As such, our findings provide the direct evidence that MP antigen-specific T cells can arise in nonlymphopenic humans.

It has been suggested that a high degree of cross-reactivity with environmental antigens is the trigger for differentiation and MP conversion [START_REF] Sprent | Normal T cell homeostasis: the conversion of naive cells into memoryphenotype cells[END_REF]. This finding has been reported for human viral pMHC restricted CD4 + T cells, in unexposed donors. While cross-reactivity is a possible explanation for our findings, we demonstrate in cured patients that the Ag-specific inexperienced cells are restored to a naive phenotype. This result favors an alternative model, where homeostatic proliferation accounts for the perturbed naive T cell repertoire in cHCV patients. Indeed, (i) several unrelated self-and non-self-populations are concerned -which goes against the hypothesis of cross-reactivity [START_REF] Welsh | Heterologous immunity between viruses[END_REF], and (ii) Mart1-specific MP T cells are being clonally expanded. These findings parallel rapid reversibility of a healthy preimmune repertoire after transient anti-CD52 (also known by alemtuzumab)-induced lymphopenia in multiple sclerosis patients [START_REF] Jones | Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation[END_REF]. Consistent with our findings, it has been indeed shown a narrowing of the Vβ repertoire and a dilution of sjTREC after this treatment, with a complete restoration of normal levels two years after therapy.

Infection and inflammation are known to lower the threshold of TCR signaling in memory T cell, making them more sensitive to activation [START_REF] Richer | Pathogen-Specific Inflammatory Milieux Tune the Antigen References Sensitivity of CD8+ T Cells by Enhancing T Cell Receptor Signaling[END_REF]. This effect is mediated by inflammatory cytokines [START_REF] Raué | Cytokine-Mediated Programmed Proliferation of Virus-Specific CD8+ Memory T Cells[END_REF]. Our results extend this concept to naive T cells and introduce CD5 downregulation as a mechanism for hyperreactivity. CD5 tunes the TCR signaling threshold in peripheral T cells, with naive cells expressing higher levels than central memory or effector T cells [START_REF] Tabbekh | T-cell modulatory properties of CD5 and its role in antitumor immune responses[END_REF]. In mice, Hawiger et al. demonstrated that anti-CD5 blocking antibodies or use of CD5 -/-transgenic MOG-specific T cells resulted in higher sensitivity to experimental autoimmune encephalitis [START_REF] Hawiger | Immunological unresponsiveness characterized by increased expression of CD5 on peripheral T cells induced by dendritic cells in vivo[END_REF]. In B cells, CD5 has also been shown to regulate activation and lower expression correlates with higher sensitivity to activation induced cell death [START_REF] Tabbekh | T-cell modulatory properties of CD5 and its role in antitumor immune responses[END_REF]. In line with these findings, we demonstrate an increased sensitivity of CD5 lo naive CD8 + T cells to TCR ligation in cHCV patients. We further provide direct evidence that this hypersensitivity phenotype can be reproduced in HD by blocking CD5. While not evaluated in our patient cohort, we suggest that elevated levels of inflammatory cytokines may be responsible for the altered CD5 expression on naive cells. Interestingly HCV and HBV induce different cytokine and chemokine signatures [START_REF] Park | Immune Responses to HCV and Other Hepatitis Viruses[END_REF], possibly explaining the differential results in these two forms of viral hepatitis. However, one caveat is that our cHBV cohort was heterogeneous in viral loads and current clinical status (immune tolerance, immune clearance or inactive carriage phase).

Further evaluation will thus be needed to confirm these initial observations in HBV chronic infection.

In mouse models, self-specific populations express higher levels of CD5 than foreign-specific ones, as a mechanism to maintain peripheral tolerance [START_REF] Grossman | Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function[END_REF]. It would be interesting to confirm these findings in humans by measuring CD5 levels on different self-and nonself-Ag-specific naive populations. As cHCV globally decreases the level of CD5 on naive cells, our results suggest a model where it will have different impacts based on the starting level of CD5 (Figure 46A):

• self-specific populations, which have the highest initial levels, will come into the range of being « easily authorized to proliferate » -which explain why they are found unspecifically expanded and which may be linked to some clinical features such as extrahepatic systemic manifestations and lymphoproliferation during the course of cHCV infection;

• non-self-specific populations, which have the lowest initial levels, will come into the range of being deleted after TCR stimulation -which could explain (i) that HCV-specific populations are detected at low frequencies in HCV patients, and (ii) that cHCV patients have poor responses to some vaccinations involving cellular immunity.

Additionally, at the level of a given naive antigenic specificity, a range of TCR avidity -and thus a range of CD5 expression, also exists and has recently been linked to some features of T cell activation in vitro [START_REF] Fulton | The TCR's sensitivity to self peptide-MHC dictates the ability of naive CD8+ T cells to respond to foreign antigens[END_REF]. With the assumption that the CD5 decreased levels observed in the context of HCV globally are homogeneously affecting cells in Ag-specific populations, this would favor the expansion of cells with the lowest affinity (highest CD5), and deletion of the highest ones (lowest CD5). This model would be in adequation with previous finding that the selection of highavidity CD8 T Cells correlates with control of hepatitis C virus infection [START_REF] Neveu | Selection of high-avidity CD8 T cells correlates with control of hepatitis C virus infection[END_REF].

Combination of low levels of CD5 and increased proportions of MP on both self and non-self specific populations may synergize, resulting in a highly reactive CD8 + T cell compartment. As such, our data fits with the concept that autoimmunity may be triggered by infection [START_REF] Jones | Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation[END_REF][START_REF] Welsh | Heterologous immunity between viruses[END_REF]. This is indeed the case for cHCV patients, were disease pathogenesis includes systemic manifestations -many of which are believed to be immune-mediated [START_REF] Lee | Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a community-based long-term prospective study[END_REF].

Further investigation in longitudinal cohorts is thus warranted, with the aim to establish a dysregulated preimmune repertoire as the cause of bystander manifestations of disease. Moreover, our data highlight the value of early cHCV treatment, as immune restoration may prevent long-term sequella.

In summary, our study indicates that naive CD8 + T cells are dysregulated during cHCV, with marked perturbations of the preimmune repertoire. Specifically, low levels of CD5 at the surface of naive T cells, and high proportions of memory-phenotype cells represent two mechanisms by which antigeninexperienced CD8 + T cells are likely to be susceptible to stimulation and cell death. These findings should be considered when designing future immunotherapeutic strategies.

IV. PROSPECTIVES

In the next months, we plan to better investigate the impact of our observations on the behavior of CD8 + T cell responses after T cell priming. For this purpose, we plan to compare the outcome of two different in vitro assays.

First, we will generate T cell libraries using the protocol developed by the group of Frederica Sallusto [START_REF] Geiger | Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells[END_REF]. In brief, from three cHCV and three HD tumor-free and CMVseronegative patients, we will sort bulk naive CD8 + T cells (2 000 cells per well). The cultures will then be stimulated by PHA in the presence of irradiated allogeneic feeder cells and Il-2, and expanded for 2 weeks. This will lead to a 1 000 -5 000 -fold expansion. Aliquots from each culture will then be tested for their capacity to proliferate in response to Mart1, hTERT, gp100, HIV and CMV antigens processed and presented by autologous monocyte-derived DCs. By comparing precursor frequency calculated at Day 0 by TAME to its estimation after library generation, we believe that this will inform us on the sensitivity of the preimmune T cells to cytokine-related expansion.

Second, we will evaluate Ag-specific T cell priming by cognate peptide. We will use an optimized 10-days protocol called ACE-CD8 [START_REF] Greenberg | Antigen-specific activation and cytokine-facilitated expansion of naive, human CD8[END_REF], that was designed to allow conclusions about the initial priming conditions. In brief, monocyte-derived DCs are matured with LPS/IFNγ (to favor IL-12 production), loaded with the peptide, and cocultured with CD57/CD45RO depleted CD8 + T cells. For the initial stage of the priming, IL-21 is the only cytokine to be added to the culture. All other cytokines required are produced by DCs or the T cells themselves. In the absence of IL-7 and IL-15, a proliferative advantage is thus provided to cells receiving a TCR stimulus, whereas non-specific T cells may be "starved" during this period. Starting at Day 3, cultures are supplemented by low amounts of IL-7 and IL-15 to favor expansion of the primed cells. No IL-2 is needed before 15 days of culture, and rather pushes the cells towards terminal differentiation. We will use this protocol on the same three cHCV and three HD patients as above, and follow both Ag-specific and non-specific T cell expansion and differentiation. This will permit to evaluate the ability of the preimmune cells to be primed, expanded, recruited into the response, and to survive after T cell activation.

Using these two assays on the same patients, plus our knowledge on their preimmune repertoire at baseline will permit to have a better idea of the consequences of naive dysregulation in cytokine and Ag-driven expansion.

As mid-term objectives, we propose to:

• Better characterize the tuning of human lymphocytes by CD5. Indeed, there are still many open questions. Is there a common pattern of CD5 expression among (i) different self-and non-self Ag-specific populations, and (ii) different donors? Is there a specific signature of CD5 hi and CD5 lo antigen-specific populations at baseline when engaged into an immune response (calcium flux, CD62L downregulation, ERK and p-38 phosphorylation, effector/memory differentiation, expansion, cell death)? Are CD5 lo antigen-specific populations more prompt to divide towards MP cells as a result of aspecific peripheral pMHC/TCR interactions? For addressing these questions, we will enrich inexperienced Agspecific populations from a cohort of 20 healthy donors, evaluate CD5 expression, sort CD5 hi and CD5 lo Mart1-specific populations, and evaluate their behavior using short, mid and long term functional assays as described previously. Cell lines derived from these assays will be later used for silencing experiments where we could specifically modulate CD5 expression and test its impact on activation and cell death following TCR stimulation;

• Test the reversibility of naive T cells alterations in cHCV patients in vitro after incubation with soluble human CD95-Fc. Recent papers indicate the rescue of T cells from TCRmediated apoptosis with soluble CD95-Fc molecules both in-vitro and in-vivo, resulting in dramatic reduction of tumor growth in CD5-deficient mice [START_REF] Tabbekh | Rescue of Tumor-Infiltrating Lymphocytes from Activation-Induced Cell Death Enhances the Antitumor CTL Response in CD5-Deficient Mice[END_REF] ;

• Evaluate same questions in the CD4 + T cell preimmune repertoire.

Finally, as long-term perspectives, we believe that our findings should be evaluated in the context of three aspects of cHCV pathogenesis:

• First, we believe that naive T cell hyperactivation is a systemic effect of cHCV, and as such could explain some of the extrahepatic manifestations observed in this disease;

• Second, patients with chronic HCV infection are at risk of developing hepatocellular carcinoma, mainly as an effect of tissue reorganization and inflammation. Our findings of MP cells in self-specific populations is intriguing, as those cells (i.e. gp100-or hTERT-specific T cells) are targeting antigens that will potentially later be over-expressed on the tumor site. One possible consequence is that those MP cellsbeing more reactive -will be an advantage for fighting the tumor when it will arise.

Alternatively, those cells that will have experienced multiple rounds of aspecific proliferation for twenty years will possibly be exhausted before any story of cancerand favor "easy" (i.e. unpressurized) tumor growth in those patients;

• Finally, cHCV patients typically have poor responses to adult vaccinations such as those against HAV and HBV ( [START_REF] Buxton | Hepatitis A and hepatitis B vaccination responses in persons with chronic hepatitis C infections: A review of the evidence and current recommendations[END_REF]. We believe that this could be related to a failure of inducing adequate CD8 + T cell responses in those patients because of an altered preimmune T cell repertoire targeting vaccine epitopes.

These three points will have to be addressed in the context of prospective cohorts. A. Impact of CD45RO-magnetic enrichment on naive/memory T cell subsets on one HD (left) and one cHCV patient (right). B. Expansion of Mart1-specific T cells from RO -fraction of HD. C. Detection of Mart1 expanded specific cells after 10 days of in vitro priming assay on enriched CD8 + T cells from one cHCV patient. Cells were detected by conventional tetramer (WT) of CD8null tetramer (Null). Two doses of Mart1 peptide were tested: low (10 -8 ) or high (10 -6 ). Cells were analysed at Day 7 or Day 11. Wells without cytokines or without peptide were used as control. D. CD5 and CD25 expression on Mart1-specific cells detected by the WT tetramer at Day 7 and 11. The activation threshold hypothesis. In healthy donors, before immunization, a given antigenspecific population contains T cells with ranges of CD5 expression (high, dark green; low, light green). In periphery, those cells are maintained by cytokines, and by low affinity pMHC contacts, and some of them may occasionally derive toward a memory-phenotype (violet). After immunization with its cognate peptide, the antigen-specific population expand largely, and express lower CD5 levels -although there is still a mix of high (dark violet) and low (light violet) expression. Some rare cells are not engaged into the response and remain as naive-phenotype into the globally experienced population. In the context of cHCV, most naive CD8 + T cells express low levels of CD5 at their cell surface. This lead to aspecific expansion and differentiation when encountering low affinity ligands in the periphery. When those cells encounter their antigen during the immunization phase, they will generate low-affinity and low-frequency specific populations.

Chapter 4. Study the impact of Cxcr3 receptor on inexperienced T cells I. INTRODUCTION

Our laboratory recently provided the first evidence for dipeptidylpeptidase 4 (DPP4) mediated chemokine antagonism in the context of chronic HCV pathogenesis (Casrouge et al., 2011b). DPP4

(also known as CD26) is a member of the family of X-prolyl dipeptidylpeptidases (DPPs) family, and is capable of enzymatically removing the N-terminal two amino acids of proteins that possess a proline or an alanine in the second position [START_REF] De Meester | CD26, let it cut or cut it down[END_REF]. Although several molecules possess the consensus motive for DPP4 recognition [START_REF] Ou | Implications of DPP4 modification of proteins that regulate stem/progenitor and more mature cell types[END_REF], only few were experimentally tested and defined as DPP4 subtracts. More importantly, the biological relevance of DPP4-mediated post-translational modifications of proteins remains poorly studied. Two notable exceptions are the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP); and the chemokine CXCL12. N-terminal truncation of GLP-1 and GIP renders these hormones incapable of inducing insulin secretion, and therefore over 30 million type II diabetes patients are being treated with DPP4 inhibitors. In analogy, processing of the chemokine CXCL12 by DPP4 diminishes the efficiency of hematopoietic cell engraftment and regulates G-CSF and GM-CSF mobilization of CXCR4-expressing hematopoietic stem cells [START_REF] Christopherson | Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells[END_REF][START_REF] Christopherson | Modulation of hematopoietic stem cell homing and engraftment by CD26[END_REF].

Although CXCL12 remains the only member of the chemokine family for which there is in vivo evidence of altered chemotactic activity induced by DPP4, in vitro studies revealed that CXCL10 is also a target of this enzyme. DPP4 truncation of CXCL10 generates a dominant negative form of the chemokine, which binds its receptor, CXCR3, but does not induce signaling (Proost et al, Blood 2001). Studies from our lab revealed that high levels of the chemokine CXCL10 correlated with the inability of HCV infected patients to respond to therapy and to achieve viral clearance. Specifically, we detected N-terminal truncation of the chemokine interferon-induced protein 10 (IP-10 or CXCL10) in cHCV patients, which correlated with higher DPP4 plasma activity, reduced trafficking of CXCR3expressing lymphocytes and poor response to treatment (Casrouge et al., 2011b;[START_REF] Riva | Truncated CXCL10 is associated with failure to achieve spontaneous clearance of acute hepatitis C infection[END_REF].

