Emmanuel Clave

Itauá Leston Araujo

Cécile Alanio

Etienne Patin

Jacob Bergstedt

Alejandra Urrutia

Silvia Lopez-Lastra

Yan Li

Bruno Charbit

Cameron Ross Macpherson

Milena Hasan

Breno Luiz Melo-Lima

Corinne Douay

Noémie Saut

Marine Germain

David-Alexandre Trégouët

Pierre-Emmanuel Morange

Magnus Fontes

Darragh Duffy

James P Di

Lluis Quintana-Murci

Matthew L Albert
email: albert.matthew@gene.com

Antoine Toubert
email: antoine.toubert@univ-paris-diderot.fr

The Milieu Intérieur ‡ Consortium

Extracellular Matrix Elements eDP early DP EMT Epithelial-Mesenchymal Transition

Keywords: polymorphisme, TREC, TCR, répertoire, diversité, thymus, sexe, âge, VDJ réarrangement, Milieu Intérieur, cohorte, souris humanisées polymorphism, TREC, TCR, repertoire, diversity, thymus, sex, age, VDJ rearrangement, Milieu Intérieur, cohort, humanized mice ABBREVIATIONS ACTB Actin, Cytoplasmic 1 AGM Aorta-Gonad-Mesonephros AIRE Autoimmune Regulator

Facteurs Environnementaux et Génétiques Déterminant la Fonction Thymique Chez l'Adulte Sain

Résumé : Le thymus est un organe clé de l'immunité, permettant tout au long de la vie la production de cellules T naïves. L'involution thymique associée à l'âge est liée à une réduction de la masse tissulaire et de la cellularité thymique, ainsi que la perte de la structure tissulaire et de l'architecture normale conduisant à une baisse de la production de cellules T naïfs. Cependant, à l'exception de l'âge, les facteurs environnementaux ou génétiques susceptibles de régir la fonction thymique chez l'Homme restent mal connus. Nous avons caractérisé la variabilité de la fonction thymique chez 1000 adultes sains stratifiés par l'âge et par sexe sur la cohorte Milieu Intérieur, en utilisant la quantification des cercles d'excision (TRECs) dans le sang périphérique comme marqueur de la thymopoïèse. L'âge et le sexe sont les seuls facteurs non héritables identifiés qui affectent la fonction thymique. Les quantités de TREC diminuent avec l'âge étant plus élevée chez les femmes de tout âge. Surtout, une étude d'association pangénomique a révélé l'existence d'un variant génétique (rs2204985) dans le locus TCRA-TCRD, situé entre les segments géniques DD2 et DD3, l'allèle G étant associé à un niveau de TRECs supérieur à l'allèle A. Cette association a été validée dans une cohorte de réplication (cohorte MARTHA). La transplantation de cellules souches hématopoïétiques humaines de foie fetal avec le génotype GG dans des souris immunodéficientes est associée à une thymopoïèse accrue avec des TRECs plus élevés, une augmentation du nombre de thymocytes et aussi une plus grande diversité du répertoire TCRA-TCRD. Notre approche d'immunologie des populations a mis en évidence un variant génétique influençant la thymopoïèse l'adulte sain. Ceci pourrait avoir un impact direct en médecine dite de précision dans les domaines du vieillissement et de la vaccination, en transplantation de cellules souches hématopoïétiques, ainsi qu'en autoimmunité. Ces travaux conduisent également à étudier plus en détail les mécanismes de la thymopoïèse précoce et des réarrangements du locus TCRA-TCRD.

Título: Determinação Genética e Ambiental da Função do Timo na Resposta Imunológica em Indivíduos Saudáveis Resumo: O timo é um órgão vital para a manutenção do sistema imunológico periférico. A involução tímica associada à idade está ligada à redução da massa tecidual e da celularidade tímica, além da perda da estrutura tecidual e arquitetura anormal; levando a um declínio na produção de novas células T. No entanto, com exceção da idade, as causas subjacentes que governam a função tímica em humanos saudáveis ainda são desconhecidas. Nós caracterizamos a variabilidade da função tímica entre 1000 adultos saudáveis estratificados por idade e sexo da coorte Milieu Intérieur, usando a quantificação de TRECs em células T, a partir do sangue periférico, como um marcador da timopoïese. A idade e o sexo foram os únicos fatores não hereditários identificados que afetaram a função tímica. Os valores de TREC diminuíram com a idade e foram maiores nas mulheres em comparação com os homens em todas as idades. Além disso, um estudo de associação genômica revelou uma variante comum (rs2204985) dentro do locus TCRA-TCRD do receptor de células T, entre os segmentos do gene DD2 e DD3, que se associou às quantidades de TREC. Essa associação foi validada em uma coorte de replicação (coorte MARTHA). Surpreendentemente, o transplante de células-tronco hematopoiéticas humanas com o genótipo GG (rs2204985) em camundongos imunodeficientes mostrou altos níveis de TRECs, aumento na contagem de timócitos e uma maior diversidade de repertório do TCR. Nossa abordagem de imunologia populacional revelou um polimorfismo que influencia a timopoïese em crianças e adultos saudáveis, com implicações potencialmente amplas na medicina de precisão, especialmente em vacinas e envelhecimento, no transplante de células tronco hematopoiéticas e autoimunidade. Esse estudo ainda necessita de alguns estudos sobre os mecanismos do rearranjo do locus TCRA-TCRD em estágios precoces da timopoiese.

Palavras-chave: polimorfismo, TREC, TCR, repertório, diversidade, timo, sexo, idade, rearranjo VDJ, Milieu Intérieur, coorte, camundongo humanizado

ACKNOWLEDGMENTS / REMERCIEMENTS

I would also like to thank my doctoral committee, Magali Irla and Michelle Rosenzwajg for their deep relecture of the manuscript, as well as Isabelle André-Schmutz, Victor Appay, Emmanuel Clave and Antoine Toubert for their participation to the jury. Your input, guidance and suggestions throughout my doctoral tenure were instrumental to my success and was greatly appreciated. I would like to thank also Emmanuel Clave and Antoine Toubert for having taken the time to read, comment and discuss my work during the entire course of my PhD.

My most sincere thanks, To Antoine Toubert, my supervisor. For having welcomed me in his research unit INSERM U1160. For his support and precious guidance in my researching, as well as for his patience and good mood while directing the team. For such a great time in your lab. It has been a

privilege for me to be part of this adventure with you at my side.

To Emmanuel Clave, my co-supervisor. I will not ever be able to thank enough. For having welcomed, encouraged, guided, helped and motivated me during this long journey. For his endless availability, for helping me developing and understanding a complex subject of great interest. For his sense of humour that renders the laboratory a joyful place to work. For his friendship. For his (our?) coffee machine.

To all the 3 rd floor of the Institut Universitaire d'Hématologie in special to Corinne Douay and Isabelle Fournier for their timeless support.

Vigorous thanks to my labmates, past and present: Marie Roelens, Marjolaine Ngollo and Odessa Fayet, Joana Félix and José Oliveira, Marialuisa Catanoso, Lucas Coelho Marliére Arruda, Breno Luiz Melo Lima, Karina Tozatto-Maio and Luciana Jarduli…thank you for your support and lots of fun.

Outside of Saint-Louis, thanks to the Pasteur Institute for the great scientific environment and all the members of the LabEx Milieu Intérieur (MI) team, in special for Darragh Duffy and Matthew Albert for the financial support necessary to finish my PhD. Special thanks to Cécile Alanio, Etienne Patin, Jacob Bergstedt, Bruno Charbit and Alexandra Urrutia, you helped me a lot…Yes!! The paper was accepted in the Science Translational Medicine, after many fingers crossed. Thanks to the members of the Immunology department, in special to Jim di Santo, Silvia Lopez-Lastra, Yan Li and Jean Marc Doisne…the humanized mice team.

I would like to thank Agata Cieslak, Isabelle André-Schmutz, Vahid Asnafi and Guillaume Hypolite…the Necker Hospital human thymus team. I thank Université Paris-Diderot for my admission as a PhD student that allowed me to engage in a scientific career.

Thanks Science Without Borders, the best national scientific program in Brazil, for the financial support during my PhD.

Most importantly, I would like to express my deepest gratitude to my family. My love thank goes to my wife, Mônica: your generosity and patience during four years of PhD -words cannot express how much I love you.

LIST OF FIGURES

Figure 1. Establishment of definitive hematopoietic stem cell (HSC) pools in mouse and human embryos. Adapted from [START_REF] Mikkola | The journey of developing hematopoietic stem cells[END_REF]Kaushansky et al., 2016;[START_REF] Ivanovs | Human haematopoietic stem cell development: from the embryo to the dish[END_REF] [START_REF] Görgens | Multipotent hematopoietic progenitors divide asymmetrically to create progenitors of the lymphomyeloid and erythromyeloid lineages[END_REF][START_REF] Velten | Human haematopoietic stem cell lineage commitment is a continuous process[END_REF][START_REF] Karamitros | Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells article[END_REF] [START_REF] Verschuren | Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF][START_REF] Yui | Developmental gene networks: a triathlon on the course to T cell identity[END_REF][START_REF] Rothenberg | Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control[END_REF]................ [START_REF] Hodges | Diagnostic role of tests for T cell receptor (TCR) genes[END_REF][START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Krangel | Mechanics of T cell receptor gene rearrangement[END_REF] [START_REF] Hodges | Diagnostic role of tests for T cell receptor (TCR) genes[END_REF][START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF].......... [START_REF] Verschuren | Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF] [START_REF] Glusman | Comparative genomics of the human and mouse T cell receptor loci[END_REF][START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Zhao | Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development[END_REF] [START_REF] Okazaki | T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining[END_REF][START_REF] Hazenberg | T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation[END_REF]

Figure 14.

Model for primary and secondary Vα to Jα recombination. Adapted from [START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF][START_REF] Hawwari | Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination[END_REF][START_REF] Zhao | Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development[END_REF]..........

Figure 15.

T cell selection and TRA expression in the thymus during migration from the cortex into the medulla. Adapted from [START_REF] Takaba | The Mechanisms of T Cell Selection in the Thymus[END_REF]....................

Figure 16. Diagram of thymic egress and phenotypic maturation of mouse and human

RTEs in secondary lymphoid organs. Adapted from [START_REF] Fink | The Biology of Recent Thymic Emigrants[END_REF]

FOXM1

Forkhead

FOREWORD

The thymus is an evolutionarily ancient primary lymphoid organ common to all vertebrates in which T cell development takes place. Fifty and a few years since the function of the thymus was discovered, the study of T cell development remains a fascinating part of immunology. A truly odyssey through the integrated network of cells, molecules and organs where each component relies on the rest to function properly. For thousands of years ago our relationship with the environment and among ourselves continues to evolve according to the new challenges. The appearance of sign language, the speech development and the invention of writing are some of the many faces of communication that allowed us to survive and create a new world. This point of view can be applied in our cells, such as the T cells that very early must use this cross-talk to develop and survive. Cross-talk refers to instances in which one or more components of one signal transduction pathway affects another. During the past three decades, molecular biologists and geneticists have deconstructed intracellular signaling pathways in individual cells, revealing a great deal of cross-talk among key signaling pathways in the animal kingdom. My interest in immunology is renewed after every meeting.

Tracing a parallel between naïve T cells and PhD students, 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. The objective of the present thesis is to overcome such practical obstacles, by giving simple and accessible experiment protocols to explore thymus biology. As a general introduction to this manuscript, I will briefly present the T cell ontogeny. Next, the second part discuss in detail the thymopoïesis and their different stages of thymocyte development, the process by which the thymic microenvironment influences the formation of the repertoire of the T cell receptors (TCRs) expressed on developing T cells. After describing what constitutes the thymopoïesis, I will present the T cell receptor excision circles (TRECs) and their importance as markers of thymus function.

Nevertheless, this has been remarkably studied in murine models and a part of the studies presented in this thesis are devoted to this central issue, underlying mechanisms of disease, as well as for translational research from bench to bedside. Thymic function has been extensively studied for its capacity to shape the adaptive immune repertoire. However, little is known about the thymopoïesis in healthy human individuals. For these reasons during the writing of this thesis I have always sought to bring the information of both species.

CHAPTER 1. GENERAL INTRODUCTION I. HEMATOPOIESIS I.1. The first waves of hematopoiesis

The lifelong production of blood cells depends on hematopoietic stem cells (HSCs) and their ability to self-renew, high capacity of proliferation and differentiation to meet the demand of blood and immune cell production during homeostasis and stress [START_REF] Weissman | Stem Cells : Units of Development , Units of Regeneration , and Units in Evolution[END_REF][START_REF] Wei | Review Niches for Hematopoietic Stem Cells and Their Progeny[END_REF]. The original pool of HSCs in mammals is formed during embryogenesis in a dynamic and complex developmental process that involves several anatomical niches-sites. In fetal tissues these niches changes rapidly where they expand in number and initiate hematopoiesis before colonize primarily to the bone marrow (BM) at birth [START_REF] Li | Using mouse models to study function of transcriptional factors in T cell development[END_REF][START_REF] Gao | The hematopoietic stem cell niche: from embryo to adult[END_REF] (Fig. 1). Hematopoiesis ontogeny can be divided into two periods, the primitive and the definitive hematopoiesis. Primitive hematopoiesis is referred as hematopoiesis taking place prior to the development of the fetal liver. The term definitive hematopoiesis is used to describe blood formation after the formation of the fetal liver [START_REF] Li | Using mouse models to study function of transcriptional factors in T cell development[END_REF]. In humans, the hematopoietic lineage arise from the mesodermal precursor (Fig. 1, in grey) through the intermediate hemogenic endothelium [START_REF] Bertrand | Haematopoietic stem cells derive directly from aortic endothelium during development[END_REF][START_REF] Kissa | Blood stem cells emerge from aortic endothelium by a novel type of cell transition[END_REF][START_REF] Crane | Adult haematopoietic stem cell niches[END_REF]. The hematopoiesis first occurs in the yolk sac (Fig. 1B, blue line) representing the primitive hematopoiesis, and then in the liver, spleen and BM by the time of birth [START_REF] Mikkola | The journey of developing hematopoietic stem cells[END_REF]) (Fig. 1B). At day 21 (Fig. 1B, red arrow), the first primitive erythroblasts can be observed inside the cardiac cavity, making the onset of blood circulation, followed by appearance of the first CD45 + cells (Tavian, [START_REF] Taub | Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo[END_REF], similar to analyses in the mouse [START_REF] Ghiaur | Rac1 is essential for intraembryonic hematopoiesis and for the initial seeding of fetal liver with definitive hematopoietic progenitor cells[END_REF]. The endothelial lining of the human dorsal aorta upregulates CD34, which later marks adult HSCs, from the day 19 [START_REF] Oberlin | Blood-forming potential of vascular endothelium in the human embryo[END_REF]. During development, cells with long-term HSC potential have first been found in an intraembryonic region called the aorta-gonadmesonephros (AGM; Fig. 1B, in green), and it has been assumed that these cells enter the blood circulation to first seed the fetal liver (Fig. 1B, yellow line), expand and generate fetal hematopoiesis there, then travel again to seed the BM, the final and lifelong site of adult hematopoiesis [START_REF] Kissa | Live imaging of emerging hematopoietic stem cells and early thymus colonization[END_REF]. At day 27 post-conception, the first CD34 + CD45 + cells in the intra-aortic haematopoietic clusters emerge in the pre-umbilical region of the dorsal aorta, and their number reaches several hundred at day 33 and disappear at day 35-38. The development of the definitive BM niche is closely linked to the invasion of cartilaginous bone by blood vessels and bone ossification. Vascular invasion facilitates seeding of BM with

I.2. Bone Marrow

The bone marrow is a highly vascular tissue [START_REF] Nombela-Arrieta | Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment[END_REF] embedded in a hypoxic microenvironment, with the lowest oxygen tensions found near sinusoids in the central cavity [START_REF] Spencer | Direct measurement of local oxygen concentration in the bone marrow of live animals[END_REF][START_REF] Zhang | Hypoxia and Metabolic Properties of Hematopoietic Stem Cells[END_REF]. In long bones, central longitudinal arteries give rise to radial arteries that in turn branch into arterioles near the endosteum [START_REF] Nombela-Arrieta | Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment[END_REF]. The transition from arterioles to venous endothelium occurs in close proximity to the endosteum characterizing the 2 stem cell niches in the bone marrow: reticular and endosteal niches (Fig. 2). Few growth factors are known to be non-cell-autonomously and required for HSC maintenance, including stem cell factor (SCF) [START_REF] Czechowicz | Efficient Transplantation via Antibody-Based Clearance of Hematopoietic Stem Cell Niches[END_REF], CX-Chemokine ligand (CXCL) 12 [START_REF] Ara | Long-term hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone marrow during ontogeny[END_REF][START_REF] Sugiyama | Maintenance of the Hematopoietic Stem Cell Pool by CXCL12-CXCR4 Chemokine Signaling in Bone Marrow Stromal Cell Niches[END_REF] and thrombopoietin [START_REF] Qian | Critical Role of Thrombopoietin in Maintaining Adult Quiescent Hematopoietic Stem Cells[END_REF][START_REF] Yoshihara | Thrombopoietin/MPL Signaling Regulates Hematopoietic Stem Cell Quiescence and Interaction with the Osteoblastic Niche[END_REF].

Quiescent HSCs preferentially localize to arterioles in the endosteal region (J. Zhu et al., 2007). Approximately 80% of dividing and non-dividing HSCs are perivascular and enriched in the endosteal region [START_REF] Sugiyama | Maintenance of the Hematopoietic Stem Cell Pool by CXCL12-CXCR4 Chemokine Signaling in Bone Marrow Stromal Cell Niches[END_REF][START_REF] Acar | Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal[END_REF][START_REF] Asada | Differential cytokine contributions of perivascular haematopoietic stem cell niches[END_REF]. This region contains a complex network of stromal cells that have been implicated in HSC maintenance including osteolineage cells, endothelial cells (both arteriolar and venous), pericytes, CXCL12-abundant reticular (CAR) cells, sympathetic nerves, and nonmyelinating Schwann cells. The remaining 20% of the phenotypic HSCs localize directly adjacent to megakaryocytes in mice. HSCs progeny (megakaryocytes) are intimately associated with the bone marrow sinusoidal endothelium, extending cytoplasmic protrusions into the sinusoids to produce platelets. Conditional ablation of megakaryocytes results in increased HSCs and HSC cycling, and data suggest that megakaryocytes promote HSC quiescence via direct mechanisms through production of CXCL4 and transforming growth factor β (TGF)-β, independent of stromal niche cells [START_REF] Taub | Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo[END_REF][START_REF] Czechowicz | Efficient Transplantation via Antibody-Based Clearance of Hematopoietic Stem Cell Niches[END_REF][START_REF] Gazit | Fgd5 identifies hematopoietic stem cells in the murine bone marrow[END_REF][START_REF] Calvi | The hematopoietic stem cell niche in homeostasis and disease[END_REF] (Fig. 2). Osteolineage cells (e.g., osteoblasts

and osteocytes) produce a number of cytokines implicated in HSC regulation, including granulocyte colony-stimulating factor (G-CSF) [START_REF] Rothenberg | Identification of a cKit(+) Colonic Crypt Base Secretory Cell That Supports Lgr5(+) Stem Cells in Mice[END_REF], thrombopoietin [START_REF] Flanagan | The <em>kit</em> ligand: A cell surface molecule altered in steel mutant fibroblasts[END_REF]) and CXCL12 [START_REF] Zsebo | Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium[END_REF]. CXCL12 promotes HSC maintenance and retention in the bone marrow by activating signaling through CX-Chemokine receptor (CXCR) type 4, which is expressed by HSCs [START_REF] Zou | Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development[END_REF].

CXCL12:CXCR4 signaling also regulates HSC engraftment after transplantation and HSC proliferation and the proliferation and retention of multiple restricted progenitor populations in the bone marrow, including various myeloid and lymphoid progenitors (Lai et al., no date;

I.3. Progenitors -from stem cell to lymphoid progenitor commitment

Human hematopoiesis produces 10x10 9 new, terminally mature blood cells daily, production that is also rapidly responsive to external change. All mature blood and immune cells are thought to derive from self-renewing and multipotent HSCs. Human stem cells are defined by their vast self-renewal and multilineage differentiation capacities to give rise to all cell types of the hematopoietic system throughout life, which place them at the top of a cellular hierarchy. According to the current model, initiation of differentiation is associated with the loss of self-renewal and generation of discrete multipotent, oligopotent and subsequently unipotent progenitor cell stages [START_REF] Chao | Establishment of a Normal Hematopoietic and Leukemia Stem Cell Hierarchy[END_REF][START_REF] Ivanovs | Human haematopoietic stem cell development: from the embryo to the dish[END_REF]. This process begins with the HSCs in the BM until the generation of common myeloid and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous multipotent population containing the capability to divide asymmetrically. This characteristic involves the polarized distribution of determinants within the mother cell and their unequal inheritance, by each daughter cell increasingly limited differentiation and self-renewal potential, to finally differentiate in mature hematopoietic cells. Such asymmetric division allows one daughter to retain an immature fate (self-renewing for the whole life of the host)

and the other to become differentiated and to proliferate into multipotent progenitors (MPPs also known as a short-term HSC) that give rise the lymphoid-primed multipotent progenitors (LMPPs) (Fig. 3). Finally LMPP give rise to unipotent multi-lymphoid progenitors (MLPs) which are committed progenitor of T cell lineage [START_REF] Görgens | Multipotent hematopoietic progenitors divide asymmetrically to create progenitors of the lymphomyeloid and erythromyeloid lineages[END_REF][START_REF] Karamitros | Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells article[END_REF] (Fig. 3). MPPs are the immediate progeny of HSCs [START_REF] Boyer | All Hematopoietic Cells Develop from Hematopoietic Stem Cells through Flk2/Flt3-Positive Progenitor Cells[END_REF] and are likely to reside in a close but potentially distinct niche than HSCs. Both are in the Lin -CD34 + CD38 - compartment, whereas more differentiated progenitors in the Lin -CD34 + CD38 + compartment [START_REF] Doulatov | Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development[END_REF][START_REF] Notta | Distinct routes of lineage development reshape the human blood hierarchy across ontogeny[END_REF]. However, the MPPs in humans have not been fully defined [START_REF] Majeti | Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood[END_REF][START_REF] Notta | Distinct routes of lineage development reshape the human blood hierarchy across ontogeny[END_REF]. In terms of lineage-potential restriction, the erythroid and megakaryocyte fates probably diverge early from other myeloid and lymphoid potentials in mice [START_REF] Månsson | Molecular Evidence for Hierarchical Transcriptional Lineage Priming in Fetal and Adult Stem Cells and Multipotent Progenitors[END_REF][START_REF] Pietras | Functionally Distinct Subsets of Lineage-Biased Multipotent Progenitors Control Blood Production in Normal and Regenerative Conditions[END_REF] and humans [START_REF] Haghverdi | Diffusion pseudotime robustly reconstructs lineage branching[END_REF][START_REF] Hoppe | Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios[END_REF] and might arise directly from either HSCs [START_REF] Notta | Isolation of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment[END_REF] or downstream MPPs [START_REF] Paul | Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors[END_REF]. Focusing on the first human lymphomyeloid progenitors downstream of HSCs and MPPs, two progenitor populations have been identified in the immature Lin -CD34 + CD38 -CD90 neg-low compartment. These include Lin - CD34 + CD38 -CD90 neg-low CD45RA + CD10 -LMPPs with granulocytic, monocytic, B cell and T cell potential but that are unable to generate erythrocytes or megakaryocytes [START_REF] Borregaard | Neutrophils, from Marrow to Microbes[END_REF]. [START_REF] Velten | Human haematopoietic stem cell lineage commitment is a continuous process[END_REF] showed that human CD34 + CD10 -cells retain lympho-myeloid potential and progressively lose myeloid potential with expression of the cell-surface marker CD10. However, single cell gene expression analysis of LMPPs revealed that these are the most primitive cells to express common lymphoid genes, and this is accompanied by loss of Megakaryocyte/ Erythroid (MegE) priming but maintenance of myeloid priming [START_REF] Månsson | Molecular Evidence for Hierarchical Transcriptional Lineage Priming in Fetal and Adult Stem Cells and Multipotent Progenitors[END_REF]. Unlike Flt3 -MPPs, less than 1% of single Flt3 + LMPPs express MegE lineage genes. They have functionally little or no capacity to produce MegE lineages [START_REF] Adolfsson | Identification of Flt3+ Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential: A Revised Road Map for Adult Blood Lineage Commitment[END_REF]. By contrast, 70% of single clonogenic LMPPs have combined lymphomyeloid differentiation capacity when grown in multilineage supportive conditions [START_REF] Adolfsson | Identification of Flt3+ Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential: A Revised Road Map for Adult Blood Lineage Commitment[END_REF]. At the molecular level, 30% of single LMPPs expressed one or more of the early lymphoid genes testedinterleukin 7 receptor (IL7R), sterile immunoglobulin heavy chain complex (IgH), or recombination activating gene 1 (RAG1) -and all of these coexpressed GM lineage-affiliated genes, suggesting that lymphomyeloid priming is functionally related to the competency of these progenitors [START_REF] Månsson | Molecular Evidence for Hierarchical Transcriptional Lineage Priming in Fetal and Adult Stem Cells and Multipotent Progenitors[END_REF]. The MLP, initially reported as Lin -CD34 + CD38 -CD90 neg-low CD45RA + CD10 + cells, have lymphoid -B/ T cells and natural killer (NK) cells, monocytic and dendritic cells (DCs) potential but cannot make granulocytes [START_REF] Doulatov | Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development[END_REF]. However, CD10 -MLP populations [START_REF] Farlik | DNA Methylation Dynamics of Human Hematopoietic Stem Cell Differentiation[END_REF] have been reported to overlap LMPPs. Finally, the human Lin -CD34 + CD38 + CD45RA + compartment also includes a CD10 + subpopulation with T cell, B cell, NK cell and DC potential but lacking myeloid potential [START_REF] Galy | Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset[END_REF] . . In postnatal mammals, using mice in combination with multicongenic fate mapping of BM-derived progenitors revealed that the adult thymus contains approximately 160 niches for seeding by thymic seeding progenitors (TSPs), 6% of which are accessible to recolonization at steady state [START_REF] Ziętara | Multicongenic fate mapping quantification of dynamics of thymus colonization[END_REF][START_REF] Krueger | T Cell Development by the Numbers[END_REF]. In humans, TSPs (Lin -CD34 + CD10 + ) candidates comprise a multiple populations of immigrant precursors broadly distributed across various hematopoietic cell subsets likely include HSCs, MPPs, LMPPs and the common lymphoid progenitors (CLPs) [START_REF] Bhandoola | Commitment and Developmental Potential of Extrathymic and Intrathymic T Cell Precursors: Plenty to Choose from[END_REF][START_REF] Schwarz | Selective thymus settling regulated by cytokine and chemokine receptors[END_REF][START_REF] Six | A human postnatal lymphoid progenitor capable of circulating and seeding the thymus[END_REF][START_REF] Engle | Signal integration and cross-talk during thymocyte migration and emigration[END_REF] M.; Mei, T-S.; Wasa, M.; [START_REF] Engle | Signal integration and cross-talk during thymocyte migration and emigration[END_REF][START_REF] Saran | Multiple extrathymic precursors contribute to T-cell development with different kinetics[END_REF][START_REF] Schlenner | Early T cell development and the pitfalls of potential[END_REF]. yellow arrow). In utero, human T cell progenitors are detected in fetal thymi as early as 9

weeks of gestation, and mature T cells appear in the thymus by 12-13 weeks and in the spleen and lymph nodes (LNs) by 24 weeks of gestation [START_REF] Haynes | Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment[END_REF][START_REF] Haddad | Dynamics of thymus-colonizing cells during human development[END_REF] followed by further phenotypic maturation of thymic epithelium mediated by Wntinduced expression of the Forkhead Box N1 (FOXN1) gene [START_REF] Balciunaite | Wnt glycoproteins regulate the expression of FoxN1, the gene defective in nude mice[END_REF]. Hence, T lymphocytes expressing either TCRαβ or TCRγδ are first detected in the thymus parenchyme. Mesodermal fibrous connective tissue surrounding vessels has begun to invaginate the thymic rudiment effecting thymic epithelial proliferation and to form discrete lobules [START_REF] Bockman | Dependence of thymus development on derivatives of the neural crest[END_REF]. The two major regions contain distinct types of stromal elements and there is a considerable degree of epithelial heterogeneity within the compartments, particularly within the medulla [START_REF] Farr | Organization of thymic medullary epithelial heterogeneity: implications for mechanisms of epithelial differentiation[END_REF]. Histologic distinction between thymus corticomedullary partitioning appears at 14 weeks. In the medulla region, keratinized epithelial cell swirls known as Hassall's bodies are derived from medullary epithelial cells at 15 and 16 weeks [START_REF] Weller | Development of the thyroid, parathyroid, and thymus glands in man[END_REF]. Finally, the definitive thymus architecture is established by gestation week 16 [START_REF] Lobach | Ontogeny of the human thymus during fetal development[END_REF]. The thymus and its specialized microenvironments play a key role for supporting the generation and maturation of thymus-derived T lymphocytes throughout life but contains no self-renewing potential.

Instead, replenishment depends on recruitment of bone marrow-derived progenitors that circulate in the blood [START_REF] Anderson | Thymic vasculature: organizer of the medullary epithelial compartment?[END_REF]. The microenvironment of the thymus at birth is composed of multiple cell types. In addition, to T lymphocytes at various stages of maturation, other cell types include thymic epithelial cells (TECs), fibroblasts, macrophages, DCs, and vascular endothelial cells as well as minor populations of myeloid cells, plasma cells basophils, and eosinophils [START_REF] Lobach | Ontogeny of the human thymus during fetal development[END_REF]. The first step in post-natal T cell production requires two distinct phases of cell migration -out of the bone marrow into the blood, and out of the blood into the thymus.

II.2. Thymopoïesis -functional trafficking during TCR acquisition

The thymic microenvironment supports the differentiation and proliferation of BM-derived progenitors as they enter and migrate through the thymus. Relatively small numbers of T cell precursors migrate into the thymus per day, but they respond to the new environment by undergoing multiple rounds of proliferation while initiating the T cell differentiation program [START_REF] Rothenberg | Stepwise specification of lymphocyte developmental lineages[END_REF][START_REF] Rothenberg | Transcriptional control of early T and B cell developmental choices[END_REF][START_REF] Petrie | Zoned out: functional mapping of stromal signaling microenvironments in the thymus[END_REF][START_REF] Rothenberg | Launching the T-cell-lineage developmental programme[END_REF][START_REF] Love | Signal integration and crosstalk during thymocyte migration and emigration[END_REF][START_REF] Naito | Transcriptional control of T-cell development[END_REF][START_REF] Thompson | On becoming a T cell, a convergence of factors kick it up a Notch along the way[END_REF][START_REF] Yui | Developmental gene networks: a triathlon on the course to T cell identity[END_REF][START_REF] Krueger | T Cell Development by the Numbers[END_REF]. From the colonization of the thymus by TSPs to the production of mature T cells, lymphoid progenitors migrate across specific regions on the thymus. Sequential interactions during the trafficking allow the immature thymocytes to receive differentiation signals transmitted by different sets of TECs.

TECs are the most abundant stromal cells in the thymus and define anatomically and functionally distinct regions involved in directing the step-wise differentiation of thymocytes.

Specialized epithelial cells of the thymic cortex (cTECs) provide positive selection signals,

while the medulla contains a unique epithelial cell population (mTECs) with promiscuous expression of tissue-restricted antigens [START_REF] Witt | Thymopoiesis in 4 dimensions[END_REF][START_REF] Petrie | Zoned out: functional mapping of stromal signaling microenvironments in the thymus[END_REF][START_REF] Love | Signal integration and crosstalk during thymocyte migration and emigration[END_REF][START_REF] Halkias | Tracking migration during human T cell development[END_REF]. Indeed, this traffic produced by interactions and signals are important in the generation and maintenance of intact thymic environments, establishing a feedback mechanism called thymic cross-talk [START_REF] Anderson | Lymphostromal interactions in thymic development and function[END_REF][START_REF] Misslitz | Thymic T cell development and progenitor localization depend on CCR7[END_REF][START_REF] Irla | Autoantigen-Specific Interactions with CD4+Thymocytes Control Mature Medullary Thymic Epithelial Cell Cellularity[END_REF][START_REF] Meireles | Thymic crosstalk restrains the pool of cortical thymic epithelial cells with progenitor properties[END_REF]. The extracellular matrix elements (ECM) is another important part of this journey and is composed by combinations of multiple collagens, and several isoforms of both laminin and fibronectin [START_REF] Gameiro | The thymus microenvironment in regulating thymocyte differentiation[END_REF]. The ECM network is well preserved between humans and mice [START_REF] Savino | Molecular mechanisms governing thymocyte migration: combined role of chemokines and extracellular matrix[END_REF], their components are important for the integrity of the tissue, the division of the thymus into lobules, and additionally provide developing T cells cues to proliferate, differentiate, and migrate. Accordingly, thymocytes demonstrate developmentally regulated expression of ECM specific receptors such as integrins, trans membrane receptors that mediate cell: cell and cell:ECM adhesion (Fig. 4).

Thymocyte motility depends on the regulation of repeated cycles of integrin-mediated adhesion and de-adhesion, along with cytoskeletal rearrangements and the generation of cell polarity. Essential to the high degree of flexibility required for cell adhesion during motility is the ability to alter the avidity of integrins for their ligands through an intracellular signaling process known as inside out signaling (Kinashi, 2005a). The interactions of thymocytesmicroenvironment are also controlled by chemokines, a family of secreted proteins that induce chemotaxis. In the human thymus, chemokines are differentially expressed by epithelial cells of the cortex, medulla, and Cortico-Medullar junction (CMJ) in a pattern of expression similar to that in the mouse [START_REF] Chantry | Macrophage-derived chemokine is localized to thymic medullary epithelial cells and is a chemoattractant for CD3(+), CD4(+), CD8(low) thymocytes[END_REF][START_REF] Griffith | Spatial Mapping of Thymic Stromal Microenvironments Reveals Unique Features Influencing T Lymphoid Differentiation[END_REF][START_REF] Farley | Dynamics of thymus organogenesis and colonization in early human development[END_REF]. Chemokines may be tethered to the cell surface or secreted into the environment and bound by the ECM to establish a gradient [START_REF] Olson | Chemokines and chemokine receptors in leukocyte trafficking[END_REF]. Additionally, chemokines are important regulators of integrin avidity through the activation of inside out signaling, which is implicated in the modulation of integrin adhesion (Kinashi, 2005a). The development of T cells from multipotent progenitors in the thymus occurs by cascades of interactions between signaling molecules and transcription factors, resulting in the loss of alternative lineage potential (plasticity) and the acquisition of the T cell functional identity. T cell specific regulatory factors are turned on in early stages of T cell development before lineage commitment requiring Notch signaling and the activity of GATA binding protein 3 (GATA3), transcriptin factor (TCF) 1, B cell lymphoma/leukemia 11b (BCL11B) and the Eprotein HEB [START_REF] Van De Walle | GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate[END_REF][START_REF] Hu | Transformation of Accessible Chromatin and 3D Nucleome Underlies Lineage Commitment of Early T Cells[END_REF][START_REF] Johnson | Lineage-Determining Transcription Factor TCF-1 Initiates the Epigenetic Identity of T Cells[END_REF]. These factors are induced in the context of a set of broadly used transcription factors: myeloblastosis (Myb),

Ikaros family (IKZF1), E-protein E2A and Run-related transcription factor (RUNX), which play numerous roles in pioneering and direct transcriptional activation in several blood lineages. These stem and progenitor specific genes become silenced in the course of T cell lineage commitment and provide a valuable window on the dynamics and mechanisms of the commitment related silencing process [START_REF] Ikawa | An essential developmental checkpoint for production of the T cell lineage[END_REF][START_REF] Yui | Fine-scale staging of T cell lineage commitment in adult mouse thymus[END_REF][START_REF] Zhang | Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity[END_REF][START_REF] Mingueneau | The transcriptional landscape of αβ T cell differentiation[END_REF][START_REF] Rothenberg | Epigenetic mechanisms and developmental choice hierarchies in Tlymphocyte development[END_REF][START_REF] Rothenberg | Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control[END_REF][START_REF] Hu | Transformation of Accessible Chromatin and 3D Nucleome Underlies Lineage Commitment of Early T Cells[END_REF]. The process involves a continuing dialogue between the differentiating cell and the signals coming from its environment. Human and mouse thymocytes are divided into multiple phenotypically distinct stages that are defined by the expression or not of CD4, CD8, and other surface markers. These stages comprise the multiple phases double negative (DN -CD4 -CD8 -) thymocytes; and late DN4 CD8 + in mice (or imature single positive thymocytes -ISP -CD4 + in humans); double positive (DP -CD4 + CD8 + ) thymocytes; and single positive (SP -CD4 -CD8 + or CD4 + CD8 -) T cells [START_REF] Hayday | Key factors in the organized chaos of early T cell development[END_REF][START_REF] Rothenberg | Launching the T-cell-lineage developmental programme[END_REF][START_REF] Naito | Transcriptional control of T-cell development[END_REF][START_REF] Yui | Developmental gene networks: a triathlon on the course to T cell identity[END_REF]) (Fig. 4). Throughout these multiple phases, thymocytes randomly generate and express a wide array of TCR. The gene complexes encoding the T cell receptor components do not contain a functional first exon. Instead, they contain multiple variable (V), diversity (D), and joining (J) genes. TCR gene assembly is an inherently dangerous process, because it involves the introduction of double-strand breaks into chromosomes directed by the lymphoid-specific recombinase RAG, composed by RAG1 and RAG2. Because of this, V(D)J gene rearrangement is tightly controlled to ensure that these lesions, once generated, are appropriately repaired during T cell development [START_REF] Krangel | Mechanics of T cell receptor gene rearrangement[END_REF]. Finally, ubiquitously expressed DNA repair proteins are responsible to couple one of each segment together to form a functional first exon and generate a gene configuration capable of expressing the functional receptors TCRγδ or TCRαβ [START_REF] Taghon | Developmental and molecular characterization of emerging beta-and gammadelta-selected pre-T cells in the adult mouse thymus[END_REF][START_REF] Van Zelm | PID comes full circle: applications of V(D)J recombination excision circles in research, diagnostics and newborn screening of primary immunodeficiency disorders[END_REF][START_REF] Del Blanco | Tcra Enhancer Activation by Inducible Transcription Factors Downstream of Pre-TCR Signaling[END_REF]. The generation of αβ T cells requires the construction of a TCR complex through a highly ordered series of somatic gene rearrangement events at the TCRB, rearranged in DN stages and TCRA loci, rearranged in DP stages during intrathymic development [START_REF] Love | Signal integration and crosstalk during thymocyte migration and emigration[END_REF][START_REF] Halkias | Tracking migration during human T cell development[END_REF][START_REF] Zhang | Trafficking to the thymus[END_REF]. Moreover, T cells with productive rearrangement of TCRD and TCRG genes concomitant with inhibition DNA binding (ID3) transcription factor functions separate pathway leads to development of several functionally distinct subsets of γδ T cells [START_REF] Lauritsen | Marked induction of the helix-loop-helix protein Id3 promotes the gammadelta T cell fate and renders their functional maturation Notch independent[END_REF][START_REF] Prinz | Functional development of γδ T cells[END_REF][START_REF] Vantourout | Six-of-the-best: unique contributions of γδ T cells to immunology[END_REF]. Once V(D)J recombination is completed the recognition of self peptides that are embedded in MHC molecules on thymic antigenpresenting cells (APCs) is crucial for determining the fate of developing αβ T cells.

Somewhat paradoxically, recognition of self can elicit diametrically opposed outcomes. On one hand, it is essential for thymocyte survival and commitment to either the SP CD4 + or SP CD8 + thymocytes (that is, for the positive selection of thymocytes). On the other hand, recognition of self can be a death verdict for thymocytes, mediating the negative selection (clonal deletion) of these cells, or it can skew cells to alternative fates, such as regulatory T (Treg) or natural killer T (NKT) cells differentiation. Its followed by export of functional, but not self-reactive, naïve T cells, regulated by the sphingosine-1-phosphate receptor 1 (S1PR1) expression [START_REF] Starr | Positive and negative selection of T cells[END_REF][START_REF] Mathis | Aire[END_REF][START_REF] Klein | Positive and negative selection of the T cell repertoire: What thymocytes see (and don't see)[END_REF].

II.2.1. Entry into the thymus: heterogeneous mixture of candidates for T cells

The BM-derived thymic progenitors enter the thymus continuing expression of a set of transcription factors used in stem and progenitor cells. Some of these factors persist and even increase in expression during T cell development: these include Myb, Gfil, RUNX1, Ikaros family (IKZF1) and the ubiquitous E-protein E2A (transcription factor 3 or TCF3). Others play a role only in the earliest stages and are downregulated, and these can be termed stemprogenitor specific factors and their patterns of expression are summarized earliest during the development [START_REF] Mingueneau | The transcriptional landscape of αβ T cell differentiation[END_REF][START_REF] Rothenberg | Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control[END_REF]) (Fig. 5). Thymus colonization by TSPs is tightly controlled by the thymic microenvironment.

Expression of essential homing receptors and the coordinated sensitivity of TSPs to cytokines, cell adhesion molecules, and chemoattractant direct TSPs to settle in the thymus after leaving the BM through the bloodstream [START_REF] Anderson | Generating intrathymic microenvironments to establish T-cell tolerance[END_REF][START_REF] Bhandoola | Commitment and Developmental Potential of Extrathymic and Intrathymic T Cell Precursors: Plenty to Choose from[END_REF][START_REF] Rothenberg | Negotiation of the T lineage fate decision by transcription-factor interplay and microenvironmental signals[END_REF][START_REF] Gonzalez-Garcia | Notch 1 signalling in human T-cell development and leukemia[END_REF][START_REF] Schlenner | Early T cell development and the pitfalls of potential[END_REF][START_REF] Koch | Mechanisms of T Cell Development and Transformation[END_REF]. Thus, it likely necessitates initiation of a rolling adhesion onto thymic microvasculature, followed by firm adhesion and cell arrest on vascular endothelium leading to extravasation of progenitors into the thymic parenchyma (Kinashi, 2005b;[START_REF] Scimone | A multistep adhesion cascade for lymphoid progenitor cell homing to the thymus[END_REF]. In mice, it is well established that TSPs enter the adult thymic stroma in a regulated signaling cascade to arrest at the large venules near the well-perfused CMJ, located at the border of the cortex and the medulla, and that progenitors entry is initiated on the dependent-interaction between P-selectin on thymic endothelium and PSGL1 on hematopoietic cells [START_REF] Rossi | Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1[END_REF]. This interaction slows down the progenitors, which allows them to respond to local chemokine gradients and to firmly adhere to the thymic endothelium by engaging intercellular adhesion molecule 1 (ICAM1) and vascular-cell adhesion molecule 1 (VCAM1) expressed by stromal cells via integrins, α4 and β2 for example, expressed by DN thymocytes [START_REF] Lepique | Characterization of vascular adhesion molecules that may facilitate progenitor homing in the post-natal mouse thymus[END_REF][START_REF] Rossi | Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1[END_REF][START_REF] Scimone | A multistep adhesion cascade for lymphoid progenitor cell homing to the thymus[END_REF]. In the mouse thymus, mesenchymal stromal cells (MSC) and TECs produce chemokines involved in T cell precursor homing such as Cc-chemokine ligand (CCL) 19, CCL21 and CCL25 (Tani-ichi et al., 2013). Chemokine receptors, particularly CCR7 and CCR9, are required for recruitment of BM-derived progenitors to the thymus [START_REF] Krueger | CC chemokine receptor 7 and 9 double-deficient hematopoietic progenitors are severely impaired in seeding the adult thymus[END_REF][START_REF] Zlotoff | CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus[END_REF][START_REF] Ziętara | Multicongenic fate mapping quantification of dynamics of thymus colonization[END_REF]. CCR9 is expressed functionally by subsets of LMPPs and CLPs; whereas HSCs and MPPs do not; and the CCR7, which is expressed by some LMPPs, is highly expressed on CLPs [START_REF] Liu | Coordination between CCR7-and CCR9-mediated chemokine signals in prevascular fetal thymus colonization[END_REF][START_REF] Schwarz | Selective thymus settling regulated by cytokine and chemokine receptors[END_REF][START_REF] Krueger | CC chemokine receptor 7 and 9 double-deficient hematopoietic progenitors are severely impaired in seeding the adult thymus[END_REF][START_REF] Zlotoff | CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus[END_REF][START_REF] Takahama | Generation of diversity in thymic epithelial cells[END_REF]. T cell precursors also express the CXCR4, which guide their entry into the thymus. This fits well with thymic stroma expression of corresponding chemokine ligands CXCL12 [START_REF] Robertson | CXCR4 and CCR5 mediate homing of primitive bone marrow-derived hematopoietic cells to the postnatal thymus[END_REF][START_REF] Zhang | Trafficking to the thymus[END_REF]. As cTECs express at least some of these factors (i.e., CXCL12 and CCL25) along the CMJ, this suggest a role in thymus colonization [START_REF] Annunziato | Chemokines and lymphopoiesis in human thymus[END_REF] (Fig. 6). Once inside the thymus, newly arrived TSPs are induced to commit to the T lineage. HSCs, MPPs, LMPPs and CLPs will each produce T lineage progeny if directly placed in the thymic environment, but only LMPPs and CLPs rapidly generate thymocytes, whereas HSCs and most MPPs are not capable of colonizing the thymus from blood [START_REF] Allman | Thymopoiesis independent of common lymphoid progenitors[END_REF][START_REF] Schwarz | Selective thymus settling regulated by cytokine and chemokine receptors[END_REF]. Earliest regulators have distinct patterns of usage in stem/progenitor cells and in other classes of lymphocytes. Patterns of expression are shown for two cytokine receptor genes -KIT and FMS-like tyrosine kinase 3 (FLT3 or CD135) (Rothenberg, Ungerbäck and Champhekar, 2016) (Fig. 5). TSPs eventually give rise to the earliest T cell precursors in the thymus, called early thymic progenitor (ETP) or Kit-high double negative 1 (Kit hi DN1, and CD44 + ). They still harbor the potential plasticity to gain access to non-T alternative fates, such as DCs, involved in negative selection events [START_REF] Naeher | A constant affinity threshold for T cell tolerance[END_REF][START_REF] Melichar | Distinct temporal patterns of T cell receptor signaling during positive versus negative selection in situ[END_REF], NK and macrophages, implicated in the clearance of dying thymocytes) [START_REF] Surh | T-cell apoptosis detected in situ during positive and negative selection in the thymus[END_REF][START_REF] Dzhagalov | Elimination of self-reactive T cells in the thymus: a timeline for negative selection[END_REF][START_REF] Krueger | T Cell Development by the Numbers[END_REF]. Interactions with surface molecules expressed by TECs and response to soluble factors produced by the same cells are mandatory events, which enable the delivery of signals, such as Notch1 that are essential for directing the progenitors along the T cell lineage commitment [START_REF] Radtke | Deficient T cell fate specification in mice with an induced inactivation of Notch1[END_REF][START_REF] Gonzalez-Garcia | Notch 1 signalling in human T-cell development and leukemia[END_REF]. ETP precursors move into the cortex whereas T cell development is initiated from the subpopulation DN [START_REF] Taghon | Notch signaling is required for proliferation but not for differentiation at a well-defined beta-selection checkpoint during human T-cell development[END_REF]. In mice, these stages are subdivided according to their expression of other surface molecules, the growth factor receptors FLT3 and KIT, the adhesion molecule CD44, and CD25 (Fig. 5). In humans, the subdivisions are made based on expression of different molecules: CD34, CD38, CD7, and CD1a [START_REF] Rothenberg | Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control[END_REF]; whereas CD34 + CD1a -CD7 -cells comprise the most immature postnatal thymocyte subset [START_REF] Six | A human postnatal lymphoid progenitor capable of circulating and seeding the thymus[END_REF][START_REF] Hao | Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7-lympho-myeloid thymic progenitors[END_REF], and CD7 upregulation specifies NK/ T cell fate [START_REF] Plum | In vitro intrathymic differentiation kinetics of human fetal liver CD34+CD38progenitors reveals a phenotypically defined dendritic/T-NK precursor split[END_REF].

II.2.2. Cortical migration of DN T cells: commitment and β-selection checkpoint

Many signals are required for migration of DN thymocytes outwards across the cortex. In mice, the majority of chemokines expressed in the thymus are more abundant in the medulla relative to the cortex. The exceptions are CCL25 and CXCL12, and the outward migration of mice DN thymocytes from the point of entry at the CMJ toward the sub-capsular zone is mediated by cortical gradients to these chemokines [START_REF] Misslitz | Thymic T cell development and progenitor localization depend on CCR7[END_REF][START_REF] Griffith | Spatial Mapping of Thymic Stromal Microenvironments Reveals Unique Features Influencing T Lymphoid Differentiation[END_REF]. Conserved cortical biased expression of CCL25 and CXCL12 point to a prominent role for CCR9 and CXCR4 signaling in guiding the migration of immature human cortical thymocytes (Fig. 6) [START_REF] Gotter | Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters[END_REF][START_REF] Farley | Dynamics of thymus organogenesis and colonization in early human development[END_REF]. Additionally, progressive differentiation of human DN thymocytes is accompanied by upregulation of CCR7, CCR9 and CXCR4 surface expression [START_REF] Zabel | Human G protein-coupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis[END_REF][START_REF] Haddad | Dynamics of thymus-colonizing cells during human development[END_REF][START_REF] Zlotoff | CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus[END_REF].

Further, it has been shown that human DN cells migrate in vitro in response to CXCL12, CCL25, and modestly to CCL21, the same three chemokines that have been suggested to play a role in the cortical migration of murine DN thymocytes [START_REF] Halkias | Opposing chemokine gradients control human thymocyte migration in situ[END_REF][START_REF] Halkias | Tracking migration during human T cell development[END_REF][START_REF] Halkias | Conserved and divergent aspects of human T-cell development and migration in humanized mice[END_REF].

In addition, the movement-migration of DN thymocytes is drived mainly by integrinmediated cross-talk that are perhaps facilitated by chemokine signaling [START_REF] Petrie | Cell migration and the control of post-natal T-cell lymphopoiesis in the thymus[END_REF]. The chemokine CXCL12 activate the integrins VLA-4/α4β1 and VLA-5/α5β1 in immature human CD34 + hematopoietic cells. Interestingly, expression of CXCL12 is highest in the human cortex, and expression of its receptors, CXCR4, is highest among human DN thymocytes which also express high levels of both α4β1 and α5β1 and adhere in fibronectin in vitro [START_REF] Mojcik | Differential expression of integrins on human thymocyte subpopulations[END_REF][START_REF] Gotter | Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters[END_REF][START_REF] Halkias | Opposing chemokine gradients control human thymocyte migration in situ[END_REF]. Thus, it is likely that chemokines not only provide directional cues to developing thymocytes but also regulate thymocyte adhesion to the ECM, contributing to the distinct migration patterns of the different thymocyte populations [START_REF] Savino | Molecular mechanisms governing thymocyte migration: combined role of chemokines and extracellular matrix[END_REF]. This interaction enforces sustained signals for lymphoid-lineage commitment including the crucial Notch signaling. The early T cell precursor development can be divided usefully into three phases (corresponding at 5% of the pool) in which the first two depend on Notch signaling and the third depends on signals from the pre-TCR complex [START_REF] Naito | Transcriptional control of T-cell development[END_REF][START_REF] Kueh | Regulatory gene network circuits underlying T cell development from multipotent progenitors[END_REF][START_REF] Van De Walle | GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate[END_REF][START_REF] Hosokawa | Cytokines, Transcription Factors, and the Initiation of T-Cell Development[END_REF]) (Fig. 5).

proliferative expansion of pre-commitment precursors [START_REF] Love | Signal integration and crosstalk during thymocyte migration and emigration[END_REF][START_REF] Yui | Developmental gene networks: a triathlon on the course to T cell identity[END_REF]; thus allowing the transition of approximately 160 TSPs to a readily detectable population of 20000 to 30000 ETP cells in ~2 weeks during the earliest phase of DN trafficking [START_REF] Krueger | T Cell Development by the Numbers[END_REF]. ETP cells compose only approximately 1% of the total thymic T cell pool and they are the most efficient progenitors at generating T cells [START_REF] Allman | Thymopoiesis independent of common lymphoid progenitors[END_REF][START_REF] Krueger | T Cell Development by the Numbers[END_REF]. The IL-7

signaling pathway may be inactive in the ETPs [START_REF] Schlenner | Early T cell development and the pitfalls of potential[END_REF], but is strongly upregulated in uncommitted DN2a progenitors [START_REF] Von Freeden-Jeffry | The earliest T lineage-committed cells depend on IL-7 for Bcl-2 expression and normal cell cycle progression[END_REF] being responsible to activate signal transducer phosphatidylinositol 3-kinase (PI3K) pathway. The PI3K pathway plays a crucial role in proliferation and survival of early T cells [START_REF] Pallard | Distinct roles of the phosphatidylinositol 3-kinase and STAT5 pathways in IL-7-mediated development of human thymocyte precursors[END_REF].

II.2.2.1.1. Notch-Dll4 checkpoint

Later T cell development also continues to make extensive use of developmental plasticity to diversify the functional assignments T cells can serve in the adaptive immune system [START_REF] Rothenberg | Lineage plasticity and commitment in T-cell development[END_REF][START_REF] Rothenberg | T cell lineage commitment: identity and renunciation[END_REF]. Notch signaling is an evolutionary conserved pathway that controls multiple cell fate decisions throughout ontogeny (Artavanis-Tsakonas, Rand and Lake, 1999; Gonzalez-Garcia and Toribio, 2009). Notch1 is necessary but not sufficient for T cell commitment under physiological circumstances [START_REF] De Smedt | Active form of Notch imposes T cell fate in human progenitor cells[END_REF][START_REF] Gonzalez-Garcia | Notch 1 signalling in human T-cell development and leukemia[END_REF]. Notch ligand expression, particularly the deltalike ligand 4 (Dll4), is tightly regulated in cTECs during human thymus ontogeny and involution. Notch:Dll4 interaction together with signals delivered by IL-7, E-protein E2A, FLT3 and SCF is essential for T cell lineage specification and for proliferation and further progression of early thymocytes along the T cell lineage (Fig. 6) [START_REF] Sambandam | Notch signaling controls the generation and differentiation of early T lineage progenitors[END_REF][START_REF] Hozumi | Delta-like 4 is indispensable in thymic environment specific for T cell development[END_REF][START_REF] Koch | Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment[END_REF][START_REF] Rothenberg | Launching the T-cell-lineage developmental programme[END_REF][START_REF] García-Ojeda | GATA-3 promotes T-cell specification by repressing B-cell potential in pro-T cells in mice[END_REF][START_REF]Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations[END_REF]. Notch signaling also initiates the expression of the crucial T cell specification factor coding genes GATA3 and TCF1 encoded by TCF7. GATA3, in contrast to TCF1, controls human T cell lineage commitment through direct regulation of three distinct processes: repression of NK cell fate, upregulation of T cell lineage genes to promote further differentiation and restraint of Notch activity. Another important element of these transcriptional changes is the activation of the six zinc-finger transcriptional repressor BCL11B in the late DN2a stage by Notch1 signaling, TCF1, RUNX1, and GATA3 [START_REF] Ikawa | An essential developmental checkpoint for production of the T cell lineage[END_REF][START_REF] Li | An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b[END_REF][START_REF] Li | An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b[END_REF][START_REF] Kueh | Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment[END_REF]. BCL11B helps to restrict the potential plasticity by downregulation of KIT expression [START_REF] Massa | Critical role for c-kit (CD117) in T cell lineage commitment and early thymocyte development in vitro[END_REF][START_REF] Ikawa | An essential developmental checkpoint for production of the T cell lineage[END_REF][START_REF] Li | An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b[END_REF] (Fig. 5). Specifically, Notch signaling promotes TCRαβ T cell commitment at the expense of TCRγδ T cell development at the DN2b-DN3a stage (Fig. 5). Furthermore, conditional inactivation of Notch1 at the DN2 stage of T cell development, in mice, results in a partial block at the subsequent stage (DN3) resulting an inefficient V to DJ recombination at the TCRB locus. In contrast, rearrangements at the TCRG and TCRD locus were not affected [START_REF] Kueh | Regulatory gene network circuits underlying T cell development from multipotent progenitors[END_REF][START_REF] Plumb | Interleukin-7 in the transition of bone marrow progenitors to the thymus[END_REF]. [START_REF] Abramson | Thymic Epithelial Cells[END_REF].

II.2.2.2. Phase 2 of DN T cellsαβ versus γδ T cell fate

The second phase is also Notch-dependent and establishes the competence of the thymocytes to express TCR complexes. DN2b cells have a slower proliferation rate and begin to be desensitized to IL-7R signaling by a mechanism mediated by E-proteins, becoming the surviving DN2b cells strictly Notch-dependent [START_REF] Masuda | T cell lineage determination precedes the initiation of TCR beta gene rearrangement[END_REF][START_REF] Wojciechowski | E2A and HEB are required to block thymocyte proliferation prior to pre-TCR expression[END_REF][START_REF] Yui | Fine-scale staging of T cell lineage commitment in adult mouse thymus[END_REF]. The ultimate T cell lineage specification occurs during the transition from the DN2b to DN3a cells when αβ versus γδ T cell fate is specified (Fig. 5). Upregulation of CD1 in DN3a marks T lineage commitment in humans and subsequent ISP CD4 + cells that first express CD8α and then CD8β as they become DP thymocytes (García-Peydró, de [START_REF] García-Peydró | Sustained Notch1 signaling instructs the earliest human intrathymic precursors to adopt a gammadelta T-cell fate in fetal thymus organ culture[END_REF][START_REF] Taghon | Notch signaling is required for proliferation but not for differentiation at a well-defined beta-selection checkpoint during human T-cell development[END_REF]. Therefore, during the transition to the next stage of T cell maturation, DN3a cells shutdown the proliferation to T cell fate even before expressing TCR on their surface, but continue to extensively rearrange their DNA at the δ, γ and β loci to express functional TCR chains followed by β-selection in the SCZ of the cortex [START_REF] Livák | Characterization of TCR Gene Rearrangements During Adult Murine T Cell Development[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF].

II.2.2.2.1. V(D)J rearrangement of the δ, γ and β gene segments TCR genes recombinations are sequential between the different genes (TCRD > TCRG > TCRB > TCRA) as well as within a particular gene (e.g., TCRD: Dδ2-Dδ3, Dδ2-Jδ1, Vδ-Jδ1) [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF]. Rearrangement of the TCRD and TCRG genes occurs during the DN stages of human development, while TCRA rearrangement occurs in the DP stage [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF].

Between the rearrangement of TCRG and TCRA genomic loci, V(D)J recombination at the TCRB locus occurs and T cell receptor β chain (TCRβ) protein is expressed. Rearrangement of the TCRB locus has been reported to begin as early as the DN3a stage, but TCRβ protein has not been detected until the ISP developmental stage, the stage between the DN and DP populations [START_REF] Blom | TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF][START_REF] Joachims | Human alpha beta and gamma delta thymocyte development: TCR gene rearrangements, intracellular TCR beta expression, and gamma delta developmental potential-differences between men and mice[END_REF][START_REF] Taghon | Notch signaling is required for proliferation but not for differentiation at a well-defined beta-selection checkpoint during human T-cell development[END_REF].

Therefore, the timing and efficiency of rearrangement of various TCR genes must be determined and is tightly regulated by modulating accessibility of V, D, and J recombination signal sequences (RSSs) to RAG enzymes [START_REF] Zhao | Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development[END_REF]. Evidence suggests that promoter and enhancer activity that is controlled by transcription factors regulate V(D)J recombination by modulating chromatin structures and rendering gene segments accessible to RAG cleavage [START_REF] Cieslak | RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement[END_REF]. RAG cleavage in vitro proceeds most efficiently with the binding of a RAG1/2 heterotetramer to one RSS, with subsequent capture of a second RSS [START_REF] Jones | Ordered assembly of the V(D)J synaptic complex ensures accurate recombination[END_REF]. RAG is recruited by epigenetic modifications and other factors to highly accessible D-J chromatin to form a recombination center (RC) and that RAG must then capture a second RSS (for example, a V segment RSS) to initiate V(D)J recombination [START_REF] Matthews | Regulation of RAG transposition[END_REF][START_REF] Ji | The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci[END_REF][START_REF] Schatz | Recombination centres and the orchestration of V(D)J recombination[END_REF]. TCRδ rearrangement occurs in developing DN thymocytes, initiating at DN2 stage and continuing into the DN3 stage during an initial period of RAG1/2 gene expression.

II.2.2.2.2. TCRγ rearrangement and γ T cell Receptor Excision Circle (γTREC)

The γδ T cells originate from DN cells that have not yet undergone β-selection [START_REF] Hayes | TCR signal strength influences alphabeta/gammadelta lineage fate[END_REF][START_REF] Von Boehmer | Unique features of the pre-T-cell receptor alpha-chain: not just a surrogate[END_REF]. In DN thymocytes both TCRG and TCRD gene segments rearrange (Vγ to Jγ and Vδ to Dδ to Jδ) without a strict order and without intermediate selection and expansion events. In this context, V segment promoters are assumed to be promoters by arrows. The region of the Jα array that depends on PDβ1 is indicated by the green bar. Adapted from [START_REF] Hodges | Diagnostic role of tests for T cell receptor (TCR) genes[END_REF][START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Krangel | Mechanics of T cell receptor gene rearrangement[END_REF].

II.2.2.2.4. Vβ segments rearrangement via TCRB locus contraction

Subsequently, one functional Vβ gene segment is joined to the previously rearranged DβJβ recombinant at the DN3 T cell stage, followed by VβDJβ-to-Cβ to give a TCRβ (Fig. 9).

Another distinctive feature is the size of the DJβ and Vβ regions. In mouse TCRB locus, the DJβ region is located within a relatively short region of 50 kb, which readily facilitates Dβ-Jβ recombination. In contrast, the Vβ region consists of a length of 670 kb contains 29 of its 31

Vβ genes in a central cluster, where almost all Vβ gene segments are regulated independently;

and are separated from the 3' proximal domain together with a potent enhancer by a 100-260 kb spacer sequence, indicating that the Vβ-DJβ recombination step must be spatially restricted [START_REF] Krangel | Mechanics of T cell receptor gene rearrangement[END_REF]) (Fig. 9). These two regions are subject to distinct control mechanisms, as chromatin changes, germline transcription, and Dβ-Jβ recombination in the 3' proximal domain strictly depend on the local enhancer, whereas chromatin accessibility and germline transcription at the Vβ genes are regulated independently of this enhancer [START_REF] Mathieu | Assessing the role of the T cell receptor β gene enhancer in regulating coding joint formation during V(D)J recombination[END_REF][START_REF] Krangel | Mechanics of T cell receptor gene rearrangement[END_REF]. These include the quality of the downstream RAG recombinase binding sites, sense, and antisense non-coding transcription, modified histones, locus compaction, and chromatin looping [START_REF] Sayegh | Visualization of looping involving the immunoglobulin heavy-chain locus in developing B cells[END_REF][START_REF] Jhunjhunwala | The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions[END_REF][START_REF] Stubbington | Non-coding transcription and large-scale nuclear organisation of immunoglobulin recombination[END_REF][START_REF] Bolland | Two Mutually Exclusive Local Chromatin States Drive Efficient V(D)J Recombination[END_REF]. CTCF promotes local looping in the distal Vβ region, while Paired box 5 (PAX5) promotes longer-range movement of local distal

Vβ domains toward the DJβ domain [START_REF] Fuxa | Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene[END_REF][START_REF] Degner | CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells[END_REF][START_REF] Le Lay | The tumor suppressor Ikaros shapes the repertoire of notch target genes in T cells[END_REF][START_REF] Ruffo | Extremely Long-Range Chromatin Loops Link Topological Domains to Facilitate a Diverse Antibody Repertoire[END_REF]. Contraction of the TCRB locus takes place only in DN cells in the thymus by looping out which likely facilitates Vβ-DJβ recombination [START_REF] Skok | Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes[END_REF].