DPP4 truncation of IP-10 generates a dominant negative form of the chemokine, which binds its receptor, CXCR3, but does not induce signaling. As a result, it suggested that DPP4 perturbs lymphocyte trafficking in the context of HCV pathogenesis, accounting for DPP4 activity being a correlate of treatment failure.

These observations inspired my work, and pushed me to explore whether disruption of chemokine gradients may alter inexperienced T cell homing in vivo. Specifically, I investigated the impact of steady-state CXCR3-expression on the homeostasis of (i) intrathymic and (ii) peripheral inexperienced CD8 + T cells in vivo. This work was initiated during the last year of my thesis and is a work-inprogress. In this Chapter, I present preliminary results from these studies, with the hope to initiate a discussion and establish a plan for completing my studies on tissue-resident inexperienced T cells.

I. IN THE THYMUS

CXCR3-expression by intrathymic T cells has been reported to direct T cell egress after full maturation [START_REF] Annunziato | Chemokines and lymphopoiesis in human thymus[END_REF][START_REF] Romagnani | Interferon-inducible protein 10, monokine induced by interferon gamma, and interferoninducible T-cell alpha chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) alphabeta+CD8+ single-positive T cells, TCRgammadelta+ T cells, and natural killer-type cells in human thymus[END_REF]. In humans, four thymocytes populations express CXCR3: (i) a subset of TCRαβ + CD3 + CD8 + SP cells; (ii) a subset of TCRαβ + CD3 + DP thymocytes; (iii) TCRγδ + and (iv) NKT cells [START_REF] Drennan | Cutting Edge: The Chemokine Receptor CXCR3 Retains Invariant NK T Cells in the Thymus[END_REF]. We confirmed this pattern of expression by phenotyping thymocytes extracted from three distinct thymi of human neonates undergoing cardiac surgery (Figure 47A). About 5 +/-2% of the CD3 + CD8SP intrathymic T cells were found to express CXCR3. We similarly studied CXCR3 expression on three thymi from C57BL/6 mice (aged 8 weeks), and observed 1 to 3% of CXCR3 + CD8SP intrathymic T cells (Figure 47B).

To investigate the function of CXCR3 on intrathymic T cells, we evaluated the effect of cxcr3 gene disruption using CXCR3-knockout (cxcr3 -/-) mice. We observed decreased percentages (M-W p=0.007) and lower absolute numbers (M-W p=0.007) of CD3 + CD8 + SP T cells in the thymus of cxcr3 - /-as compared to control WT mice (Figure 47C andD). By contrast, absolute numbers of DN, DP and CD3 + CD4 + SP cells were unchanged (data not shown).

When considering additional phenotypic markers, we identified a subset of about 5.5% of CD3 + thymocytes that included both CD4 + and CD8 + cells, and expressed high levels of CD44 and no CD62L (Figure 47E andF). This subset was significantly less abundant in cxcr3 -/-mice, and could account for the loss of at least some of the CD8 + SP thymocytes observed in these mice. In control mice, the CD62L -CD44 hi subset of T cells expressed higher levels of CXCR3 and lower levels of CD3

and TCRβ than the other thymocytes (Figure 47G). As detailed in the introduction, intrathymic T cells contain both T cells in early stages of development, and activated T cells rehoming to this organ.

To discrimination between the two, we used RAG2p-GFP mice, where GFP expression is under the promoter of Rag2 and stain T cells under the process of development [START_REF] Yu | Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5' of RAG2[END_REF]. We observed that CD62L -CD44 hi cells were GFP neg , which indicates that they are T cells rehoming to the thymus (Figure 47H). We thus hypothesized that they could be intrathymic resident memory T cells [START_REF] Mackay | Tissue-resident memory T cells: local guards of the thymus[END_REF]. Accordingly, we found that those cells expressed high levels of CD69 and CD103 (Figure 47I).

Together, our data suggest that the expression of CXCR3 is necessary for the rehoming of CD8 + T lymphocytes into the thymus, where they constitute a subset of CXCR3-expressing resident memory T cells.

II. IN PERIPHERY

A. MP cells in the blood of non-immunized cxcr3 -/-animals I next analyzed the number and phenotype of T lymphocytes in the peripheral blood, again comparing WT and cxcr3 -/-mice. We observed similar numbers of CD3 + CD8 + (Figure 48A) and CD3 + CD4 + T cells (data not shown) in cxcr3 -/-mice as compared to WT controls. However, we found increased proportions (Figure 48B andC) and absolute numbers of CD62L -CD44 + memory-phenotype (MP)

CD3 + CD8 + T cells circulating in the blood of cxcr3-deficient mice (Figure 48D). Of note, similar observations were made in the CD3 + CD4 + T cell subset (data not shown). To evaluate the impact of these perturbations on antigen-specific populations, we developed cxcr3 -/-OTI transgenic mice. OTI mice contain transgenic inserts for mouse TCRα-V2 and TCRβ-V5 genes, driving the expression of a transgenic TCR that recognize ovalbumin residues 257-264 in the context of H2-Kb molecules. As cxcr3 is located on the X chromosome, we bred OTI tg/tg (Rag-competent) males (cxcr3 +/0 ) with cxcr3 -/- females. We utilized in our experiments (i) F1 males (OTI tg/0 -Rag competent CXCR3 -/0 , later referred to as OTI-CXCR3KO) for analysis, and (ii) F0 animals (later referred to as OTI-WT) as controls.

When examining the peripheral blood of OTI-CXCR3KO mice, we found comparable absolute numbers of OTI cells as in OTI-WT animals, but increased proportion of MP cells (Figure 48E

B. Distinct TCR activation pattern

As presented in the Introduction, MP cells in the preimmune repertoire can have distinct threshold for TCR activation as compared to naïve-phenotype cells. To evaluate the threshold of TCR activation of cxcr3-sufficient and cxcr3-deficient Ag-specific CD8 + T cells, we examined expression of surface activation markers in OTI-WT and OTI-CXCR3KO blood cells 24 hours after in vitro stimulation with their cognate SIINFEKL peptide. We tested two distinct concentrations of peptide (C1= 1ug/ml, C2= 5ng/ml), and used CD3/CD28-coated beads and PMA-Ionomycin as positive controls for T cell activation. We found OTI-CXCR3KO cells expressing lower levels of CD69 and CD25 after stimulation with different concentrations of the SIINFEKL peptide (Figure 49A andB). These lower levels were related to the presence of two distinct patterns of activation in OTI-CXR3KO populations, rather than to a global decrease (Figure 49C). When examining cell phenotype, we identified a population of CD62L -CD8 + T cells (blue) associated with lower levels of CD69-expression as compared to CD62L + T cells (red) (Figure 49D). These results suggest that the CD62L -CD44 + MP population (overrepresented in cxcr3 -/-mice) displays a different response to peptide-induced stimulation, and might play distinct immune functions from its naïve counterparts.

These data highlight the impact of the heterogeneity in the preimmune repertoire of OTI-CXCR3KO animals on TCR-induced activation and cell death.

C. MP OTI cells in the skin of unimmunized WT animals

The results presented so far revealed that CXCR3-deficiency results in an altered phenotype of CD8 + T cells in blood and spleen of antigen-specific OTI transgenic mice. We therefore became interested to study the phenotype of CD8 + T cell populations in other lymphoid and non-lymphoid organs of OTI-CXCR3KO and OTI-WT mice. To address this question, we investigated the presence of Ova-specific tetramer-positive T cells in lymph nodes, bone marrow, liver and skin of transgenic mice. Our first analysis revealed an elevated number of transgenic OT1 cells the bone marrow, spleen and liver of OTI-CXCR3KO mice, as compared to WT mice (Figure 50A, red gate). Inversely, OT1 cells were less frequent in the skin of Cxcr3-deficient animals (blue gate). When considering the phenotype of cells harvested from OTI-WT tissues, we observed that circulating OTI cells (i.e. cells localizing in the blood and the spleen) were predominantly of naïve-phenotype (NP). In contrast, OTI cells harvested from the skin contained high proportions of memory-phenotype (MP) cells (Figure 50B, left). Additionally, these cells expressed high levels of CD103 and CD69 (Figure 50C), suggesting that they are resident memory T cells [START_REF] Clark | Resident memory T cells in human health and disease[END_REF]. Of note, MP cells were still detectable -although less frequent, in Rag-deficient OTI animals (Figure 50D). This suggests that non-exclusive α-chain TCR rearrangement account for part -but not all -of the OTI skin phenotype. When examining Cxcr3-deficient animals, OTI skin cells were rare, and displaying a similar phenotype as compared to the controls (Figure 50B, right).

In the above experiments, we compared the distribution and phenotype of OTI transgenic CD8 + T cells in organs from F0 OTI tg/tg cxcr3 +/0 (OTI-WT) and F1 OTI tg/0 cxcr3 -/0 (OTI-CXCR3KO) mice. We therefore tested if our results were dependent on the number of OTI transgene copies present in the different genetically modified mice. For this, we enumerated skin OTI cells in cxcr3-sufficient transgenic mice harboring different OT1 transgene copies: OTI WT (2 transgene copies); OTI tg/0 WT (1 transgene copy) and OTI tg/tg Rag -/-WT (2 transgene copies). We found similar numbers of skin OTI cells in the three cxcr3-sufficient OT1 mouse strains, and those numbers were significantly higher than in OTI-CXCR3KO animals (Figure 50E). We therefore excluded transgene-related bias as a reason for OT1 accumulation in the skin.

D. MP OTI cells arise in homeostatic environments

The altered number and phenotype of CD8 + T cells found in cxcr3-deficient mice led us to hypothesize that the expression of this chemokine receptor might be important for the steady-state migration and/or retention of CD8 + T cells into peripheral tissues, such as skin. To evaluate the behavior of OTI-WT and OTI-CXCR3KO cells in a unique homeostatic environment, we created mixed bone marrow chimeras (Figure 51A). We reconstituted irradiated WT CD45.1 C57/Bl6 mice with a mix (ratio 1:1) of bone marrow from OTI-Rag -/--GFP Ub (OTI-WT:GFP) and from OTI-CXCR3KO (CD45.2). After 2 months, we harvested the thymus, blood, spleen and skin of reconstituted animals for enumeration and phenotypic analysis of OTI cells. In the blood, we observed similar proportions and absolute numbers of OTI-WT and OTI-CXCR3KO specific cells (Figure 51B and C), suggesting that cxcr3-deficient or sufficient bone marrow was equally engrafted upon transfer. However, circulating OTI-CXCR3KO population contained higher proportions of CD62L - CD44 + MP cells as compared to OTI-WT (Figure 51B andD). Same phenotype was observed in the spleens of those animals (data not shown). Confirming our previous results, circulating MP cells presented a distinct pattern of activation after activation with their cognate peptide (Figure 51E). In the skin, however, we observed similar phenotype and number of reconstituted WT or cxcr3-deficient OTI cells. These preliminary observations indicate that circulating memory phenotype OTI cells do not arise as a result of possible microenvironment differences, but rather relate to intrinsic T cell properties. Additionally, we interpret the discrepancy between our skin observations in single KO animals and bone marrow chimeras as indicating that T cell depletion observed in OTI-CXCR3KO animals reflects the depletion of non-cognate crossreactive resident tissue-memory cells, rather than a distinct engraftment capacity. While these experiments need to be repeated, one should notice that the OT1 WT bone marrow used in these experiments presents at least two differences as compared to the OTI-CXCR3KO bone marrow: they have two copies of the transgene, and they are Rag-deficient.

Therefore, we are planning experiments using OTI tg/o animals, as source of cxcr3-sufficient bone marrow.

Our results suggest that the identification of steady-state inexperienced MP CD8 + T cells into tissues relates to them being tissue resident-memory cells, which localization is dependent on the expression of the chemokine receptor CXCR3 (Figure 52). Whether abrogation of CXCR3-mediated migration results in a higher number of Ag-specific MP cells circulating in the blood is unclear.

III. DISCUSSION

Our work confirms that MP Ag-specific inexperienced T cells do exist in unimmunized animals [START_REF] Goldrath | Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation[END_REF] and identifies three situations: (i) the skin, where Ag-specific inexperienced T cells do localize at steady-state, resembling resident memory T cells [START_REF] Ariotti | Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert[END_REF], (ii) the thymus, where cxcr3 + CD8 + T cells likely correspond to rehoming memory T cells trafficking through CXCR3-mediated signaling [START_REF] Mackay | Tissue-resident memory T cells: local guards of the thymus[END_REF], and (iii) the blood, where high numbers of MP cells circulate in the context of Cxcr3-deficiency.

Although preliminary, our results identify T cells residing into tissues as a source of inexperienced T cells. Further experiments will be needed to test whether those cells are deeply into the tissues, or trapped into the vasculature [START_REF] Anderson | Cutting Edge: Intravascular Staining Redefines Lung CD8 T Cell Responses[END_REF]. In the skin, CXCL9 and CXCL10

(both being ligands for CXCR3) are quickly and strongly upregulated after stimulation [START_REF] Villarroel | CXCR3-Mediated Skin Homing of AutoreactiveCD8 T Cells Is a Key Determinant in MurineGraft-Versus-Host Disease[END_REF]. Increased basal levels of CXCL10 have been implicated in several T-cell mediated skin diseases such as alopecia [START_REF] Mcphee | Increased Expression of Cxcr3 and Its Ligands, Cxcl9 and Cxcl10, during the Development of Alopecia Areata in the Mouse[END_REF], erythema migrans [START_REF] Mullegger | Chemokine Signatures in the Skin Disorders of Lyme Borreliosis in Europe: Predominance of CXCL9 and CXCL10 in Erythema Migrans and Acrodermatitis and CXCL13 in Lymphocytoma[END_REF], as well as contact hypersensitivity [START_REF] Suga | CXCR3 Deficiency Prolongs Th1-Type Contact Hypersensitivity[END_REF]. On the other hand, cxcr3 deficiency results in reduced control of cutaneous Leishmania infection [START_REF] Rosas | CXCR3-/mice mount an efficient Th1 response but fail to controlLeishmania major infection[END_REF], as well as less severe acute skin GVHD lesions in a murine Ova-specific model of acute skin GVHD [START_REF] Villarroel | CXCR3-Mediated Skin Homing of AutoreactiveCD8 T Cells Is a Key Determinant in MurineGraft-Versus-Host Disease[END_REF]. Here, we observed memory-phenotype (MP) inexperienced Ag-specific T cells (i) localizing into this tissue, (ii) depleted in cxcr3 -/-mice, (iii) but not in mixed bone marrow chimeras. Our interpretation is that MP cells correspond to skin resident memory T (T RM ) cells that have the potential to be crossreactive for specificities of the preimmune repertoire. T RM cells have been identified in parabiosis and transplant experiments as resting tissue memory CD8 + T cells with specific non-circulating properties [START_REF] Shin | Tissue-resident memory T cells[END_REF]. They are conserved across species (mouse, rat, pigs), and found in multiple tissues such as small intestine, vaginal mucosa, brain, lung, salivary glands, and thymus [START_REF] Mackay | Tissue-resident memory T cells: local guards of the thymus[END_REF][START_REF] Mueller | Tissue-resident T cells: dynamic players in skin immunity[END_REF]. Their recruitment into the skin, lungs, and vaginal mucosa, is dependent on cell-surface expression of CXCR3 (Mackay et al., 2013). This has been demonstrated by a depleted T RM compartment in cxcr3 -/-animals. Their behavior after sublethal irradiation is unknown, and it is not excluded that -as happening for some DCs subsets [START_REF] Bogunovic | Identification of a radio-resistant and cycling dermal dendritic cell population in mice and men[END_REF]) -they could be radioresistant. This could explain why we do lose our skin phenotype in our mixed bone marrow chimeras experiments. T RM are characterized by high expression levels of αE integrin (CD103) and CD69 [START_REF] Mueller | Tissue-resident T cells: dynamic players in skin immunity[END_REF], and a specific gene-expression signature [START_REF] Ariotti | Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert[END_REF]. They are fully functional, quickly producing IFNγ upon local peptide stimulation, and triggering a state of tissue-wide pathogen alert [START_REF] Ariotti | Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert[END_REF]. In the thymus, T RM have recently been identified, and demonstrated as important in local immunity [START_REF] Mackay | Tissue-resident memory T cells: local guards of the thymus[END_REF]. Our results suggest that, similar to the skin situation, trafficking and/or retention of those cells into the thymus is dependent on cxcr3.