The combined action of these transcription factors brings all Vβ genes into close proximity with the DJβ segment, providing equal spatial opportunity for all to participate in V(D)J recombination [START_REF] Majumder | Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element[END_REF][START_REF] Bolland | Two Mutually Exclusive Local Chromatin States Drive Efficient V(D)J Recombination[END_REF]. Indeed, following the transcription of the VβDJβ rearranged gene and the translation of the RNA products, the TCRβ in association with the pre-TCR proteins forms, in the absence of rearranged TCRα, and also the CD3 components, forms the pre-TCR complex [START_REF] Allman | Thymopoiesis independent of common lymphoid progenitors[END_REF][START_REF] Hayes | TCR signal strength influences alphabeta/gammadelta lineage fate[END_REF][START_REF] García-León | Dynamic regulation of NOTCH1 activation and Notch ligand expression in human thymus development[END_REF]. Upon assembly, the pre-TCR signals downregulate RAG activity, mediate TCRβ allelic exclusion, activate cell-survivalproliferation and thymocyte develop toward the DN4/ISP cell stage and subsequently, to DP cell stage [START_REF] Dion | HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation[END_REF]) (Fig. 9). βTREC quantification will be discussed in details in the following chapter.

II.2.2.2.6. β-selection checkpoint -allelic exclusion and highway to αβ T cell fate

Developing T cells that enter the β-selection checkpoint orchestrate seemingly conflicting processes: genomic recombination for TCR rearrangement, cell proliferation with and without (self-renewal) differentiation. The developmental stages before and after the β-selection checkpoint are termed DN3a and DN3b stages, respectively, and the transition between these stages involves dramatic changes in transcriptional profile, with abrupt shutdown or initiation of key fate regulators (Fig. 5) [START_REF] Maillard | The requirement for Notch signaling at the beta-selection checkpoint in vivo is absolute and independent of the pre-T cell receptor[END_REF][START_REF] Taghon | Developmental and molecular characterization of emerging beta-and gammadelta-selected pre-T cells in the adult mouse thymus[END_REF][START_REF] Carpenter | Decision checkpoints in the thymus[END_REF][START_REF] Yui | Developmental gene networks: a triathlon on the course to T cell identity[END_REF]. The β-selection and the initiation of TCRα rearrangement occurs at the similar timing in both mice (CD27 lo to CD27 hi ) and humans (CD28 neg to CD28 pos ) [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF][START_REF] Taghon | Notch signaling is required for proliferation but not for differentiation at a well-defined beta-selection checkpoint during human T-cell development[END_REF]. Because of the deletion and untemplated addition of nucleotides during RAG mediated recombination, most rearrangements are out-of-frame and fail to give rise to genes encoding a functional protein [START_REF] Carpenter | Decision checkpoints in the thymus[END_REF]. The β-selection checkpoint precisely verifies proper TCR gene rearrangement. For αβ precursors, it requires thymocytes to signal through a pre-TCR complex.

II.2.2.2.7. Allelic exclusion at the TCRB locus

Allelic exclusion of antigen-receptor genes is ensured primarily by monoallelic locus activation upon rearrangement and subsequently by feedback inhibition of continued rearrangement. Allelic exclusion limits productive antigen-receptor rearrangement to a single chromosome at most loci [START_REF] Khor | Allelic exclusion at the TCRbeta locus[END_REF][START_REF] Krangel | Mechanics of T cell receptor gene rearrangement[END_REF]. The feedback inhibition provokes downregulation of RAG1/2 expression, thus DN3 thymocytes entry into the cell cycle and initiate the differentiation to DP stage [START_REF] Michie | Regulation of thymocyte differentiation: pre-TCR signals and beta-selection[END_REF].

Productive Vβ-DJβ rearrangement leads to the expression of the TCRβ chain in association with the pre-TCRα chain as a pre-TCR complex, transmitting the signals to initiate proliferation and developmental progression toward CD4 + CD8 + DP thymocytes, and to inhibit continued Vβ-DJβ rearrangement. In addition, this inhibition of rearrangement is associated with reduced germline transcription, nucleosome remodeling, DNA methylation and/or histone acetylation of the Vβ genes [START_REF] Tripathi | A change in the structure of Vbeta chromatin associated with TCR beta allelic exclusion[END_REF]. Thus, the feedback signal is enforced primarily at the level of Vβ chromatin accessibility. The molecular mechanisms underlying monoallelic locus activation and reduced accessibility mediated by pre-TCR signaling have remained largely unknown. To ensure allelic exclusion, there must then be feedback inhibition by a productively rearranged allele to avoid further rearrangement on the other allele that will only proceed if the first attempt of rearrangement was unsuccessful. Cells that succeed after this second attempt (i.e., one allele unproductively rearranged and the other productively rearranged [V(D)J -/V(D)J + ]) as well as V(D)J + /DJ cells that were productive on the first attempt can continue to differentiate and, depending on the affinity properties of these functional receptors, become mature T cells. Approximately 60% of mature T lymphocytes are of the V(D)J + /DJ genotype, while 40% are of the V(D)J -/V(D)J + genotype [START_REF] Aifantis | Essential role of the pre-T cell receptor in allelic exclusion of the T cell receptor beta locus[END_REF][START_REF] Gärtner | Immature thymocytes employ distinct signaling pathways for allelic exclusion versus differentiation and expansion[END_REF]. Thus, both asynchronous onset and feedback inhibition are essential elements for allelic exclusion of the TCRB locus [START_REF] Von Boehmer | Crucial function of the pre-T-cell receptor (TCR) in TCR beta selection, TCR beta allelic exclusion and alpha beta versus gamma delta lineage commitment[END_REF][START_REF] Von Boehmer | Unique features of the pre-T-cell receptor alpha-chain: not just a surrogate[END_REF]. Finally, cells that fail to produce a functional receptor after both rearrangements (V(D)J -/V(D)J -) are eliminated during the β-selection [START_REF] Malissen | Regulation of TCR alpha and beta gene allelic exclusion during T-cell development[END_REF]. This recombination-directed combinatorial process generates the remarkable receptor diversity observed in the αβ T cells that comprise ~ 95% of adult T lymphocytes [START_REF] Scott | Patterns of membrane TcR alpha beta and TcR gamma delta chain expression by normal blood CD4+CD8-, CD4-CD8+, CD4-CD8dim+ and CD4-CD8-lymphocytes[END_REF].

II.2.2.3. Phase 3 of DN thymocytes -cell proliferation, survival and differentiation

In both humans and mice, successful expression of TCRβ leads to proliferation, survival, and differentiation [START_REF] Michie | Regulation of thymocyte differentiation: pre-TCR signals and beta-selection[END_REF], processes that must be tightly regulated to balance the expansion of TCRβ thymocytes with the prevention of leukemia. A family of transcription factors that is likely to regulate β-selection is the Ikaros family [START_REF] Mitchell | Expression and splicing of Ikaros family members in murine and human thymocytes[END_REF]. DN3a cells signaling through the newly expressed pre-TCR triggers another shift in the gene regulatory network, transitioning into phase 3, strongly and rapidly turning off Notch target genes in an Ikaros-dependent manner becoming DN3b (Fig. 5) [START_REF] Chari | Ikaros regulates Notch target gene expression in developing thymocytes[END_REF][START_REF] Le Lay | The tumor suppressor Ikaros shapes the repertoire of notch target genes in T cells[END_REF]. Also, the pre-TCR signaling promotes cell survival metabolism through activation of the PI3K and serine/threonine kinase (Akt)

pathway. This induces a very rapid cell proliferation and contributes to overcome the differentiation block enforced in DN3b thymocytes to the DN4 stage by E2A proteins [START_REF] Garbe | Differential synergy of Notch and T cell receptor signaling determines alphabeta versus gammadelta lineage fate[END_REF][START_REF] Maillard | The requirement for Notch signaling at the beta-selection checkpoint in vivo is absolute and independent of the pre-T cell receptor[END_REF]. The strong proliferation allows the amplification of the clones with productive TCRβ rearrangements to maximize the chance for productive TCRαβ diversity [START_REF] Dose | c-Myc mediates pre-TCR-induced proliferation but not developmental progression[END_REF]. At this point, thymocytes not only begin to express the CD4 and CD8 co-receptors in response a combination of TCF1 and β-catenin [START_REF] Yu | TCF1 and beta-catenin regulate T cell development and function[END_REF], with a required input from Myb transcription factor, but the cells also distinctively activate expression of new transcription factors: RAR-related orphan receptor gamma (RORγt) and the Ikaros zinc-finger family member Helios and Aiolos (IKZF3). Ikaros, Helios, and Aiolos mRNA levels increase as human thymocytes progress from the DN to the DP developmental stage (Mitchell, Seng andYankee, 2016, 2017).

II.2.3. Cortical DP thymocytes -TCRA-TCRD locus and positive selection

After reaching the subcapsular zone during the DN stages, thymocytes turn around and begin to migrate once again back into the cortex (Fig. 4). While murine thymocytes mature via a CD8 DN4 cells between the DN and DP stages, human thymocytes traverse a CD4 ISP stage following the acquisition of CD1a [START_REF] Joachims | Human alpha beta and gamma delta thymocyte development: TCR gene rearrangements, intracellular TCR beta expression, and gamma delta developmental potential-differences between men and mice[END_REF]. In addition, upregulation of CD69 marks the early stages of positive selection in both human and mouse [START_REF] Halkias | Tracking migration during human T cell development[END_REF] (Fig. 10). En route within the outer cortex, the thymocytes cells with productive TCRβ chain upregulate CD4 and CD8 on their surface after pre-TCR signaling thereby achieving the DP (CD4 + CD8 + ) stage of maturation within 19 days of the arrival of thymocytes in the thymus [START_REF] Porritt | Kinetics of steady-state differentiation and mapping of intrathymic-signaling environments by stem cell transplantation in nonirradiated mice[END_REF]. Furthermore, two DP subpopulations can be distinguished based on the expression of CD71: early DP (eDP, CD71 + ) and late DP (lDP, CD71 -) thymocytes in mice [START_REF] Brekelmans | Transferrin receptor expression as a marker of immature cycling thymocytes in the mouse[END_REF][START_REF] Hernández-Munain | Recent insights into the transcriptional control of the Tcra/Tcrd locus by distant enhancers during the development of T-lymphocytes[END_REF] (Fig. 10). The eDP thymocytes cells comprise 5-10% of total thymic-resident cells [START_REF] Hernández-Munain | Recent insights into the transcriptional control of the Tcra/Tcrd locus by distant enhancers during the development of T-lymphocytes[END_REF] and express both CXCR4 and CCR9 and can migrate to CXCL12 and CCL25 gradient [START_REF] Norment | Murine CCR9, a chemokine receptor for thymus-expressed chemokine that is up-regulated following pre-TCR signaling[END_REF][START_REF] Plotkin | Critical role for CXCR4 signaling in progenitor localization and T cell differentiation in the postnatal thymus[END_REF][START_REF] Misslitz | Thymic T cell development and progenitor localization depend on CCR7[END_REF]. Indeed, most lDP cells move randomly at low motility rates and the inward movement of DP cells may be facilitated by interstitial fluid currents that flow directionally from the subcapsular zone to the medulla [START_REF] Witt | Thymopoiesis in 4 dimensions[END_REF][START_REF] Rothenberg | Launching the T-cell-lineage developmental programme[END_REF]. At this point, RAG genes are reexpressed to rearrange the TCRA locus and followed by additional selection steps [START_REF] Koch | Mechanisms of T Cell Development and Transformation[END_REF][START_REF] Abramson | Thymic Epithelial Cells[END_REF][START_REF] Krueger | T Cell Development by the Numbers[END_REF] (Fig. 10). After, rearrangement of the TCRA locus a robust proliferation generate a large pool of lDP cortex-resident cells that comprise 75-80% of total thymic-resident cells [START_REF] Hernández-Munain | Recent insights into the transcriptional control of the Tcra/Tcrd locus by distant enhancers during the development of T-lymphocytes[END_REF].

of which lie in a proximal unique region upstream of the Dδ, and one Vδ of which is in an inverted orientation downstream of Cδ (Vδ5) adjacent to the Jα array that is comprised of 60 gene segments. Other Vδ, also used by TCRα, lie at greater distances from Dδ [START_REF] Carico | Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires[END_REF]. In humans the TCRA-TCRD locus spans 1.1 Mb and is very similar in overall organization to that of the murine locus (Fig. 11B). The major structural difference between the murine and human loci is the absence of recent Vα/Vδ duplication events in humans, resulting in fewer Vα/Vδ gene segments (55) [START_REF] Glusman | Comparative genomics of the human and mouse T cell receptor loci[END_REF]. In addition, the human TCRA-TCRD locus carries three Dδ and four Jδ gene segments (Fig. 11). . The region of the Jα array that depends on TEA is indicated by the green bar. Cen., centromere; Tel., telomere. Adapted from [START_REF] Glusman | Comparative genomics of the human and mouse T cell receptor loci[END_REF][START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Zhao | Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development[END_REF].

II.2.3.2. TCRA-TCRD locus chromatin remodeling and accessibility to V(D)J segments

Gene expression programs operate within the confines of chromatin and hence chromatin organization influences key aspects of mammalian development. Major factors that affect chromatin organization are DNA methylation, covalent modification of histones and ATP-dependent alterations to chromatin. ATP-dependent chromatin alteration is accomplished by several multi-subunit chromatin-remodeling complexes that utilize energy derived from ATP hydrolysis to alter nucleosome structure or conformation and, thereby, regulate the access of transcription factors to their cognate DNA binding sites [START_REF] Ho | Chromatin remodelling during development[END_REF]. The subsequent higher order packing of nucleosomes within chromatin would limit the ability of RAG recombinase to bind and cleave at RSSs imposing an important barrier to V(D)J rearrangement [START_REF] Carico | Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires[END_REF]. The chromatin barrier to the RAG recombinase is relieved during lymphocyte development through the activities of transcriptional control elements in TCR loci. Chromatin remodeling is important because it increases the locus accessibility by nuclear enzymes to alter the state allowing V(D)J recombination [START_REF] Lorch | Mechanism of chromatin remodeling[END_REF][START_REF] Narlikar | Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes[END_REF]). The SWI/SNF complex moves nucleosomes around to expose or hide specific DNA sequences. A DNase I hypersensitive site (DHS) is a region that is characterized by elevated DNase I cleavage and is considered as an hallmark of regulatory DNA. DHSs presumably reflect acute alterations of localized chromatin structure by such enzymes [START_REF] Bossen | The chromatin remodeler Brg1 activates enhancer repertoires to establish B cell identity and modulate cell growth[END_REF].

Essentially, all transcriptional enhancers coincide with DHS in cell types where the enhancer is active. Generation of DNase I-sensitive chromatin domains as well as induction of DHS indicate that transcriptionally active genes undergo remodeling of their chromatin state. The unrepressed chromatin structure permits transcription factors and RNA polymerase II (RNAP II) to access genes to initiate transcription. Histone modification states modulate the efficiency of chromatin remodeling complexes to accessibility [START_REF] Lalonde | Histone target selection within chromatin: an exemplary case of teamwork[END_REF]. Recombination also depends directly on the histone modification H3 lysine 4 trimethylation (H3K4me3), since this modification is specifically recognized by the RAG2 domain [START_REF] Matthews | RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination[END_REF] and can stimulate RAG cleavage [START_REF] Shimazaki | H3K4me3 stimulates the V(D)J RAG complex for both nicking and hairpinning in trans in addition to tethering in cis: implications for translocations[END_REF]. The recruitment of chromatin remodelers and the deposition of histone modifications such as H3K4me3 are associated with promoter activation and the initiation of transcription. Transcriptional elongation by RNAP II deposits histone modifications such as H3ac and H3K4me3 across the 50 portions of transcription units (Abarrategui andKrangel, 2007, 2009), disrupts nucleosome organization, may provoke nucleosome removal [START_REF] Kondilis-Mangum | Transcription-dependent mobilization of nucleosomes at accessible TCR gene segments in vivo[END_REF] and causes transient eviction of one copy each of H2A and H2B from histone octamers, exposing 35-40 bp of nucleosomal DNA to RAG [START_REF] Bevington | Transcription-coupled eviction of histones H2A/H2B governs V(D)J recombination[END_REF]. Binding of RAG1 likely depends on the exposure of nucleosomal RSSs whereas binding of RAG2 depends critically on nucleosomal H3K4me3. RAG1/2 therefore appear to be co-recruited to RSSs associated with strong promoter activity, high H3K4me3, and other signatures of highly accessible chromatin [START_REF] Matthews | RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination[END_REF][START_REF] Ji | The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci[END_REF].

II.2.3.3. TCRA-TCRD locus conformation and loop organization

The spatial organization of genes within the nucleus is an important determinant of gene expression, and would therefore be expected to influence the dynamics of V(D)J recombination at antigen TCRA-TCRD locus (Fig. 12). Within the interphase nucleus, individual chromosomes occupy discrete regions, or chromosome territories, and genomic regions can loop out from those territories in a developmentally regulated fashion [START_REF] Fraser | Nuclear organization of the genome and the potential for gene regulation[END_REF][START_REF] Sutherland | Transcription factories: gene expression in unions?[END_REF]. Antigen receptors loci are demarcated by chromatin loops that juxtapose distant segments of DNA. Such looping may facilitate interactions with foci of RNAP II or RNA splicing factors as well as with other actively transcribed genes. The primary mediator of chromatin looping at TCRA and TCRB is the CCCTC-factor binding factor (CTCF) [START_REF] Degner | CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells[END_REF][START_REF] Shih | Tcra gene recombination is supported by a Tcra enhancer-and CTCFdependent chromatin hub[END_REF][START_REF] Majumder | Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element[END_REF]. CTCF is a highly conserved, ubiquitously expressed, zinc-finger-containing transcription factor that binds throughout the genome and mediates long-distance looping between CTCF-binding elements (CBEs) [START_REF] Ong | CTCF: an architectural protein bridging genome topology and function[END_REF]. CTCF can block, or insulate, enhancer activity by creating DNA loops that separate enhancers from promoters, or can facilitate gene expression by creating DNA loops that juxtapose enhancers and promoters (Fig. 12). These two mechanisms account for the known roles of CTCF in V(D)J recombination at antigen receptors loci. CTCF and cohesin bind to a large number of sites in the TCRA-TCRD locus and play critical roles in long-distance interactions, transcription, and V(D)J recombination. At the TCRA-TCRD locus, CTCF and cohesin mark the majority of cisregulatory elements in the locus, including the Eα, the T-early-alpha (TEA) promoter that is situated upstream of Jα61, and the promoters of most Jα-proximal V gene segments [START_REF] Shih | Tcra gene recombination is supported by a Tcra enhancer-and CTCFdependent chromatin hub[END_REF]. These interactions promote transcription, accessibility and recombination of these Vα and Jα gene segments during primary TCRα recombination. Deletion of CTCF in the DP thymocytes causes substantial defects in both primary and secondary Vα to Jα recombination by reduced interactions between Eα and the TEA promoter, between Eα and proximal Vα gene segments, and between the TEA promoter and proximal Vα gene segments [START_REF] Shih | Tcra gene recombination is supported by a Tcra enhancer-and CTCFdependent chromatin hub[END_REF] (Fig. 11). TCRD locus also contains a pair of intergenic CBEs (INT1 and INT2) between the Vδ and Dδ segments, and INT2 makes a chromatin interaction loop (CIL) with a downstream convergently oriented CBE within the TEA region (Fig. 11). The INT1-2 CBEs have been implicated in regulating TCRδ and TCRα V(D)J recombination (L. Chen et al.,

II.2.3.4.1. Enhancer δ role

Eδ is thought to be activated in DN thymocytes by the binding of RUNX, Myb and GATA3 transcription factors, and then inactivated in DP thymocytes by loss of transcription factor occupancy [START_REF] Ko | Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer[END_REF][START_REF] Redondo | Identification of an essential site for transcriptional activation within the human T-cell receptor delta enhancer[END_REF][START_REF] Hernández-Munain | A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation[END_REF][START_REF] Cieslak | RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement[END_REF][START_REF] Hernández-Munain | Recent insights into the transcriptional control of the Tcra/Tcrd locus by distant enhancers during the development of T-lymphocytes[END_REF]. RUNX1 is expressed in DN thymocytes and downregulated in DP thymocytes [START_REF] Taniuchi | Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development[END_REF]. Eδ promotes accessibility of Dδ and Jδ segments by influencing the activities of promoters associated with these gene segments (Fig. 11). Germline transcripts initiate within murine Dδ2 and upstream of and within murine Jδ1 in DN thymocytes [START_REF] Carabana | Regulation of the murine Ddelta2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb[END_REF] and these sites display high levels of histone modifications H3ac, H4ac, H3 lysine 4 bimethylation (H3K4me2), and H3K4me3 [START_REF] Hao | Long-distance regulation of fetal V(δ) gene segment TRDV4 by the Tcrd enhancer[END_REF][START_REF] Proudhon | Long-Range Regulation of V(D)J Recombination[END_REF].

II.2.3.4.2. Dδ and Jδ rearrangement

Developmental regulation of RAG accessibility at the TCRδ recombination center (Dδ2-Jδ1) is directed by the activated Eδ [START_REF] Monroe | Developmental regulation of TCRδ locus accessibility and expression by the TCRδ enhancer[END_REF][START_REF] Ji | The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci[END_REF], which is located in the Jδ3-Cδ intron in humans and the Jδ2-Cδ intron in mice [START_REF] Capone | Kinetics of T cell receptor beta, gamma, and delta rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44(+)CD25(+) Pro-T thymocytes[END_REF][START_REF] Livák | Characterization of TCR Gene Rearrangements During Adult Murine T Cell Development[END_REF][START_REF] Carabana | Regulation of the murine Ddelta2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb[END_REF][START_REF] Carico | Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires[END_REF]. Unlike the other loci that contain D gene segments (TCRβ), TCRδ is not subjected to ordered rearrangement in mice, whereas its ordered in humans [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF][START_REF] Cieslak | RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement[END_REF].

Thus, Vδ-to-Dδ and Dδ-to-Jδ joins occur at the same time, which enables Dδ gene segments to recombine together to form DDδ gene rearrangements [START_REF] Monroe | Developmental regulation of TCRδ locus accessibility and expression by the TCRδ enhancer[END_REF][START_REF] Carico | Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires[END_REF]. This ordering is dependent on the binding of RUNX1 to the human Dδ2 23

bp RSS that recruit RAG1 to the Dδ2 23 bp RSS. RUNX1 was required to enforce a classical Dδ2-to-Dδ3 join to form a chromosomal Dδ2-Dδ3 coding joint [START_REF] Cieslak | RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement[END_REF] (Fig. 11).

II.2.3.4.3. Vδ rearrangement

The pool of Vδ segments is limited, only a small fraction of the V gene segments located in the proximal portion of the V array are exclusively used in TCRδ recombination, contributing to the TCRδ repertoire. There are nominally 16 Vδ gene segments interspersed across the Vα-Vδ array [START_REF] Naik | Specification of Vδ and Vα Usage by Tcra/Tcrd Locus V Gene Segment Promoters[END_REF]. In humans, complete Vδ1-Jδ1 or Vδ2-Jδ1 rearrangements were detected first in late DN2 phase, increased to peak levels in ISPCD4 cells, and declined thereafter when the majority of thymocytes differentiated further into the TCRαβ lineage [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF]. At the TCRA-TCRD locus such regulation could dictate that Vδ 23 bp RSSs are intrinsically biased to rearrange to Dδ 12 bp RSSs, whereas Vα 23 bp

RSSs are intrinsically biased to rearrange to Jα 12 bp RSSs. In addition, Vδ 23 bp RSSs have a strong preference to rearrange to the Jδ1 12 bp RSS, which would contribute to the appropriate ordering of TCRD gene rearrangement events [START_REF] Lee | Differential utilization of T cell receptor TCR alpha/TCR delta locus variable region gene segments is mediated by accessibility[END_REF]. Vδ gene segments may be unique among TCRA-TCRD locus V gene segments in being accessible for RAG cleavage in DN thymocytes when Dδ and Jδ segments are also made accessible for recombination [START_REF] Hawwari | Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure[END_REF]. Complete δ rearrangements occur on both alleles in as many as 70% of murine γδ hybridomas, and a substantial fraction of these carry two in-frame TCRδ rearrangements, suggesting that δ rearrangement is not subject to allelic exclusion (Hernández-Munain, [START_REF] Hernández-Munain | A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation[END_REF]. For human γδ cell clones, depending on the Vδ gene segment used, only 34% or 18% had complete TCRδ rearrangements on both alleles, suggesting an allelic exclusion based on a feedback inhibition mechanism [START_REF] Brady | Position-dependent silencing of germline Vß segments on TCRß alleles containing preassembled VßDJßCß1 genes[END_REF].

II.2.3.4.4. Enhancer α role

Enhancer α as a critical regulator of TCRA locus rearrangements during the thymocyte development. Eα is inactive in DN1 to DN3a thymocytes when they attempt to successfully rearrange their TCRD locus; its activity is first detected after pre-TCR signaling in DN4 thymocytes, coinciding with the detection of TCRA germinal transcripts (Hernández-Munain, [START_REF] Hernández-Munain | A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation[END_REF][START_REF] Taghon | Developmental and molecular characterization of emerging beta-and gammadelta-selected pre-T cells in the adult mouse thymus[END_REF]. Eα influences chromatin structure across a 500-kb region that includes the 65-kb Jα array and the proximal third part of the 1.5megabase Vα array [START_REF] Hawwari | Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure[END_REF] (Fig. 11).

II.2.3.4.5. Jα rearrangement

The activation of Eα stimulates TCRA germline transcription and histone modifications across Jα chromatin in DP thymocytes. All Vα-to-Jα rearrangements, with the exception of a few Vα2 rearrangements that occur in DN cells by the action of the Eδ [START_REF] Aifantis | The E delta enhancer controls the generation of CD4-CD8-alphabetaTCRexpressing T cells that can give rise to different lineages of alphabeta T cells[END_REF], depend on the Eα [START_REF] Sleckman | Function of the TCR alpha enhancer in alphabeta and gammadelta T cells[END_REF]. TEA promoter activation plays a central role in targeting primary Vα-to-Jα rearrangements into the 5' portion of the Jα array (Fig. 11 and Fig. 14B). Moreover, the accessible chromatin state created by TEA and Eα is directly linked to transcriptional elongation across the 5' Jα gene segments [START_REF] Abarrategui | Noncoding transcription controls downstream promoters to regulate T-cell receptor alpha recombination[END_REF][START_REF] Ji | The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci[END_REF][START_REF] Del Blanco | Tcra Enhancer Activation by Inducible Transcription Factors Downstream of Pre-TCR Signaling[END_REF]. All TEA transcripts include the TEA exon; additional germline transcripts initiate upstream of several of the 5' Jα gene segments: Jα58, Jα57, Jα56, and Jα49; these transcripts also extend the length of the Jα array and are spliced from Jα-to-Cα [START_REF] Hawwari | Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure[END_REF]. Transcriptional elongation from the TEA promoter also appears to facilitate transcription initiating at Jα58-Jα56, perhaps as a consequence of chromatin remodeling [START_REF] Hawwari | Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure[END_REF][START_REF] Abarrategui | Noncoding transcription controls downstream promoters to regulate T-cell receptor alpha recombination[END_REF].

Chromatin remodeling and RAG recruitment are presumably highest across the most 5' Jα gene segments because this is the region with the highest density of elongating RNAP II [START_REF] Abarrategui | Regulation of T cell receptor-alpha gene recombination by transcription[END_REF]. TCRα alleles that have undergone primary Vα-to-Jα recombination physiologically delete the TEA promoter, and in some instances may delete the Jα49 promoter as well; and subsequent rearrangements are directed in stepwise fashion to successively more 3' Jα gene segments by chromatin remodeling directed by successively introduced Vα promoters (Hawwari and Krangel, 2007) (Fig. 14).

II.2.3.4.6. Generation of TCRα TRECs and Delta (δ) locus deletion

During TCRA gene rearrangements in DP thymocytes, the rearranged TCRD gene is deleted from the TCRA-TCRD gene complex [START_REF] Verschuren | Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF], which results

in the formation of a TREC that may contain δ gene rearrangements [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF]. The TCRD locus is deleted, preferentially via the δREC-ΨJα and δREC-Jα58 rearrangements, that represents, respectively, 68% and 23% of all rearrangements [START_REF] Verschuren | Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool[END_REF]. This rearrangement results in a coding joint (cj) δREC-ΨJα present in the genomic DNA. DNA fragments between rearranging V, D, and J gene segments are deleted as circular excision products, a signal joint (sj) δREC-ΨJα on the corresponding TREC (Fig. 13). These initiating events are followed by multiple, consecutive Vα-Jα rearrangements in DP thymocytes [START_REF] Sleckman | Function of the TCR alpha enhancer in alphabeta and gammadelta T cells[END_REF][START_REF] Verschuren | Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool[END_REF][START_REF] Capone | Kinetics of T cell receptor beta, gamma, and delta rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44(+)CD25(+) Pro-T thymocytes[END_REF][START_REF] Hawwari | Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure[END_REF][START_REF] Hao | Long-distance regulation of fetal V(δ) gene segment TRDV4 by the Tcrd enhancer[END_REF][START_REF] Carico | Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires[END_REF]. Due to ongoing Vα-Jα recombination the cj δREC-ΨJα will also be excised and become a signal joint on the corresponding TREC [START_REF] Verschuren | Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool[END_REF][START_REF] Hazenberg | T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation[END_REF].

Constitutive activation of α4β1 in pre-selection lDP (CD3 lo CD69 -) thymocytes mediates firm adhesion to fibronectin in vitro and results in a stationary state, whereas post-selection DP (CD3 hi CD69 + ) thymocytes do not adhere to fibronectin despite equivalent levels of α4β1 surface expression [START_REF] Crisa | Cell adhesion and migration are regulated at distinct stages of thymic T cell development: the roles of fibronectin, VLA4, and VLA5[END_REF].

II.2.3.6.1. Cortical positive selection and acquisition of MHC restriction

The αβ and γδ TCRs are noncovalently associated with complex of molecules known as the CD3 complex. This complex is essential for signal transduction by the TCRs and is only expressed on the cell surface if either αβ or γδ TCRs are expressed. The CD3 molecule is detectable intracellularly in all thymocytes and at the surface of positively selected lDP thymocytes and also in all later stages of T cell development. DN3 cells express the invariant signaling components of the TCR complex CD3 -γ, -δ, -ε (epsilon), and -ζ (zeta) which, in mature T cells, require association with both TCRα and TCRβ chains to be assembled into a functional signaling complex [START_REF] Brodeur | Critical and multiple roles for the CD3epsilon intracytoplasmic tail in double negative to double positive thymocyte differentiation[END_REF]. Genetic programs initiated in T cell progenitors induce TCRB expression, which is sufficient to provide the signals required for the expression of the co-receptors CD4 and CD8, as well as TCRA gene rearrangement, without any ligand engagement. When DP thymocytes recognize the complex peptide-MHC molecule presented on cTECs, they receive critical survival signals and differentiate into SP CD4 and SP CD8 thymocytes according to their affinity to MHC class II and I, respectively [START_REF] Anderson | Generating intrathymic microenvironments to establish T-cell tolerance[END_REF][START_REF] Takahama | β5t-containing thymoproteasome: specific expression in thymic cortical epithelial cells and role in positive selection of CD8+ T cells[END_REF][START_REF] Abramson | Thymic Epithelial Cells[END_REF]. Gene expression programs operate the differentiation into SP CD4 or SP CD8

thymocytes. BAF complexes play essential role in the expression control of T cell coreceptors on the surface of T cells, where they remodel chromatin in response to different signals and regulate transcription [START_REF] Chi | Sequential roles of Brg, the ATPase subunit of BAF chromatin remodeling complexes, in thymocyte development[END_REF][START_REF] Gebuhr | The role of Brg1, a catalytic subunit of mammalian chromatin-remodeling complexes, in T cell development[END_REF]. Mature T cells express either CD4 or CD8 co-receptors, and Brahma-related gene-1 (BRG1) regulates CD8 activation, whereas both BRG1 and BAF57 are involved in silencing of CD4 [START_REF] Chi | Reciprocal regulation of CD4/CD8 expression by SWI/SNF-like BAF complexes[END_REF]. CD4 repression depends on a 434 bp transcriptional silencer region, and both BRG1 and BAF57 increase chromatin accessibility by reducing H1 linker histone content and facilitating the binding of RUNX1, a transcriptional repressor, to this silencer region [START_REF] Wan | Molecular basis of CD4 repression by the Swi/Snf-like BAF chromatin remodeling complex[END_REF]. BRG1 is also involved in CD4 activation and regulatory T cell activation, which partially require its chromatin-remodeling ability and physical interactions between BRG1

and the Cd4 locus [START_REF] Jani | A novel genetic strategy reveals unexpected roles of the Swi-Snf-like chromatin-remodeling BAF complex in thymocyte development[END_REF][START_REF] Chaiyachati | BRG1-mediated immune tolerance: facilitation of Treg activation and partial independence of chromatin remodelling[END_REF]. DP CD3 hi thymocytes are capable to generate low-intensity activating signals following interaction with self-ligands undergo positive selection in the cortex. They transition into either MHC class I-restricted SP CD8 and MHC class II-restricted SP CD4 thymocytes migrate out of the cortex and enter the medulla [START_REF] He | The role of ThPOK in control of CD4/CD8 lineage commitment[END_REF][START_REF] Abramson | Thymic Epithelial Cells[END_REF].

II.2.4. Medullary SP thymocytes and negative selection

SP CD4 and SP CD8 T cells migrate into the thymic medulla, and interact with medullary TECs, which present the peptide of tissue-restricted antigen (TRA) in the context of MHC [START_REF] St-Pierre | Differential Features of AIRE-Induced and AIRE-Independent Promiscuous Gene Expression in Thymic Epithelial Cells[END_REF][START_REF] Takaba | The Mechanisms of T Cell Selection in the Thymus[END_REF]) (Fig. 15). Most autoreactive T cells are eliminated by apoptosis during the negative selection. However, some of these cells differentiate into thymic regulatory T cells or natural killer T cells -by agonist selectioncontributing to the establishment of self-tolerance in T cells [START_REF] Halkias | Opposing chemokine gradients control human thymocyte migration in situ[END_REF][START_REF] Klein | Positive and negative selection of the T cell repertoire: What thymocytes see (and don't see)[END_REF]. TRAs are promiscuously expressed under the control of the autoimmune regulator (AIRE), but also regulated by the brain-specific transcription factor FEZ famiy zinc finger protein 2 (FEZF2) in mTECs, leading to the elimination of T cells that respond to selfantigens [START_REF] Takaba | Fezf2 Orchestrates a Thymic Program of Self-Antigen Expression for Immune Tolerance[END_REF][START_REF] Takaba | The Mechanisms of T Cell Selection in the Thymus[END_REF]) (Fig. 15). TRA genes are induced by AIRE interacting with histone H3 or directly regulated by FEZF2. TRAs expressed by mTEC are taken up by the DCs, which also contribute to the TRA presentation to T cells [START_REF] Hubert | Aire regulates the transfer of antigen from mTECs to dendritic cells for induction of thymic tolerance[END_REF].

II.2.4.1. AIRE -promotion of clonal deletion of self-reactive thymocytes

Feedback signaling from SP thymocytes is important for this effect, as it induces the expression of the AIRE in mTECs, through interactions between thymocyte-expressed receptor activator of NF-κB (RANK), and its ligand RANKL on epithelial cells. In mouse thymus, AIRE is selectively expressed in approximately 30% of mTECs and a subset of B cells [START_REF] Yamano | Thymic B Cells Are Licensed to Present Self Antigens for Central T Cell Tolerance Induction[END_REF], but not in cTECs. AIRE regulates the thymic expression and presentation of TRA and thereby controls thymocyte tolerization and consequently autoimmunity (Fig. 15). AIRE also regulates the expression of a range of non-TRA genes in mTECs, either positively or negatively [START_REF] Sato | Aire downregulates multiple molecules that have contradicting immuneenhancing and immune-suppressive functions[END_REF], for example, mTEC maturation [START_REF] Irla | Autoantigen-Specific Interactions with CD4+Thymocytes Control Mature Medullary Thymic Epithelial Cell Cellularity[END_REF][START_REF] Nishikawa | Temporal lineage tracing of Aire-expressing cells reveals a requirement for Aire in their maturation program[END_REF] and CCR7 and CCR4 ligands expression [START_REF] Laan | Autoimmune regulator deficiency results in decreased expression of CCR4 and CCR7 ligands and in delayed migration of CD4+ thymocytes[END_REF]. Patients with an AIRE mutation present with an autoimmune disease called APECED [START_REF] Nagamine | Positional cloning of the APECED gene[END_REF]. APECED patients exhibit inflammatory cell infiltration into specific peripheral tissues such as the parathyroid and adrenal cortex, and produce autoantibodies against self-components including cytokines [START_REF] Browne | Anticytokine autoantibody-associated immunodeficiency[END_REF]. Each AIREdeficient patient has a unique repertoire of autoantibody reactivity. These observations expressing cortical thymocytes, a non-polymorphic major MHC class I-like antigenpresenting molecule [START_REF] Benlagha | A thymic precursor to the NK T cell lineage[END_REF][START_REF] Tuttle | TCR signal strength controls thymic differentiation of iNKT cell subsets[END_REF]. These self-lipid/CD1d complexes are expressed by both DP thymocytes and mTECs in the thymus. INKT cells are positively selected at the DP stage by CD1d-expressing DP cells themselves, instead of the mTECs that drive the selection of conventional T cells [START_REF] Bendelac | Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes[END_REF]. Developmental [START_REF] Aschenbrenner | Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ medullary thymic epithelial cells[END_REF][START_REF] Takaba | The Mechanisms of T Cell Selection in the Thymus[END_REF].

II.2.4.3.2. FOXP3 + T regulatory (Treg) cell development

Naturally occurring CD25 + CD4 + Treg cells, which constitutively express the transcription factor forkhead box P3 (FOXP3), are indispensable for the maintenance of immune selftolerance and homeostasis by suppressing aberrant or excessive immune responses harmful to the host [START_REF] Sakaguchi | Regulatory T cells and immune tolerance[END_REF][START_REF] Rudensky | Regulatory T cells and Foxp3[END_REF]. Use of peptide: MHC tetramers has demonstrated clonal deletion of antigen-specific thymocytes within the wild-type TCRαβ repertoire [START_REF] Malhotra | Tolerance is established in polyclonal CD4(+) T cells by distinct mechanisms, according to self-peptide expression patterns[END_REF]. The importance of the thymus medulla for tolerance induction appears to be explained by the ability of mTECs to support negative selection of conventional αβ T cells alongside generation of FOXP3 + Tregs [START_REF] Aschenbrenner | Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ medullary thymic epithelial cells[END_REF].

Treg development occurs via two-step process. In step one, strong TCR signaling induces expression of CD25 leading to a CD25 + FOXP3 -Treg progenitor. In step two, exposure of this Treg progenitor to IL-2 results in conversion to a CD25 + FOXP3 + Treg [START_REF] Coquet | Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway[END_REF][START_REF] Farrar | Development of regulatory T cells in the thymus -two paths to the same goal?[END_REF]. However, expression of FOXP3 alone is not sufficient for conferring and maintaining Treg cell function and phenotype. Complementing the insufficiency, Treg-cell-specific epigenetic changes are also critical in the process of Treg cell specification, in regulating its potential plasticity, and hence in establishing a stable lineage [START_REF] Ohkura | Development and maintenance of regulatory T cells[END_REF]. Mutations of human FOXP3 result in impaired development or dysfunction of Treg cells and, consequently, the occurrence of Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome accompanying severe autoimmune diseases, inflammatory bowel disease, and allergy [START_REF] Bennett | The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3[END_REF]. Likewise, mice that carry a mutation or genetic deletion of FOXP3 are deficient in Treg cells and develop fatal systemic autoimmunity [START_REF] Fontenot | Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[END_REF]. Adapted from [START_REF] Fink | The Biology of Recent Thymic Emigrants[END_REF].

II.2.5. Thymocyte Emigration -"au revoir" thymus

These selection events eventually result in the elimination of 90% of all thymocytes [START_REF] Staal | Transcriptional control of t lymphocyte differentiation[END_REF][START_REF] Koch | Mechanisms of T Cell Development and Transformation[END_REF]. During their time within the medulla, mature thymocytes progressively acquire "egress competence" through a program of post-selection maturation, which enables them to exit the thymus via post-capillary venules in the medulla to establish the peripheral naïve T cell pool. The egress competence program includes expression of the transcription factor Kruppel-like factor 2 (KLF2) [START_REF] Mccaughtry | Thymic emigration revisited[END_REF] and the cell surface receptor S1PR1 that is essential for thymus emigration at the CMJ [START_REF] Matloubian | Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1[END_REF][START_REF] Zachariah | Neural crest-derived pericytes promote egress of mature thymocytes at the corticomedullary junction[END_REF]. In mice, S1PR1 governs the migration of SP thymocytes toward an S1P gradient in part formed by the combined activity of pericytes and DCs surrounding blood vessels at the CMJ, where the concentration of S1P ligand is higher [START_REF] Matloubian | Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1[END_REF][START_REF] Zachariah | Neural crest-derived pericytes promote egress of mature thymocytes at the corticomedullary junction[END_REF]) (Fig. 16). Lymphotoxin-β receptor (LTβR) controls thymus emigration by its influence on thymic epithelium but also also endothelial cells [START_REF] James | Endothelial cells act as gatekeepers for LTβR-dependent thymocyte emigration[END_REF]. Mice lacking LTβR show increased mature SP thymocytes, which correlates with medullary disorganization and impaired mTEC development [START_REF] Martins | Ltbetar signaling does not regulate Aire-dependent transcripts in medullary thymic epithelial cells[END_REF]. Indeed, CXCL12 is implicated in this process in both humans and mice, while the timing of thymocyte egress was shown to be tightly regulated by the effects of TCR signaling and CD69 expression on surface expression of S1PR1 [START_REF] Schwab | Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients[END_REF][START_REF] Shiow | CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs[END_REF][START_REF] Zachariah | Neural crest-derived pericytes promote egress of mature thymocytes at the corticomedullary junction[END_REF]) (Fig. 16).

Finally, a reverse trans-endothelial-migration in which mature SP thymocytes exit from the perivascular space (PVS) and enter into the blood stream by migrating across thymic blood endothelium takes roughly 3 minutes [START_REF] Zachariah | Neural crest-derived pericytes promote egress of mature thymocytes at the corticomedullary junction[END_REF], and through this process, approximately one million mature thymocytes exit daily to seed the lymphoid periphery in a young adult mouse. The estimate of RTE production in humans between 20-to-25 years old is 1.7-17 X 10 7 cells daily (Vrisekoop et al., 2008;[START_REF] Den Braber | Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans[END_REF][START_REF] Fink | The Biology of Recent Thymic Emigrants[END_REF]. It is estimated that approximately 1% to 5% of thymocytes produced in both mice and humans thymus survive the complex maturation process to finally join in the peripheral T cell pool as a recent thymic emigrants (RTEs), a heterogeneous subset within the naïve T cells pool that undergoes further post-thymic maturation [START_REF] Fink | Post-thymic maturation: young T cells assert their individuality[END_REF].

II.2.5.1. Recent Thymic Emigrants

Recent thymic emigrants constitute the youngest T cells in the lymphoid periphery that have most recently completed intrathymic development and egress the thymus [START_REF] Fink | Post-thymic maturation: young T cells assert their individuality[END_REF]. The expression of CD45 isoforms has been most widely used for the enumeration of the naïve (CD45RA + ) T cells, although not always reliably [START_REF] Michie | Lifespan of human lymphocyte subsets defined by CD45 isoforms[END_REF][START_REF] Kohler | Life after the thymus: CD31+ and CD31-human naive CD4+ T-cell subsets[END_REF]. The use of TRECs and additional surface markers such as CD31 [START_REF] Douaisi | CD31, a Valuable Marker to Identify Early and Late Stages of T Cell Differentiation in the Human Thymus[END_REF] and protein tyrosine kinase (PTK) 7 in conjunction with the CD45RA, has improved discrimination between RTE and peripheral naïve T cells in humans (Fig. 16). The appearance of TRECs is used to identify human RTEs [START_REF] Livàk | T-cell receptor alpha locus V(D)J recombination by-products are abundant in thymocytes and mature T cells[END_REF][START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF]. These TCR products are generated during TCR rearrangement and are enriched in RTEs. Because TRECs are not replicated during mitosis, the TREC content per cell in the progeny cells is diminished with each cell division. As the high sjTREC content of thymic naïve CD4 T cells was only slightly reduced compared with thymocytes, it strongly supports the assumption that this subset contains RTE. However, the drastically diminished sjTREC content of central naïve CD4 T cells strongly implies peripheral proliferation. Also, CD31 expression can be maintained during cytokine-driven proliferation of CD4 T cells, limiting its use as a specific marker of thymopoïesis [START_REF] Kohler | Life after the thymus: CD31+ and CD31-human naive CD4+ T-cell subsets[END_REF]. The PTK7 was also proposed as a marker for human CD4 RTEs. Consistent with their recent thymic origin, human PTK7 + RTEs contained higher levels of signal joint TCR gene excision circles and were more responsive to IL-7 compared with PTK7 -naïve CD4 T cells, and rapidly decreased after complete thymectomy [START_REF] Haines | Human CD4+ T cell recent thymic emigrants are identified by protein tyrosine kinase 7 and have reduced immune function[END_REF]. Lower IL-7Rα, CD28, Qa2, Ly6C and CD45RB, and higher CD24, CD3, Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and PD1 cell surface expression characterize RTEs from mice of all ages [START_REF] Fink | The Biology of Recent Thymic Emigrants[END_REF]. RTEs (Fig.

II.2.5.2. TCR-self-pMHC interactions

The TCR signaling of T cells maintain cell viability, but in its absence, T cells die within several weeks [START_REF] Sprent | Normal T cell homeostasis: the conversion of naive cells into memory-phenotype cells[END_REF]. This signal also affects multiple aspects of T cell function, including antigen reactivity and migration [START_REF] Takada | Naive T cell homeostasis: from awareness of space to a sense of place[END_REF]. 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 in the late step of maturation [START_REF] Grossman | Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function[END_REF]. This sets a threshold for TCR activation just above the level required for T cell maintenance in the periphery, thereby keeping the cells in a self-tolerant state.

II.2.5.3. The essential role of IL-7 in T cell development and survival

Interleukin-7 has a central role in supporting T cell survival, proliferation and migration through Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and PI3K-AKT signaling pathway [START_REF] Swainson | IL-7-induced proliferation of recent thymic emigrants requires activation of the PI3K pathway[END_REF][START_REF] Takada | Naive T cell homeostasis: from awareness of space to a sense of place[END_REF]. This cytokine is synthesized by stromal cells, and is primarily produced in the thymus, lymph nodes and bone marrow. IL-7 responsiveness is controlled by modulation of expression of IL-7Rα and this expression is regulated via a negative feedback loop following receptor engagement, using notably Foxo1. Continuous interaction with the stromal cell network in secondary lymphoid organs is essential for naïve T cells to optimally encounter and respond to homeostatic factors [START_REF] Takada | Naive T cell homeostasis: from awareness of space to a sense of place[END_REF]. IL-7Rα turn on after positive selection at the SP stage, and its expression continues to increase with maturation as RTEs transit into peripheral naïve T cells. Decreased expression or function of IL-7 or IL-7Rα leads to a defect in peripheral T cell homeostasis [START_REF] Kerdiles | Foxo1 links homing and survival of naive T cells by regulating Lselectin, CCR7 and interleukin 7 receptor[END_REF][START_REF] Hogquist | T Cell Adolescence: Maturation Events Beyond Positive Selection[END_REF].

RTEs from neonates, but not from adults, proliferate in response to the key homeostatic cytokine IL-7 in the absence of T cell receptor stimulation [START_REF] Haines | Human CD4+ T cell recent thymic emigrants are identified by protein tyrosine kinase 7 and have reduced immune function[END_REF].

II.2.6. Generation of T cell receptor repertoire diversity

The intricate process of T cell development in the thymus is essential in the formation and maintenance of the peripheral T cell compartment with a diverse TCR repertoire [START_REF] Bains | Quantifying Thymic Export: Combining Models of Naive T Cell Proliferation and TCR Excision Circle Dynamics Gives an Explicit Measure of Thymic Output[END_REF]. However, vertebrate genomes are not large enough to encode an immune system capable of recognizing all possible threats. Instead, diversity in T cell receptor genes is generated by DNA rearrangements and mutations, beginning with the assembly of these genes from pools of interchangeable parts, via V(D)J recombination [START_REF] Ru | DNA melting initiates the RAG catalytic pathway[END_REF]. A TCR heterodimer composed of α and β chain, engaging peptide-MHC (pMHC). TCRβ chain consists of V, D, and J segments, while the TCRα chain are comprised of only V and J segments. In TCRα chains the J region is followed by the V region, whereas in TCRβ chain, a D region is located between the V and J regions. In the human TCR loci, there are 42 Vβ, 2

Dβ, 12 Jβ, 43 Vα and 58 Jα functional gene segments. In the mouse TCR loci, there are 35

Vβ, 2 Dβ, 12 Jβ, 71 Vα and 51 Jα functional gene segments [START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF]. The discontinuous localization and multiplicity of D, J, and especially V gene segments, combined with the randomness of their recombination, is responsible for the diversity of the immune repertoire at its disposal [START_REF] Ebert | Spatial Regulation of V-(D)J Recombination at Antigen Receptor Loci. 1st ed[END_REF]. As we saw previously, during T cell development, V(D)J gene segments recombine and are spliced together with the constant region to form the functional TCRαβ, with each T cell expressing only one type of recombined receptor complex [START_REF] Turner | Structural determinants of T-cell receptor bias in immunity[END_REF]. Two processes define the potential diversity of the TCR: II.2.6.1. Combinatorial diversity, as the rearranged allele includes a single copy of each V, D or J segment, out of the many present in the germline and II.2.6.2.

Junctional diversity, resulting from nucleotide deletions and non-templated N and P (palindromic) nucleotides additions made during the joining process to the original DNA breaks. The RAG complex mediates combinatorial synapsis and cleavage of discontinuous gene segments during lymphocyte development, thereby initiating the V(D)J recombination reaction that generates the large repertoire of TCR genes for adaptive immunity [START_REF] Ru | DNA melting initiates the RAG catalytic pathway[END_REF]. Consequently, the process of antigen receptor generation entirely depends on accurate regulation of the accessibility of RSSs for the RAG endonuclease (Fig. 17). While V(D)J gene rearrangement is believed to be random [START_REF] Venturi | Sharing of T cell receptors in antigen-specific responses is driven by convergent recombination[END_REF], some clonotypes are produced more commonly than others [START_REF] Venturi | A mechanism for TCR sharing between T cell subsets and individuals revealed by pyrosequencing[END_REF][START_REF] Bianconi | An estimation of the number of cells in the human body[END_REF], leading to unequal frequencies of naïve T cell clonotypes and to public clonotypes (i.e. clonotypes shared between people). This unequal frequency distribution is believed to be due to a process known as convergent recombination, whereby certain nucleotide sequences can be produced using a greater variety of recombination events; certain amino acid sequences can be made by a greater number of nucleotide triplets; and certain TCRs require fewer nucleotide insertions, deletions or substitutions [START_REF] Quigley | Convergent recombination shapes the clonotypic landscape of the naive T-cell repertoire[END_REF]. Finally, the successful of V(D)J rearrangement is clearly useful in terms of antigen recognition, and it is absolutely required for the development and survival of T cells [START_REF] Market | V(D)J recombination and the evolution of the adaptive immune system[END_REF].

II.2.6.1. Combinatorial diversity

Combinatorial diversity of variable regions is obtained by random recombination between V, D, and J gene segments. Pairing between α and β, or γ and δ chains for TCR adds another level of diversity, while the number of each type of segments within the chains allows for a large but finite combinatorial possibility in rearrangement.

II.2.6.2. V(D)J recombination mechanism

V(D)J gene segments present in each TCR loci are cut-and-pasted together through a DNA recombination mechanism reminiscent of DNA transposition [START_REF] Fugmann | The RAG proteins and V(D)J recombination: complexes, ends, and transposition[END_REF]. This mechanism is initiated by RAG1/2 proteins, which are responsible to mediate the recognition and splicing of V, D and J segments. Together form the RAG recombinase and carry out the first enzymatic step of the reaction-site-specific cleavage of DNA [START_REF] Market | V(D)J recombination and the evolution of the adaptive immune system[END_REF]. V(D)J recombination is governed by the 12/23 rule: RSSs adjacent to each gene demarcate the site of cleavage, which lies immediately between the RSS and its partner gene segment. Each RSS begins with a heptamer followed by a spacer of 12 or 23 base pairs, and ends with a nonamer. RAG1 binds to specific RSS present as 23-RSS or 12-RSS sequences flanking each gene segment and then recruits RAG2, a cofactor that is essential for recombinase activity. For recombination, the RSSs partner in an asymmetrical fashion, such that high-efficiency cleavage and rejoining occurs only between a 12/23 RSS pair (Drejer-Teel, [START_REF] Drejer-Teel | The beyond 12/23 restriction is imposed at the nicking and pairing steps of DNA cleavage during V(D)J recombination[END_REF][START_REF] Banerjee | Synapsis Alters RAG-Mediated Nicking at Tcrb Recombination Signal Sequences: Implications for the 'Beyond 12/23' Rule[END_REF]. RAG introduces DNA doublestrand breaks (DSBs) specifically at the borders between two coding segments and their flanking recombination signal sequences (Fig. 17). The capture of a second RSS through synapsis leads to the formation of a paired complex, within which DNA cleavage is completed. After cleavage, RAG is retained at DSBs in a post-cleavage complex, where it plays a role in directing repair the broken ends -containing the V, (D), or J gene segmentsand RSSs, joining to one another through nonhomologous end-joining (NHEJ) to form coding and signal joints, respectively (Fig. 17) [START_REF] Coster | A dual interaction between the DNA damage response protein MDC1 and the RAG1 subunit of the V(D)J recombinase[END_REF][START_REF] Helmink | The response to and repair of RAG-mediated DNA double-strand breaks[END_REF].

array to permit otherwise disfavored Vα-Jα combinations (Fig. 18) [START_REF] Fuschiotti | Analysis of the TCR α-chain rearrangement profile in human T lymphocytes[END_REF][START_REF] Carico | Tcrd Rearrangement Redirects a Processive Tcra Recombination Program to Expand the Tcra Repertoire[END_REF]. Therefore, recombination events at two developmental stages with distinct chromatin conformations synergize to promote TCRα repertoire diversity. The result of V(D)J recombination is a clonally diverse population of immature DP thymocytes that expresses a hugely diverse TCRαβ repertoire.

II.2.6.3. Junctional diversity

Junctional diversity is an additional mechanism to generate TCR diversity and arises when the fusion of V, (D), and J segments is not exact. Such "imprecise joining" of gene segments can result from the deletion of nucleotides in the joint region, and/or the addition of P and/or N nucleotides during V(D)J recombination [START_REF] Cabaniols | Most alpha/beta T cell receptor diversity is due to terminal deoxynucleotidyl transferase[END_REF][START_REF] Wang | αβ T-cell receptor bias in disease and therapy (Review)[END_REF]. The deletion of nucleotides occurs in the last stages of V(D)J recombination, the strands of the nicked hairpins are paired and then trimmed by exonuclease prior to repair by DNA ligases.

Very often, the exonuclease trims into the coding sequence itself, eliminating "templated" nucleotides (Fig. 19, in red). (far left panel), the nucleotide triplets "CCC" and "TCC" are eventually transcribed into the mRNA codons "CCC" and "UCC" and no junctional diversity occurs. However, prior to DNA ligation, the processes of deletion (in red), P nucleotide addition (in yellow), and/or N nucleotide addition (in purple) may result in alterations to the mRNA coding sequence.

Adapted from [START_REF] Mak | Primer to The Immune Response: Second Edition[END_REF].

Random addition of P and/or N nucleotides at region junctions (V-(P/N)-J, V-(P/N)-D and/or D-(P/N)-J). P nucleotide addition (Fig. 19, in yellow) occurs if the hairpin loops arising from RSS alignment are nicked exactly at the ends of the coding sequences, blunt ends are formed and the coding joint is exact. However, if the loops are nicked in the intervening DNA between gene segments, one strand on each gene segment will have a recessed end while the other strand will have an overhang. The gaps on both strands are filled by standard exonuclease trimming, DNA repair, and ligation to form an intact coding joint. The insertion of P nucleotides into the coding joint can change the amino acid sequence of the TCR protein, making this process a minor source of TCR repertoire diversity. Another source of junctional diversity is N nucleotide addition (Fig. 19, in purple). These N nucleotides ("non-template nucleotides") are added randomly by the terminal dideoxy transferase (TdT) enzyme on the ends of the nicked hairpins before final repair and ligation. TdT is likely recruited to the site of recombination by Ku (Fig. 17). TdT driven junctional diversity is crucial for the TCRαβ repertoire because the limited number of available gene segments reduces the combinatorial diversity [START_REF] Mak | Primer to The Immune Response: Second Edition[END_REF].

II.2.6.4. Complementarity Determining Regions (CDRs)

Following transcription, the sequence between the recombined V(D)J regions and the gene encoding the constant (C) region is removed by splicing. The antigen-binding site of TCRαβ and TCRγδ consists of six CDRs generated as a consequence of the combinatorial and junctional mechanisms during V(D)J rearrangement. CDRs are the most variable part of the TCR and complement an antigen-major histocompatibility complex's shape. CDRs loops of TCRs only recognize processed fragments of antigens that are presented by MHC molecules on APCs [START_REF] Davis | T-cell antigen receptor genes and T-cell recognition[END_REF][START_REF] Wang | αβ T-cell receptor bias in disease and therapy (Review)[END_REF]. Three defined TCR hypervariable regions -CDR1, CDR2 and CDR3 -have been found in the variable regions of β and α chains (Fig. 20). CDR1α, CDR1β, CDR2α and CDR2β are entirely encoded by the V region in germline DNA segments [START_REF] Turner | Structural determinants of T-cell receptor bias in immunity[END_REF]. The CDR3 domain, approximately 45 nucleotides long, comprises parts of the V and J segments (for TCRα) and, a part of the V segment and the entire D and J segments (for TCRβ) junction [START_REF] Woodsworth | Sequence analysis of T-cell repertoires in health and disease[END_REF][START_REF] Toor | On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments[END_REF]. Color gradients at junctions represent the regions encoded by arbitrary, untemplated nucleotides introduced during somatic recombination, and which represent a primary source of sequence diversification and TCR variability (Fig. 20a and20c).

In addition, the D segment insertion occurs in CDR3 of the TCRA-TCRD locus, thus, yielding a broader TCRβ repertoire for antigen recognition compared with TCRα and TCRγ. The CDR3 loop size distributions of the TCR γ and δ chains are relatively broad and dissimilar [START_REF] Pannetier | The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments[END_REF]. Furthermore, the greater diversity of the CDR3β loop (relative to the CDR3α loop) might indicate that the TCR Vβ chain is the main factor in determining TCR usage bias (Fig. 20c) [START_REF] Turner | Structural determinants of T-cell receptor bias in immunity[END_REF]. Relative to CDR1 and CDR2 loops, CDR3 loops are the highly polymorphic main domains and mediate interacts between TCR and the antigenic peptide-MHC complex (Fig. 20a) [START_REF] Woodsworth | Sequence analysis of T-cell repertoires in health and disease[END_REF]. These antigens that are recognized by TCR family members include peptides, glycoproteins, lipids and small molecule metabolites. As this region determines the antigen specificity of a T cell, each CDR3 represents a single T cell clone. Characterization of CDR3 sequence variation therefore provides a measure of the size of T cell diversity spectratype or Immunoscope methods [START_REF] Pannetier | The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments[END_REF][START_REF] Turner | Structural determinants of T-cell receptor bias in immunity[END_REF][START_REF] Woodsworth | Sequence analysis of T-cell repertoires in health and disease[END_REF]. The extreme diversity of the TCR repertoire represents a major analytical challenge;

this has led to the development of specialized methods, which aim to characterize the TCR repertoire in-depth. Recently, next generation sequencing (NGS) based technologies provides a more comprehensive approach to T cell repertoire diversity (Nikolich-Zugich, Slifka and [START_REF] Nikolich-Zugich | The many important facets of T-cell repertoire diversity[END_REF][START_REF] Six | The past, present, and future of immune repertoire biology -the rise of nextgeneration repertoire analysis[END_REF][START_REF] Woodsworth | Sequence analysis of T-cell repertoires in health and disease[END_REF][START_REF] Calis | Characterizing immune repertoires by high throughput sequencing: Strategies and applications[END_REF][START_REF] Han | Immune repertoire: A potential biomarker and therapeutic for hepatocellular carcinoma[END_REF][START_REF] Hou | Current status and recent advances of next generation sequencing techniques in immunological repertoire[END_REF][START_REF] Rosati | Overview of methodologies for T-cell receptor repertoire analysis[END_REF][START_REF] Zhang | 3D: diversity, dynamics, differential testing -a proposed pipeline for analysis of next-generation sequencing T cell repertoire data[END_REF][START_REF] Mahe | T cell clonality assessment: past, present and future[END_REF].