In the blood, we highlighted a dysregulated preimmune repertoire in OTI-CXCR3KO mice, with high proportions of MP cells. High proportions of MP cells have been described into inexperienced OTI-Rag -/-populations in mice when those cells were adoptively transferred into sublethally irradiated animals. This effect reflected homeostatic-driven proliferation, and was transient, with a recovery of a naive T cell phenotype within 120 days after the adoptive transfer [START_REF] Goldrath | Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation[END_REF]. In line with this, in our hands, OTI-Rag [START_REF] Kurachi | Chemokine receptor CXCR3 facilitates CD8+ T cell differentiation into short-lived effector cells leading to memory degeneration[END_REF]. Whatever the mechanism, we believe that increased numbers of antigen-specific memory T cells reported in the latter study is a direct consequence of high numbers of MP cells in the preimmune repertoire of OTI-CXCR3KO mice.

Finally, OTI T cells deficient in Cxcr3 presented a distinct pattern of TCR activation as compared to OTI-WT cells, related to the presence of MP cells. This confirms the influence of preimmune repertoire diversity in early kinetics of activation. In the VV-Ova and LM-Ova models, other groups observed a faster downregulation of CD25 and CD69 markers on OTI-CXCR3KO cells as compared to OTI-WT [START_REF] Kurachi | Chemokine receptor CXCR3 facilitates CD8+ T cell differentiation into short-lived effector cells leading to memory degeneration[END_REF]. Our results of lower CD25 and CD69 after 24 hours of activation could also reflect higher sensitivity to TCR activation, but investigation of additional earlier time points will be required to formally document this effect. For which concerns the impact of preimmune repertoire perturbations on long-term immune responses, we believe that our observations might account for the documented skewing of OTI-CXCR3KO cells towards a high potential for memory precursor effector-cells (MPECs) generation. Further studies will be needed to validate this assumption.

IV. PROSPECTIVES

Altogether, our interpretation of these preliminary results is that (i) CXCR3-dependent tissue resident memory T cells can be crossreactive for preimmune repertoire specificities, (ii) CXCR3expression might facilitate the homeostatic clearance of MP inexperienced Ag-specific T cells, and

(iii) MP preimmune cells can impact early, mid, and long term aspects of the immune response.

In the next months, we will study the functionality of MP circulating T cells, with (i) PhosFlow at early time points (t=5min), and (ii) precise kinetics of activation and cell death events during the first 24-48 hours after stimulation with SIINFEKL peptide. These experiments will allow us to test the hypothesis that OTI-CXCR3KO cells have a decreased threshold for TCR activation.

Additionally, we will document the sensitivity of Cxcr3-deficient cells to TCR-induced cell death.

In parallel, we will evaluate if some of the MP cells circulate in OTI-CXCR3KO mice because they are lacking ability to access their target tissue. We plan to investigate this by comparing geneexpression patterns of FACS-sorted (i) skin MP OTI cells from OTI-WT, (ii) spleen MP OTI cells from OTI-CXCR3KO mice, and (iii) thymus GFP -CD3 + CD8 + T cells. We will focus on genes previously identified as differentially expressed on tissue resident memory cells [START_REF] Ariotti | Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert[END_REF].

We will compare those subsets to "true memory" cells, i.e. OTI cells recovered >40 days after in vivo infection with LM-Ova. These experiments will evaluate the distance between those different subsets of T cells, providing important informations on their nature. Our hypothesis is that skin and thymic subsets will be close, and different from both the spleen MP OTI-CXCR3KO, and "true memory" cells.

We also plan to repeat bone marrow chimeras experiments, with grafting OTI tg/0 -cxcr3 +/0 -WT and OTI tg/0 -cxcr3 -/0 (OTI-CXCR3KO) Rag-competent cells into WT and cxcr3 -/-hosts. These experiments will provide additional information towards the hypothesis of skin resident memory T cells crossreacting with the preimmune repertoire.

Finally, we propose to enrich Ova-, LCMV-, and MCMV-specific inexperienced populations from the thymus of WT and cxcr3 -/-RAG-GFP unimmunized animals. Enumerating and phenotyping those cells will provide informations about the residency of inexperienced T cells into the thymus.

Same analysis on animals infected by LM-Ova will provide additional information on memory T cells rehoming into this tissue, as well as their potential for crossreactivity.

Ultimately, our work indicates that CXCR3 blockade before immunization could be a useful strategy for shaping the preimmune repertoire towards higher reactivity -therefore improving vaccine efficacy. To test this, we will evaluate the impact of i.p. injections of blocking anti-CXCR3 antibodies on the T cell preimmune repertoire of OTI-WT mice, and in early kinetics of the immune response after LM-Ova challenges. Blocking anti-CCR5 antibodies, as well as relevant isotype will be used as controls. These experiments will bring innovative insights into the possibility of artificially shaping the T cell preimmune repertoire, and dictating the magnitude and/or qualitative aspects of the later immune responses. From our results, we propose that MP might be resident memory T cells that (i) seed into the skin (and maybe the thymus) tissues at steady state in unimmunized animals under the influence of CXCR3/ligands gradients; and (ii) might have the potential to crossreact with unknown specificities of the pre-immune repertoire. Whether high numbers of MP Ag-inexperienced T cells are circulating into the blood of cxcr3-deficient animals reflects (i) resident-memory T cells lacking their ability to reach target tissues, and/or (ii) a direct effect of Cxcr3 is unclear and needs additional investigations.

Chapter 5. General Discussion

I. IMMUNOPHENOTYPING CHALLENGES

When I joined the lab, my project was to enumerate and characterize Ag-specific naive T cells from the blood of healthy humans. I thus immediately dived into the reading of previous studies on naive and memory T cell compartments -and found myself lucky to work on naive T cells and not memory as the diversity in the latter is quite intimidating. I selected phenotypic markers that I knew will I am trained as a medical biologist, and as such had the opportunity to participate to the immunomonitoring of several clinical trials in different hospital sites. Most of the analyses that we were doing on the blood samples of the patients were phenotypic analysis, with different FACS panels to enumerate and characterize subsets of hematopoietic cells. One of them usually referred to naive and memory T cells, with two or three markers coupled to CD3/4/8 antibodies. Others and we were hoping to observe some perturbations of these compartments that might correlate with clinical outcome, and to be able to later utilize those biomarkers to predict vaccine efficacy. Others and we were always disappointed to see no evidence of correlation, although it was clear that CD8 + T cell differentiation was clue. After my PhD way of the cross, I think the unexpected diversity and migration properties of the preimmune repertoire might be one of the reason of this failure, leading to a lot of confusion in what we thought were clear-cut markers.

So how do we move forward?

First I think we have to be more aware of the limits of immunophenotyping, and more critic (in a positive way) for immunomonitoring studies.

Then, we have to consider immunity in the tissue. Until know, we have largely underestimated its importance, especially in homeostatic conditions. However, as we get more and more evidence that for many situations blood is not enough informative, then we have to find out a solution, even if that sounds like a big challenge. A.Einstein was saying: « La folie, c'est de faire toujours la même chose et de s'attendre à un résultat différent. ». I think it fits pretty well with my actual vision of immunomonitoring studies, which are technically heavy, time-consuming and costly. Epigenetic imprinting, isolation of rare circulating tissue-cells, optimization of organ dissociations or immunohistochemical protocols, studies on deceased organ donors, or more systematic immunoscope studies might be some tracks to evolve it in a more productive way.

Finally, we also need to admit that we cannot do without tracking Ag-specific T cell populations with tetramers. There have been some fabulous improvements over the past decade, especially as a result of the creativity of Ton Schumacher's group with this technology, but we need to do better, and implement them in routine analysis. This will require significant efforts from the community for largescale production, generation of appropriate quality controls, and spreading of analysis expertise. This is feasible, as illustrated by the standardization work of the CIMT group with CIP proficiency panels (http://www.cimt.eu/workgroups/cip/proficiency), but for practical reasons, this will probably require to have some specific sites dedicated to this expertise. On our side, we propose to adapt the combicolor coding technology to the Sony spectral analyzer that we have in place, and couple it to tetramer enrichment. This will permit to recover a large number of information from very limited amount of sample. And we hope it will also permit to efficiently analyze tissue pieces by diminishing the constraints relating to autofluorescence and background staining. An important last point is that all of these efforts take time, much more than repeating standard analysis on blood of patient's cohorts, and that needs to be sufficiently valorized by the community.

II. PREIMMUNE REPERTOIRE AND PERSONALIZED MEDICINE

Beside the pure phenotypic aspects, our work evidences that the preimmune repertoire of both mice and humans is more complex than anticipated. Not all naive T cells are created equal. Not all Agspecific inexperienced T cell populations are the same, both in frequency and composition. They do not have a similar inherent ability to be recruited into the subsequent response. And all of this is not fixed, but can vary as a result of environmental modifications. Like « memory T cells » includes different subsets with distinct behavior and functions, « naive T cells » (or inexperienced T cells) now refers to very diverse subpopulations. And this diversity has to be taken into account while interpreting any T cell priming experiment. My belief is that the T cell response to a given antigen is the sum of the behavior of individual subsets that are part of Ag-specific inexperienced populations.

And that the composition of the preimmune repertoire is a point of regulation of CD8 + T cell immune responses. Whether our observations also hold true for CD4 + T cells is unclear, and will need additional explorations. Identifying markers for those subsets, and understanding their contribution to the global function will be clue.

One example of the subset we need to better identify is recent thymic emigrants (RTEs). Cord and newborn bloods are expected to contain higher numbers of these cells than later in life, but we miss phenotypic markers for tracking them. We suggest we take advantage of this age-stratified evolution to find biomarkers for identification of this population. From microarrays datas that we have on CD8SP neonate thymocytes, we will select 80 genes defining a transcriptomic signature of intrathymic cells that distinguishes them from naive peripheral T cells. We will then collect, and FACS-sort (i) single and (ii) bulk naive T cells from cord and peripheral blood of age-stratified healthy children. On bulk cells, we will evaluate the proportion of sjTRECs in the naive CD8 + T cell population. On single cells, we will evaluate gene expression levels for the 80 pre-selected genes. Assuming that sjTRECs do identify RTEs -at least in children, we will then be able to know how many of the 100 sorted single cells are RTEs, and to identify them as bearing the more thymus-like profile. By this strategy, we hope to identify tracking markers for RTEs, which would be extremely useful for example in studies on childhood vaccinations. Similar studies could be proposed for the other aspects of preimmune repertoire diversity. At the end, the dream would be to be able to provide for each patient a cartography of its preimmune repertoire for different antigenic specificities. That would be a basis for personalized medicine where specific immunotherapies could be proposed on a patient-per-patient basis.

III. PREIMMUNE REPERTOIRE AND PERSISTENT

VIRUSES

Finally, we believe that our findings of dysregulated preimmmune repertoire during HCV chronic infection are not unique to this virus, and could also happen in the context of other persistent infections. Since birth -and even before, we all are in permanent contact with lots of pathogens that infect us, usually driving asymptomatic to mild-severe infections, and later persist for years or life in a resting state in our organism. These hosts that inhabits us constitute the « virome », which includes viruses that infect eukaryotic cells (eukaryotic virome), bacteriophages that infect bacteria (bacterial virome), viruses that infect archaea (archaeal virome), and virus-derived genetic elements in host chromosomes, as well as the mycobiome and other members of the meiofauna such as protists and metazoans [START_REF] Virgin | The Virome in Mammalian Physiology and Disease[END_REF]. They can be « hidden » into neurons, hematopoietic cells, stem cells, vascular endothelial cells, and likely many additional cell types. Specifically, there are chronic viruses that may be considered commensals, and those viruses are thought to alter disease susceptibility via significant effects on their host, independent of their role as pathogens. Our hypothesis is that these viruses could shape the preimmune repertoire, and explain some of the diversity in immune responses observed among individuals. One example is the cytomegalovirus, which infects half of a healthy adult population, and has dramatic effects on CD8 + naive/memory T cell phenotypes.

In the context of a French scientific initiative, financed by the Investissement d'Avenir as part of a Laboratoire d'Excellence (LabEx) research program, the Milieu Intérieur Consortium was developed with the objective to define the determinants of human immune variance. The aim was to define the genetic and environmental factors that contribute to the observed heterogeneity in immune responses.

To this purpose, 1000 healthy volunteers, descendants of mainland French persons for at least three generations, splited equally by sex (1: 1 sex ratio) and stratified across five-decades of life were recruited. For which concern analysis of environmental factors, the study included a large questionnaire with detailed questions addressing sociodemographic factors, personal and familial history of disease, smoking and alcohol habits, sport activities, as well as some nutritional aspects. In term of viral history, we know the serologic status of the donors for viruses that were defined as excluding factors (i.e. IgG anti-HIV1/2, IgG anti-HCV, and anti-HbS antigen to exclude ongoing HBV chronic infection), as well as IgG titers for CMV, HTLV1 and Influenza. We are now planning to extend this analysis to (i) other persistent pathogens such as EBV (IgG EBNA, IgG VCA), HSV1/2, VZV, toxoplasmosis, helicobacter pilori, and (ii) pathogens that are included into vaccines commonly used in the French population (HBV IgG anti-HbS and anti-HbC, Smallpox). The aim is to generate a personalized map of previous stimulations with either pathogens or vaccines, and to get insights into the impact of these events -as well as their sequential order -on phenotypic and functional aspects of the immune system.

I. GENERATION OF PMHC CLASS I TETRAMERS THROUGH LIGAND EXCHANGE.

This unit presents a set of linked protocols from Ton Schumacher's laboratory, adapted by Cécile Alanio and Matthew Buckwalter with the help of Fabrice Lemaitre and Philippe Bousso One support protocol is included that describe the measurement of biotinylation efficiency. The technology that is described in this unit has been developed for the human MHC alleles HLA-A*0101, -A*0201, -A*0301, -A*1101 and -B*0702 [START_REF] Toebes | Design and use of conditional MHC class I ligands[END_REF][START_REF] Toebes | Design and use of conditional MHC class I ligands[END_REF][START_REF] Bakker | Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7[END_REF] and the mouse alleles H-2Db, Kb and Ld [START_REF] Toebes | Design and use of conditional MHC class I ligands[END_REF]Frickel et al., 2008;Grotenbreg et al., 2008), and it should be possible to extend it to other MHC alleles. HLA molecules that are loaded with UV sensitive peptide are also available through commercial sources (see background information), providing an alternative to some of the protocols described in this Unit.

BASIC PROTOCOL 1 BACTERIAL EXPRESSION AND PURIFICATION OF HLA CLASS I HEAVY CHAINS AND HUMAN β2M (4 DAYS)

A soluble version of the MHC class I heavy chain containing a COOH-terminal biotinylation sequence (biotag) accepted by the Bir A biotin ligase and hβ2m are both produced by expression in E coli and are isolated as inclusion bodies. The procedure detailed below for HLA-A*0201 is based on that first described by Garboczi et al. (Garboczi et al., 1992) and can be used for different human and mouse MHC class I alleles. For 500ml : 25mL Tris-HCl 1M pH8 (50mM) + 125g saccharose (25%) + 1mL EDTA 0,5M (1mM) + 5mL azide 10% (0,1%) + eau QSP 500mL.