II.2.6.5 Size of TCR repertoire diversity

The sum of all TCRs by the T cells of one individual is termed the TCR repertoire or TCR profile. The size and diversity of the available human TCRαβ repertoire (the range of different TCRs expressed) are crucial in shaping immune responses to a given antigen [START_REF] Fuschiotti | Analysis of the TCR α-chain rearrangement profile in human T lymphocytes[END_REF][START_REF] Rosati | Overview of methodologies for T-cell receptor repertoire analysis[END_REF]. TCR diversity is associated with the effective control of viral infections and other pathogens [START_REF] Messaoudi | Direct Link Between mhc Polymorphism, T Cell Avidity, and Diversity in Immune Defense[END_REF][START_REF] Chen | TCR clonotypes modulate the protective effect of HLA class I molecules in HIV-1 infection[END_REF]. The number of clonotypes observed in the blood in one person has been reported to decrease with age [START_REF] Naylor | The influence of age on T cell generation and TCR diversity[END_REF][START_REF] Yager | Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to influenza virus[END_REF][START_REF] Boyd | Human lymphocyte repertoires in ageing[END_REF], viral challenge [START_REF] Chen | TCR clonotypes modulate the protective effect of HLA class I molecules in HIV-1 infection[END_REF], immunization [START_REF] Höhn | Longitudinal analysis of the T-cell receptor (TCR)-VA and -VB repertoire in CD8+ T cells from individuals immunized with recombinant hepatitis B surface antigen[END_REF] and as a result of immune suppression, for example after hematopoietic stem cell transplantation (HSCT) [START_REF] Muraro | T cell repertoire following autologous stem cell transplantation for multiple sclerosis[END_REF]. Moreover, the main challenge while studying the immune repertoire is its diversity. Accurate quantification of diversity is important to assess the extent of immune convergence (sharing of clonotypes between people) [START_REF] Venturi | A mechanism for TCR sharing between T cell subsets and individuals revealed by pyrosequencing[END_REF][START_REF] Madi | T-cell receptor repertoires share a restricted set of public and abundant CDR3 sequences that are associated with self-related immunity[END_REF][START_REF] Laydon | Estimating T-cell repertoire diversity: Limitations of classical estimators and a new approach[END_REF]. By combinatorial and junctional mechanisms, the small set of genes that encode the TCRαβ has the potential to create an estimated theoretical diversity between 10 12 to 10 18 TCR unique clonotypes (a clonotype is a population of T cells that carry an identical TCR) in humans [START_REF] Davis | T-cell antigen receptor genes and T-cell recognition[END_REF][START_REF] Arstila | A direct estimate of the human alphabeta T cell receptor diversity[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF][START_REF] Sewell | Why must T cells be cross-reactive?[END_REF]X. Hou et al., 2016) and 10 15 to 10 20 in mice [START_REF] Davis | T-cell antigen receptor genes and T-cell recognition[END_REF][START_REF] Kyewski | A central role for central tolerance[END_REF]. Current techniques do not allow direct quantification of the diversity of the lymphocyte compartment. In the absence of direct measures, various indirect means for estimating diversity have been used such as by extrapolation from molecular measurements of TCR diversity using CDR3 length analysis [START_REF] Pannetier | T-cell repertoire diversity and clonal expansions in normal and clinical samples[END_REF]. As CDR3 interacts most closely with the antigenic peptide, the diversity of CDR3 amino acid sequences provides a measure of T cell diversity in an antigen-selected T cell repertoire. The analysis of TCR usage, especially regarding CDR3 and the TCRβ V family, is currently an essential tool for deciphering mechanisms of autoimmunity, transplantation, cancer therapy, and infectious diseases (X. Hou et al., 2016). These analyses mainly focused on the β chain diversity because of the complexity of the Vα chain locus. [START_REF] Turner | Structural determinants of T-cell receptor bias in immunity[END_REF][START_REF] Woodsworth | Sequence analysis of T-cell repertoires in health and disease[END_REF]. The estimates were for about 10 6 different β chains in the human blood [START_REF] Calis | Characterizing immune repertoires by high throughput sequencing: Strategies and applications[END_REF], each pairing with at least 25 different α chains, giving a total TCRβ diversity of 2.5x10 7 [START_REF] Arstila | A direct estimate of the human alphabeta T cell receptor diversity[END_REF]. In contrast to TCRB, the TCRA locus is unusually complex, rendering it difficult to directly establish repertoire diversity. Not only does the TCRA locus contain the entire TCRD locus but the combined TCRA locus spans 1000 kb. Thus, human TCRA genes extend over a large region of the genome, rendering it technically difficult to detect all possible rearrangements. The human TCRA locus consists of 43-54 Vα genes including 8 pseudogenes, 61 Jα genes as well as 3 known J pseudogenes (Jα 51, 59, 60) and a unique Cα gene, whereas in mouse TCRB locus has 71 Vα and 51 Jα functional gene segments [START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF][START_REF] Glusman | Comparative genomics of the human and mouse T cell receptor loci[END_REF]. Human TCRα repertoire diversity estimated using theoretical approaches based on Immunoscope analyses and NGS sequencing has of the order of 2.5x10 7 . The extent of the diversity of actual biological repertoires falls dramatically short of these theoretical estimates because of bias in generation frequency (many clonotypes are of high abundance due to strong selection forces)

and the depletion by thymic selection (positive and negative selection and antigen specificity)

of T cells bearing non-productively rearranged TCRs. In addition, there are only an estimated 10 13 lymphocytes in human body [START_REF] Bianconi | An estimation of the number of cells in the human body[END_REF]; and the most of those specificities will never be used during an individual's life. The fact that only a fraction of the complete lymphocyte repertoire may be present in peripheral blood [START_REF] Warren | Exhaustive T-cell repertoire sequencing of human peripheral blood samples reveals signatures of antigen selection and a directly measured repertoire size of at least 1 million clonotypes[END_REF][START_REF] Calis | Characterizing immune repertoires by high throughput sequencing: Strategies and applications[END_REF].

CHAPTER 2. EVALUATE THYMIC FUNCTION

The production of T lymphocytes by the thymus is an important indicator of immune system functioning. Interestingly enough, their immunological function was first documented only in 1961 by using neonatally thymectomized mice [START_REF] Miller | Immunological function of the thymus[END_REF]Miller 2011) and since then some techniques were developed to evaluate the thymic function, for example, by naïve T cells (CD45RA, CD62L, CCR7), TRECs or novel markers such as CD31 (PECAM-1) and PTK7. One of these techniques involves the quantification of extrachromosomal circularized DNA molecules in T cells of the thymus, cord blood (CB) or more commonly peripheral blood. The presence of TRECs molecules coming from the V(D)J rearrangement at the TCRβ chain was first demonstrated by Okazaki & Sakano (Okazaki and Sakano, 1987). Previously detected from Vα-to-Jα joining by Fujimoto & Yamagishi [START_REF] Fujimoto | Isolation of an excision product of T-cell receptor alpha-chain gene rearrangements[END_REF],

TRECs have been characterized from the TCRA-TCRD gene region by [START_REF] Okazaki | Thymocyte circular DNA excised from T cell receptor alphadelta gene complex[END_REF]. The rearrangement of the TCRA genes has the peculiarity of involving the excision of delta-coding segments that, being nestled in the TCRA locus between the Vα and Jα genes. These delta segments must be removed in order to allow the generation of the TCRα protein. More than 30 years after its first description by [START_REF] Fujimoto | Isolation of an excision product of T-cell receptor alpha-chain gene rearrangements[END_REF], this method has been developped to measure thymic output in the context of aging [START_REF] Sempowski | Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy[END_REF][START_REF] Weinberg | Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation[END_REF]Mitchell, Lang andAspinall, 2010), Congenital Immunodeficiencies (van Zelm et al., 2011;[START_REF] Tagliaferri | Newborn screening for severe combined immunodeficiency using a novel and simplified method to measure T-cell excision circles (TREC)[END_REF], HIV infection [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF][START_REF] Dion | HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation[END_REF][START_REF] Drylewicz | Reconciling Longitudinal Naive T-Cell and TREC Dynamics during HIV-1 Infection[END_REF], HSCT [START_REF] Hazenberg | T-cell receptor excision circle and T-cell dynamics after allogeneic stem cell transplantation are related to clinical events[END_REF][START_REF] Talvensaari | A broad T-cell repertoire diversity and an efficient thymic function indicate a favorable long-term immune reconstitution after cord blood stem cell transplantation[END_REF][START_REF] Clave | Prognostic value of pretransplantation host thymic function in HLA-identical sibling hematopoietic stem cell transplantation[END_REF][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] Clave | Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse[END_REF][START_REF] Clave | Lenalidomide consolidation and maintenance therapy after autologous stem cell transplant for multiple myeloma induces persistent changes in T-cell homeostasis[END_REF][START_REF] Thiel | Direct assessment of thymic reactivation after autologous stem cell transplantation[END_REF][START_REF] Mensen | Utilization of TREC and KREC quantification for the monitoring of early Tand B-cell neogenesis in adult patients after allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Da Rocha | Thymopoiesis in Pre-and Post-Hematopoietic Stem Cell Transplantation[END_REF] and from patients using different ethnic groups [START_REF] Cho | Age estimation via quantification of signal-joint T cell receptor excision circles in Koreans[END_REF][START_REF] Ibrahim | Age Estimation in Living Egyptians Using Signal Joint T-cell Receptor Excision Circle Rearrangement[END_REF]Clave et al., 2018).

TREC assay -a surrogate marker of thymic function

These stable small episomal DNA circles do not replicate during mitosis and appears to be a relevant surrogate marker at thymic function because the majority of thymocyte expansion occurs before this TREC is formed. Its detection is so an important tool for quantification of RTEs export in the periphery since they can persist for a long time in peripheral T cells [START_REF] Douek | Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution[END_REF][START_REF] Hazenberg | T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation[END_REF][START_REF] Hazenberg | Thymic output: A bad TREC record[END_REF][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] Van Zelm | PID comes full circle: applications of V(D)J recombination excision circles in research, diagnostics and newborn screening of primary immunodeficiency disorders[END_REF][START_REF] Toubert | Thymus and immune reconstitution after allogeneic hematopoietic stem cell transplantation in humans: Never say never again[END_REF][START_REF] Arruda | Immune rebound associates with a favorable clinical response to autologous HSCT in systemic sclerosis patients[END_REF]. Importantly, TREC results should be carefully interpreted to avoid erroneous conclusions, particularly since sjTREC levels are also influenced by other biological parameters such as longevity of naïve T cells, peripheral expansion or apoptosis of T cells and intracellular degradation [START_REF] Hazenberg | Thymic output: A bad TREC record[END_REF][START_REF] Jiménez | Clinical factors influencing T-cell receptor excision circle (TRECs) counts following allogeneic stem cell transplantation in adults[END_REF][START_REF] Ringhoffer | T-cell reconstitution after allogeneic stem cell transplantation: assessment by measurement of the sjTREC/βTREC ratio and thymic naive T cells[END_REF]. Furthermore, in contrast to genomic DNA and as mentioned above, sjTRECs do not duplicate during cell proliferation, consequently, they will dilute out during consecutive cell divisions [START_REF] Fujimoto | Isolation of an excision product of T-cell receptor alpha-chain gene rearrangements[END_REF][START_REF] Takeshita | Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus[END_REF]. SjTRECs are the excised DNA circles formed during the process of TCRα chain VJ (or δREC-ΨJα) recombination and TCRA locus excision [START_REF] Serana | Use of V(D)J recombination excision circles to identify T-and B-cell defects and to monitor the treatment in primary and acquired immunodeficiencies[END_REF]. In order to overcome this limitation, Dion et al., [START_REF] Dion | HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation[END_REF]) developed a novel approach that allows diminishing the effect of peripheral expansion. In their technique, two types of TRECs in peripheral T cells are measured simultaneously and the sjTREC/ βTREC ratio was determined: those generated early (at DN3 stage) in thymocyte maturation during TCRB DJ locus rearrangement (DβJβ TREC or βTREC), and another, the sjTREC generated at a later stage (at DP stage) after many rounds of intrathymic proliferation during the TCRA locus rearrangement and the deletion of the TCRδ region [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF][START_REF] Dion | Estimating Thymic Function Through Quantification of T-Cell Receptor Excision Circles[END_REF]. Given the dilution of βTRECs at each cell division between βTREC and sjTREC generation, the log2 transformation of the sjTREC/ βTREC ratio gives an estimate of the number of TRECs and Intrathymic divisions (n) occurring between DN and DP stages was calculated using the following formulas (from 150,000 cells) [START_REF] Dion | HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation[END_REF]:

Although this methodology is more informative, quantifying the sjTREC/ βTREC ratio is laborious and time consuming since 11 multiplex PCR reactions (10 X DβJβ + 1 X sjTREC) [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] Clave | Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse[END_REF]. Besides, in a single experiment a vast quantity of DNA, not always available, needs to be used [START_REF] Dion | HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation[END_REF][START_REF] Ferrando-Martínez | A reliable and simplified sj/beta-TREC ratio quantification method for human thymic output measurement[END_REF]. Because the β chain recombines before the α chain, βTRECs are much less abundant than sjTRECs in the periphery and frequently fall below the detection threshold in quantitative PCR. To overcome the DNA limitation in quantifying mainly the βTRECs, a multiplex PCR preamplification step that precedes the quantitative PCR was included [START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF]. Thus the ratio between the two TRECs, which becomes fixed once the sjTREC is generated is a measure of the intrathymic proliferation, which is in turn a primary determinant of thymic function [START_REF] Dion | HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation[END_REF].

Methods to detect-express TREC

The TREC quantitative assay, initially proposed by Douek et al. [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF], has been later modified in different ways and TREC number has been evaluated in different biological samples or calculated by different approaches, resulting into hardly comparable results between laboratories. In fact, a more accurate TREC measurement has been obtained

when the quantification was performed relative to a housekeeping gene for the genomic copy number, such as chemokine CCR5, Albumin, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and/or TRA constant (TRAC) gene [START_REF] Zhang | Measuring recent thymic emigrants in blood of normal and HIV-1-infected individuals before and after effective therapy[END_REF][START_REF] Hazenberg | Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection[END_REF][START_REF] Ponchel | Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions[END_REF][START_REF] Zubakov | Estimating human age from T-cell DNA rearrangements[END_REF]. Furthermore, TREC content in peripheral blood has been reported either relative to peripheral blood mononuclear cells (PBMC), or quantified within sorted individual T cell subsets, such as in purified CD3 + T cells, purified CD4 + and CD8 + T cells, and also CD45RA + CD27 + or CD31 + T cells [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF][START_REF] Hazenberg | Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection[END_REF][START_REF] Ponchel | Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions[END_REF][START_REF] Junge | Correlation between recent thymic emigrants and CD31+ (PECAM-1) CD4+ T cells in normal individuals during aging and in lymphopenic children[END_REF][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] Ringhoffer | T-cell reconstitution after allogeneic stem cell transplantation: assessment by measurement of the sjTREC/βTREC ratio and thymic naive T cells[END_REF][START_REF] Adams | A comparison of TRECs and flow cytometry for naive T cell quantification[END_REF]. In other approaches, the quantity of TRECs has been expressed either as number of TREC molecules per µg of DNA within PBMC or T lymphocytes [START_REF] Zhang | Measuring recent thymic emigrants in blood of normal and HIV-1-infected individuals before and after effective therapy[END_REF][START_REF] Nobile | De novo T-cell generation in patients at different ages and stages of HIV-1 disease[END_REF], or per 10 6 cells, on the basis of the theoretical recovery of 1 µg of DNA from approximately 150,000 cells [START_REF] Hazenberg | T-cell receptor excision circle and T-cell dynamics after allogeneic stem cell transplantation are related to clinical events[END_REF][START_REF] Bains | Quantifying Thymic Export: Combining Models of Naive T Cell Proliferation and TCR Excision Circle Dynamics Gives an Explicit Measure of Thymic Output[END_REF][START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF]. Because in adults' samples TREC calculation per 10 6 PBMC can lead to erroneous

or n = log (sjTREC/ βTREC) 2 n = βTREC sjTREC log(2)
interpretations due to the diluting effect of peripheral T cell divisions, this limitation has been, at least in part, overcome by expressing TRECs per mL of blood [START_REF] Ribeiro | Determining thymic output quantitatively: using models to interpret experimental T-cell receptor excision circle (TREC) data[END_REF][START_REF] Bains | Quantifying Thymic Export: Combining Models of Naive T Cell Proliferation and TCR Excision Circle Dynamics Gives an Explicit Measure of Thymic Output[END_REF][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] Krenger | Thymic T-cell development in allogeneic stem cell transplantation[END_REF]. TREC and gene control copy number were determined by extrapolating the values from a unique standard curve which was obtained by the amplification of serial dilutions of a triple-insert plasmid (10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 ), which encodes one gene copy of TREC and gene control each [START_REF] Hazenberg | T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation[END_REF]. The unique standard curve enabled the elimination of variability related to direct DNA quantificaton. Assessment of housekeeping gene served as a control for the quality and quantity of genomic DNA in the sample. The number of TREC copies per mL or µL of blood was calculated with the following formula in PBMCs [START_REF] Mensen | Utilization of TREC and KREC quantification for the monitoring of early Tand B-cell neogenesis in adult patients after allogeneic hematopoietic stem cell transplantation[END_REF]:

Clinical applications of TREC

In recent years, numerous reports have described associations between TREC levels and variables related to treatment procedure and patient characteristics where T lymphocytes are essential for clinical evaluation and/or are involved in disease pathophysiology.

Quantification of sjTREC and βTREC molecules in peripheral blood cells has provided useful information in studies addressing the issues of 2. Hematopoietic stem cell transplantation [START_REF] Krenger | Thymic T-cell development in allogeneic stem cell transplantation[END_REF][START_REF] Clave | Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse[END_REF].

Losing TREC with age

TREC levels are elevated in T cell preparations of children and then decline with increasing age due to thymic involution. About 70% of all newly produced naïve T cells are TREC positive [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF]. Nevertheless, TREC levels can still be detected in elderly people indicating that the thymus partly retains its function in old age [START_REF] Douek | Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution[END_REF]. It has been evidenced by the decrease of the number of 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][START_REF] Sempowski | Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy[END_REF]. In addition, TRECs are also temporal dynamics within the expression of CD31 on naïve CD4 + T cell pool. The percentage of CD31 + cells in the naïve CD4 + T cell compartment

Mean TREC quantity

Mean gene control quantity/2 X (lymphocyte + monocyte count) / ml decreases, while the percentage of CD31 -naïve CD4 + T cells increases during healthy aging [START_REF] Kimmig | Two subsets of naive T helper cells with distinct T cell receptor excision circle content in human adult peripheral blood[END_REF][START_REF] Kilpatrick | Homeostasis of the naive CD4+ T cell compartment during aging[END_REF][START_REF] Kohler | Life after the thymus: CD31+ and CD31-human naive CD4+ T-cell subsets[END_REF]. In contrast, during cytokine-induced in vitro proliferation, CD31 is expressed in a stable manner by CD31 + thymic naïve CD4 + T cells. The coexistence of 2 distinct subsets of naïve CD4 + T cells in human adult peripheral blood implicates that current models of naïve CD4 + T cells homeostasis are insufficient [START_REF] Kohler | Post-thymic in vivo proliferation of naive CD4+ T cells constrains the TCR repertoire in healthy human adults[END_REF][START_REF] Kohler | Life after the thymus: CD31+ and CD31-human naive CD4+ T-cell subsets[END_REF]. When we compare humans and mice aging, the lifespan of mice is approximately two years, whereas humans live much longer. This reflects in a different lifespans of naïve T cells in mice and humans might indicate distinct mechanisms to maintain naïve T cell populations [START_REF] Zhang | Losing TREC with age[END_REF]. The naïve T cells of mice have comparable TREC content whether they are obtained from young or old mice. In humans, in contrast, the TREC content of T cells with naïve phenotype is high in neonates but declines with age. Estimating the fraction of cells that were originally thymically derived using the average TREC content of the naïve T cell population indicates that up to 90% of human "naïve" T cells are generated through proliferation in the periphery in aged individuals. Hence, thymic output maintains naïve T cell populations in mice, and thymic involution and loss of thymic output result in reductions in frequencies of naïve T cells in the periphery. In contrast, human T cells may divide in the periphery without losing their naïve phenotype based on current markers [START_REF] Kilpatrick | Homeostasis of the naive CD4+ T cell compartment during aging[END_REF]. The consequences of immune system aging includes a diminished output of new, naïve T cells, with immune sequelae that include diminished responses to novel pathogenic challenge, especially viral infection (including a well characterized loss of immunity against influenza A epitopes) diminished vaccine responsiveness [START_REF] Yager | Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to influenza virus[END_REF] and decreasing TCR repertoire diversity with age [START_REF] Cepeda | Thymic stromal cells: Roles in atrophy and age-associated dysfunction of the thymus[END_REF]. In addition, young adults who were thymectomized as children (during cardiac surgery) show signs of premature immunosenescence, including lower T cell counts, especially of the naïve T cell pool, and increased pools of oligoclonal memory T cells [START_REF] Sauce | Evidence of premature immune aging in patients thymectomized during early childhood[END_REF]. These data likely underestimate the impact of the thymus on maintaining the peripheral T cell pool in humans,

given that thymic fragments likely remain after childhood surgery (Appay, Sauce and Prelog, 2010).

Human immunodeficiency virus HIV infection

As mentioned previously, thymic involution, which peaks at puberty [START_REF] Lynch | Thymic involution and immune reconstitution[END_REF], leads to a decreased but heterogeneous thymic function during adulthood and old age [START_REF] Ferrando-Martínez | Thymopoiesis in elderly human is associated with systemic inflammatory status[END_REF]. Alterations in thymic structure due to infection can alter T cell export in other ways. A major consequence of HIV infection is reduced export of recent thymic emigrants; an effect confirmed by TREC analysis [START_REF] Douek | Evidence for increased T cell turnover and decreased thymic output in HIV infection[END_REF]. As thymic activity is essential to maintain or reconstitute a functional peripheral T cell pool, interventions that enhance this process may potentiate the beneficial effects of antiretroviral therapy on immunity [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF][START_REF] Corbeau | Immune reconstitution under antiretroviral therapy: the new challenge in HIV-1 infection[END_REF]. A rapid increase in numbers of naïve CD4 + T cells and detectable TRECs in peripheral cells following administration of highly active antiretroviral therapy (HAART) to HIV-infected adults indicates de novo lymphopoiesis [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF]. A recent longitudinal study demonstrated that both CD4 + and CD8 + naïve T cell TREC contents declined biphasically, with a rapid loss during the first year and a much slower loss during the chronic phase of

Severe combined immunodeficiency (SCID) diagnosis

Severe combined immunodeficiency is a life-threatening condition of newborns and infants caused by defects in genes involved in T cell development. Newborn screening (NBS) for SCID using the TREC assay began in Wisconsin in 2008 and has been adopted or is being implemented by all states in 2017. Adaptation for dried blood spotted onto filter paper made possible a TREC assay for SCID and other conditions with T cell lymphopenia (TCL) (Puck, 2012). In SCID after bone marrow transplantation therapy, the rise of T cells and naïve CD4 + T lymphocytes is associated with an increase in TRECs [START_REF] Patel | Thymic function after hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency[END_REF]. In addition, TREC analysis is capable of detecting known genetic causes of SCID, including IL2RG, RAG1, ADA, IL7R, JAK3, DCLRE1C, and CD3D deficiencies [START_REF] Kwan | Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States[END_REF][START_REF] Audrain | Newborn Screening for Severe Combined Immunodeficiency: Analytic and Clinical Performance of the T Cell Receptor Excision Circle Assay in France (DEPISTREC Study)[END_REF]. It has been established that NBS using the TREC assay is extremely sensitive to detect SCID in the newborn period [START_REF] Thakar | A Practical Approach to Newborn Screening for Severe Combined Immunodeficiency Using the T Cell Receptor Excision Circle Assay[END_REF].

TREC monitoring after hematopoietic stem cell transplantation

TREC has been extensively used to study different parameters that affect thymic recovery after HSCT [START_REF] Douek | Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution[END_REF]. Clinical studies found a positive correlation between TREC content and frequency of CD4 + CD31 + naïve T cells in adult autologous and pediatric allogeneic HSCT patients [START_REF] Weinberg | Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Hazenberg | T-cell receptor excision circle and T-cell dynamics after allogeneic stem cell transplantation are related to clinical events[END_REF][START_REF] Junge | Correlation between recent thymic emigrants and CD31+ (PECAM-1) CD4+ T cells in normal individuals during aging and in lymphopenic children[END_REF][START_REF] Thiel | Direct assessment of thymic reactivation after autologous stem cell transplantation[END_REF][START_REF] Mensen | Utilization of TREC and KREC quantification for the monitoring of early Tand B-cell neogenesis in adult patients after allogeneic hematopoietic stem cell transplantation[END_REF]. Allogeneic HSCT is the only curative option for many hematological malignancies. However, the development of graft-versus-host disease (GVHD) limits allo-HSCT success. GVHD frequency depends on factors such as recipient age, conditioning regimen, haematopoietic graft source and GVHD prophylaxis. GVHD is fatal to ~15% of patients [START_REF] Blazar | Advances in graft-versus-host disease biology and therapy[END_REF]. TREC analysis has been proposed as a predictive marker for complications and clinical outcome after allogeneic HSCT. Clinical manifestations of GVHD are associated with changes to both the lymphocyte and stromal compartments of the thymus, leading to acute involution. These include loss of cortical and medullary thymocytes, loss of TECs, decreased demarcation of the corticomedullary junction and disrupted architecture of the gland [START_REF] Seddik | T cell functional defect associated with thymid epithelial cell injury induced by a graft-versus-host reaction[END_REF]. Such changes have been shown to lead to decreased thymic output of naïve T cells, as indicated by TRECs [START_REF] Przybylski | No recovery of T-cell receptor excision circles (TRECs) after nonmyeloablative allogeneic hematopoietic stem cell transplantation is correlated with the onset of GvHD[END_REF]. Long-term T cell reconstitution after HSCT is dependent on patient thymic function and affected by acute and chronic GVHD [START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF]. Young patients affected by acute GVHD presented reduced levels of TRECs at the first 6 months after allo-HSCT, which the after recovered demonstrating the impact of the GVHD on thymic function and the plasticity of the thymic function in young adults [START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF]. Also, patients with SCID after allo-HSCT showed decreased long-term T-cell reconstitution. T-cell recovery was already poor early after HSCT, demonstrating that longterm immune failure could be caused by poor early grafting [START_REF] Borghans | Early determinants of long-term T-cell reconstitution after hematopoietic stem cell transplantation for severe combined immunodeficiency[END_REF]. The number of TRECs early after HSCT was most predictive of long-term T cell reconstitution and may thus serve to timely identify patients who will fail to reconstitute properly and who may need additional treatment.

THYMIC INVOLUTION THROUGHOUT LIFE

Thymic architecture disruption associated with age and fat accumulation

In vertebrates, the thymus develops during fetal life, reaches its maximum output during adolescence and, after which it shrinks and generates fewer and fewer T cells. After the first year of life, naïve T cell production in the thymus starts to decline, resulting in a low production rate in healthy adults [START_REF] Bains | Quantifying Thymic Export: Combining Models of Naive T Cell Proliferation and TCR Excision Circle Dynamics Gives an Explicit Measure of Thymic Output[END_REF][START_REF] Mitchell | Tracing thymic output in older individuals[END_REF][START_REF] Bains | Heterogeneity in Thymic Emigrants: Implications for Thymectomy and Immunosenescence[END_REF]. Physiological age-related involution of the thymus and failure to recover after injury are associated with impaired cellular immunity [START_REF] Chinn | Changes in primary lymphoid organs with aging[END_REF][START_REF] Boehm | Thymus involution and regeneration: Two sides of the same coin?[END_REF]. Crucial to this observation was the appreciation of dual components of the human thymus, the thymic epithelial space (TES) in which thymopoiesis occurs and the nonepithelial, nonthymopoietic PVS [START_REF] Steinmann | The involution of the ageing human thymic epithelium is independent of puberty. A morphometric study[END_REF]. The TES (cortex and medulla) contains keratin-positive thymic epithelium that nurtures developing thymocytes, but the PVS (adipocytes, peripheral lymphocytes, and stroma) are keratin negative and does not harbor thymopoiesis (Fig. 21) [START_REF] Hale | Histologic and molecular assessment of human thymus[END_REF]. Indeed, aged thymi display marked perturbations in stromal cellularity, characterized by dramatic TECs reduction [START_REF] Anderson | Projection of an immunological self shadow within the thymus by the aire protein[END_REF][START_REF] Zhu | Lymphohematopoietic progenitors do not have a synchronized defect with agerelated thymic involution[END_REF][START_REF] Gui | Thymus Size and Age-related Thymic Involution: Early Programming, Sexual Dimorphism, Progenitors and Stroma[END_REF] and expansion of adipocytes (Yang et al., 2009;[START_REF] Manley | Structure and function of the thymic microenvironment[END_REF] and fibroblasts [START_REF] Gray | Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells[END_REF][START_REF] Yang | Inhibition of thymic adipogenesis by caloric restriction is coupled with reduction in age-related thymic involution[END_REF], resulting in progressive fibrosis and fattening of thymic tissue (Fig. 21). The involution starts in the periphery of lobules with the invasion of adipose tissue.

PVS between blood vessels and the surrounding cells in the thymus increases and it is infiltrated by adipose tissue, lymphoid cells and phagocytized erythrocytes. Young and aged mice thymi differ greatly in the proliferative and self-renewing capacity of TECs.

Specifically, whereas during the perinatal period ∼30% of all TECs are proliferating, only 5% are proliferating at the age of 4 weeks [START_REF] Gray | Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells[END_REF]. The loss of a clear morphological demarcation reflects the disruption of TECs regions and thymocyte cross-talk. It corresponds to changes in progressive loss of thymic cellularity and macroscopic architecture leads to a gradual decline in naïve T cell output [START_REF] Abramson | Thymic Epithelial Cells[END_REF]. Even if thymic involution in humans begins shortly after birth, with a 3%/yr decrease through middle age (35-45 years of age), thymopoiesis persists into at least the fifth decade of life (Fig. 21) [START_REF] Hazenberg | T-cell receptor excision circle and T-cell dynamics after allogeneic stem cell transplantation are related to clinical events[END_REF][START_REF] Westera | Lymphocyte maintenance during healthy aging requires no substantial alterations in cellular turnover[END_REF], however, the thymus involutes and continues to decrease ∼1%/yr throughout the rest of life; eventually resulting in strongly diminished thymic output (van der [START_REF] Van Der Weerd | Combined TCRG and TCRA TREC analysis reveals increased peripheral T-lymphocyte but constant intra-thymic proliferative history upon ageing[END_REF]. To compensate for this, in contrast to what is seen in mice, most of the peripheral naïve T cell population in humans is maintained by homeostatic proliferation [START_REF] Den Braber | Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans[END_REF]. Due to increased homeostatic proliferation or decreased cell death, only a marginal decrease of the overall size of the naïve T cell pool is observed during aging [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF][START_REF] Lynch | Thymic involution and immune reconstitution[END_REF][START_REF] Wertheimer | Aging and cytomegalovirus infection differentially and jointly affect distinct circulating T cell subsets in humans[END_REF]. The expansion of the PVS with age results in a shift in the ratio of true TES to PVS, of note, by 50 years of age approximately 80% of the TES is dysfunctional and composed of adipose tissue (Fig. 21) and TES shrinks to less than 10% of the total thymus tissue by 70 years of age 2010; [START_REF] Chaudhry | Thymus: the next (re)generation[END_REF]. Significant reciprocal feedback occurs between the thymus and endocrine system (Montecino-Rodriquez, Min and Dorshkind, 2005).

Steroid hormones modulate the immune system

Age-related thymic involution is often described as being sex hormone dependent: both androgens (testosterone) and, progesterone and estrogens, male and female sex hormones respectively, have been directly associated with thymic involution [START_REF] Brelińska | Thymic epithelial cells in age-dependent involution[END_REF][START_REF] Montecino-Rodriguez | Causes, consequences, and reversal of immune system aging[END_REF][START_REF] Ventevogel | Thymic rejuvenation and aging[END_REF]. Their effect is most obviously manifest during puberty, during which the rate of thymic involution increases rapidly [START_REF] Steinmann | The involution of the ageing human thymic epithelium is independent of puberty. A morphometric study[END_REF][START_REF] Lynch | Thymic involution and immune reconstitution[END_REF][START_REF] Chaudhry | Thymus: the next (re)generation[END_REF] and the progress is more pronounced in males compared with in females [START_REF] Mitchell | Tracing thymic output in older individuals[END_REF][START_REF] Olsen | Evidence that androgens modulate human thymic T cell output[END_REF][START_REF] Dumont-Lagacé | Sex hormones have pervasive effects on thymic epithelial cells[END_REF] suggesting that increasing levels of sex hormones contribute to the involution process. Similarly, the effects of sex steroids on thymus size are remarkable, including atrophy of the thymus during pregnancy. Progesterone receptor (PR) null mutant and wild-type female recipients, demonstrated that thymic stromal PR blocks very early T cell lymphopoiesis during pregnancy and is essential for normal fertility [START_REF] Tibbetts | Progesterone receptors in the thymus are required for thymic involution during pregnancy and for normal fertility[END_REF][START_REF] Dixit | Gonadotropin-releasing hormone attenuates pregnancy-associated thymic involution and modulates the expression of antiproliferative gene product prohibitin[END_REF]. Sex steroids have multiple effects to reduce the lymphocyte pool within the thymus. TECs express a functional androgen receptor (AR), and AR knock-down in this population partially abrogates sex steroid-induced thymic involution, further implicating TECs as important targets of androgens [START_REF] Lai | Targeting thymic epithelia AR enhances T-cell reconstitution and bone marrow transplant grafting efficacy[END_REF]. Androgens in particular exert considerable influence on the size and composition of the thymus [START_REF] Chen | Thymus-derived glucocorticoids mediate androgen effects on thymocyte homeostasis[END_REF].

Androgens directly inhibit thymopoietic factors in TECs, including CCL25, a ligand critical for entry of T cell progenitors into the thymus, and Dll4, a Notch ligand crucial for the specification, commitment and differentiation of T cell progenitors in a dose-dependent manner [START_REF] Williams | CCL25 increases thymopoiesis after androgen withdrawal[END_REF][START_REF] Velardi | Sex steroid blockade enhances thymopoiesis by modulating Notch signaling[END_REF]. Furthermore, through their effects on both HSCs and the bone marrow microenvironment, sex steroids decrease lymphoid differentiation, reducing the number of available T cell progenitors in the thymus [START_REF] Dudakov | Withdrawal of sex steroids reverses age-and chemotherapy-related defects in bone marrow lymphopoiesis[END_REF]. Lymphoid component in PVS undergoes a process of reduction in 20-to-25-yearold humans and at the same time PVS left by thymocytes is populated by adipocytes which concur to enlarge this area, enlarging also PVS [START_REF] Haynes | The human thymus during aging[END_REF] (Fig. 21). Thus, relative to males, females usually display stronger immune response to vaccination and infection but suffer a higher propensity to many autoimmune diseases [START_REF] Danska | Sex matters for mechanism[END_REF][START_REF] Markle | SeXX matters in immunity[END_REF]. Sex hormones can affect immune cells directly or via modulation of the gut microbiome, which in turn influences the immune response [START_REF] Markle | Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity[END_REF](Markle et al., , 2014;;[START_REF] Furman | Systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination[END_REF]. Sex hormones have pervasive effects on TEC biology. In adult mice, males accumulate more cTECs than females, but male cTECs display low proliferation rates and show evidence of functional impairment [START_REF] Dumont-Lagacé | Adult thymic epithelium contains nonsenescent label-retaining cells[END_REF]. Furthermore, following acute ablation in a transgenic mouse model, cTECs were found to regenerate in females and castrated males [START_REF] Sutherland | Activation of thymic regeneration in mice and humans following androgen blockade[END_REF][START_REF] Dooley | Molecular control over thymic involution: from cytokines and microRNA to aging and adipose tissue[END_REF][START_REF] Griffith | Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth[END_REF] but not in males or androgen-treated females (Dumont-Lagacé, St-Pierre and Perreault, 2015). In addition, relative to castrated males, mTECs from males and females showed decreased expression of promiscuously expressed genes (Dumont-Lagacé, St-Pierre and Perreault, 2015). Moreover, it is known that sex hormone ablation by chemical or surgical castration reverses age-related thymic involution and restores thymic function with a decrease in the rate of thymocyte apoptosis and increased proliferation of both T cells and TECs.

Similarly, treatment with estrogen also leads to a reduction in thymic cellularity [START_REF] Marotti | In vivo effect of progesteron and estrogen on thymus mass and T-cell functions in female mice[END_REF]. In contrast to castration, removal of the ovaries does not lead to dramatic changes in thymus size or cellularity [START_REF] Bodey | Involution of the mammalian thymus, one of the leading regulators of aging[END_REF][START_REF] Taub | Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo[END_REF].

Other hormones implicated in the thymic function

FGF21

The fibroblast growth factors (FGFs) constitute a family of 22 proteins that regulate diverse biological processes such as growth, development, differentiation, and wound repair (Y. [START_REF] Zhang | Losing TREC with age[END_REF]. FGF21 is predominantly secreted from liver but is also expressed in thymus [START_REF] Nishimura | Identification of a novel FGF, FGF-21, preferentially expressed in the liver[END_REF]. FGF21 is a prolongevity hormone that elicits it biological effects by binding to βKlotho in complex with FGF receptor [START_REF] Badman | Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states[END_REF][START_REF] Zhang | The starvation hormone, fibroblast growth factor-21, extends lifespan in mice[END_REF]. FGF21 supports host survival during states of energy deficit by increasing ketogenesis and fuel utilization through mitochondrial fatty acid oxidation [START_REF] Badman | Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states[END_REF]; Y. [START_REF] Zhang | Losing TREC with age[END_REF]. Interestingly, caloric restriction could block the age-related increase in adipocyte accumulation and maintains thymopoiesis in aged mice [START_REF] Yang | Inhibition of thymic adipogenesis by caloric restriction is coupled with reduction in age-related thymic involution[END_REF]. Indeed, coexpressed in TECs, FGF21 and βKlotho could maintain T cell diversity in models of aging and HSCT by enhancing thymic function [START_REF] Youm | Prolongevity hormone FGF21 protects against immune senescence by delaying age-related thymic involution[END_REF].

However, TECs progressively lose expression of FGF21, which plays a critical role in preventing TEC degeneration in old mice [START_REF] Youm | Prolongevity hormone FGF21 protects against immune senescence by delaying age-related thymic involution[END_REF]. Indeed, mutation of the FGF21 obligate coreceptor, βKlotho, promotes thymic atrophy [START_REF] Kuro-O | Mutation of the mouse klotho gene leads to a syndrome resembling ageing[END_REF]. Klotho deficiency causes thymic involution via systemic effects rather than being TEC intrinsic [START_REF] Xing | Thymic Epithelial Cell Support of Thymopoiesis Does Not Require Klotho[END_REF]. This is correlated with a reduction in the export of new T cells into the periphery.

Ghrelin

Calorie restriction has been shown to partially mitigate age-associated thymic atrophy [START_REF] Yang | Inhibition of thymic adipogenesis by caloric restriction is coupled with reduction in age-related thymic involution[END_REF]. Conversely, obesity has been associated with decreases in thymus output [START_REF] Howard | Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice[END_REF]. Adipogenesis within the thymus and thymic atrophy are promoted by age-associated decreases in expression of ghrelin, an orexigenic and antiinflammatory peptide, and ghrelin receptor (growth hormone secretagogue receptor -GHS-R)

in thymic stromal cells [START_REF] Dixit | Ghrelin promotes thymopoiesis during aging[END_REF][START_REF] Youm | Deficient ghrelin receptor-mediated signaling compromises thymic stromal cell microenvironment by accelerating thymic adiposity[END_REF][START_REF] Auton | A global reference for human genetic variation[END_REF]. Ghrelin and GHS-R are expressed by peripheral resting and activated human T cells [START_REF] Dixit | Ghrelin inhibits leptin-and activation-induced proinflammatory cytokine expression by human monocytes and T cells[END_REF] and have been shown to mediate anti-inflammatory signals in several cells types [START_REF] Dixit | Ghrelin and immunity: a young player in an old field[END_REF], including mononuclear cells and in various murine models of acute and chronic inflammation [START_REF] Dixit | Ghrelin and immunity: a young player in an old field[END_REF][START_REF] Granado | Anti-inflammatory effect of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) in arthritic rats[END_REF]. Involution of the thymus is accompanied by replacement of the T cell compartment via infiltration of adipocytes and an increase in proinflammatory cytokines such as IL-6 within the thymic microenvironment [START_REF] Sempowski | Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy[END_REF]. Mechanistic links between aging, thymic adiposity, and thymic atrophy have been revealed that lipotoxic "danger-associated molecular patterns" (DAMPs), such as ceramide and free cholesterol, increase during aging [START_REF] Youm | The Nlrp3 inflammasome promotes age-related thymic demise and immunosenescence[END_REF][START_REF] De Mello-Coelho | Age-associated alterations in the levels of cytotoxic lipid molecular species and oxidative stress in the murine thymus are reduced by growth hormone treatment[END_REF], and can initiate NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome signaling and IL-1β production in thymic myeloid cells [START_REF] Youm | The Nlrp3 inflammasome promotes age-related thymic demise and immunosenescence[END_REF]. Consistent with the idea that TECs are highly sensitive to damage induced by aerobic metabolisms is the observation that TEC degeneration can be prevented by reduced metabolic activity (e.g., by caloric restriction) [START_REF] Yang | Inhibition of thymic adipogenesis by caloric restriction is coupled with reduction in age-related thymic involution[END_REF] but is accelerated in obese or leptin-resistant individuals [START_REF] Dixit | Thymic fatness and approaches to enhance thymopoietic fitness in aging[END_REF]. Relatedly, recent studies demonstrate that some TECs can transdifferentiate into adipogenic fibroblasts via the activation of an epithelial-mesenchymal transition (EMT) process in the aged thymus [START_REF] Youm | Deficient ghrelin receptor-mediated signaling compromises thymic stromal cell microenvironment by accelerating thymic adiposity[END_REF], offering a possible explanation for the progressive loss of TECs, with a concomitant increase in thymic fibroblasts and adipocytes [START_REF] Abramson | Thymic Epithelial Cells[END_REF].

Leptin

The fat that accumulates in the PVS with age substitutes TECs in TES with advancing age contributing to thymocyte loss and structural changes within the aging thymus. In humans, fat infiltration of the thymus can be observed as early as 2 years after birth and progressively accumulates throughout adulthood. Leptin, an anorexigenic hormone that exerts significant effects on immune responses and induces proinflammatory cytokine expression [START_REF] Fantuzzi | Leptin in the regulation of immunity, inflammation, and hematopoiesis[END_REF][START_REF] Dixit | Ghrelin inhibits leptin-and activation-induced proinflammatory cytokine expression by human monocytes and T cells[END_REF], is predominantly produced by fat cells and controls food intake and energy balance. Given the proinflammatory nature of leptin and its ability to induce the production of IL-6, IL-1β, tumor necrosis factor (TNF)-α, interferon-γ, and a number of other cytokines and growth factors by both immune and nonimmune populations, increases in thymic adipose tissue would result in an increase in intrathymic leptin possibly resulting in greater thymic loss. Systemically, circulating leptin levels tend to correlate with the body mass index (BMI) of the subject and increasing in the circulation of subjects with age-associated disease states [START_REF] Fantuzzi | Leptin in the regulation of immunity, inflammation, and hematopoiesis[END_REF][START_REF] Dixit | Ghrelin inhibits leptin-and activation-induced proinflammatory cytokine expression by human monocytes and T cells[END_REF]. Moreover, leptin and GHS-R ligands appear to counterregulate each other's activities, and thus, possibly increased leptin levels may hinder the beneficial effects of intrathymic and systemic GHS-R ligands (e.g. ghrelin) [START_REF] Dixit | Ghrelin inhibits leptin-and activation-induced proinflammatory cytokine expression by human monocytes and T cells[END_REF].

RESEARCH PLAN

The immune system plays a key role in maintaining a healthy state by preventing infection and malignant transformation but also by participating in the steady-state tissue homeostasis.

Numerous studies using human or animal models, have described the pathways involved in different pathological models (e.g., infectious diseases, autoimmunity, genetic polymorphism) that can be used as reference values by the community. To this purpose, extended epidemiological and biological data was collected from 1,000 healthy volunteers with a homogeneous ethnic background (French persons for at least three generations), splited equally by sex (50% men/women) and stratified across five-decades of life age (20 to 69 years). 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 (Fig. 22) [START_REF] Thomas | The Milieu Intérieur study -An integrative approach for study of human immunological variance[END_REF]. Indeed, the diversity of protocols used between studies makes it difficult to directly compare between datasets. In order to implement accurate comparisons to minimize pre-analytical biases, it becomes increasingly important to standardize the different steps of these systemic approaches (Fig. 22). In this context, further studies were developed using this unbiased "system immunology" approach permitting an 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][START_REF] Chen | Automated flow cytometric analysis across large numbers of samples and cell types[END_REF][START_REF] Duffy | Milieu intérieur: Defining the boundaries of a healthy immune response for improved vaccination strategies[END_REF]. Other studies were developed through the MI consortium program at different approaches [START_REF] Kilens | Parallel derivation of isogenic human primed and naive induced pluripotent stem cells[END_REF][START_REF] Patin | Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors[END_REF][START_REF] Piasecka | Distinctive roles of age, sex, and genetics in shaping transcriptional variation of human immune responses to microbial challenges[END_REF]. In the context of this cohort collection, my PhD thesis has focused around two main studies.

The first study was driven by two principal aims:

I. Provide a clear picture of the thymic function in healthy human conditions in a large-scale study and validate the TRECs as a safe and efficient mechanism to quantify the thymic output by the Biomark HD system (Fluidigm France, Paris, France). Moreover, quantitative determination of the intrathymic proliferative history within the peripheral blood T lymphocyte compartment might in provide an additional marker of healthy thymic function.

II. Apart from age, environmental or genetic parameters governing human thymic function have been still poorly defined. We evaluated the TRECs in this unbiased "system immunology" approach combining demographic, phenotypic and molecular analysis (genome-wide associtation studies -GWAS) to identify potential non-heritable and heritable factors tested in the MI study that could impact the thymic function in a healthy population.

Following the discovery of a genetic polymorphism in the TCRA-TCRD locus (MI study) we aimed in the second study at getting more mechanistic insights of the role of this genetic variation in human thymopoïesis. I took advantage of a partnership with the Laboratory of Onco-Hematology at Necker-Children-Sick Hospital (Paris, France). This study is still ongoing and the data presented will be completed after my PhD. The thymus is the primary lymphoid organ where naïve T cells are generated; however, with the exception of age, the parameters that govern its function in healthy humans remain unknown. We characterized the variability of thymic function among 1000 age-and sexstratified healthy adults of the Milieu Intérieur cohort, using quantification of T cell receptor excision circles (TRECs) in peripheral blood T cells as a surrogate marker of thymopoïesis.

RESULTS

Part

Using a unique combination of state of the art techniques in mice and humans, we have directly quantified the contribution of de novo T cell production by the thymus and peripheral T cell division in mice and humans, respectively. Age and sex were the only nonheritable factors identified that affect thymic function. TREC amounts decreased with age and were higher in women compared to men. In addition, a genome-wide association study revealed a common variant (rs2204985) within the T cell receptor TCRA-TCRD locus, between the DD2 and DD3 gene segments, which associated with TREC amounts. Strikingly, transplantation of human hematopoietic stem cells with the rs2204985 GG genotype into immunodeficient mice led to thymopoïesis with higher TRECs, increased thymocyte counts, and a higher TCR repertoire diversity. Our population immunology approach revealed a genetic locus that influences thymopoïesis in healthy adults, with potentially broad implications in precision medicine.

INTRODUCTION

In healthy individuals, continuous production of naïve self-tolerant T cells by the thymus ensures potent immune responses toward newly encountered antigens and contributes to maintenance of the naïve T cell repertoire (1). Thymic function has been extensively studied for its capacity to shape the adaptive immune repertoire through positive and negative selection [START_REF] Stritesky | Selection of self-reactive T cells in the thymus[END_REF]3). However, little is known about the environmental or genetic determinants of thymopoiesis in healthy individuals. Such insights would be relevant for optimizing regenerative strategies (4), especially in conditions where thymic function is altered, such as aging [START_REF] Palmer | Thymic involution and rising disease incidence with age[END_REF][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], HIV infection [START_REF] Dion | HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation[END_REF], or allogeneic hematopoietic stem cell transplantation (allo-HSCT) [START_REF] Krenger | Thymic T-cell development in allogeneic stem cell transplantation[END_REF].

A bilateral cross-talk between thymocytes and thymic stromal cells directs sequential intrathymic T cell development and helps maintain activity of thymic stromal niches [START_REF] Abramson | Thymic epithelial cells[END_REF][START_REF] Kurd | T-cell selection in the thymus: A spatial and temporal perspective[END_REF]. Thymocyte progenitors receive signals from cortical thymic epithelial cells (TECs) for their commitment to the T cell lineage via the engagement of the NOTCH1 receptor with Delta-like 4 ligand, a major Forkhead box protein (FOX)N1 target in the thymic epithelium [START_REF] Žuklys | Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells[END_REF]. The medulla, via medullary TECs (mTECs) and dendritic cells, has a critical role in establishing self-tolerance by negative selection and induction of regulatory T cells (T regs ), especially but not exclusively via mTECs expressing the autoimmune regulator gene (AIRE) [START_REF] Abramson | Thymic epithelial cells[END_REF]. Naïve T cells are hetero geneous and include so-called recent thymic emigrants (RTEs), a subset that undergoes further post-thymic maturation (12). Some phenotypic markers have been proposed to identify RTEs, such as CD31 (PECAM-1) in CD4 + T cells. However, CD31 expression can be maintained during cytokine-driven proliferation of CD4 + T cells, limiting its use as a specific marker of thymopoiesis. RTEs are enriched in T cell receptor excision circles (TRECs), which are produced during thymic TCR somatic recombination [START_REF] Kohler | Life after the thymus: CD31 + and CD31human naive CD4 + T-cell subsets[END_REF]. TRECs persist within mature T cells as episomal DNA ( 14), cannot replicate, and are diluted out by peripheral cell divisions. Their quantification in peripheral blood provides a noninvasive surrogate marker of thymopoiesis, especially relevant in steady-state homeostatic conditions of the T cell compartment.

Signal joint TRECs (sjTRECs) are generated during the recombination of the TCRa chain, in double-positive (DP) CD4 + CD8 + thymocytes, before positive and negative selection and lineage commitment [START_REF] De Villartay | Deletion of the human T-cell receptor d-gene by a site-specific recombination[END_REF]. Polymerase chain reaction (PCR)-based quantification of sjTRECs is used in clinical laboratories as a diagnostic test for the recovery of the naïve T cell repertoire during HIV treatment, after allo-HSCT, and in the screening of severe combined immunodeficiencies in newborns [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF][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] Puck | Laboratory technology for population-based screening for severe combined immunodeficiency in neonates: The winner is T-cell receptor excision circles[END_REF]. Similarly, assays are available to measure bTRECs generated during the TCRb chain recombination at the CD4 -CD8 -double-negative (DN) 3 stage. Because the b chain recombines before the a chain, bTRECs are much less abundant than sjTRECs in the periphery and frequently fall below the detection threshold in quantitative PCR. Given the dilution of bTRECs at each cell division between bTREC and sjTREC generation, the log 2 transformation of the sjTREC/bTREC ratio gives an estimate of the number of intrathymic divisions occurring between DN and DP stages [START_REF] Dion | HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation[END_REF].

Here, we quantified TRECs from the peripheral blood of 1000 healthy individuals of western European ancestry [the Milieu Intérieur (MI) cohort] at immunological steady state, stratified by sex and age across five decades of life from 20 to 69 years (18). This population immunology approach revealed determinants of heterogeneity in human thymic function and identified a common genetic variation within the TCRA-TCRD locus directly affecting thymopoiesis.

RESULTS

Validation of TRECs as surrogate markers of thymic function in the MI cohort

We first standardized and validated sjTREC and bTREC highthroughput assays (Fig. 1A and fig. S1) and then applied them to DNA from the 1000 MI donors. sjTREC counts normalized per 150,000 whole blood cells were used in subsequent analyses and correlated (r 2 = 0.99, P < 10 -16 ) with sjTRECs calculated as absolute numbers per microliter of blood (fig. S1C), the latter being not affected by T cell peripheral divisions. Log 10 -transformed values of sjTRECs (log 10 sjTRECs) showed a normal distribution (kurtosis test, P = 0.25), with a mean of 2.4 ± 0.03 (minimum to maximum range, 0.2 to 4.1; fig. S1D). By contrast, log 10 -transformed values of bTRECs (log 10 bTRECs) showed a bimodal distribution, with 368 donors having samples below the limit of assay detection. In donors with detectable bTRECs in whole blood, log 10 bTRECs followed a normal distribution (kurtosis test, P = 0.70), with a mean of 1.75 ± 0.06 (minimum to maximum range, 0 to 3.1). Finally, the number of intrathymic divisions was also normally distributed across the healthy donors with detectable bTRECs (kurtosis test, P = 0.72), with a mean of 3.0 ± 0.21 (minimum to maximum range, -4.3 to 10.6; fig. S1D).

We evaluated whether TRECs are associated with any of 173 immune cell variables, defined through 10 eight-color immunophenotyping flow cytometry panels [START_REF] Patin | Milieu Intérieur Consortium, Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors[END_REF]. Using a generalized linear mixed model approach controlling for potential confounders and batch effects, sjTRECs were found to be strongly associated with naïve CD8 + and CD4 + T cell counts or other cell types that are known to develop within the thymus, including naïve T regs and invariant natural killer T (iNKT) cells (Fig. 1B and fig. S2). Naïve CD8 + T cell counts doubled with a 10-fold increase in sjTRECs [confidence interval (CI), 78 to 136%; Kenward-Rogers (K-R) approximate F test, adjusted P = 3 × 10 -47 ], whereas naïve CD4 + T cell, NKT cell, and naïve T reg counts showed 63% (CI, 40 to 88%; adjusted P = 3 × 10 -21 ), 40% (CI, 6 to 84%; adjusted P = 7 × 10 -3 ), and 44% (CI, 25 to 65%; adjusted P = 5 × 10 -13 ) increases, respectively, per 10-fold increase in sjTRECs (Fig. 1C). We also found significant associations of T cell compartments with bTRECs (fig. S3). Naïve CD8 + T cell, naïve CD4 + T cell, and naïve T reg counts showed 14% (CI, 6 to 22%, adjusted P = 9 × 10 -6 ), 11% (CI, 3 to 19%; adjusted P = 6 × 10 -4 ), and 8% (CI, 1 to 16%; adjusted P = 0.008) increases, respectively, per 10-fold increase in bTRECs (fig. S3).

Nonheritable factors associated with TREC amounts in the MI cohort

Not only several nonheritable factors have been previously identified as affecting thymic function, in particular aging [START_REF] Mitchell | Tracing thymic output in older individuals[END_REF], but also endocrine factors such as sex steroid and growth hormones, body mass index, and metabolic syndrome [START_REF] Taub | Insights into thymic aging and regeneration[END_REF][START_REF] Yang | Obesity accelerates thymic aging[END_REF][START_REF] Youm | Prolongevity hormone FGF21 protects against immune senescence by delaying age-related thymic involution[END_REF]. Subjects included in the MI cohort were surveyed for a large number of variables related to nutrition, sleep, smoking, vaccination, and medical history (18). From these, we selected 56 candidate variables that potentially affect thymic function (table S1 and Supplementary Materials and Methods) and applied linear mixed models, controlling for potential confounders and batch effects, to identify factors that contribute to variance in thymic function. We estimated with power simulations that our false-negative rate was <5% if the candidate variable explained >2.5% of the variance. We found no significant effects of cytomegalovirus (CMV) seropositivity, influenza A serostatus, metabolic score index, or C-reactive protein on sjTRECs and bTRECs (figs. S4 to S7). By contrast, age had a strong effect on sjTRECs and, to a lesser extent, on bTRECs and the number of intrathymic divisions (Fig. 2, A to C, and figs. S4 to S6). sjTRECs showed a decrease of 4.9% per year (CI, 4.3 to 5.6%; K-R F test, adjusted P = 4 × 10 -104 ; Fig. 2A) yet remained detectable in >95% of 60-to 69-year-old donors. Among donors with detectable bTRECs, we detected a 2% decrease per year (CI, 0.7 to 3.4%; adjusted P = 8 × 10 -5 ; Fig. 2B). We also observed fewer donors with detectable amounts of bTRECs as a function of age [odds of having detectable amounts decreased by 3.36% per year; CI, 1.76 to 4.99%; likelihood ratio test (LRT), adjusted P = 6 × 10 -10 ; fig. S5D]. Finally, we estimated a decrease of 0.26 intrathymic divisions every 10 years of age (CI, 0.03 to 0.5; K-R F test, adjusted P = 9 × 10 -3 ). Strikingly, sex also showed a strong effect on sjTREC amounts with 67% (CI, 38 to 102%; adjusted P = 2 × 10 -15 ) higher sjTREC amounts in women of all ages, relative to men (Fig. 2D). In contrast, no associations were found between sex and either the probability of having detectable bTRECs, bTREC amounts for donors with detectable bTRECs, or the number of intrathymic divisions (adjusted P > 0.05; figs. S5 and S6).

Association of a genetic variation at the TCRA-TCRD locus with sjTRECs

We next conducted a genome-wide association study of log 10transformed sjTREC numbers on 5,699,237 common single-nucleotide polymorphisms (SNPs) with a linear mixed model adjusted for age, sex, genetic relatedness, and other covariates selected using a datadriven variable selection scheme [START_REF] Zhou | Efficient multivariate linear mixed model algorithms for genome-wide association studies[END_REF]. No association was detected at genome-wide significance (LRT, P < 5.0 × 10 -8 ). Nevertheless, seven independent genomic regions on chromosomes 2, 4, 5, 10, 11, 14, and 17 showed suggestive evidence for association (LRT, P < 1.0 × 10 -5 ; Fig. 3A). To test for replication of these suggestive associations, we measured sjTRECs in an independent cohort, the Marseille Thrombosis Association study (MARTHA) cohort, which includes 612 unrelated patients of European descent affected with venous thromboembolism [START_REF] Morange | KNG1 Ile581Thr and susceptibility to venous thrombosis[END_REF]. We validated in this cohort the association of decreased sjTRECs with increasing age (4.05% per year; CI, 3.55 to 4.56%; K-R F test, P = 5 × 10 -45 ; fig. S8A), and their higher abundance in women, relative to men (86%; CI, 60 to 116%; P = 1.6 × 10 -15 ; fig. S8B). Among 14 SNPs tagging the seven suggestively associated loci, only variants on chromosome 14 showed statistical evidence for replication in the MARTHA cohort (table S2). Clave et al., Sci. Transl. Med. 10, eaao2966 (2018) Clave et al., Sci. Transl. Med. 10, eaao2966 (2018) 5 September 2018

SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

of 11

These variants all mapped within a 25-kb region included in the TCRA-TCRD locus (Fig. 3B).

To fine-map the signal, we genotyped the eight most informative imputed SNPs within this region in the MI cohort and combined these data with array-based or imputed genotype data from the MARTHA cohort. This led us to identify four SNPs in linkage disequilibrium (rs8013419, rs10873018, rs12147006, and rs2204985) with genome-wide statistical significance (DerSimonian and Laird meta-analysis, P < 2 × 10 -8 ; table S2) located in the intergenic DD2 and DD3 segments (Fig. 3C). Among them, rs2204985 (located 472 bases upstream of DD3) was considered the most likely candidate variant (effect allele frequency of 0.49; meta-analysis, P = 1.9 × 10 -8 ; table S2) because it is located in an open-chromatin region targeted by the transcription factors Runt-related transcription factor 3 (RUNX3), E74-like factor 1 (ELF1), FOXM1, and RNA polymerase II according to the Encyclopedia of DNA Elements (ENCODE) consortium reference data set (Fig. 3D) [START_REF]An integrated encyclopedia of DNA elements in the human genome[END_REF].

Influence of the TCRA-TCRD genetic polymorphism on T cell development in immunodeficient mice

Immunodeficient mice engrafted with human hematopoietic stem cells (HSCs) are able to develop a diverse repertoire of thymusdependent human T cells [START_REF] Halkias | Conserved and divergent aspects of human T-cell development and migration in humanized mice[END_REF]. To directly evaluate in vivo the impact of the rs2204985 polymorphism on thymopoiesis, we reconstituted immunodeficient Balb/c Rag2 -/-Il2rg -/-Sirpa NOD (BRGS) mice [START_REF] Lopez-Lastra | A functional DC cross talk promotes human ILC homeostasis in humanized mice[END_REF] with human CD34 + hematopoietic progenitors harvested from fetal livers having different genotypes for the rs2204985 variant (Fig. 4A). Controlling for mouse recipient sex in a linear model, we observed significantly higher sjTRECs (multiplicative effect size CI, 1.24 to 2.16; t test, P = 7 × 10 -4 ; Fig. 4B) and total CD3 + thymocyte numbers (multiplicative effect size CI, 1.63 to 3.92; P = 9 × 10 -5 ; Fig. 4C) in thymi of mice reconstituted with CD34 + progenitors of the rs2204985 GG genotype, as compared to mice reconstituted with AA or GA genotypes. Significant results were also obtained when controlling for the origin of the human fetal liver sample (1.6 times increase in sjTRECs: CI, 1.1 to 2.5; K-R F test, P = 0.047; 2.5 times increase in thymocytes: CI, 1.54 to 4.25; P = 6.6 × 10 -3 ). We next studied thymocyte developmental stages on human CD45 + cells by flow cytometry (fig. S9). We observed that mice grafted with cells from rs2204985 genotype GG donors had larger thymocyte counts at all stages, starting as early as the CD3 -CD4 -CD8 -DN population (Fig. 4D). These data support the hypothesis of a T cell-intrinsic effect of the identified genetic variant, which associates with thymocyte counts.

As shown in fig. S1, sjTRECs are produced by the dRec-YJa(Ja61) recombination leading to the prominent TCRD locus deletion. However, there are alternative rearrangements including the one between dRec and Ja58 gene segments that represents 23% of total dRec rearrangement (fig. S1B) [START_REF] Verschuren | Preferential rearrangements of the T cell receptor-delta-deleting elements in human T cells[END_REF]. We found a similar effect of the rs2204985 genotype on the alternative dRec-Ja58 rearrangement as on sjTRECs (fig. S10), excluding an effect of the genetic variant on the Ja segment usage during primary TCRA rearrangements. Evaluating the TCRA-TCRD repertoire diversity according to rs2204985 genotypes (table S3), we found that the numbers of total and productive rearrangements did not differ (Mann-Whitney U test, P > 0.05). We found no specific overlap of TCRA-TCRD clonotypes, as calculated by the Morisita index, between mice grafted with the same fetal liver CD34 + cells or even with the same rs2204985 genotype (fig. S11). Conversely, repertoire diversity, as quantified by productive clonality or Shannon equitability indexes, was significantly greater in mice grafted with cells of the GG genotype (Mann-Whitney U test, P = 0.016 and P = 0.003, respectively; table S3). Whereas no differences in TCRAV and TCRAJ gene segment usage were observed among mice grafted with cells of the AA or GG genotypes (Mann-Whitney U test, P > 0.05; Fig. 5, A andB), large differences were found in TCRDV and TCRDJ usage, with a preferential usage of gene segments close to the variant region (DJ, DV2, and DV3) in rs2204985 AA individuals (adjusted P < 0.05; Fig. 5B). Accordingly, the calculated frequency of T cells carrying a productive TCRD rearrangement was higher in AA individuals (Mann-Whitney U test, P = 0.012; Fig. 5C). A more detailed analysis of TCRDV and TCRDJ usage restricted to productive TCRD rearrangements showed that DV1, DD2, and DJ1 segments were used preferentially in GG, whereas DV2, DD3, and DJ3 were used preferentially in AA individuals (Fig. 5D), confirming that the rs2204985 variant locally affects TCRD rearrangements.

Modeling the variance of thymic function in healthy adults

Finally, we developed a model that estimates TREC content in healthy adults as a function of the rs2204985 genotype, age, and sex. We combined data of the MI and MARTHA cohorts in a mixed model, controlling for population stratification and batch variables. We found a 43% increase of sjTRECs in rs2204985 GG homozygotes, relative to AA homozygotes in the MI cohort (marginal CI, 22 to 69%; Fig. 6A). Similarly, in the MARTHA cohort, we found a 44% increase of sjTRECs in rs2204985 GG homozygotes, relative to AA homozygotes (marginal CI, 21 to 71%; Fig. 6B). The relative contribution of age, sex, and the rs2204985 variant to the variance of log 10 sjTRECs was estimated to be 37.8,4.78,and 1.32% in the MI cohort and 25.6,8.5,and 1.3% in the MARTHA cohort, respectively (Fig. 6C). There was no indication that the effect of age on sjTRECs S2). Variants that are significantly associated at the genome-wide level are indicated in red. (D) Physical position of the four most strongly associated variants, relative to active transcription activity [START_REF]An integrated encyclopedia of DNA elements in the human genome[END_REF]. The position of Dd3 is indicated. Clave et al., Sci. Transl. Med. 10, eaao2966 (2018) was dependent on rs2204985 genotypes (CI: 0.94 to 0.95, 0.94 to 0.96, and 0.94 to 0.96 for AA, GA, and GG, respectively, in MI; CI: 0.95 to 0.97, 0.95 to 0.96, and 0.95 to 0.98 for AA, GA, and GG, respectively, in MARTHA). We next sought to express the effect of the TCRA-TCRD genetic variation as a function of "thymic age," defined as the age of a male carrying the AA genotype with sjTRECs equal to those predicted by a linear model fitted on age, sex, and the rs2204985 genotype, using combined data of the MI and MARTHA cohorts. We then estimated the difference between actual age and thymic age for women and men carrying the GG genotype of 18.5 years (CI, 15 to 22.2) and 7.3 years (CI, 4.57 to 10.1), respectively (Fig. 6D). To support the application of rs2204985 genotyping in future clinical studies, we have developed the Shiny application allowing interactive visualization of the MI data (https://mithymus.pasteur.fr) (fig. S12).