Materials:

•• Luria-Bertani (LB) medium (6L, RT) •• Carbenicillin (diluted in
•• Detergent buffer: 50 mM Tris-HCl pH8, 100mM NaCl, 1 mM EDTA, 0,5% Triton-X-100, 0,1 % azide, DTT 1mM.

For 500mL : 25mL Tris-HCl 1M pH8 (50mM) + 50mL NaCl 1M (100mM) + 1mL EDTA 0,5M (1mM) + 5mL azide 10% (0,1%) + 2,5mL Triton-X-100 (0,5%) + eau QSP 500mL. Add 50 µl/50mL of 1M DTT (1mM) just before use.

•• Wash buffer: idem detergent buffer but without Triton.

•• 1-10 ng/µl MHC-BSP expression constructs, reconstituted in water or TE buffer (10mM Tris-HCl pH8, 1mM EDTA).

The cDNA sequence encoding amino acid residue 1-100 of human β2m is cloned in pET3a. The cDNA sequence encoding the MHC class I heavy chain without the signal peptide, transmembrane domain and cytoplasmic domain (amino acid residue 1-276 for HLA-A*0201) is cloned into pMBio5, a derivative of pET3a that encodes an in-frame COOH terminal peptide tag (GSGGSGGSAGGGLNDIFEAQKIEW) that serves as a substrate for BirA biotin ligase.

Novagen Competent Cells : Transformation Protocol

-Remove 2 Single Cell Kits (50ul reaction) from the freezer. Immediately place the tubes on ice, so that all but the cap is immersed in ice. Allow the cells to thaw on ice for 2-5 min. Gently finger-flick (never vortex) the tube 1-2 times.

-Add 1 and 2 ul of 1-10ng/ul purified plasmid 1mM EDTA,pH8) directly to each of the two aliquots of cells. Stir gently to mix, return the tube to ice, and incubate for 5 min.

-Heat the tubes for exactly 30s in a 42°C water bath. Do not shake.

-Place the tubes on ice for 2 min.

-Add 250ul of RT SOC medium to each tube. Keep the tubes on ice until all have received SOC.

-Incubate for 1 hour at 37° while shaking at 250rpm.

-Prepare 3 dilutions for each plasmid condition (pure, 1/10, 1/100). Plate on LB medium containing antibiotics for the plasmid-encoded drug resistance (approx. 50uL per plate, 6 plates in total).

-Incubate all plates, cover-slide down, overnight at 37°.

(Day 2

) Inoculate 1-2 colonies in 60 ml LB plus antibiotics (60µl carbenicillin at 50mg/ml ; 60µl chloramphenicol at 34mg/ml). Place at 37°, 200rpm, until OD 600nm is approx. 0.8 (37°C, approx. 4 hrs). Place at 4°C overnight. C(mg/mL) = OD / [E x L (cm) ], where L is the lenght of the cuvette (usually = 1 cm).

22.

Aliquot per 500 nmoles for A2, and 3 000 nmoles for β2m (17mg for heavy chains ; 41mg for β2m) in polypropylene tubes. Keep the tubes on ice while working.

The use of polypropylene tubes minimizes sticking of peptide or protein to the tube.

23.

Snap-freeze aliquots, and store at -80°.

Inclusion bodies can be stored at -80°C for at least 1 year.

BASIC PROTOCOL 2

REFOLDING, BIOTINYLATION AND PURIFICATION OF MHC CLASS I COMPLEXES WITH CONDITIONAL LIGANDS

The refolding of HLA A*0201 complexes occupied with conditional ligand is largely based on the procedures established by Garboczi et al. (Garboczi et al., 1992). The conditional ligand used for HLA-A*0201 is the sequence KILGFVFJV, in which J indicates the UV sensitive amino acid (See Support protocol 1). •• Amicon ultrafiltration membrane (Mw cut-off 30 kDa) for pressure filtration.

•• Amicon stirred ultrafiltration cells for pressure filtration (Model 8400).

•• Nitrogen.

•• Amicon Ultra-4 PLTK Ultracel-PL Membrane, 30KDa

•• Amicon Ultra-15 PLTK Ultracel-PL Membrane, 30KDa To each 500 ml refolding, an aliquot of HLA class I heavy chain corresponding to a 1uM final concentration is added on 3 consecutive days (resulting in an MHC class I heavy chain concentration of 3 uM on day 3). Thus, a total of 3 aliquots of 500 nmoles is required to perform a 500 mL refolding.

HLA class I heavy chain fractions to be added on Day 2 and 3 should be stored at -20° in 8M guanidine-HCl until use.

2. Prepare 500mL dilution buffer.

3. Add 0,6mL conditional ligand dissolved in 100 % DMSO (50mM stock ; 60µM final).

6.

Carefully dispose Amicon ultrafiltration membranes into their support, and prepare the two Amicon ultrafiltration cells. Fill each with 2 x 250mL filtered refolding reaction.

7.

Concentrate the refolding reaction to approximately 2 x 10 mL by nitrogen flow concentration (1 bar) over the Amicon ultrafiltration membrane (stir continuously during concentration) in two Amicon stirred ultrafiltration cells.

8. Rinse filter twice with 1 mL of PBS to give about 2 x 12 mL in total. Pool.

9.

Wash an Ultra-50 PLTK Ultracel-PL Membrane (Mw cut-off 30 kDa) by placing 3 mL milliQ H2O on the filter and spin at 4°C, 4,500 rpm for 2 min. Concentrate the flow through fraction to 1.8mL in the Amicon. Add antiproteases (100ul of the 10X cocktail stock; 20ul of PMSF 100X stocks).

The desired end volume depends on the HPLC or FPLC system used for subsequent purification.

10. Purify by gel-filtration HPLC : wash the column with PBS at 1mL/min, then inject refolding reaction using one 2 mL injection on the column with a 2 mL loop (normally max 5mL/run). Collect relevant fractions (approximately 15 mL) and keep the sample on ice. 16. Wash an Ultra-4 PLTK Ultracel-PL Membrane (Mw cut-off 30 kDa) by placing 4 mL milliQ H2O on the filter and centrifugation at 4°C, 4,500 rpm for 2 min. Discard H2O, add sample (2 times 4 mL?) and concentrate the sample on the membrane filter to a volume of 3 mL.

17.

Recover concentrated sample, rinse the filter once with 50 µL PBS, and add to concentrated sample.

18.

Measure protein concentration at OD 280nm .

From a 500 ml refolding, a final amount of around 5mg biotinylated monomer is usually obtained, depending on the refolding efficiency. -Then immediately add diluted photocleavable monomer (from step 2) on the top, and pipette up and down 5 times.

As peptides are in very small volumes, they evaporate, so you should do this procedure 3 peptides at a time (pipette 3 peptides, add monomers, then 3 more peptides, etc…)

4. Place the 96-well plate under a UV lamp (366nm) for 1 hr in the cold room, with a distance between the UV lamp and sample of approximately 5 cm.

Temperature of the solution may rise to approximately 30 °C, avoid overheating.

5.

Spin the plate (with parafilm) at 4500rpm for 15 minutes (4°). Transfer supernatant (keep the plate at an angle to avoid transferring any pellet) to a new polypropylen 96 well plate for downstream applications.

Be aware that only 60-80% of the starting volume will be left due to evaporation.

Resulting MHC complexes may be used to determine exchange efficiency by MHC ELISA (Basic protocol 4), or to produce MHC tetramers for T cell detection (Basic protocol 5).

BASIC PROTOCOL 4 MEASURING PEPTIDE-MEDIATED MHC STABILIZATION BY HLA ELISA

This protocol describes an ELISA that measures the concentration of correctly folded HLA class I molecules.

This assay can be used to verify the success of routine peptide exchange reactions, i.e. to ensure that the HLA ligand of choice indeed rescued the HLA class I complex, but can also be used for high-throughput screening of potential MHC ligands. Depending on the number of epitopes that is screened, the assay can be performed both in a 96 (described below) or a 384 well format. A 96 well-based assay is also available commercially (see Background information).

The We have used directly conjugated rabbit anti-human b2m antibody from DakoCytomation (cat. #: P0174), but alternatives may be used.

•• Developing reagent:

-10x substrate buffer -50x 2',2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) solution -100x H2O2 solution

Stop buffer

We have used custom-produced developing reagent and stop buffer from Sanquin Reagents. Alternatives are available but have not been tested.

1.

In a 96 well plate, add 100 µL of 2 µg/mL streptavidin in PBS per well.

17. Measure absorbance at 405 nm in a plate reader.

A 384-well ELISA may also be used to measure HLA class I stabilization by added ligands. In our hands the assay is somewhat more sensitive in this format (possibly a reflection of a different dynamic range of the plate reader used) and pHLA class I complexes should be diluted to a final concentration of 1.6 nM. All reaction steps are performed using 25 uL/well, blocking and washing steps using 100 uL/well, and stop buffer is added in 12.5 uL/well.

BASIC PROTOCOL 5 TETRAMERIZATION OF MHC CLASS I MOLECULES

MHC class I complexes may be complexed with fluorophore-labeled streptavidin to form MHC class I tetramers for T cell analysis. Commonly used fluorophores include allophycocyanin and phycoerythrin, and the formation of MHC tetramers with these conjugates is described below. However, streptavidin-coated quantum dots may also be used to prepare MHC tetramers for T cell detection.

We strongly recommend to first determine the efficiency of biotinylation of the UV-sensitive HLA class I batch, as described in Support protocol 3. In case biotinylation is found to be complete or near complete, tetramerization of peptide-MHC class I complexes obtained by UV-induced exchange is performed as described below.

Materials:

•• PE-streptavidin solution 1 mg/mL (cat. #: S866, Molecular Probes) or any fluorochrome-linked streptavidin solution at 1 mg/mL (APC, PE-Cy7, Qdots,…)

If Streptavidin-Qdots are used, spin the mat 10,000rpm, 2min, 4° before use, and take the top portion.

•• Polypropylene plates with exchanged MHC class I complexes prepared according to Basic protocol 3, containing 100 µg/mL of exchanged pMHC in a given volume/well.

1. Add appropriate amount of streptavidin-fluorochrome into each well of a new polypropylene plate.

Appropiate amounts are ideally calculated following titration of the streptavidin-fluorochrome on CD8-biotin antibodies. Usually we use these reagents at 1/10 ratio, meaning 1ul in 10ul pMHC volume.

2.

Immediately add exchanged monomer on top, pipette up and down 5 times.

3.

Cover the plates with aluminium.

Incubate 30mn (or longer is fine).

It may be better to add the total streptavidin amount in four different time-points (10mn intervals), or in ten different time-points (5mn intervals), instead of only once, but one addition works.

SUPPORT PROTOCOL

DETERMINATION OF BIOTINYLATION EFFICIENCY

The degree of biotinylation of MHC class I molecules can be determined by incubation of biotinylated MHC class I molecules with streptavidin and subsequent analysis of samples by SDS-Page electrophoresis. In case of full biotinylation, all MHC class I molecules should be bound to streptavidin when an excess of streptavidin is used (more than one streptavidin molecule per four MHC class I molecules), resulting in a shift in retention time of streptavidin and the complete disappearance of the monomeric MHC class I band in the gel (Fig. 3).

Materials:

•• PE-streptavidin solution 1 mg/mL (cat. #: S866, Molecular Probes) or APC-streptavidin solution 1 mg/mL

II. TAME PROTOCOL Purpose

To achieve a 100-fold enrichment of antigen specific T cells from human peripheral blood. More details about the procedure can be found in [START_REF] Alanio | Tracking antigen-specific CD8⁷ T cells using MHC class I multimers[END_REF]. Depending on expected precursor frequency, use 10 -200 million PBMCs (for a typical pulldown of naive cells, 100 million will give you a sensitivity of 2. Wash cells once at 1300rpm for 8mins (4°) and resuspend in 80µl pulldown buffer (PB).

Materials

3. Add 10µl of FcR Blocking Reagent (Miltenyi) .

4.

Vortex and incubate for 15mins at 4° .

5.

Add Tetramers at Cf=20nM (10µl per 100µl PB for our preparations)

If the experiment includes several tetramers in different colors, add all of them at this step, and ajust PB volumes accordingly.

6.

Vortex gently and incubate for 30mins at 4° in the dark.

7.

Wash once in 2mL PB, spinning at 1300rpm for 8mins (4°).

8.

Resuspend in 400µL PB.

9. Take a 5µL aliquot and place it in 5ml FACS tubes (« Before » fraction).

10. Add 100µL of anti-PE microbeads (Miltenyi).

11. Vortex and incubate for 20mins at 4° in the dark.

12. Wash twice in 2mL PB at 1300rpm for 8mn (4°).

13.

During the wash steps, prepare MS columns (one per Falcon15 tube) on a magnet support. Clean each column with 1000µL of PB (throw away the first elution). Prepare Falcon15 tubes for collecting first flow through fraction.

14.

Resuspend each sample in 1000µL PB.

  ABBREVIATIONS ............................................................................................................................................... 17! FOREWORD ........................................................................................................................................................ 21! RESEARCH PLAN .............................................................................................................................................. 23! I.! DEVELOP TOOLS FOR SCREENING RARE ANTIGEN-SPECIFIC RESPONSES IN HUMANS .... 24! II.! EVALUATE THE IMPACT OF CHRONIC INFECTION ON THE PREIMMUNE REPERTOIRE .... 24! III.! STUDY THE IMPACT OF CXCR3 RECEPTOR ON INEXPERIENCED T CELLS .......................... 25! CHAPTER 1. GENERAL INTRODUCTION ................................................................................................... 27! I.! PREIMMUNE REPERTOIRE ................................................................................................................... 31! A.! THYMIC DEVELOPMENT ................................................................................................................ 31! 1)! Thymus anatomy and function ......................................................................................................... 31! 2)! Early thymocyte development .......................................................................................................... 34! 3)! Thymic selection of the MHC-restricted preimmune repertoire ...................................................... 34! 4)! Maturation and emigration of mature SP thymocytes ...................................................................... 36! B.! PERIPHERAL NAIVE T CELLS ........................................................................................................ 38! 1)! Recent Thymic Emigrants ................................................................................................................ 38! 2)! Population size and TCR repertoire ................................................................................................. 39! 3)! Homeostasis and homeostatic proliferation ..................................................................................... 43! 4)! CD5 expression and dynamic tuning of lymphocytes ...................................................................... 45! 5)! Activation, differentiation and activation-induced cell death .......................................................... 47! 6)! Phenotypic and functional diversity ................................................................................................. 50! 7)! Impact of precursor frequency and diversity ................................................................................... 51! II.! CHRONIC INFLAMMATION ................................................................................................................. 54! 1)! General introduction ......................................................................................................................... 54! 2)! HCV as a model of chronic inflammation ....................................................................................... 56! (a)! Epidemiology ............................................................................................................................. 57!

  67! 3)! HBV as a comparative model of chronic hepatitis ........................................................................... 72!

Figure 1 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :

 1345678910 Figure 1: Location, morphological aspect and anatomic structure of the thymus……………………………… Figure 2: Chemokine-driven trafficking and maturation of T cells into the thymus…………………………… Figure 3: Emigration of SP thymocytes from thymus…………………………..……………………………… Figure 4: Comparison of recent thymic emigrants and mature naïve T cell function..…………….…………… Figure 5: Antigen specificity and tetramer staining…………………………….…………….………………… Figure 6: Naïve T cell phenotype, homeostasis, and homeostatic proliferation...……………………………… Figure 7: Dual role of CD5 in lymphocyte activation and survival……………..……………………………… Figure 8: Sequence of critical events after TCR activation……………………..……………………………… Figure 9: Composition of the preimmune repertoire…………………………………………………………… Figure 10: Segregation of TLR agonists based on their induced protein signatures…………………………… Figure 11: Prevalence and distribution of HCV genotypes……………………..……………………………… Figure 12: HCV genome organization, and polyprotein processing…………………………………………… 59

Figure 13 :Figure 14 :Figure 15 :Figure 16 :

 13141516 Figure 13: Life cycle of the Hepatitis C Virus (HCV) and targets of therapy….……………………………… 61 Figure 14: Clinical, virological, and immunological course of acute HCV infection……..…………………… Figure 15: Extrahepatic manifestations related to HCV………………………...……………………………… Figure 16: Molecular processes that signal the host response to HCV infection.……………………………….

Figure 17 :Figure 22 :

 1722 Figure 17: Weak CD8 T cell responses during cHCV………………………….………………………………. 71 Figure 18: Natural course of HBV chronic infection…………………………………………………………… 72 Figure 19: Bystander infection negatively impacts effector to memory transition…..…………………………. 76 Figure 20: Human chemokines and their receptors……………………………...……………………………… Figure 21: Memory T cell heterogeneity in the blood and in tissues…………....……………………………… 83 Figure 22: Shematic model of chemokine antagonism………………………….……………………………… Figure 23: Preparation of pMHCI tetramers……….…………………………………………………………..

Figure 24 :

 24 Figure 24: Peptide exchange technology……………………………………………..……………………….

Figure 25 :

 25 Figure 25: Development of Tetramer Associated Magnetic Enrichment (TAME)………..………………….

Figure 26 :

 26 Figure 26: TAME increases detection of antigen-specific T cells by up to 100-fold...………………………...

Figure 27 :

 27 Figure 27: Comparison of CCR7 and CD27-based naïve gating strategies…….……………………………… 111 Figure 28: Naïve CD8 T cells specific for HIV Gag p17 77-85 can be detected on healthy donors……………...

Figure 29 :

 29 Figure 29: Enumeration of epitope-specific CD8 T cells reveals conserved precursor frequency among HD...

Figure 30 :

 30 Figure 30: Schematic representation of critical steps in immune-mediated clearance of HCV infection…..….

Figure 31 :

 31 Figure 31: Validation of CD45RA/CD27 gating strategy for identifying naïve CD8 T cells in cHCV………..

Figure 32 :

 32 Figure 32: Perturbed naïve CD8 T cell repertoire during chronic infection…………....……………………… Figure 33: Increased peripheral expansion of selected naïve population………………….…………………… 139 Figure 34: Mart1-specific CD8 T cell MP during chronic infection may be reversed by viral clearance……... 141 Figure 35: Sensitivity of sjTRECs and Vb repertoire assays………….……………..………………………… Figure 36: MP cells are a common feature of self and non-self antigen-inexperienced populations………..… Figure 37: CD5 expression on T cells during homeostatic or pathologic conditions………..…………………

Figure 39 :

 39 Figure 39: Schematic representation of the impact of low levels of CD5 on T cell activation………………... 151 Figure 40: Lower threshold for TCR activation in cHCV patients……………….………..…………………...

Figure 41 :

 41 Figure 41: Increased blastogenesis and CD25 expression after TCR activation in cHCV patients...…………..

Figure 42 :

 42 Figure 42: Increased AICD after TCR activation in cHCV patients……...…….……………………………... 157 Figure 43: Anti-CD5 blocking Abs recapitulate the hypersensitivity phenotype……………………………… Figure 44: Cytokines levels detected in plasma of cHCV, and SVR patients, as compared to HD…………… 161 Figure 45: In vitro priming of Mart1-specific CD8 T cells…………………………………………….…..….. 163 Figure 46: Proposed models for dysregulation of naïve CD8 T cells during HCV chronic infection……...…..

Figure 47 :

 47 Figure 47: cxcr3 -/-mice have lower numbers of CD8SP thymocytes…………....………………………..…... 177 Figure 48: cxcr3 -/-mice present increased numbers of circulating MP CD8 T cells…………………………..

Figure 49 :

 49 Figure 49: Activation profile of OTI-CXCR3KO cells after stimulation with the Ova peptide………..……...

Figure 50 :

 50 Figure 50: MP cells are found into the skin of OTI-WT transgenic unimmunized animals………….………...

Figure 51 :

 51 Figure 51: MP cells are present in OTI-CXCR3KO cells of mixed bone marrow chimeras ……………….….

Figure 52 :

 52 Figure 52: Proposed model of MP Ag-specific inexperienced T cells migration…………….………..……….

Table 1 :

 1 Naive and memory phenotype of CD8 T cells in humans and mice. ....................................................... 29! Table 2. Mouse and human adults preimmune repertoire numbers. ....................................................................... 40! Table 3. Diversity of the CD8 T cell preimmune repertoire. .................................................................................. 51! Table 4. Hepatotropic viruses. ................................................................................................................................ 56! Table 5. Calculation of epitope-specific T cell numbers, and validation. .............................................................. 94! Table 6. Description of the patients included in the Tetra cohort. ....................................................................... 121! I would like to thank Marion Pepper and Marc Bonneville for their deep relecture of the manuscript, as well as

Figure 9 .

 9 Figure 9. Composition of the preimmune repertoire.The preimmune repertoire is composed of mature naive-phenotype cells (NP, dark blue), recent thymic emigrants (RTEs, light blue), and memory-phenotype cells (MP, red) that can arise by homeostatic proliferation (HP), cross-reactivity (CR, one black point), or be virtual memory cells (VM, two black points). A. Precursor frequency positively correlates with the magnitude of subsequent responses. In this simple situation, considering other parameters influencing immunodominance are controlled, epitope 2 will drive bigger expansion than epitope 1. B. However, the composition of a given antigen-specific population is likely to be more complex, and based on specific functional features of each of these subsets, likely influence the behavior of the population during and after expansion.

  (b) Virus genome HCV is a small enveloped virus belonging to the flaviviridae family, hepacivirus gender. Its genomic structure is composed of a 9.6 kb single-strand RNA of positive polarity (Halliday et al., 2011). It contains a single open-frame reading encoding for a 3000 amino-acid (aa) polyprotein (Penin et al., 2004). Non-coding areas contains highly conserved regions which are important for replication and translation of the virus genome. The polyprotein is cleaved by both host and virus proteases into the endoplasmic reticulum (RE) membrane, leading to the generation of 10 mature proteins (Figure 12): • the N-terminal region encodes structural proteins: the core, and the envelop glycoproteins E1 and E2, which are essential for ligation of the virus to membrane receptors at the surface of host target cells; • the C-terminal region encodes for non-structural proteins (NS): p7, NS2, NS3, NS4A, NS5A and NS5B. These proteins are not incorporated into viral particles. They participate to intracellular replication of the virus. In particular, NS5B is a RNA-dependent RNApolymerase, which permits synthesis of a negative strand from the positive strand of the transmitted virus. Importantly, detection of negative strand of the RNA then sign active replication of the virus. After viral replication, viral particles, composed of viral genome, core protein, and E1 and E2 glycoproteins, are assembled by budding of the RE, and transported to the membrane by the host secretion system before exocytosis. In the blood, circulating HCV particles show heterogeneous and lower density than expected from its putative viral structure. This is likely related to binding of the virion to very-low-(VLDL) or low-(LDL) density lipoproteins

Figure 14 .

 14 Figure 14. Clinical, Virological, and Immunological Course of Acute HCV Infection.A. Acute hepatitis C followed by clinical recovery. The acute phase is marked by elevated alanine aminotransferase (ALT) levels and the onset of HCV-specific CD4 + and CD8 + T cell responses around weeks 8 to 12. HCV-specific antibodies are detectable by enzyme immunoassay (ELISA) a few weeks later, but the impact of low titers of strain-specific neutralizing antibodies is still controversial. After spontaneous resolution of acute HCV infection, HCV-specific memory T cell responses remained detectable for decades whereas antibodies disappear from the circulation. B. Acute hepatitis C followed by chronic infection. HCV RNA titers decrease 2-3 log10 after the acute phase and remain relative stable in the chronic phase. Selection of HCV escape mutants is mostly driven by T cells in the acute and early chronic phases of infection and by increasing titers of broadly targeted neutralizing antibodies in the chronic phase. The intensity of the shaded background indicates the degree of intrahepatic inflammation in the different phases of hepatitis. From Park et al, Immunity 2014.

  administration. Its mechanism of action is uncertain, but is likely multiple, with (i) immunomodulation in favor of Th1 lymphocytes; (ii) incorporation into the viral genome -thus limiting its replication;(iii) direct inhibition of viral polymerase NS5B; (iv) mutagenesis during viral replication; and (v) modulation of IFN-induced gene expression[START_REF] Feld | Mechanism of action of interferon and ribavirin in treatment of hepatitis C[END_REF]. Development of pegylated interferon (peg-IFNα) permitted to reduce the number of SC to 1 per week. Combination of peg-IFNα and RBV (800 mg/day for genotypes 2 or 3, or adapted to the weight [13-15 mg/kg/day] for genotypes 1 or 4) was for long the reference treatment. It permits clearance of the virus in 45% of genotype 1, 85 % of genotype 2, 70 % of genotype 3, 65 % of genotype 4, and 75% of rare genotypes 5 and 6. Factors influencing duration and modality of treatment are viral genotype, initial viremia, presence of extensive fibrosis or cirrhosis, IL28B genotype, response to a previous treatment, and viral kinetic at 4 and 12 weeks of treatment. Typically, patients are treated 24 (genotype 2 and 3) to 48-72 weeks (genotype 1, 4, 5).

  HCV RNA structures are recognized by cytosolic pattern recognition receptors. The HCV internal ribosomal entry site is recognized by RNA-dependent protein kinase R (PKR), whereas double-strand RNA is recognized by cytosolic retinoic-acid-inducible gene I (RIG-I) and TLR3. This leads to phosphorylation, dimerization and nuclear translocation of IRF3, which induces IFNβ synthesis. IFN-β binds to the IFN-α/β receptor and signals through activation of the associated Tyk2and Jak1 protein kinases to direct the phosphorylation and assembly of a STAT1-STAT2 heterodimer and trimeric ISGF3 complex containing IRF-9. The ISGF3 complex locates to the cell nucleus, where it binds to the ISRE on target genes and drives the expression of numerous Interferon Stimulated Genes (ISGs). This includes: (i) synthesis of IRF-7, that lead to production of IFN-α subtypes and amplifies ISG expression, (ii) IFNα and β themselves, and (iii) pro-inflammatory cytokines such as TNFα or CXCL10 (IP-10). However, viral proteins largely interfere with these pathways, and reduce dramatically IFNα/β production to levels that are undetectable in HCV-infected patients. As such, type I interferons are usually barely detectable at the RNA level in the liver, and usually undetectable at the protein level in the blood of acutely infected patients. In contrast, type III interferons are robustly detected in the two organs, and their expression parallel viremia and ISGs levels[START_REF] Park | IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection[END_REF].

Figure 17 .

 17 Figure 17. Weak CD8 T cell responses during cHCV. A. Example of tetramer-based detection of HCV-specific T cells before enrichment (left) and after enrichment (right) in one cHCV and one SVR patient. B. Schematic representation of results obtained from a large screen of in silico predicted epitopes from the entire sequence of the HCV virus for one cHCV (up) and one SVR (bottom) patients. Responses detected after enrichment are represented by a black and white gradient as indicated in the legend: negative responses (� 0.1% of CD8 + T cells), very low (0.1-1%), low (1-5%), medium (5-10%) or high (�10%) frequencies. C. Immunophenotyping of cell lines of HCV-specific T cells from HCV-seropositive and seronegative donors. Of interest is the apparent heterogeneity of the cell lines obtained from the cHCV patients, with dual aspect of expression of CD3 -and thus likely dual level of affinity. From Neveu et al, Hepatology 2008 (Neveu et al. 2008). D. Analysis of functional and TCR avidity of NS31073-specific T cells from resolved (R), chronic HCV (C), or healthy donors (H), as expressed as peptide concentration (nM) necessary to achieve 50% of maximal target-cell lysis. From Neveu et al, Hepatology 2008.

  . Of the three CXCR3 ligands, CXCL11 has the highest receptor affinity and is the most potent agonist in chemotaxis and calcium-mobilization assays. The binding of chemokines causes a conformational change of the receptor, activating an associated G protein that initiates cAMP and phosphatidylinositol signal transduction pathways. In parallel, the binding leads to a rapid concentration-dependent internalization of the chemokine receptor, which is not observed after the binding of antagonists. By preventing association of G proteins with the seven-transmambrane domain of the receptor, pertussis toxin is a natural inhibitor of G protein-coupled receptor.Naive T cells do not express CXCR3. The expression of this chemokine receptor on T cells is quickly upregulated after T cell priming, and later present on up to 40% of CD4 + -preferentially on T helper type 1, and and 60% of CD8 memory T cells. It is also expressed by natural killer cells, B lymphocytes, endothelial cells, mast cells, and thymocytes. In T lymphocytes, the stimulation of CXCR3 by its agonists leads to elevation of intracellular calcium as well as activation of phosphoinositide 3-kinase (PI3K)/Akt-dependent signaling and p44/42 extracellular signal-regulated kinase (ERK) pathways. CXCR3 activation has also been shown to induce rapid tyrosine phosphorylation of several proteins including zeta-associated protein of 70 000 MW (ZAP-70), linker

  CXCR3-binding chemokines are often expressed in inflamed tissues, and mediate local recruitment of primed CD4 + and CD8 + effector T cells that do express CXCR3 after having been primed into the draining lymph node. These chemokines have different temporal and spatial patterns of expression, relating to (i) their production by distinct cell types during the course of an immune response, and (ii) intracellular transcriptional regulation. Whereas CXCL9 is strongly induced by IFN-γ but not type I interferons (IFNα/β), CXCL11 is induced by both IFN-γ and β, but not α, and CXCL10 is strongly induced by all three, as well as by TNF-α. Through activation of IRF3 and the release of IFN-α, IP10 is the only ligand also induced by innate sensors, such as the TLRs and the RNA helicases (RIG-I and MDA-5).Locally, effector cells secrete IFNγ, and trigger an inflammatory amplification loop that further increases the recruitment of other lymphocytes into the site of inflammation.Besides playing a crucial role in lymphocyte trafficking to inflamed peripheral tissues, recent studies indicate that CXCR3-ligands are also expressed in SLOs during T cell activation. Here, engagement of CXCR3 on T cells promotes their intranodal migration into areas where Ag-loaded DCs accumulate (e.g. medullary region of lymph nodes), permitting an efficient initiation of adaptive immune

  inhibitors such as sitagliptin (FDA approved 2006, Merck and co, as Januvia) exist, and are clinically approved alone or in combination with other oral antihyperglycemic agents in the context of type II diabetis treatment. They have been developed based on their capacity to prevent GLP1 (Glucagonlike-peptide-1) and GIP (Glucose-dependent insulinotropic polypeptide) inactivation, leading to an increased insulin secretion. To date, the effect of DPP4 inhibitors on chemokine and lymphocyte trafficking has not been studied. Using in vivo models, our group has recently demonstrated DPP4 inhibition as a way to protect the biologically active form of CXCL10 -leading to an increased local recruitment of lymphocytes and improvement of adjuvant-based immunotherapy, adoptive T cell transfer and checkpoint blockade (Barreira Da Silva, Nat Immunol 2015, In press). These findings support the use of DPP4 inhibitors as a strategy to enhance tumor immunotherapy.