DISCUSSION

The thymus is the primary lymphoid organ where T lymphocytes are generated in the adaptive immune system of all vertebrates, through spatiotemporal interactions between thymocytes and specialized microenvironments [START_REF] Abramson | Thymic epithelial cells[END_REF]. The thymus is sensitive to insults received throughout life upon inflammation and infections, reflected in its functional decline with age [START_REF] Palmer | Thymic involution and rising disease incidence with age[END_REF][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][START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF]. It is, however, an extremely plastic tissue endowed with endogenous regenerative capacities after an acute damage during chemotherapies or irradiation (4,[START_REF] Lopes | Administration of RANKL boosts thymic regeneration upon bone marrow transplantation[END_REF][START_REF] Wertheimer | Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration[END_REF]. However, the parameters that control the levels of thymic function in homeostatic conditions remain largely unknown, an unmet need to develop precision and regenerative medicine. Here, by combining TREC quantification and a population immuno logy approach, we report the assessment of nongenetic and genetic determinants of thymic function in healthy adults.

sjTRECs are produced by the thymus and diluted out during T cell divisions [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF]. Taking into account the dynamics of TRECs and peripheral cell division in young healthy individuals, an average of 4% per year involution in thymic output was previously estimated on the basis of the dynamics of TRECs and peripheral cell division in young healthy individuals [START_REF] Bains | Quantifying thymic export: Combining models of naive T cell proliferation and TCR excision circle dynamics gives an explicit measure of thymic output[END_REF], which is in line with our values of a 4.9 and 4% decrease per year in sjTREC amounts in the MI and MARTHA cohorts, respectively. In addition, our study in immunodeficient mice reconstituted with human CD34 + HSC allowed a direct investigation of the impact of TCRA-TCRD genetic variation on the developing thymocytes independent of any peripheral dilution of TRECs. The only nonheritable factors that we found strongly affecting thymopoiesis in the healthy population were age and sex, with a higher thymic function in women relative to men. Previous studies reported higher thymic mass, as measured by computed tomography in young (20 to 30 years old) women relative to young men [START_REF] Ackman | Sex difference in normal thymic appearance in adults 20-30 years of age[END_REF]. We demonstrate that the impact of sex on sjTRECs is observed during all of adulthood. In mice, androgens have a direct detrimental effect on stromal TECs [START_REF] Rode | Regenerative capacity of adult cortical thymic epithelial cells[END_REF], and male cortical TECs express low levels of genes implicated in thymocyte expansion and positive selection [START_REF] Dumont-Lagacé | Sex hormones have pervasive effects on thymic epithelial cells[END_REF]. We suggest that the sex differences observed in our study could similarly reflect sex differences in TEC function, resulting in a more efficient bilateral cross-talk between thymocytes and thymic stroma and higher thymopoiesis in women [START_REF] Abramson | Thymic epithelial cells[END_REF]. Overall, the strong and replicated effect of sex on TREC content reinforces the need of stratifying immunological studies by sex [START_REF] Markle | SeXX matters in immunity[END_REF].

Twin studies reported that RTE numbers are highly heritable, although no genetic associations have been found so far [START_REF] Roederer | The genetic architecture of the human immune system: A bioresource for autoimmunity and disease pathogenesis[END_REF]. In addition, naïve CD27 + CD4 T cell counts have a high estimated heritability in healthy twins [START_REF] Brodin | Variation in the human immune system is largely driven by non-heritable influences[END_REF] and in the MI cohort [START_REF] Patin | Milieu Intérieur Consortium, Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors[END_REF]. Collectively, these studies estimated a higher heritability of naïve rather than A/D V genes A/D J genes 0 50 100 % of DJ genes/total J genes % of use Alpha genes Alpha/delta genes Delta genes Fig. 5. Effects of TCRA-TCRD human genetic variation on thymic TCR repertoire in humanized immunodeficient mice. The human TCRA-TCRD locus was sequenced using genomic DNA from 8 (3 males and 5 females) and 12 (4 males and 8 females) immunodeficient mice thymi grafted with rs2204985 AA (orange) and GG (purple) human fetal livers, respectively (table S3). (A) Effects of the donor genotype on V (left) and J (right) gene usage, among TCRa and TCRd productive rearrangements. (B) Ratio of median percentage of V (left) or J (right) gene usage in GG-grafted mice, over that in AA-grafted mice. Gene segments used specifically by TCRd are indicated in red, by TCRa in gray, and shared by both in cyan. Whiskers indicate bias-corrected and accelerated bootstrap 95% CIs. (C) Effect of genotypes on the percentage of TCRD specific J genes (TCRDJ01 to 04) among total TCRD and TCRA J genes used in productive rearrangements. (D) Effect of genotypes on the percentages of DV (left), DD (center), and DJ (right) genes usages among TCRD productive rearrangements. Genes are ordered according to their genomic location (see Fig. 3B). Blue asterisks indicate P < 0.05 obtained using nonparametric Mann-Whitney U test, adjusted for multiple testing using the false discovery rate as error rate. differentiated T cells in the adaptive compartment and suggest that T cell generation could be under genetic control. We found that TRECs, used as the closest readout of TCR rearrangements, are influenced by genetic variation at the TCRA-TCRD locus, which offers insights into the TCR locus function. The TCRA-TCRD locus is organized in a single genetic locus contributing to two different TCR specificities, TCRgd and TCRab. It therefore requires a complex program to regulate chromatin accessibility of TCRA and TCRD gene segments to the recombination machinery at two different developmental stages [START_REF] Carico | Chromatin dynamics and the development of the TCRa and TCRd repertoires[END_REF]. The four most associated variants are located in a short segment spanning 4 kb within the DD2 and DD3 intergenic region, in a close 5′ position to the TCRd enhancer (Ed). Our best candidate variant, rs2204985, is located in an openchromatin region [START_REF]An integrated encyclopedia of DNA elements in the human genome[END_REF] close to a CCCTC-binding factor binding site, a critical element mediating chromatin looping and the access of the recombination machinery to the chromatin [START_REF] Carico | Chromatin dynamics and the development of the TCRa and TCRd repertoires[END_REF][START_REF] Chen | A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRd and TCRa repertoires[END_REF].

DV genes DJ genes T C R D J 1 T C R D J 4 T C R D J 2 T C R D J 3 T C R D V 3 T C R D V 2 T C R D V 8 T C R D V 7 T C R D V 5 T C R D V 1 T C R D V 6 T C R D V 4 A AA GG CD 5
Although the precise molecular mechanisms underlying the observed association of sjTRECs with genetic polymorphism will require further studies, the data collected in immunodeficient mice experiments allow generation of some hypotheses. The TCRd rearrangement is the first to occur at the earliest CD34 + CD38 -CD1a - DN stage [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF] and is tightly ordered in humans, DD2-DD3 rearrangements occurring before DD2-DJ1 rearrangements [START_REF] Cieslak | RUNX1-dependent RAG1 deposition instigates human TCR-d locus rearrangement[END_REF]. dREC-yJa rearrangements measured with sjTRECs are first detected in immature single-positive cells and reach peak levels in singlepositive thymocytes [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF]. We show in our study the effect of the rs2204985 variant, or a close genetic element in linkage disequilibrium, on DN thymocyte numbers before sjTREC generation and on DV and DJ usages. This supports a direct role of the genetic variation at an early stage of thymocyte differentiation. Our interpretation is that the higher sjTRECs and TCR diversity in mice engrafted with the rs2204985 GG genotype could relate to a higher rate of T cell generation starting at the early DN stage. The higher usage of DV1, as well as sjTRECs, in GG as compared to AA genotypes excluded a possible effect of the genetic variation on the reciprocal usage of DV1 and dREC. The Ed element is a major regulator of TCRD accessibility in DN thymocytes [START_REF] Monroe | Developmental regulation of TCR delta locus accessibility and expression by the TCR delta enhancer[END_REF], functioning over a limited chromosomal distance [START_REF] Bassing | T cell receptor (TCR) a/d locus enhancer identity and position are critical for the assembly of TCR d and a variable region genes[END_REF]. It has been suggested that Ed may require additional upstream elements to promote TCRD accessibility [START_REF] Bassing | T cell receptor (TCR) a/d locus enhancer identity and position are critical for the assembly of TCR d and a variable region genes[END_REF]. Hence, we hypothesize that the rs2204985 variant, or a genetic element nearby, could influence chromatin conformation and TCRD accessibility directly or through the binding of transcription factors and participate in the regulation of the TCRD recombination center [START_REF] Zhao | Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development[END_REF]. It will be interesting to investigate whether this polymorphism affects the generation of the different TCRgd T cell subsets [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF][START_REF] Cieslak | RUNX1-dependent RAG1 deposition instigates human TCR-d locus rearrangement[END_REF]. It remains also to be explained how the TCR genetic polymorphism could be linked to thymocyte survival or thymocyte proliferation at the DN stage. Notably, physiological DNA doublestrand breaks generated in developing lymphocytes activate a broad transcriptional program [START_REF] Bredemeyer | DNA double-strand breaks activate a multi-functional genetic program in developing lymphocytes[END_REF], some of them promoting lymphocyte survival via, for instance, the activation of p38MAPK in DN thymocytes [START_REF] Pedraza-Alva | Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint[END_REF]. In addition, transcription factors binding the rs2204985 genomic region might affect DN survival/proliferation, such as FOXM1, which is required for cellular proliferation in normal cells [START_REF] Bella | FOXM1: A key oncofoetal transcription factor in health and disease[END_REF]. It is intriguing to find evidence for genetic control of T cell generation in loci deleted in all mature peripheral T cells, TCRgd through TCRD rearrangements and TCRab T cells through sjTREC generation. This suggests selective pressure at a critical step in T cell development, which might be otherwise unnecessary or possibly harmful if functional in the periphery. In support of the pathogenic potential of this genomic region is its proposed involvement during oncogene activation in T cell acute lymphoblastic leukemia [START_REF] Le Noir | Extensive molecular mapping of TCRa/d-and TCRb-involved chromosomal translocations reveals distinct mechanisms of oncogene activation in T-ALL[END_REF].

By providing reference values of thymic function in a large healthy population via a key genetic control, our data provide a resource that may be useful in the context of precision medicine and regenerative strategies for diverse diseases. This study contributes to a better understanding of aging of the immune system, a major public health concern (50). We showed that the decrease in thymic output with age was different in men and women and was independent of several environmental factors, including latent CMV infection, previously shown to associate with exhaustion of differentiated T cells [START_REF] Patin | Milieu Intérieur Consortium, Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors[END_REF][START_REF] Nikolich-Zugich | Known unknowns: How might the persistent herpesvirome shape immunity and aging?[END_REF]. About 50% of the variance in sjTREC numbers remained unexplained, suggesting a role for still unknown environmental or genetic factors. Nonetheless, we showed differences in healthy thymic function depending on the TCRA-TCRD genetic variation in two independent cohorts of western European origin. It is important to estimate this impact in other ethnic groups, especially given the differences in frequency of the rs2204985 G allele across populations, ranging from 25% in East Asia to >80% in South America (52). ). Regression lines were fitted using linear regression. P values were obtained with a mixed model of log 10 (sjTRECs), including rs2204985 genotypes as predictor; covariates were selected using a data-driven variable selection scheme; and correcting for population stratification was performed using the genetic relatedness matrix (GRM) as a random effect. Considering the clinical implications of our findings, we anticipate that there may be settings where it would be beneficial to achieve a higher potential for T cell production. This would be the case, for instance, in an uncomplicated allo-HSCT setting or in the recovery of lymphopenic conditions in young patients. In contrast, it would be detrimental to fuel the system if the thymic environment is damaged as, for instance, in older individuals, in graft versus host disease in allo-HSCT [START_REF] Krenger | Thymic T-cell development in allogeneic stem cell transplantation[END_REF][START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF] or in autoimmune conditions where women are known to have an overall higher susceptibility [START_REF] Dragin | Estrogen-mediated downregulation of AIRE influences sexual dimorphism in autoimmune diseases[END_REF]. Such cases could result in the generation of T cells defective in their selection process with an autoreactive potential which could be pathogenic.

MATERIALS AND METHODS

Study design MI cohort

The 1000 healthy donors of the MI cohort were recruited from September 2012 to August 2013 by Biotrial, stratified by sex (500 men and 500 women) and age (200 individuals from each decade between 20 and 69 years of age). Donors were selected on the basis of inclusion and exclusion criteria detailed elsewhere (18). To avoid the influence of hormonal fluctuations in women during the perimenopausal phase, only pre-or postmenopausal women were included. To avoid issues related to population stratification, the study was restricted to French citizens with Metropolitan French origin for three generations. The clinical study was approved by the Comité de Protection des Personnes-Ouest 6 on 13 June 2012 and by the French Agence Nationale de Sécurité du Médicament on 22 June 2012. The study is sponsored by the Institut Pasteur (Pasteur ID-RCB number: 2012-A00238-35) and was conducted as a single center study without any investigational product. The protocol is registered under ClinicalTrials.gov (study number NCT01699893). Primary data for the humanized mouse experiments are shown in table S5.

Replication cohort

Our replication cohort included 612 patients from the MARTHA cohort [START_REF] Morange | KNG1 Ile581Thr and susceptibility to venous thrombosis[END_REF]. Donors were all of European descent and were examined between January 1994 and October 2005 for having suffered a single venous thrombosis event, without detectable cause. The study was approved by the Institutional Ethics Committee ("Département Santé de la Direction Générale de la Recherche et de l'Innovation"; Projects DC: 2008-880 & 09.576), and written informed consent was obtained from each subject. MARTHA biobank is hosted by the HEMOVASC bioresource center (CRB APHM). sjTRECs of all donors were quantified in DNA extracted from blood. Genotypes for candidate variants were obtained from the Illumina Human610-Quad SNP array [START_REF] Morange | KNG1 Ile581Thr and susceptibility to venous thrombosis[END_REF] or probe-based genotyping.

Statistical analysis

We tested for association between TRECs and immunophenotypes, and TRECs and nonheritable factors by fitting linear mixed models, using the mmi R package (https://github.com/jacobbergstedt/ mmi). The CIs were false coverage-adjusted intervals designed to keep the rate of false coverage at 5%. Hypothesis tests were done using K-R F tests with the false discovery rate as error rate. Impact of nonheritable factors on bTREC detection status was analyzed using logistic regression and LRTs. Genome-wide association studies were conducted using linear mixed models controlling for nonher-itable variables and using the GRM as one of the correlation matrices. A similar model was used to compute effect sizes and 95% CIs for the rs2204985 polymorphism, age, and sex, with respect to sjTRECs in the MI cohort. For the MARTHA cohort, the four principal components of the genotype matrix that explained most variance were used instead of the GRM, and the hypothesis test was conducted using the K-R F test. The DerSimonian and Laird method was used to compute the meta-analysis P values. Both linear regression models and linear mixed models were used to compute 95% CIs and P values for the effect of the rs2204985 polymorphism on sjTREC numbers and thymus T cell progenitors in humanized mice. For gene segment usage, nonparametric 95% CIs were estimated by a bootstrap procedure. Thymic age and proportion of variance was estimated from a linear regression model with log 10 -transformed sjTRECs as response and age, sex, and the rs2204985 polymorphism as predictors. Details on these analyses can be found in the Supplementary Materials. Clave et al., Sci. Transl. Med. 10, eaao2966 (2018) T. hold a patent on "Common genetic variations at the TCRA-TCRD locus control thymic function in humans" (PCT/EP2018/055873). All other authors declare that they have no competing interests. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials. TCR repertoire data are accessible through ImmunACCESS database under the "Genetic of Thymic function in hMice" name project (https://clients.adaptivebiotech.com/ immuneaccess). The SNP array data have been deposited in the European Genome-Phenome Archive with the accession code EGAS00001002460. The flow cytometric data and the code implementing statistical analyses can be downloaded as an R package (http://github.com/JacobBergstedt/mmi) and explored with the online Shiny application (http://milieu-interieur.cytogwas.pasteur.fr/).

Composition of the Milieu Intérieur Consortium:

The MI Consortium is composed of the following team leaders: Laurent Abel (Institut Imagine, Paris); Andres Alcover, Hugues Aschard, and Kalle Åström (Lund University, Lund); Philippe Bousso, Pierre Bruhns, Ana Cumano, Caroline Demangel, Ludovic Deriano, James Di Santo, Françoise Dromer, Darragh Duffy, Gérard Eberl, Jost Enninga, and Jacques Fellay (EPFL, Lausanne); Magnus Fontes, Antonio Freitas, Odile Gelpi, Ivo Gomperts-Boneca, Milena Hasan

Materials and Methods

DNA extraction from human whole blood

Blood was collected in 5 ml EDTA tube and was kept at room temperature (18-25°C) until processing. DNA extraction was performed using the Nucleon BACC3 kit (GE-Healthcare). Upon arrival at the processing site, blood was transferred into a 50 ml polypropylene tube. 20 ml of sterile Reagent A 1x (lysis buffer) were added to the blood sample in aseptic conditions and mixed by rotation for 4 minutes at room temperature.

After red blood cell lysis, the tube was centrifuged 1300g for 5 minutes and the supernatant was discarded. The cell pellet was resuspended with 40 µl of PBS1X, transferred to a 0.5 ml 2D-cap tube and stored at -80°C before processing. After thawing, 1 ml of sodium Reagent B was added directly to the cell pellet for resuspension before transfer to a 15ml screw capped propropylene centrifuge tube. 250 μl of sodium perchlorate solution were then added for deproteinisation and the tube was mixed by inverting the tube at least 7 times. 1 ml of chloroform was then added to the tube, which was mixed by inversion.

Without remixing the phases, 150 μl of Nucleon resin were added and the tube, which was then centrifuged at 1300g for 3 minutes. Without disturbing the Nucleon resin layer, the upper phase was transferred to a clean 15 ml tube and 2 volumes of cold absolute ethanol were added. The tube was then mixed by inversion until the DNA precipitate appeared.

Using a heat-sealed Pasteur pipette, the precipitated DNA was hooked out and placed into a clean 1.5 ml microcentrifugation tube. 1 ml of cold 70% ethanol was added, the DNA was washed and the supernatant was discarded after centrifugation at 4000g for 5 minutes.

3 After DNA pellet air dry for 10 minutes, 400 µl of deionized water were added and the tube kept overnight at 4°C to complete the resuspension before DNA quantification.

T-cell receptor excision circle (TREC) assays

sjTRECs and βTRECs are episomal circular DNAs generated during TCR α and β chain recombination, respectively (Fig. 1a and fig. S1). We implemented the protocol from Clave et al. (16), to quantify sjTRECs and βTRECs. The protocol is based on a quantitative PCR of genomic DNA extracted from whole blood, using the Biomark HD system (Fluidigm France, Paris, France). 1 to 2 µg of genomic DNA was preamplified for 3 minutes at 95°C and then 18 cycles of 95°C for 15 seconds, 60°C for 30 seconds and 68°C for 30 seconds, in a 50 µl reaction that contained the primers listed in table S4, Assay Biomark that contains only the 2 primers and the probe specific for each assay. The sum of the 10 βTRECs was multiplied by 1.3 in order to take into account the 3 Dβ-Jβ that were not quantified (Dβ2-Jβ2. 5, 2.6 and 2.7). sjTRECs and βTRECs were normalized to 150 000 cells (around 1 µg of DNA) using the Albumin gene quantification. As log10transformed values of βTRECs (log10 βTRECs) showed a bimodal distribution, we analyzed it as either a binary variable (0 if undetectable; 1 if detectable), or a quantitative variable in donors with detectable βTRECs only (log10 βTRECs>0). When comparing βTRECs and flow cytometry measures, we included donors with levels below detection limit, and put their value at the detection limit. The number of intrathymic division was obtained using the log2 of sjTRECs/βTRECs ratio, and could thus be calculated in donors with detectable βTRECs only. When indicated, a shorter probe (sjTREC-LNA) was used for sjTREC quantification, which contains only the 26 first nucleotides of the standard probe and 4 Locked Nucleic Acids (LNA) in order to keep the same Tm as the original probe (table S4). From the 1000 MI donors, 979 DNA were available for TREC analysis and 10 were further excluded because of low Albumin quantification (less than 50,000 copies).

DNA genotyping and imputation

The 1,000 subjects were genotyped at 719,665 SNPs by the HumanOmniExpress-24 BeadChip (Illumina, California) [START_REF] Patin | Milieu Intérieur Consortium, Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors[END_REF]. To increase coverage of rare and potentially functional variation, 966 of the 1,000 donors were also genotyped at 245,766 exonic SNPs by the California). A total of 945,213 unique SNPs were thus genotyped. SNP quality-control filters yielded a total of 661,332 and 87,960 SNPs for the HumanOmniExpress and HumanExome BeadChips, respectively. The two datasets were then merged. Average concordance rate for the [START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF]753 SNPs shared between the two genotyping platforms was 99.9925%. The final dataset included 732,341 QCfiltered genotyped SNPs. Genotype imputation was performed by IMPUTE v.2 (54), considering 1-Mb windows and a buffer region of 1 Mb. After quality-control filters, a total of 11,395,554 high-quality SNPs were obtained, which were further filtered for minor allele frequencies >5%, yielding a final set of 5,699,237 SNPs for association analyses.

Probe-based genotyping of candidate variants

Eight SNPs (table S2) were selected for confirmation and fine mapping, on the basis of the availability of accurate TaqMan© assays and their strength of association.

Genotyping was done on the Biomark HD (Fluidigm, San Francisco). Briefly, 60 to 120 ng of genomic DNA were preamplified with 0.2X of the 8 TaqMan Genotyping Assays (ThermoFisher Scientific, Massachusetts) and 1x Preamp Master Mix (Fluidigm) for 14 cycles (95°C for 15 sec, 60°C for 60 sec). The FLEXsix Genotyping IFCs (Fluidigm) were loaded with 1/50th dilution of the preamplified product, 2X Takyon Low Rox Probe MM (Eurogentec) and 40X TaqMan Genotyping Assay (ThermoFisher Scientific) according to manufacturer's instructions.

Reconstitution of Human Immune System (HIS) mice

Mouse strain

HIS mice were generated in Balb/c Rag2 -/-Il2rg -/-Sirpa NOD (BRGS) recipients using human fetal liver hematopoietic stem cells as previously described [START_REF] Lopez-Lastra | A functional DC cross talk promotes human ILC homeostasis in humanized mice[END_REF]. Briefly, newborn mice (3 to 5 days of age) received sublethal irradiation (3 Gy) and were injected intrahepatically with the equivalent of 2.0x10 5 CD34 + CD38 -human fetal liver cells. A total of 92 HIS mice in 15 independent experiments (4-10 mice per experiment) were analyzed at 8-29 weeks of age. Thymocytes and splenocytes were mechanically dissociated using a Cell Strainer (100µm nylon Falcon ® ). Cells (5.0x10 5 ) were frozen as dry pellet. DNA was prepared using the Proteinase K method (54°C for 1 min, 95°C for 10 min).

Human and mouse sex determination and 1 unit of HiFi Taq Platinum (Thermofisher). Cycling conditions were 94°C for 5 min and 40 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 2 min. PCR product was subsequently loaded on a 0.8% agarose gel giving a 1329 pb band for ZFX and a 906 pb band for ZFY. Sexing of mouse recipient was made single amplification of the SRY/IL3 genes in 25 µl PCR using mSRY and mIL3 primer pairs, 200 µM each dNTPs, 1.5 mM MgSO4 and 1 unit of HiFi Taq Platinum (Thermofisher). Cycling conditions were 94°C for 3 min and 35 cycles of: 94°C for 15 sec, 57°C for 15 sec and 72°C for 30 sec, and, 72°C for 5 min. PCR product was subsequently loaded on a 1.5% agarose gel giving a 402 pb band for SRY and a 544 pb band for IL3.

Quantification of T-cell Receptor (TCR) Excision Circles (TRECs) in HIS mice

TREC quantification adapted from Clave et al. [START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF] was performed in all 92 HIS mice samples. Real time quantification was made using ViiA7 (Applied Biosystems by Life Technologies, Austin, TX, USA) in 384-well plates loaded with 20 µl containing 5 µl of DNA (0.5 to 1 µg of genomic DNA), 10 µl of 2x Takyon Low Rox Probe MM (Eurogentec) and 5 µl of specific primer-probe mix (table S4). In addition, the quantification of the alternative rearrangement in the TCRδ locus (called δRec-Jα58) of TRECs in HIS mice was made using ViiA7 in 384-well plates loaded with 20 µl containing 5 µl of DNA (0.5 to 1 µg of genomic DNA), 10 µl of 2x Takyon Low Rox Probe MM (Eurogentec) and 5 µl of specific primer-probe mix (table S4). sjTRECs were normalized to 150 000 cells using the Albumin gene quantification. The samples were genotyped to the SNPs rs2204985 and rs10873018 with 5 µl containing 2 µl of DNA (10 to 20ng of genomic DNA), 0.25 µl of 2x Takyon Low Rox Probe MM (Eurogentec) and 2.5 µl of 40X TaqMan Genotyping Assay (ThermoFisher Scientific) according to manufacturer's instructions.

DNA genotyping in HIS mice

Immunophenotyping of human thymocytes in HIS mice

Flow cytometry was performed in 49 of the 99 mice (22 males, 27 females), reconstituted from 8 human liver donors (3 males, 5 females) (table S5). Total thymus cell number was determined by trypan blue cell staining. Then, 10 6 cells were labelled with human specific CD45 PERCP-Cy5.5, CD8 PE, CD4 APC H7, CD3 V500 AmCyan and CD1a FITC monoclonal antibodies (All from BD Biosciences), then read on a FACSCanto II flow-cytometer. Data were analyzed using FACSDiva software (BD Biosciences).

Next Generation sequencing of the TCRAD locus recombination

Sequencing of TCRAD locus in HIS mouse thymi was performed on genomic DNA at Adaptive Biotechnologies (Seattle, USA) using the immunoSEQ assay at the survey level. Numbers of sequenced rearrangements are listed in table S3. Analysis of diversity was made using the ImmunoSEQ software tool and Morisita's index (fig S11) using the R tcR package.

Association between TREC measurements and immunophenotypes

Immunophenotyping was conducted on whole blood from all donors. Details on technical procedures and complete results are available in Patin et al. (19). Ten 8-color flow cytometry panels were developed, allowing for the measurement of 168 traits, including 76 cell counts, 89 MFI and 3 ratios. Five additional measurements were obtained from flow cytometry raw data, to test the association of TRECs with HLA-DR-memory T-cells. The minimum, median and maximum proportion of missing observations for the 173 variables were respectively, 0%, 7% and 25%. Missing data was imputed using the missForest R package [START_REF] Stekhoven | MissForest-Non-parametric missing value imputation for mixed-type data[END_REF].

We investigated the relationship between these immunophenotypes and sjTRECs and βTRECs as well as and the number of intrathymic divisions, by fitting linear mixed models. Substantial batch effects were observed in the flow cytometry data. To control for those batch effects, we included the day of blood draw as a random effect in the model. We also controlled for age, sex, cytomegalovirus (CMV) serostatus and smoking, the four factors having previously been shown to impact immunophenotypes [START_REF] Patin | Milieu Intérieur Consortium, Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors[END_REF]. Specifically, let y be one of the 173 immunophenotypes, x be either log10(sjTRECs), log10(βTRECs) or the number of intrathymic divisions. Then we fitted models that assume the following linear relationship for the i th individual: CMVi, Femalei, ExSmokeri and Smokeri are indicator functions (e.g., = if individual i is CMV + ). βTRECs below the limit of detection were set to the detection limit.

Hypothesis tests on the parameters were performed using Kenward-Rogers approximated F-tests, implemented in the pbkrtest R package [START_REF] Halekoh | A Kenward-Roger approximation and parametric bootstrap methods for tests in linear mixed models-The R Package pbkrtest[END_REF]. The 173 x 3 = 519 tests conducted at this stage were considered a multiple testing family, and corresponding Pvalues were corrected for multiple testing accordingly, using the false discovery rate (FDR) as error rate (figs. S2A and S3A). Confidence intervals of significantly-associated parameters (adj. P<0.05) were constructed using the profile likelihood as implemented in the lme4 package (Fig. 1B). The confidence intervals were adjusted for this selection to control the rate of false coverage over the selected intervals at 0.05 (57). All the flow cytometric data, together with source code used to perform analyses can be found in the mmi R package (github.com/jacobbergstedt/mmi).

Impact of non-genetic factors on TREC measurements

56 physiological and demographic variables (table S1) that potentially affect thymic function as suggested in the literature and that showed a sufficient frequency for analysis in our cohort were selected from the detailed dataset available in the MI cohort (18) (accessible on http://www.synapse.org/MilieuInterieur). Association between the 56 demographic variables and log10-transformed sjTRECs, log10-transformed βTRECs (in donors with detectable amounts), and the number of intrathymic divisions, was tested with linear mixed models. We estimated the false negative rate (1power) of these tests with the pwr.f2.test function of the pwr R package, assuming a sample size of n=969, a significance level of P=0.05/56=9x10 -4 and proportions of variance explained by a demographic variable ranging from 0 to 1.

Because of their strong impact on TRECs and other candidate demographic variables, age and sex were included in all models. Specifically, let y be either log10(sjTRECs), log10(βTRECs) or the number of intrathymic divisions, and x one of the candidate demographic variables, except age and sex. Then we fitted mixed models that assume the following relationship for the i th individual:

= + + + + + , (1)
10 with ~ , , ∈ { , … ,7} and ~ , , and where is either a linear term when takes ordered values, i.e., = , or a standard corner point parametrization with K-1 parameters, when is a categorical variable with K levels.

Removing the term related to gives the model used for age and sex. For βTREC levels, only donors with detectable amounts were included, leaving 464 observations. A total of 56 x 3 = 168 hypothesis tests were performed, using the Kenward-Rogers F-test approximation implemented in the pbkrtest R package.

We also investigated the impact of non-genetic variables on the βTREC detection status, defined as a binary variable that equals 0 if βTRECs were not detected, or 1 if βTRECs were detected. Specifically, let y be the βTREC detection status, and x one of the candidate demographic variables, except age and sex. Then we fitted logistic regression models:

ℙ = = + + + ,
where = log -, using the glm function in R. Removing the term related to gives the model used for age and sex. A total of 56 chi-square likelihood ratio tests were conducted using these models. In total 56 + 168 = 224 hypothesis tests were done. All these tests were considered as one multiple testing family, and corresponding P-values were corrected for multiple testing accordingly, using the FDR as error rate.

Genotype-wide association studies

Univariate genome-wide association study of log10-transformed sjTRECs was conducted using the linear mixed model implemented in GEMMA [START_REF] Zhou | Efficient multivariate linear mixed model algorithms for genome-wide association studies[END_REF]. Genetic relatedness matrix was estimated with GEMMA, using a leave-one-chromosome approach.

11

Because GEMMA cannot fit more random effect terms than the GRM, batch effects were not considered as random effects, as above, but as indicator terms. Covariates were selected using a variable selection scheme based around the elastic net and stability selection (58).

Among all demographic and batch effect variables, only age, sex and two TREC processing plates were selected and included as covariates in the model. The effect of a SNP on log10(sjTRECs) was tested with a likelihood ratio statistic, and considered to be genomewide significant if P<5x10 -8 .