Figure 23 .

 23 Figure 23. Preparation of pMHCI tetramers.A. Schematic description of the generation of MHC-peptide tetramers (from[START_REF] Bousso | Generation of MHC-peptide tetramers: a new opportunity for dissecting T-cell immune responses[END_REF]. B. Control of the production of HLA-A0201 heavy chains and bêta-2-microglobulin molecules by SDS/PAGE electrophoresis (NuPage 4-12% gel). C. Representative chromatogram from size-exclusion chromatography after HIV Gag p1777-85 HLA-A0201 monomer refolding. D. Control of monomers biotinylation by SDS/PAGE electrophoresis (NuPage 4-12% gel). In presence of an excess of avidin, biotinylated pMHCI monomers associate and form complexes of molecules. Disapearance of pMHCI band is proportional to the extent of monomer biotinylation. E. After UV-exhange, monomers are incubated with fluorochrome-labeled streptavidin, then used for staining cells together with monoclonal antibodies. Dump chanel includes CD4, CD14, CD16, and CD19. Tetramer positive cells are defined as FSC-W lo Live/Dead neg LymphocytesDump -CD8 + Tetramer + events.

Figure 24 .

 24 Figure 24. Peptide exchange technology.A. UV-exchange facilitates tetramer production. Influenza A M1 epitope has been engineered to be able to disintegrate upon UV exposure. As such, in its entire form, it permits stabilization, thus large production, of class I monomers. And when needed, it can be cleaved and exchanged upon short UV exposure. Similar technology are in place in the lab for the alleles indicated in the blue box. (Adapted fromHadrup et al, Meth Mol Biol 2009). B. UV-exchange permits in vitro evaluation of peptide MHC binding properties by streptavidin-based sandwich ELISA. Exchanged biotinylated monomers to be tested are added to the streptavidin-coated wells. HRP-conjugated anti-bêta-2microglobulin antibody recognizes pMHCI complexes bound to the well. ABTS is added as a substrate for HRP and gives a yellow-colored oxidation product. C. Example of an ELISA read-out. No UV, non-irradiated conditional MHC complex; no peptide, irradiation without rescue peptide; CMV, cytomegalovirus pp65(495-503); flu, influenza A Matrix 1(58-66); Mart-1 A2L, melanocyte lineage-specific protein A(26-35 A2L); Mart-1, melanocyte lineage-specific protein A[START_REF]Each time, pull each in Falcon15 tubes , add 2mL of PB to the upper fraction of the column, push the piston, and discard the colum. You obtain the Enriched Fraction. 27[END_REF][START_REF]Stain them and analyse on FACS machine using standard procedures[END_REF](28)(29)(30)(31)(32)(33)(34)(35); gp100 T2M, human melanoma associated antigen gp100(209-217 T2M); gp100, gp100(209-217); A3, HLA-A3 restricted gp100[START_REF]Discard the pale orange supernatant. Resuspend in Detergent buffer. First add 2 mL to each tube, vortex or dissociate with a glass stick and transfer to the plastic beacker. Add buffer until 15mL. Add 50 µl/50mL of 1M DTT (1mM) just before use. Sonicate, then ultra-centrifuge 10min at 10 000 rpm[END_REF](18)(19)(20)(21)[START_REF]PB to the initial Falcon15 tube (Wash step to get every last cell)[END_REF](23)[START_REF]The flow through is your Depleted Fraction[END_REF](25). (Adapted from Rodenko et al, Nat Protocol 2010).

Figure 25 .

 25 Figure 25. Development of Tetramer Associated Magnetic Enrichment (TAME).A. Buffy coat is defined as a thin central layer after centrifugation of heparinized human plasma. B. HLA-A2 typing was performed on whole blood on each buffy coat. Histograms present representative A2-positive (left) and A2-negative (right) results gated on lymphocytes. C. Schematic representation of TAME. D. Gating strategy to reduce background. 100x10 6 PBMC from healthy individuals were analyzed by eight-color flow cytometry following staining and TAME with a Flu-Matrix1 HLA-A0201 tetramer labelled with PE. Flu-specific CD8 + T cells from the enriched (bound) fraction were identified as side-scatter low dump -CD3 + CD8 + Tet + events. The tetramerpositive gate was set using the CD8 -gated events as a negative control. Represented results show 10 6 collected events.

Figure 26 .Figure 27 .

 2627 Figure 26. TAME Increases Detection of Antigen-specific T cells by up to 100-fold. A. 100x10 6 PBMC from HD were stained with Flu-M158-66, CMVpp65495-503 and MART126-35 HLA-A2.1 tetramers labelled with PE. Enrichment was performed by addition of anti-PE microbeads and MS magnetic column. « Pre-enriched », « Enriched », and « Depleted » fractions were analyzed on a LSRII cytometer. Represented results show 2x10 6 collected events for « Before » and « Depleted » fraction, and acquisition of all sample for « Enriched » fraction. Plots are gated in total CD3 + T cells. B. Eight color flow cytometry on « Enriched » specific populations accurately discriminate naive (MART126-35) and memory (Flu-M158-66, CMVpp65495-503) situations. C. 2x10 5 PBMC were plated from « Before » and « Depleted » fraction for Flu-M158-66 or MART126-35 MHCI tetramer pulldown for IFNg-Elispot, and cells were stimulated by each corresponding peptide. Absence of Flu reactive cells in Flu-depleted fraction as compared with Before confirms efficient depletion of antigenspecific cells. Absence of IFNg producing cells in the Before fraction for MART126-35 is consistent with their naive phenotype. D. Comparison of phenotype and precursor frequencies on fresh versus frozen samples. Frequencies represent the number of epitope-specific T cell within CD8 + .

Figure 28 .Figure 29 .

 2829 Figure 28. Naive CD8 + T cells Specific for HIV Gag p1777-85 can be Detected on Healthy donors. A. TAME enrichment on 400 millions healthy PBMCs with HIV Gag p1777-85 and MART126-35 HLA-A0201 tetramers labelled with PE. B. Phenotype of HIV Gag and Mart1-specific populations enriched in A (black) as compared to bulk CD8 T cells (grey). C. 2 000, 200, 20, and 2 lymphocytes specific for HIV Gag p1777-85 epitope were spiked into 100x10 6 PBMC of one single healthy donor.Samples were stained with HIV Gag p1777-85 HLA-A0201 tetramer labelled with PE, and analyzed before or after tetramer enrichment. D. PBMCs were enriched in CD8 using magnetic microbeads, and enriched cells were cocultured for 7 days with DCs loaded with MART126-35 peptide. T cells were then harvested and stained with MART126-35 and HIV Gag p1777-85 tetramers. E. A panel including MART126-35, HIV Gag p1777-85, CMVpp65495-503, and Flu-M158-66 HLA-A0201 tetramers was developed. All tetramers were labeled with PE, and a second tetramer labeled by a unique fluorochrome was used, as indicated. Simultaneous enrichment was performed using anti-PE microbeads. Within the global PE-enriched tetramer-positive population, each specificity was identified using the second tetramer label, and cell phenotype was analyzed using anti-CD45RA and anti-CD11a antibodies.

Figure 30 .

 30 Figure 30. Schematic representation of critical steps in immune-mediated clearance of HCV infection. 1. Viral antigens uptake by local DCs leads to their maturation while migrating to the draining lymph node. 2. Presentation of viral epitopes to corresponding T cell precursors. 3. Trafficking of activated T cells back to the infected tissue. 4. Direct and/or indirect killing of infected cells by T cells. All of these four step are well recognized as critical for successful clearance of the infection. We propose to explore the step 5, about the availability and quality of precursor T cells, that we think are also critical points.

Figure 31 .

 31 Figure 31. Validation of CD45RA/CD27 gating strategy for identifying naive CD8 + T cells in cHCV.Example of CD45RA/CD27 gating strategy in one cHCV patient. CD8 + naive T cells are identified as CD45RA + CD27 + . Pattern of CD127, CD45RO and CCR7 expression of each identified population is displayed on histogram overlays (black line represents the subpopulation of interest; grey ones the total CD8 + population).

Figure 32 .

 32 Figure 32. Perturbed naive CD8 + T cell repertoire during chronic infection. A. Representative examples of CD3 + CD8 + compartment in Healthy Donor (HD), Sustained Virologic Responder (SVR), chronic HCV (cHCV), and chronic HBV (cHBV) patients. FACS plots are gated into Live FSC lo SSC lo Peripheral Blood Mononuclear Cells (PBMCs). B. Combined data from the patient cohort is shown. C. Absolute numbers (G/L) of CD3 + (left), CD8 + (middle), and CD4 + (right) T cells in the cHCV, SVR, and HD patients subsets. D. Representative examples of CD45RA + CD27 + naive compartments in HD, SVR, cHCV, and cHBV patients. FACS plots are gated on Live FSC lo SSC lo CD3 + CD8 + T cells. E. Percentages of naive CD8 + T cells in the four donor subsets. F/.Absolute numbers (G/L) of naive CD8 + and CD4 + T cells in the cHCV, SVR, and HD patients subsets. ns (not significant, p>0.05), * (p≤0.05), ** (p≤0.01), and *** (p≤0.001) refer to Dunn's multiple comparison test of each subset toward healthy donors.

Figure 33 .

 33 Figure 33. Increased peripheral expansion of selected naive populations. A. Normalized numbers of signal-joint T cell receptor excision circle (sjTREC) per 150 000 CD3 + CD8 + cells in HD and cHCV samples. *, p≤0.05. B. Normalized numbers of sjTRECs per 150 000 naive CD8 + in HD and cHCV samples. *, p≤0.05. C. Representative example of the distribution of 24 FACS-screened Vβ families in naive CD8 + T cells from one cHCV and one HD sample. Families are ordered by increasing size in both individuals. D. Lorenz curves representing the mean distribution of % of usage of 24 FACS-screened Vβ families from 7 cHCV and 7 HD patients. Mean corresponding Gini coefficients and standard deviations are indicated. E/ Individual Gini coefficients of all tested samples are represented for cHCV and HD subgroups.

Figure 34 .

 34 Figure 34. Mart1-specific CD8 + T cell MP during chronic infection may be reversed by viral clearance. A. Representative examples of Mart1-specific populations in HD, SVR, cHCV, and cHBV patients.. B. Mart1 precursor frequency in the four donor subsets. ns, ***, ** and * refer to Dunn's multiple comparison test of each subset toward healthy donors. C. Representative examples of the CD45RA/ CD27 phenotype of Mart1-specific populations in patients subsets as in A. FACS plots are gated on TAME-enriched Mart1-specific populations. D. Percentages of naive-phenotype cells in Mart1specific populations in the four donor subsets. ns (not significant, p>0.05), * (p≤0.05), ** (p≤0.01), and *** (p≤0.001) refer to Dunn's multiple comparison test of each subset toward healthy donors. E. Follow up of one patient successfully treated for cHCV. On treatment (on tx), immediately posttreatment (post-tx), 4 months (m) and 18m post-tx data are shown. At each time point, an overlay of TAME-enriched Mart1-specific T cells (black) projected onto total CD8+ T cells from the same patient (grey), and the CD45RA/CD27 phenotype of the above antigen-specific population are displayed. F. Percentages of naive-phenotype cells in the Mart1-specific populations vs. time elapsed since clearance of the virus in SVR patients (time-stratified, in years). G. Detection of two Mart1specific populations in one single SVR patient.

Figure 35 .

 35 Figure 35. Sensitivity of sjTRECs and Vbêta repertoire assays. sjTRECs detection (A), Vbêta repertoire (B) and CDR3 profiles (C) in titrated amounts of naive T cells. D. Immunoscope of Mart1 naive and memory phenotype cells.

Figure 36 .

 36 Figure 36. MP cells are a common feature of self and non-self antigen-inexperienced populations. A. Representative examples of Mart1-, hTERT-, and CMV-specific populations are shown for HD, SVR, and cHCV patients. Enriched tetramer-specific populations are overlaid on total live FSC lo SSC lo CD3 + CD8 + PBMCs. B. Representative examples of CD45RA/CD27 phenotype of Mart1-, hTERT-, and CMV-specific populations in HD, SVR, and cHCV patients. FACS plots are gated into TAME-enriched antigen-specific populations. C. Cumulative representation of percentages of naive-phenotype cells into hTERT-and CMV-specific populations in the three precited subsets of donors. D. Ratio of percentages of cells with IFNg-positive staining after Mart1-, hTERT-, and CMV-in vitro restimulation over unstimulated condition. E. Naive/memory phenotype of EBV BMLF1-specific CD8 + T cells.

Figure 37 .

 37 Figure 37. CD5 expression on T cells during homeostatic or pathologic conditions. A. CD5 gradually increases during T cell development. B. Representative overlay of histograms of CD5 in T cell differentiation subsets (HD). C. Example of CD5 levels of Mart1-, NS31073-and CMVpp65-specific T cell populations (black) as compared to those of bulk CD8 + T cells (grey) in one CMV-seropositive healthy donor. D. CD5 profiles in bulk CD8 and CD4 populations (black histograms), and related subsets of differentiation (colored histograms) in spondylarthritis (SPA), and cHCV patients, or healthy donors (HD). E. Representative histograms of CD5 levels in CD8 + T cells subsets in HD and cHCV patients. F. MFI of CD5 on CD8 + T cell differentiation subsets of 3 HD and 3 cHCV donors (color code as in E).

Figure 38 .

 38 Figure 38. Decreased cell surface expression of CD5 on naive CD8 + T cells from cHCV patients. A. Representative histograms of CD5 levels on naive CD8 + T cells from one HD and one cHCV patient. MFI of CD5 on the surface of naive CD8 + T cells (B) or CD4 + T cells (C) from HD, SVR, cHCV, and cHBV patients. ns, ***, ** and * refer to Dunn's multiple comparison test of each subset toward healthy donors. D. Representative histograms of CD5 levels on naive-phenotype Mart1specific CD8 + T cells from one HD and one cHCV patient. E. MFI of CD5 on the surface of naivephenotype Mart1-specific CD8 + T cells from HD, SVR, cHCV, and cHBV patients. ns, ***, ** and * refer to Dunn's multiple comparison test of each subset toward healthy donors.

Figure 39 .

 39 Figure 39. Schematic representation of the impact of low levels of CD5 on T cell activation.A. CD5 is a negative regulator of TCR signaling. As such, it permits TCR signaling cascade to occur only in the context of a high-affinity interaction between TCR and pMCH complexes. B. Low levels of CD5 at the cell surface likely favor the expansion and differentiation of the cell even for lowaffinity (non-cognate) peripheral interactions.

Figure 40 .

 40 Figure 40. Lower threshold for TCR activation in cHCV patients. A. Percentages of naive, CM, EM, and EMRA CD8 + T cells with detectable intracellular phosphorylation of ERK (P-ERK, upper) and p38 (P-p38, lower), over time after CD3/CD28 platebound stimulation. B. MFI of P-ERK signals in same subsets from one HD, one SVR and one cHCV after CD3/CD28 stimulation. C. Representative overlay of histograms of phospho-ERK (p-ERK) signal at different time points following TCR stimulation from one HD and one cHCV patient. Plots are gated on naive CD8 + T cell populations. D. Representation of fold-changes in percentages of p-ERK positive cells (t=5 mins) as compared to unstimulated condition in naive CD8 + populations in HD and cHCV samples.

Figure 41 .

 41 Figure 41. Increased blastogenesis and CD25 expression after TCR activation in cHCV patients. A. Increased blastogenesis of CD8 + T cells at baseline and after TCR activation in cHCV patients (arrow). B. Blasting cells at 24 hours express CD25. C. Kinetics of fold-changes of % CD25 in naive CD8 + T cells following CD3/CD28 stimulation. D. Representative overlay of histograms of CD25 expression, detected at 24h after TCR stimulation from one HD, one cHBV and one cHCV patient. Plots are gated on naive CD8 + T cell populations. E. Fold-change in percentage of CD25 + cells, as compared to unstimulated condition in naive CD8 + population from HD, cHCV and cHBV patients.

Figure 42 .

 42 Figure 42. Increased AICD after TCR activation in cHCV patients. A. Increased blastogenesis of CD8 + T cells at baseline and after TCR activation in cHCV patients (arrow). Phenotype of blasting cells at 10 hours. B. Cumulative representation of % of active (cleaved) caspase-3 (aCasp3) signal in gated live naive CD8 T cells after 24 hours of TCR stimulation as compared to unstimulated condition in 4 HD, 4 cHCV, and 3 HBV patients. C. % of input cells recovered after TCR-activation culture.

Figure 43 .

 43 Figure 43. Anti-CD5 blocking Abs recapitulate the hypersensitivity phenotype. A. CD25, CD69 and a-casp3 expression at baseline, after low doses (TCR) or high dose (TCRx5) of CD3/CD28 stimulation, or after incubation with PMA. B. Representative overlay of histograms of p-ERK signal detection in naive CD8 + T cells in a HD 5 minutes after TCR stimulation with or without one hour of preincubation with anti-CD5 blocking Abs (bCD5). C. Percentages of p-ERK detected in naive CD8 + T cells in ten HD 5 minutes after TCR stimulation with or without one hour of preincubation with bCD5. D. Representative overlay of histograms of CD25 signal detection in naive CD8 + T cells in a HD after TCR stimulation with or without one hour preincubation with bCD5. E. Percentages of CD25 cells in naive CD8 + T cells in HD (n = 8), after TCR stimulation with or without one hour preincubation with bCD5 antibodies. F. Representation of % of active (cleaved) caspase-3 (aCasp3) in gated live naive CD8 T cells in HD (n = 4) after TCR stimulation as compared to unstimulated condition with or without one hour preincubation with bCD5.

Figure 44 .

 44 Figure 44. Cytokines levels detected in plasma of cHCV, and SVR patients, as compared to HD. Cytokines levels detected in plasma of HD, SVR, and cHCV patients. Squares indicate significant differences as compared to healthy donors.

Figure 45 .

 45 Figure 45. In vitro priming of Mart1-specific CD8 + T cells.A. Impact of CD45RO-magnetic enrichment on naive/memory T cell subsets on one HD (left) and one cHCV patient (right). B. Expansion of Mart1-specific T cells from RO -fraction of HD. C. Detection of Mart1 expanded specific cells after 10 days of in vitro priming assay on enriched CD8 + T cells from one cHCV patient. Cells were detected by conventional tetramer (WT) of CD8null tetramer (Null). Two doses of Mart1 peptide were tested: low(10 -8 ) or high(10 -6 ). Cells were analysed at Day 7 or Day 11. Wells without cytokines or without peptide were used as control. D. CD5 and CD25 expression on Mart1-specific cells detected by the WT tetramer at Day 7 and 11.

Figure 46 .

 46 Figure 46. Proposed models for dysregulation of naive CD8 + T cells during cHCV chronic infection.The activation threshold hypothesis. In healthy donors, before immunization, a given antigenspecific population contains T cells with ranges of CD5 expression (high, dark green; low, light green). In periphery, those cells are maintained by cytokines, and by low affinity pMHC contacts, and some of them may occasionally derive toward a memory-phenotype (violet). After immunization with its cognate peptide, the antigen-specific population expand largely, and express lower CD5 levels -although there is still a mix of high (dark violet) and low (light violet) expression. Some rare cells are not engaged into the response and remain as naive-phenotype into the globally experienced population. In the context of cHCV, most naive CD8 + T cells express low levels of CD5 at their cell surface. This lead to aspecific expansion and differentiation when encountering low affinity ligands in the periphery. When those cells encounter their antigen during the immunization phase, they will generate low-affinity and low-frequency specific populations.

  Similar observations were made in the spleen (data not shown). Together, our results argue for a dysregulated T cell preimmune repertoire in the context of Cxcr3deficiency, with increased representation of memory-phenotype (MP) cells into Ag-specific inexperienced populations.

Figure 47 .

 47 Figure 47. CXCR3KO mice have lower numbers of CD8SP thymocytes. A. Human CXCR3-expressing thymocytes (blue) within all thymocytes (left, red) or CD8SP. B. Mouse CXCR3-expressing thymocytes (blue) within all thymocytes (left, red) or CD8SP. B and C. Cxcr3 -/-mice present lower percentages and absolute numbers of intrathymic CD8SP. E and F. Decreased proportions of CD62L -CD44 hi T CD8SP cells in Cxcr3 -/-animals. G. Phenotype of gated CD62L -CD44 hi (blue) intrathymic T cells as compared to other thymocytes (red) and DN population (grey). H. CD44 expression by GFP + and GFP -T cell populations in Rag2p-GFP mice. I. Phenotype of CD44 hi GFP -(blue) as compared to developmental thymocytes (red) and DN population (grey).

Figure 48 .

 48 Figure 48. Cxcr3 -/-mice present increased numbers of circulating MP CD8 + T cells. A. Normal numbers of CD8 + T cells into the blood of Cxcr3 -/-mice. B, C and D. High proportions and absolute numbers of memory-phenotype (MP) CD8 + T cells in the blood of Cxcr3 -/-mice. E and F. High proportions of MP CD8 + T cells in the blood of OTI-CXCR3KO mice.

Figure 49 .

 49 Figure 49. Activation profile of OTI-CXCR3KO cells after stimulation with the Ova peptide.A and B. CD69 and CD25 expression on OTI-WT (white) and OTI-CXCR3KO (red) cells after 24h in vitro stimulation with SIINFEKL peptide. C. Overlay of CD69 histograms after 24h in vitro stimulation with SIINFEKL peptide. D. CD69/CD62L expression of OTI-WT cells (black, left), NP OTI-CXCR3KO (red) and MP OTI-CXCR3KO (blue) after 24h of stimulation with SIINFEKL peptide, as compared to unstimulated OTI-WT cells (grey).

Figure 50 .

 50 Figure 50. MP cells are found into the skin of OTI-WT transgenic unimmunized animals. A. Absolute numbers of OTI cells detected into tissues of OTI-WT (white) and OTI-CXCR3KO (black) mice. B. Detection and CD62L/CD44 phenotyping of OTI cells in the blood, spleen and skin from OTI-WT (left) and OTI-CXCR3KO (right) animals. C. Expression of CD62L, CD44, CD103, CD69 by skin OTI cells harvested from OTI-WT mice. D. Percentages of MP OTI cells into the skin of OTI-Rag competent (white circle) and OTI-Rag deficient animals (white square). E. Number of OTI cells into the skin of OTI tg/tg -WT, OTI tg/0 -WT, OTI tg/0 -CXCR3KO, and OTI tg/tg Rag -/--WT mice.

Figure 51 .

 51 Figure 51. MP cells are present in OTI-CXCR3KO cells of mixed bone marrow chimeras.A. Mixed bone marrow chimeras were generated by reconstituting irradiated WT (CD45.1) animals with a 1:1 mix of OTI tg/tg Rag -/-GFP Ub (CD45.2) and OTI-CXCR3KO (CD45.2) bone marrows. B. Identification and CD62L/CD44 phenotyping of OTI cells arising from OTI-WT and OTI-CXCR3KO bone marrows in blood and skin of grafted animals. C. Absolute numbers of OTI-WT and OTI-CXCR3KO cells recovered into the blood and skin of grafted animals. D. Percentages of MP cells into OTI-WT and OTI-CXCR3KO cells circulating in the blood of grafted mice. E. Expression of CD69 and CD25 activation markers on OTI-WT (dark grey) and OTI-CXCR3KO (red) cells after 24 hours of in vitro stimulation with SIINFEKL peptide, as compared to unstimulated conditions (light grey).

Figure 52 .

 52 Figure 52. Proposed model of MP Ag-specific inexperienced T cells migration.From our results, we propose that MP might be resident memory T cells that (i) seed into the skin (and maybe the thymus) tissues at steady state in unimmunized animals under the influence of CXCR3/ligands gradients; and (ii) might have the potential to crossreact with unknown specificities of the pre-immune repertoire. Whether high numbers of MP Ag-inexperienced T cells are circulating into the blood of cxcr3-deficient animals reflects (i) resident-memory T cells lacking their ability to reach target tissues, and/or (ii) a direct effect of Cxcr3 is unclear and needs additional investigations.

  identify naive cells, and developed my work without too much concerns. When we came to HCV studies, and the observation of sometimes large numbers of MP cells into inexperienced populations, I turned pale. When I gate on bulk T cells with naive/memory markers, how do I know the proportion of cells that are MP but part of inexperienced T cells vs. true memory? How can I interpret an increase in memory cells if I don't know which of these subset is concerned? While I was working on HCV and naive T cells, several research papers were published about the new subset of tissue resident memory cells -and again I found myself released not to have to deal with such an issue in humans: at least naive cells are circulating in the blood… When I started studies on mice, and observed MP Agspecific inexperienced T cells hidden into the organs at steady state, I became very confused. If similar things happen in humans, what is the relevance of my observations in peripheral blood? How much circulating cells are representative of the body capacity? Maybe I am looking at only a very small subset of what can be part of antigen-inexperienced populations, leading to important biases in my interpretations. Now I get extremely cautious when I need to interpret CD45RA/CD27 FACS plots.

  , and later optimized by Vincent Bondet. It can be used to create large collections of recombinant MHC class I molecules loaded with peptides of interest. The collections of peptide-MHC (pMHC) complexes that can be produced by this method are useful for two major purposes. First, the measurement of MHC binding affinity of a set of potential MHC ligands. Second, the production of large collections of MHC tetramers to measure antigen-specific T cell responses by MHC tetramer flow cytometry. The first basic protocol describes the expression and purification of recombinant MHC class I heavy and light chains. The second basic protocol describes the refolding and biotinylation of MHC class I complexes that are occupied with a conditional ligand. The third protocol describes how such MHC class I complexes that are occupied with conditional ligands can be utilized to produce pMHC complexes of interest in UV-induced peptide exchange reactions. The fourth basic protocol describes an ELISA-based strategy to measure the binding of peptides of interest to 'empty' MHC complexes generated by UV exposure. The fifth basic protocol describes how MHC complexes generated by ligand exchange can be used to produce MHC tetramers for T cell staining.

  water, 1 000X stock 50mg/mL at -20°) •• Chloramphenicol (diluted in ethanol, 1 000X stock 34mg/mL at -20°) •• LB plates + antibiotics (Cf = 50 µg/mL carbenicillin, 34 µg/mL chloramphenicol; 500mL = 16 plaques; 4°) •• Singles Competent Cells (50 µl reactions, -80°): E.Coli expression strain BL21 (DE3) pLysS •• SOC medium (4°) •• 1-S-Isopropyl-β-(D)-thiogalactopyranoside (IPTG) 1M: 2,38g in 10mL H2O (-20°) •• Lysozyme (powder, -20°) •• DL-Dithiothreitol (DTT) 1M: 1,54g in 10mL H2O (-HCl 1M pH8 (60,6g Trizma base in 500mL H20, +HCl until pH8, stored at RT) •• Sodium Azide 10% (RT) •• NaCl 1M (29,22g in 500mL H20, RT) •• Guanidine-HCl 8M (RT) •• Glycerol (RT) •• MilliQ H2O (RT) •• DNAse I 2 mg/mL : 16mg DNAse I + 4 mL glycerol (50 %) + 600 µl NaCl 1M (75mM) + 3,4mL H2O ; stored at -20°C. •• Resuspension buffer: 50 mM Tris-HCl pH8, 25% sucrose, 1 mM EDTA, 0 ,1% azide, DTT 10mM.

•

  Day 1) MHC class I heavy chains and β2m are expressed separately in the bacterial strain Bl21 (DE3) pLysS. Transform bacteria according to supplier's protocol (for Novagen Competent Cells, see below) and grow overnight on Luria-Bertani (LB) plates in the presence of 50 µg/ml carbenicillin and 34 µg/mL chloramphenicol.

19.

  Resuspend in 20mL Wash buffer. Add 20 µl/20mL of 1M DTT (1mM). Sonicate.20.Centrifuge at 10,000 rpm, 10min, 4°C. Discard supernatant. Resuspend the pellet in 3,5mL H2O, then add 7,5mL 8M guanidine-HCl.21.Spin for 30min at 14,000 rpm. Take supernatant. Measure OD 280nm (1/100 dilution in 8M guanidine-HCl). e (280 nm) of HLA A*0201 (1-276 pMBio) with biotag = 80 750 M-1cm-1. e (280 nm) of β2m (1-100) = 19 180 M-1cm-1. E (280nm) = ((1280 x n Tyr) + (5690 x n Trp) + (120 x n Cys) / MW (Da) MW β2m = 13 705 Da ; MW HLA A*0201 (1-276 pMBio) with biotag = 33 910 Da E β2m = 1.4 ; E HLA A*0201 (1-276 pMBio) with biotag = 2.18 HLA-A*01:01 E = 2,018 HLA-A*03:01 E = 2,189 HLA-A*11:01 E = 2,216 HLA-B*08:01 E = 2,183 C(M) = OD / [e (M-1cm-1) x L (cm)], where L is the lenght of the cuvette (usually = 1 cm).

  -HCl 10mM, pH8 (RT) •• Protease inhibitor cocktail (10X stock : 5 tablets in 50mL H20 ; -20° ; stock tablets at 4°) •• EDTA 0,5M (RT) ligase : either biotin ligase obtained by E coli expression (B ; see Support protocol 2), either bulk BirA, Avidity, Denver, USA (A) (-80°) •• D-Biotin (B) (5 mM dissolved in 100 mM phosphate buffer, pH 7

4 .Day 4 : 5 .

 445 Set up the 500mL refolding reaction by adding the following components to cold refolding buffer in the critical order indicated below. a) Add 5mL of 100mM PMSF (1mM final). PMSF is very unstable in aqueous solution. b) Add slowly the aliquot of 3000 nmoles hβ2m (6µM final) to the border of the vortex. c) Proceed similarly for HLA class I heavy chain (1 aliquot of 500 nmol; 1 µM final). d) Stir slowly O/N at 4° (cold room). Keep dark. e) (Day 2) Repeat addition of PMSF. f) Repeat addition of HLA class I heavy chain (1 aliquot of 500 nmol; 1 µM final). g) Stir slowly O/N at 4° (cold room). Keep dark. h) (Day 3) Repeat addition of PMSF. i) Repeat addition of HLA class I heavy chain (1 aliquot of 500 nmol; 1 µM final). j) Stir for 48hrs. Keep dark. Prepare the concentration step by washing two Amicon ultrafiltration cells with distilled water. Dispose two Amicon ultrafiltration membrane (MW cut-off 30kDa) in two Petri dishes filled with sterile water (glossy part up). (Day 5) Spin refolding reaction in Falcon 50 at 4500 rpm to sediment aggregates, and filter supernatant on 0.22 µm 500mL Stericups.

12 . 13 .

 1213 Following purification, the sample is generally used immediately for biotinylation. The use of MHC class I complexes stored in glycerol is not recommended as glycerol affects biotinylation efficiency. However, MHC class I complexes occupied by conditional ligand may be stored by snap-freezing in liquid nitrogen without the addition of glycerol. 11. Wash 6 PD10 columns with 25mL Tris-HCl 10mM pH8. Pass 2,5mL refolding on each column. Discard the flow through. Eluate with 3,5mL of Tris-HCl 10mM pH8. You need to exchange PBS to Tris because biotin ligase is inhibited by high concentrations of NaCl and Tris. See also the Avidity website: http://www.avidity.com/tech-protein.html. Wash an Ultra-15 PLTK Ultracel-PL Membrane (Mw cut-off 30 kDa) by placing 3 mL milliQ H2O on the filter and spin at 4°C, 4,500 rpm for 2 min. Concentrate the 21ml eluate fraction to 1.2mL in the Amicon (4To biotinylate the biotag of the HLA class I heavy chain, you can either use commercially available bulk BirA (A), or either use biotin ligase obtained by E coli expression (Option B). Option A: Mix 7 volumes of Tris-monomer (1200ul) with 1 volume of Biomix A buffer (171,4), 1 volume Biomix B buffer (171,4), 1 volume Biotin (171,4), and 5µl BirA enzyme (desired final volume < 2ml). Option B: Add 1 volume of freshly prepared biotinylation solution to 1 volume of pMHC class I monomer, using 0.03 µM ligase for 1 µM of MHC class I.

19.

  Aliquot at 1mg/mL (50µg in 50µl final volume) in PBS in polypropylene Starstedt tubes. Biotinylated MHC class I molecules (Mw 47 kDa) with a final concentration of approximately 1 mg/mL can be stored at -20°C for up to 1 year, or at -80° and LN for years. Biotinylation reactions performed according to the protocol above should give complete or near-complete biotinylation of the tagged MHC class I heavy chain. It is nevertheless recommended to verify the degree of biotinylation for every batch of MHC class I before further use. See Support protocol 3.

  HLA ELISA (described here for HLA-A*0201) measures the concentration of HLA class I complexes in the range of 0.5 to 10 nM. It is recommended to include a standard MHC titration curve, to ensure that the obtained absorbance values are within the linear range of the assay. UV-irradiation of the UV-sensitive HLA A*0201-KILGFVFJV complex leads to a reduction of the absorbance signal of approximately 70-80% and an estimated reduction in concentration of folded HLA of 85%. This reduction is comparable to the reduction in HLA concentration as detected by gel-filtration chromatography. It is important to note that this assay will generally underestimate the extent of peptide cleavage, which is usually complete (see anticipated results). Inclusion of a high affinity HLA A*0201 restricted ligand in UV-mediated exchange reactions leads to recovery of 80-100% of the original ELISA signal. Lower rescue is observed using peptides with a moderate to low affinity for HLA Aimmuno plate 96 wells (cat. #: 439454, F96 CERT Maxisorp, Nunc) •• Absorbance plate reader •• Coating solution: 2 µg/mL streptavidin (cat. #: S888, Invitrogen, Molecular probes) in PBS •• Wash buffer: 0.05% Tween 20 (cat. #: P1379, Sigma-Aldrich) in PBS •• Blocking buffer: 2% BSA (cat. #: A-7030, Sigma-Aldrich) in PBS •• Horseradish peroxidase (HRP)-conjugated anti-human β2m antibody

(

  cat. #: S868, Molecular Probes) •• 10x UV-sensitive MHC monomer (250 µg/mL; ~5 µM) •• PBS •• polypropylene microtubes 1.5 mL with cap (cat. #: 72692, Sarstedt) •• SDS-Page electrophoresis material 1. To determine the optimal ratio of streptavidin conjugate and HLA class I complex, prepare the following 4 samples in a total volume of 100ul in 1.5 mL tubes : 1. UV-sensitive HLA class I monomer (Cf = 5 nM) 2. UV-sensitive HLA class I monomer (Cf = 5 nM) + streptavidin-PE solution 1/100 3. UV-sensitive HLA class I monomer (Cf = 5 nM) + streptavidin-PE solution 2/100 4. UV-sensitive HLA class I monomer (Cf = 5 nM) + streptavidin-PE solution 4/100 2. Analyze each sample on SDS-Page electrophoresis under reducing and non denaturing conditions.This analysis will reveal the fraction of HLA class I that is maximally bound by streptavidin and this number can be used to calculate the optimal ratio of HLA class I and streptavidin conjugate, that gives high order tetramers, while leaving little residual monomeric HLA class I present. It is noted that an excess of fluorochromestreptavidin over HLA class I results in the formation of HLA class I tetramers with a lower valency and the binding of such lower order tetramers to antigen-specific T cells is poor. In addition, a failure to saturate the available biotin-binding sites can lead to spurious background signal in case biotinylated antibodies are used for co-staining.Time ConsiderationsThe time required to produce a batch of refolded MHC class I occupied with UV sensitive ligand (Basic protocol 1 and 2) is fairly significant, but not different from that required to produce a batch of MHC class I occupied with any other peptide ligand. The actual ligand exchange process (Basic protocol 3) is extremely straightforward, allowing one to produce collections of hundreds of different peptide-MHC complexes in a timespan of hours. The time required for the measurement of MHC class I stabilization by ELISA (Basic protocol 4) is restricted to several hours, and formation of MHC tetramers (Basic protocol 5) can likewise be completed within hours.

•Protocol 1 .

 1 • Fresh or Frozen human PBMCs of known haplotype •• PE -labeled tetramers •• Anti-PE microbeads (Miltenyi Biotec) •• Pulldown buffer (PB): PBS 1X (Gibco)+ 5% Human Serum Albumin 20% (PAA Laboratories) + 5% Citrate Dextrose Anticoagulant (ACD; Sigma-Aldrich) •• MS MACS Separation columns, magnets, stands (Miltenyi Biotec) •• FcR Blocking Reagent (Miltenyi Biotec) •• BD Falcon Cell Strainer 70µm (BD Biosciences) •• DUMP channel mix of antibodies (CD14, CD16, CD19, CD56-PacBlue) •• Dapi Nucleic Acid Stain (Invitrogen) •• Cocktail of antibodies for characterization of T cells (For example CD8 Amcyan, CD11a FITC, CD3 PerCP, CD45RA PE-Cy7) Count and aliquot cells into 15ml Falcon tubes.

15 .

 15 Load columns with 1mL of cells. Cécile Alanio, F.Lemaitre, H.Law, M.Hasan, Matthew L. Albert. Enumeration of human antigen-specific naive CD8 + T cells reveals conserved precursor frequencies. Blood 2010; 115 (18):3718-3725. This manuscript contains results presented in Chapter 2. Cécile Alanio, I.Bouvier, H.Jusforgues-Saklani, Matthew L. Albert. Tracking antigen-specific T cells using MHC Class I tetramers. Methods in Molecular Biology 2013; 960:309-26. This book chapter complete results presented in Chapter 2. Cécile Alanio, Sultanik P, Flecken T, Perot B, Gowry M, Duffy D, Lim A, Clave E, van Buuren MM, Schnuriger A, Garbarg-Chenon A, Bousquet L, Mottez E, Schumacher TN, Toubert A, Appay V, Heshmati F, Thimme R, Pol S, Mallet V, Albert ML. Bystander hyperactivation of preimmune CD8 + T cells in chronic HCV patients can be reversed by viral clearance. Submitted. This manuscript contains results presented in Chapter 3. Philippe Sultanik, Casrouge A, Alanio C, Mottez E, Rosa-Hézode I, Hézode C, Renard P, Bousquet L, Uzé G, Pol S, Albert ML, Mallet V. Baseline sensitivity of T cells to alpha-interferon correlates with sustained virological response to alpha-IFN-based triple therapy in chronic hepatitis C virus infection -ANRS 11416. J Viral Hepatitis 2014. Stéphanie Thomas, Rouilly V, Patin E, Alanio C, Dubois A, Delval C, Marquier LG, Fauchoux N, Sayegrih S, Vray M, Duffy D, Quintana-Murci L, Albert ML, for The Milieu Intérieur Consortium. The Milieu Intérieur study -a resource for study of human immunologic variance. Clinical Immunology 2

Table of contents

 of

  , and risk factors are gender (male), ethnicity (african origin), age (> 40yrs at the time of infection), coinfection with HBV, immunosuppression (including HIV co-infection), metabolic syndrome, including diabetes, obesity, insulino-resistance and steatosis, and alcohol consumption. Main risk factor for evolution of HCV patients toward HCC is alcohol consumption. Of note, HIV co-infected

patients have also an increased progression towards cirrhosis, an increased liver-related mortality, and earlier and more agressive HCC. Mortality related to HCV account for 86 000 deaths per year in Europe in 2002, within 35% related to cirrhosis, and 32% to HCC. After diagnosis of HCC, 33% of patients die within one year. When cirrhosis and its complications are established, the only curative treatment is the liver transplantation.

  -/-cells harvested from BMC hosts 2 months after the allograft were of naive phenotype. Presence of MP cells into the OTI-CXCR3KO population thus could indicate (i) an inability of those cells to reach target tissues, or (ii) a lack of clearance of cells generated by homeostatic proliferation. Results from our mixed bone marrow chimeras experiments argue in defavor of a trafficking issue, with comparable skin engraftment of OTI-WT and OTI-CXCR3KO cells. It is possible that our results reflects a defective clearance of MP inexperienced T cells as a result of Cxcr3 deficiency. This would be in line with observations of attenuated contraction and resistance to apoptosis of cxcr3 -/-memory OTI CD8 + T cells in models of acute viral (Ova-expressing vaccinia virus, VV) and bacterial (LM-Ova) infections

Table 1

 1 Conditionalligands for a series of other MHC class I alleles developed by us and others are listed in Table1. Conditional ligands described to date. It is likely that most of these ligands can also be utilized for related HLA allele subtypes with the same or a very similar ligand preference (e.g. KILGFVFJV for HLA A*0202, A*0203 etc.), but this has not been tested.Molecular weight of the conditional ligand peptides is calculated by adding 89.004 Da to the aminoacid at the UV sensitive position. For example, A2 KILGFVFCV MW is 1025.3, so A2 photocleavable peptide molecular weight is 11114.3. But we always assume our 9-mers are MW 1000, so that 10mg/ml corresponds to 10mM, and

	HLA A*0101	STAPGJLEY	Bakker et al., 2008
	HLA A*0201	KILGFVFJV	Toebes et al., 2006
	HLA A*0301	RIYRJGATR	Bakker et al., 2008
	HLA B*0702	AARGJTLAM	Bakker et al., 2008
	HLA A*1101	RVFAJSFIK	Bakker et al., 2008
	H2-D	b	ASNENJETM	Toebes et al., 2006
	H2-K	b	FAPGNYJAL	Grotenbreg et al, 2008
	H2-L	d	YPNVNIHJF	Frickel et al., 2008

  •• HPLC (or FPLC) system with a gel-filtration column, e.g. a HiLoad 16/600 Superdex 200pg. •• PBS pH=7.4 (Day 1) Thaw 1x500 nmol MHC Class I heavy chain (3x1 aliquot over 3 days; 3µM final) and 1x3 000 nmol hβ2m (1 aliquot; 6µM final). Resuspend each in 1 mL 8M guanidine-HCl.

	1.
	for running buffer
	•• Centrifuge
	•• 0.22 µm 500mL Stericups
	Reagent	Amount	Final concentration
	1 M Tris-HCl pH 8	50 ml	100 mM Tris-HCl pH 8
	L-Arginine	34,8 g	400 mM L-arginine
	Reduced glutathione	0,61g	5 mM reduced glutathione
	Oxidized glutathione	0,15 g	0.5 mM oxidized glutathione
	0.5 M EDTA	2 ml	2 mM EDTA
	10X Protease inhibitor cocktail	50 ml	Not applicable
	H	2	O (milliQ)	350 ml	Adjust to 500mL final volume

•• Dilution buffer (B ; see recipe below ; must be prepared fresh).

  Prepared fresh and ensure that ATP is dissolved in Tris buffer.14. Incubate overnight at RT.15. (Day 6)Purify by gel-filtration HPLC (or FPLC) : wash the column with PBS at 1mL/min, then inject refolding reaction using one 2 mL injection on the column with a 2 mL loop (normally max 5mL/run). Collect the biotinylated HLA class I molecules. Keep the sample on ice.

	Reagent	Amount
	D-biotin (5 mM dissolved in 100 mM phosphate buffer, pH 7.5)	0.04 vol
	ATP (0.5 M dissolved in 1 M Tris pH 9.5)	0.04 vol
	BirA his-tagged biotin ligase (6.6 µM)	0.01 vol
	10x ligase buffer	0.2 vol
	EDTA-free protease inhibitor cocktail (25x: 1 tablet in 2 mL H 2 O or 0.08 vol
	exchange buffer)
	H 2 O (MilliQ)	0.63 vol

Remerciements

To my three naïve T cells, with all my love

Remerciements welcomed me at the 5 th floor quite a number of time... You were extremely supportive, inside and outside the lab, and I now count you as my friends. Viva Italia!!

Abbreviations

Methods

BASIC PROTOCOL 3 UV MEDIATED PEPTIDE EXCHANGE

UV-mediated cleavage of the conditional ligand is time dependent. With the set-up described below, peptide cleavage is detected after 1 min and is essentially complete after approximately 15 min. A 30 to 60 min incubation time is normally used to ensure optimal exchange of the conditional ligand with the peptide of interest. Protein concentration may influence the rate of UV-mediated cleavage, as both the nitrophenyl moiety and the reaction product absorb long wavelength UV light. In addition, path length may affect the reaction speed.

Empty, peptide receptive MHC molecules that are formed upon UV exposure can be rescued by performing the UV-mediated cleavage in the presence of an MHC ligand of interest. In most experiments, a 100 fold molar excess of peptide over MHC is used. UV-induced peptide exchange is routinely performed using 25 µg/mL of UV sensitive MHC class I complexes. However, peptide exchange reactions have been performed with MHC class I concentrations up to 100 µg/mL.

Materials:

•• 96 well plates (polypropylene microplate 96 well V sharp, BD)

Use polypropylene material to avoid sticking of peptide or protein to the plate.

•• UV-lamp 366 nm CAMAG UV fitted with UV Lamp long-wave UV, 366 nm, 2x8 W The use of short wavelength (254 nm) or broad-band UV lamps is detrimental to the MHC complex and should be avoided at all times.

•• Centrifuge with rotor for microtiter plates.

Work on ice to avoid proteases + dark during all the steps. Work on polypropylene plates to avoid sticking of peptide or protein to the plate.

1. Calculate total volume of monomer you will need to exchange (Ci=400nM ; Cf = 20nM ; so usually 5ul in 100ul for 1 staining).

Be aware that only 60-80% of the starting volume will be left at the end due to evaporation.

Prepare appropriate quantity of p*monomer + 1X PBS mix :

i.e. Vi= Cf (0.1µM = 0.1mg/ml) x Vf (that you need) / Ci (look on the aliquot : usually around 1mg/ml) For more detailed protocol, see « 2012-04-01 Protocol 7 colors CA » and related files.

3.

In a 96 well plate, add the following reagents to each well:

-Exchange Peptide (10mM stock in DMSO, 50X, -20° ; Cf=200uM) 9-mers peptides can be approximated to have a Mw = 1000, so 10mM stocks = 10mg/mL. They are reconstituted in 100% DMSO.

They are used at 1/50 dilution, so 0.1ul of the stock in each 5ul p* monomer volume.

Manuscripts