Impact of the rs2204985 polymorphism on sjTRECs and thymocytes in humanized mice

To investigate the relationship between the rs2204985 polymorphism and sjTREC and thymocyte levels in mice we fitted the following linear models controlling for mouse sex:

log = + + + , and
log + + = + + + ,
where for both models are normally distributed residuals. In this section we assume an additive genotype model, (i.e. counts the number of G alleles. Inference for these models was done using Wald tests and confidence intervals.

Liver donor determines the genotype of the mice. It could also potentially affect phenotype values. The liver series origin could thus be a confounder, especially considering that we observed variability in sjTRECs and thymocyte counts between samples of different liver sample origin. We therefore also tested the impact of the rs2204985 genotype on mice sjTREC and thymocyte values when controlling for liver series origin. We did this by estimating a mean for each human liver donor using a random effect. This random effect 12 incorporates possible differences in age and sex of the human liver samples. Since mouse sex has no significant effect on sjTRECs in mice (P=0.288) we did not include it in this model. We therefore fitted the model:

log = + + + ,
where ~ , 2 is the mean for the human liver sample grafted in mouse i, ∈ { , … , 6} and ~ , 2 .

In contrast to sjTRECs, mouse sex showed an impact on thymocyte counts. This association remained even after controlling for human liver sample origin. To investigate the impact of the polymorphism on CD3 + CD45 + values we therefore fitted a similar mixed model as above but included mouse sex as a covariate. All models were fitted using the lme4 R package. Confidence intervals were constructed using the profile likelihood based method implemented in that package. For hypothesis tests on , because of the low number of degrees of freedom for these data and models, we chose not to rely on large sample approximations, and used instead parametric bootstrapping of likelihood ratios.

Effect of the rs2204985 SNP on sjTRECs in the MI and MARTHA cohorts

To estimate the effect of the rs2204985 polymorphism on sjTRECs, we chose to compute its effect size and its confidence interval by fitting a new model, including both ancestry and batch variables as random effects. This was done in R using the lme4qtl package (59). The computations in this package is performed by the lme4 package, which does not factorize the GRM prior to fitting models and is therefore not suitable for GWAS.

Because our initial analyses showed that age and sex are the main non-genetic factors impacting sjTRECs, it was natural to fit the following model to compute the effect size of the SNP in the MI cohort: Here G is the GRM. The lme4qtl package makes it possible to include a random effect term correlated according to a constant matrix like the ancestry term in the model above in a usual lme4 model object. However, possibly due to this added functionality, we were not able to compute the confidence interval of using the usual profile likelihood methods implemented in lme4. Instead we used a large sample Wald approximation.

We could not access the GRM matrix for the MARTHA cohort, and used instead the four principal components that explain the most variance of the SNP array data. To compute the effect size of the SNP in the MARTHA cohort, we therefore used the following model: The "thymic age" of an individual i is defined as the age of the AA male, i.e, , when {log | , , , } -= {log ( )| } -By the first two equations of this section this is equivalent to

+ ℎ = + + + + ⟺ -ℎ = 1 + +
which gives an expression for the difference between actual age and the thymic age. The 95% CI for this quantity was computed by parametric bootstrapping.

Proportion of sjTRECs variance estimation

The contribution of rs2204985 genotypes, age and sex to the explained variance of The proportion of variance explained by a particular predictor was estimated by averaging the sum of squares for that particular variable over different orderings in the regression model. The estimation was done using the relaimpo R package.

R Shiny interactive Web application

The interactive web application was developed using the Shiny package for the R statistical environment. The purpose of the webapp is to allow exploration of study results

around the SNP rs2204985. The models developed in the study were exposed in the app to give users the ability to make their own predictions using either real or synthetic data. All predictions are stored temporarily during the user's session and may be downloaded at any (A) 500 healthy men and 500 healthy women, stratified over 5 decades of life, were recruited in the MI study. For each donor, the circulating levels of sjTRECs and βTRECs were quantified by RT-PCR on DNA extracted from whole blood. In parallel, hundreds of demographic, medical and lifestyle variables were collected and extensive immunophenotyping and genome-wide genotyping were performed. (B) Genomic location in the TCRA-TCRD locus of primers (sjTREC-F and sjTREC-R, in red) and probe (sjTREC-P, in green) used for the sjTREC assay. Sequences of primers and probe are provided in table S1. (C) TRECs were normalized "per 150,000 cells", an estimate of 1μg

DNA content (n=969). Numbers of cells were evaluated using an RT-PCR targeting the Albumin gene, obtained simultaneously as TREC quantification. TRECs can also be normalized per μl of blood, using the absolute number of CD45+ cells obtained by flow cytometry (n=892). We confirmed in our cohort that the two normalized measures are tightly correlated for both sjTRECs and βTRECs (r 2 =0.99), and used the "number of TRECs per 150,000 cells" measurement in all subsequent analyses. (D) Distributions of log10-transformed sjTRECs (purple, n=969), log10-transformed βTRECs (blue, n=969), and the number of intrathymic divisions (green, n=506 because the ratio could not be calculated on the 463 donors negative for βTRECs) in the MI cohort. Over the 969 healthy donors, log10-transformed sjTRECs were approximately normally distributed. In contrast, βTRECs showed a bimodal distribution, with 463 donors under the threshold of detection of the assay (βTREC=0). Among donors with measurable whole-blood βTRECs, the log10transformed values approximately followed a normal distribution. The number of intrathymic divisions was estimated as the log2-transformation of the ratio between sjTRECs and βTRECs, and was approximately normally distributed in the cohort. (E) To

assess the reproducibility of the TREC assay on Biomark HD, technical replicates we made using 43 random donors from the cohort: first, two independent Biomark quantifications were done on the same preamplification plate (PCR1 and PCR2, r 2 =0.99) and secondly, two independent preamplifications were done on the same donors (PAmp 1 and PAmp 2; S3).

Although the A allele has a frequency below 8% and does not influence the binding of the probe at 60°C (as predicted in silico), we designed a shorter probe that contains only the 26 first nucleotides, thus excluding the rs76132819 SNP (table S1). Same Tm as original probe was obtained by using 4 Locked Nucleic Acids (LNA). We quantified sjTRECs on 58 MI donors. A high correlation was found between the two measures (r 2 =0.8). Regression lines were fitted using simple linear regression. Adjusted P-values were obtained using the mixed model described in (A). (A) Significance of the effect of demographic, medical and lifestyle variables (table S2) on βTREC levels, as estimated by the -log10

AA AA AA AA AA AA AA AA GG GG GG GG GG GG GG GG GG GG GG GG 45605T3RD 45609T3RD 45615T3RD 45742T3RD 47139T5TH47140T5TH47143T5TH47145T5TH47692T13TH 47693T13TH 47694T13TH 47695T13TH 47697T13TH 47702T13TH 47707T13TH 62T14TH 64T14TH 65T14TH 68T14TH 69T14TH AA 45605T3RD NA
0,08219 0,15453 0,36750 0,01368 0,08937 0,06151 0,04373 0,10516 0,01729 0,12829 0,12062 0,18010 0,18677 0,16889 0,53319 0,29839 0,05639 0,29951 0,21629 AA 45609T3RD 0,08219 NA 0,23644 0,11159 0,02027 0,15318 0,12258 0,11364 0,04756 0,02959 0,04026 0,03973 0,04860 0,05057 0,04956 0,05777 0,05498 0,05058 0,05634 0,05615 AA 45615T3RD 0,15453 0,23644 NA 0,22060 0,02318 0,22536 0,15961 0,09764 0,07293 0,03917 0,06848 0,06901 0,08736 0,09608 0,08187 0,11386 0,10061 0,07474 0,10395 0,08604 AA 45742T3RD 0,36750 0,11159 0,22060 NA 0,01442 0,11476 0,07355 0,05082 0,09241 0,02055 0,10748 0,10364 0,15118 0,15541 0,14029 0,32693 0,22199 0,05852 0,22348 0,16821 AA 47139T5TH 0,01368 0,02027 0,02318 0,01442 NA 0,03319 0,04447 0,03760 0,00872 0,00943 0,00911 0,00939 0,01075 0,00915 0,01082 0,01063 0,01039 0,01442 0,01049 0,00924 AA 47140T5TH 0,08937 0,15318 0,22536 0,11476 0,03319 NA 0,17450 0,09200 0,05342 0,03984 0,04770 0,04828 0,05994 0,05559 0,05701 0,06368 0,06117 0,06319 0,06011 0,05406 AA 47143T5TH 0,06151 0,12258 0,15961 0,07355 0,04447 0,17450 NA 0,13742 0,03905 0,03740 0,03367 0,03521 0,03969 0,04183 0,04297 0,04409 0,04242 0,06483 0,04370 0,03953 AA 47145T5TH 0,04373 0,11364 0,09764 0,05082 0,03760 0,09200 0,13742 NA 0,02634 0,02309 0,02362 0,02348 0,02282 0,03156 0,02865 0,02874 0,03022 0,03715 0,02782 0,03331 GG 47692T13TH 0,10516 0,04756 0,07293 0,09241 0,00872 0,05342 0,03905 0,02634 NA 0,01371 0,05920 0,05683 0,07165 0,06878 0,06953 0,09824 0,08057 0,03476 0,08470 0,07089 GG 47693T13TH 0,01729 0,02959 0,03917 0,02055 0,00943 0,03984 0,03740 0,02309 0,01371 NA 0,01226 0,01134 0,01416 0,01331 0,01294 0,01391 0,01471 0,01605 0,01364 0,01186 GG 47694T13TH 0,12829 0,04026 0,06848 0,10748 0,00911 0,04770 0,03367 0,02362 0,05920 0,01226 NA 0,06004 0,07926 0,07993 0,07588 0,12268 0,10124 0,03493 0,10182 0,08531 GG 47695T13TH 0,12062 0,03973 0,06901 0,10364 0,00939 0,04828 0,03521 0,02348 0,05683 0,01134 0,06004 NA 0,07736 0,07690 0,07532 0,11481 0,09434 0,03038 0,09573 0,07809 GG 47697T13TH 0,18010 0,04860 0,08736 0,15118 0,01075 0,05994 0,03969 0,02282 0,07165 0,01416 0,07926 0,07736 NA 0,10082 0,10107 0,17285 0,12986 0,03901 0,13740 0,10532 GG 47702T13TH 0,18677 0,05057 0,09608 0,15541 0,00915 0,05559 0,04183 0,03156 0,06878 0,01331 0,07993 0,07690 0,10082 NA 0,09734 0,18036 0,13906 0,03834 0,14226 0,11139 GG 47707T13TH 0,16889 0,04956 0,08187 0,14029 0,01082 0,05701 0,04297 0,02865 0,06953 0,01294 0,07588 0,07532 0,10107 0,09734 NA 0,16065 0,12769 0,04071 0,13078 0,10470 GG 62T14TH 0,53319 0,05777 0,11386 0,32693 0,01063 0,06368 0,04409 0,02874 0,09824 0,01391 0,12268 0,11481 0,17285 0,18036 0,16065 NA 0,30206 0,04945 0,30945 0,21547 GG 64T14TH 0,29839 0,05498 0,10061 0,22199 0,01039 0,06117 0,04242 0,03022 0,08057 0,01471 0,10124 0,09434 0,12986 0,13906 0,12769 0,30206 NA 0,04811 0,21370 0,16154 GG 65T14TH 0,05639 0,05058 0,07474 0,05852 0,01442 0,06319 0,06483 0,03715 0,03476 0,01605 0,03493 0,03038 0,03901 0,03834 0,04071 0,04945 0,04811 NA 0,04622 0,04376 GG 68T14TH 0,29951 0,05634 0,10395 0,22348 0,01049 0,06011 0,04370 0,02782 0,08470 0,01364 0,10182 0,09573 0,13740 0,14226 0,13078 0,30945 0,21370 0,04622 NA 0,16237 GG 69T14TH 0,21629 0,05615 0,08604 0,16821 0,00924 0,05406 0,03953 0,03331 0,07089 0,01186 0,08531 0,07809 0,10532 0,11139 0,10470 0,21547 0,16154 0,04376 0,16237 NA Fig. S10. Effect of the SNP rs2204985 polymorphism on the TCRD locus deletion alternative rearrangement δRec-Jα58. δRec-Jα58 rearrangement excision circles (fig. S1B) were evaluated in reconstituted thymi from 8-29 weeks old immunodeficient Balb/c Rag2 -/-Il2rg -/-Sirpa NOD (BRGS) mice reconstituted with human CD34+ hematopoietic progenitors harvested from fetal livers with rs2204985 genotype AA (orange, n=10), GA (brown, n=21) or GG (purple, n=12). Values were log10-transformed and P-value of a Kruskal-Wallis test is indicated. Productive clonality is calculated by normalizing productive entropy using the total number of unique productive rearrangements and subtracting the result from 1. The max productive frequency is the maximum productive frequency value found within a sample. Shannon Equitability (or Pielou Evenness) is a normalized Shannon's entropy. was demonstrated that the only non-heritable factors strongly impacting thymopoiesis in the healthy population were age and sex, with a higher thymic function in women relative to men.

Also, the effect of sex on sjTRECs generation is observed during the entire adulthood. More importantly, it was demonstrated that TREC production, used as the closest readout of TCR rearrangements, is indeed influenced by a common genetic variation at the TCRA-TCRD locus, the single nucleotide polymorphism SNP rs2204985. We observed increased levels of sjTRECs in rs2204985 GG homozygotes relative to AA homozygotes in human peripheral blood. Similar result was observed in immunodeficient mice reconstituted with human CD34 + HSC (human stem cell), which showed increased sjTRECs in mice engrafted with the rs2204985 GG genotype (Clave et al., 2018). This prompted us to further investigate how the

DNA Genotyping and TREC quantification

Thymocytes (5x10 6 ) were frozen as dry pellet at -80°C after 1 min in liquid nitrogen. DNA was prepared using the Proteinase K digestion method (54°C for 1 min, 95°C for 10 min). We implemented the protocols from [START_REF] Clave | Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation[END_REF] to determinate the genotypes and to quantify the TREC levels in 68 of the 76 human samples. The samples were genotyped and the real time PCR quantification were made using ViiA7 (Applied Biosystems by Life Technologies, Austin, TX, USA).

ChIP assay

All steps in this protocol from fixation, lysis and sonication (Diagenode Bioruptor ® Pico)

were standardized to obtain the best results using human thymocytes using the iDeal ChIP-seq kit for Transcription Factors protocol by Diagenode.

Cells and chromatin preparation

Freshly isolated thymocyte suspension cells (1x10 8 ) were cross-linked using 1% formaldehyde (FA) and gentle shaking for 10 min at room temperature. Crosslinking was stopped by dropwise addition of glycine and gentle shaking for 5 min at room temperature.

Immediately, the fixed cells were dry pelleted and stored at -80°C after 1 min in liquid nitrogen. Overall the ChIP experiments were performed and validated in 24 of the 76 human thymic samples covering at least 3 samples of each genotype.

Antibodies and chromatin immunopreciptation

The Immunoprecipitation was performed with antibodies against proteins related to remodeling of chromatin structure: CTCF, Rad21 (cohesin) and H3K4me3; transcription machinery: RNAP II, and transcription factors involved in the differentiation and proliferation of thymocytes: RUNX3 and Forkhead box protein M1 (FOXM1). For IP, we calculated 12.5x10 6 of cells in comparison with the 4x10 6 used at the Transcription Factors protocol by Diagenode.

Quantitative-PCR and data analysis

Once the reverse cross-linking and DNA purification were performed, the gene segments associated with these antibodies were then amplified by real time PCR quantification were made using ViiA7 (Applied Biosystems by Life Technologies, Austin, TX, USA) using specific primers (Forward 5'-GGGCTACTGGCCTGAATTTAGCA-3' and Reverse 5'-GCTTCTGAATCACCTCTTCCTGCTT-3') for a region of 107 bp comprising the SNP

DISCUSSION AND PERSPECTIVES

The thymus is the primary lymphoid organ where T lymphocytes are generated in the adaptive immune system of all vertebrates, through spatiotemporal interactions between thymocytes and specialized microenvironments [START_REF] Abramson | Thymic Epithelial Cells[END_REF]. However, the parameters that control the levels of thymic function in homeostatic conditions remain largely unknown, urging for an unmet needs to develop precision and regenerative medicine.

Age-associated thymic involution is associated with a reduction in tissue mass and thymic cellularity, a loss of tissue structure and an abnormal architecture. This is thought to contribute to the reduction in T cell diversity seen in older individuals and linked with an increased susceptibility to infection, autoimmune disease, and cancer [START_REF] Palmer | The effect of age on thymic function[END_REF]. Also, the thymus is sensitive to insults received throughout life upon inflammation and infections, leading to a decline in naïve T cell output with age [START_REF] George | Thymic involution with ageing: obsolescence or good housekeeping?[END_REF][START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF][START_REF] Taub | Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo[END_REF][START_REF] Lynch | Thymic involution and immune reconstitution[END_REF][START_REF] Ferrando-Martinez | Thymic function failure is associated with human immunodeficiency virus disease progression[END_REF][START_REF] Palmer | Thymic involution and rising disease incidence with age[END_REF]. It is, however, an extremely plastic tissue endowed with endogenous regenerative capacities after an acute damage during chemotherapies or irradiation [START_REF] Boehm | Thymus involution and regeneration: Two sides of the same coin?[END_REF][START_REF] Lopes | Administration of RANKL boosts thymic regeneration upon bone marrow transplantation[END_REF][START_REF] Wertheimer | Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration[END_REF]. In humans, the relationship between thymic activity and naïve T cell homeostasis is a matter of debate, with the recent observations that peripheral proliferation and not thymic output contributes to the maintenance of naïve T cells in young adults [START_REF] Den Braber | Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans[END_REF]. Twin studies reported that RTE numbers are highly heritable, although no genetic associations have been found so far [START_REF] Roederer | The genetic architecture of the human immune system: A bioresource for autoimmunity and disease pathogenesis[END_REF]. In addition, naïve CD27 + CD4 T cell counts have a high estimated heritability in healthy twins [START_REF] Brodin | Variation in the human immune system is largely driven by non-heritable influences[END_REF] and in the MI cohort [START_REF] Patin | Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors[END_REF].

Collectively, these studies estimated a higher heritability of naïve rather than differentiated T cells in the adaptive compartment and suggested that T cell generation could be under genetic control. Here, by combining TREC quantification and a population immunology approach.

We report the assessment of nongenetic and genetic determinants of thymic function in healthy adults. SjTRECs are produced by the thymus and diluted out during T cell divisions [START_REF] Douek | Changes in thymic function with age and during the treatment of HIV infection[END_REF]. Taking into account the dynamics of TRECs and peripheral cell division in young healthy individuals, involution appears to proceed at a steady rate. An average of 4% per year involution in thymic output was previously estimated on the basis of the dynamics of TRECs and peripheral cell division in young healthy individuals [START_REF] Bains | Quantifying Thymic Export: Combining Models of Naive T Cell Proliferation and TCR Excision Circle Dynamics Gives an Explicit Measure of Thymic Output[END_REF], which is in line with our values of a 4.9 and 4% decrease per year in sjTREC amounts in the MI and MARTHA cohorts, respectively. Studies examining human thymus suggesting a rate of 3% of thymic tissue is lost per year until middle age, followed by a rate of 1% per year [START_REF] Steinmann | Changes in the human thymus during aging[END_REF][START_REF] George | Thymic involution with ageing: obsolescence or good housekeeping?[END_REF] which perhaps may cease in later life with studies showing sjTREC levels being barely detectable in individuals over the age of 85 years [START_REF] Mitchell | Tracing thymic output in older individuals[END_REF][START_REF] Ferrando-Martínez | Age-related deregulation of naive T cell homeostasis in elderly humans[END_REF]. Considering the effect of the age on the thymic function will be essential on the future to study cohorts from elderly and at the opposite children and adolescents. The age range analyzed in our studies was between 20 to 69 years old, and therefore, does not include individuals before the puberty and individuals over 70 years old. The only nonheritable factors that we found strongly affecting thymopoiesis in the healthy population were age and sex, with a higher thymic function in women relative to men. Previous studies reported higher thymic mass, as measured by computed tomography in young (20 to 30 years old) women relative to young men [START_REF] Ackman | sex Difference in normal Thymic appearance in adults 20-30 Years of age[END_REF]. We demonstrate that the impact of sex on sjTRECs is observed during all of adulthood. In mice, thymic cellularity begins to decrease within the first few weeks after birth [START_REF] Ortman | Molecular characterization of the mouse involuted thymus: aberrations in expression of transcription regulators in thymocyte and epithelial compartments[END_REF][START_REF] Aw | It's not all equal: a multiphasic theory of thymic involution[END_REF] and a similar picture is evident in human thymus [START_REF] Steinmann | The involution of the ageing human thymic epithelium is independent of puberty. A morphometric study[END_REF][START_REF] Jamieson | Generation of functional thymocytes in the human adult[END_REF]. In addition, the androgens have a direct detrimental effect on stromal TECs [START_REF] Rode | Regenerative capacity of adult cortical thymic epithelial cells[END_REF], and male cortical TECs express low levels of genes implicated in thymocyte expansion and positive selection [START_REF] Dumont-Lagacé | Sex hormones have pervasive effects on thymic epithelial cells[END_REF]. We suggest that the sex differences during all of adulthood observed in our study could similarly reflect sex differences in TEC function due the maintenance of the tissue structure and prolonged "normal" architecture, resulting in a higher survival rate of TECs and a more efficient bilateral cross-talk between thymocytes and thymic stroma during migration and, finally, a higher thymopoiesis in women [START_REF] Abramson | Thymic Epithelial Cells[END_REF]). An often held view is that thymic involution is triggered during puberty. This is based on studies showing that sex steroids have a detrimental effect on thymocytes and that chemical or surgical castration in older rodents is able to restore thymic size [START_REF] Heng | Effects of castration on thymocyte development in two different models of thymic involution[END_REF][START_REF] Sutherland | Activation of thymic regeneration in mice and humans following androgen blockade[END_REF][START_REF] Griffith | Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth[END_REF]. We report in our study based in thymus from healthy children no impact of sex on sjTRECs during early childhood (data not showed). The young age of the individuals in this study (~ 20 weeks old) suggests that the exposure to sex hormones during pubertal development is implicated in the sex differences in the thymic function. However, the role of these steroids being responsible for initiating thymic involution is now being questioned [START_REF] Montecino-Rodriquez | Reevaluating current models of thymic involution[END_REF]. Indeed, several studies using a variety of thymic indices (cellularity, epithelial space, number of recent thymic emigrants) have observed that thymic involution occurs early in life, prior to puberty and that the rate of decline is not linear, but appears to be phasic, something which would need to be reevaluated in cohorts of children. Our most salient result is that TRECs, used as the closest readout of thymopoïesis, are influenced by genetic variation at the TCRA-TCRD locus, which offers newly insights into the TCR locus function. The TCRA-TCRD locus is organized in a single genetic locus contributing to two different TCR specificities, TCRγδ and TCRαβ. It therefore requires a complex program to regulate chromatin accessibility of TCRA and TCRD gene segments to the recombination machinery at two different developmental stages [START_REF] Carico | Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires[END_REF]. The four most associated variants are located in a short segment spanning 4 kb within the DD2 and DD3 intergenic region, in a close 5′ position to the TCRδ enhancer (Eδ). Our best candidate variant, rs2204985, is located in an open-chromatin region (ENCODE Project Consortium, 2012) close to a CCCTC-binding factor binding site, a critical element mediating chromatin looping and the access of the recombination machinery to the chromatin [START_REF] Carico | Chromatin Dynamics and the Development of the TCRα and TCRδ Repertoires[END_REF][START_REF] Chen | A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRd and TCRa repertoires[END_REF]. Although the precise molecular mechanisms underlying the observed association of sjTRECs with genetic polymorphism will require further studies, the data collected in immunodeficient mice experiments allow generation of some hypotheses. The TCRδ rearrangement is the first to occur at the earliest CD34 + CD38 -CD1a -DN stage [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF] and is tightly ordered in humans, DD2-DD3 rearrangements occurring before DD2-DJ1 rearrangements [START_REF] Cieslak | RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement[END_REF]. δREC-ΨJα

rearrangements measured with sjTRECs are first detected in immature single-positive cells and reach peak levels in single-positive thymocytes [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF]. We show in our study the effect of the rs2204985 variant, or a close genetic element in linkage disequilibrium, on DN thymocyte numbers before sjTREC generation and on DV and DJ usages. This supports a direct role of the genetic variation at an early stage of thymocyte differentiation. Our interpretation is that the higher sjTRECs and TCR diversity in mice engrafted with the rs2204985 GG genotype could relate to a higher rate of T cell generation starting at the early DN stage. The higher usage of DV1, as well as sjTRECs, in GG as compared to AA genotypes excluded a possible effect of the genetic variation on the reciprocal usage of DV1 and δREC. The Eδ element is a major regulator of TCRD accessibility in DN thymocytes [START_REF] Monroe | Developmental regulation of TCRδ locus accessibility and expression by the TCRδ enhancer[END_REF], functioning over a limited chromosomal distance [START_REF] Bassing | T cell receptor (TCR) locus enhancer identity and position are critical for the assembly of TCR and variable region genes[END_REF]. It has been suggested that Eδ may require additional upstream elements to promote TCRD accessibility [START_REF] Bassing | T cell receptor (TCR) locus enhancer identity and position are critical for the assembly of TCR and variable region genes[END_REF]. Hence, we hypothesize that the rs2204985 variant, or a genetic element nearby, could influence chromatin conformation and TCRD accessibility directly or through the binding of transcription factors and participate in the regulation of the TCRD recombination center [START_REF] Zhao | Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development[END_REF]. It will be interesting to investigate whether this polymorphism affects the generation of the different TCRγδ T cell subsets [START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF][START_REF] Cieslak | RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement[END_REF]. It remains also to be explained how the TCR genetic polymorphism could be linked to thymocyte survival or thymocyte proliferation at the DN stage. Notably, physiological DNA double-strand breaks generated in developing lymphocytes activate a broad transcriptional program [START_REF] Bredemeyer | DNA double-strand breaks activate a multi-functional genetic program in developing lymphocytes[END_REF], some of them promoting lymphocyte survival via, for instance, the activation of p38MAPK in DN thymocytes [START_REF] Pedraza-Alva | Activation of p38 MAP kinase by DNA double-strand breaks in V(D)J recombination induces a G2/M cell cycle checkpoint[END_REF]. We found that the GG homozygotes contribute to resource that may be useful in the context of precision medicine and regenerative strategies for diverse diseases. However, about 50% of the variance in sjTREC numbers remained unexplained, suggesting a role for still unknown environmental or genetic factors. We showed differences in healthy thymic function depending on the TCRA-TCRD genetic variation in two independent cohorts of western European origin. It will be interesting to investigate whether this polymorphism affects the T cell development in other ethnic groups [START_REF] Cho | Age estimation via quantification of signal-joint T cell receptor excision circles in Koreans[END_REF][START_REF] Loret De Mola | The Effect of Fetal and Childhood Growth over Depression in Early Adulthood in a Southern Brazilian Birth Cohort[END_REF][START_REF] Andrade | Conditions of gestation, childbirth and childhood associated with Cpeptide in young adults in the 1982 Birth Cohort in Pelotas-RS; Brazil[END_REF][START_REF] Silveira | Low birthweight and preterm birth: trends and inequalities in four population-based birth cohorts in Pelotas, Brazil, 1982-2015[END_REF], especially given the differences in frequency of the rs2204985 G allele across populations, ranging from 25% in East Asia to >80% in South America [START_REF] Auton | A global reference for human genetic variation[END_REF]. Considering the clinical implications of our findings, we anticipate that there may be settings where it would be beneficial to achieve a higher potential for T cell production. A better definition of immune status during aging, in patients undergoing immunosuppressive therapies, in patients treated for acquired immunodeficiencies (HIV, immune reconstitution after hematopoietic stem cell transplantation). This would be the case, for instance, in an uncomplicated allo-HSCT setting or in the recovery of lymphopenic conditions in young patients. Also, it will be interesting to evaluate the rs2204985 variant as a biomarker predicting immune reconstitution in patients with HIV chronic infection. A recent longitudinal study demonstrated that both CD4 + and

CD8 + naïve T cell TREC contents declined biphasically, with a rapid loss during the first year and a much slower loss during the chronic phase of infection [START_REF] Drylewicz | Reconciling Longitudinal Naive T-Cell and TREC Dynamics during HIV-1 Infection[END_REF]. This test may be also potentially useful to predict the quality of response to vaccination in an aging population, which correlates with a reduced ability of the immune system to generate antigen specific responses to pathogens and vaccination. In contrast, it would be detrimental to fuel the system if the thymic environment is damaged as, for instance, in older individuals, in graft-versus host disease in allo-HSCT [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] Krenger | Thymic T-cell development in allogeneic stem cell transplantation[END_REF] or in autoimmune conditions where women are known to have an overall higher susceptibility [START_REF] Dragin | Estrogen-mediated downregulation of AIRE influences sexual dimorphism in autoimmune diseases[END_REF]. Such cases could result in the generation of T cells defective in their selection process with an autoreactive potential, which could be pathogenic.

  Figure 1. Establishment of definitive hematopoietic stem cell (HSC) pools in mouse and human embryos. Adapted from[START_REF] Mikkola | The journey of developing hematopoietic stem cells[END_REF]Kaushansky et al., 2016;[START_REF] Ivanovs | Human haematopoietic stem cell development: from the embryo to the dish[END_REF]................................................................................................

Figure 2 .

 2 Figure 2. Potential Bone Marrow Niche Constituents of the Hematopoietic Progenitors. Adapted from (Wei and Frenette, 2018).......................................................

Figure 3 .

 3 Figure3. Overview of Human Hematopoietic T Lineage Hierarchy. Adapted from[START_REF] Görgens | Multipotent hematopoietic progenitors divide asymmetrically to create progenitors of the lymphomyeloid and erythromyeloid lineages[END_REF][START_REF] Velten | Human haematopoietic stem cell lineage commitment is a continuous process[END_REF][START_REF] Karamitros | Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells article[END_REF].................................

  Figure3. Overview of Human Hematopoietic T Lineage Hierarchy. Adapted from[START_REF] Görgens | Multipotent hematopoietic progenitors divide asymmetrically to create progenitors of the lymphomyeloid and erythromyeloid lineages[END_REF][START_REF] Velten | Human haematopoietic stem cell lineage commitment is a continuous process[END_REF][START_REF] Karamitros | Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells article[END_REF].................................

Figure 4 .

 4 Figure 4. Overview of regulated migration events during T cell development. Adapted from (Love and Bhandoola, 2011).....................................................................................

Figure 5 .

 5 Figure 5. T cell development during DN phases: T progenitor plasticity, TCR V(D)J rearrangement, surface markers and transcription factors. Adapted from[START_REF] Verschuren | Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF][START_REF] Yui | Developmental gene networks: a triathlon on the course to T cell identity[END_REF][START_REF] Rothenberg | Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control[END_REF]................

Figure 6 .

 6 Figure 6. Cortical TECs play a critical role in multiple stages of T cell development. Adapted from (Abramson and Anderson, 2017)...............................................................

Figure 7 .

 7 Figure 7. The germline of the Tcrγ locus has on the chromosome 7. Adapted from (van der Weerd et al., 2013)......................................................................................................

Figure 8 .

 8 Figure 8. Schematic of TCRB locus on chromosome 7. Adapted from[START_REF] Hodges | Diagnostic role of tests for T cell receptor (TCR) genes[END_REF][START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Krangel | Mechanics of T cell receptor gene rearrangement[END_REF]...............................................................................................

  Figure 8. Schematic of TCRB locus on chromosome 7. Adapted from[START_REF] Hodges | Diagnostic role of tests for T cell receptor (TCR) genes[END_REF][START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Krangel | Mechanics of T cell receptor gene rearrangement[END_REF]...............................................................................................

Figure 9 .

 9 Figure 9. Model of human V-D-J recombination of the TCRβ chain DNA in DN thymocytes. Adapted from[START_REF] Hodges | Diagnostic role of tests for T cell receptor (TCR) genes[END_REF][START_REF] Krangel | Gene segment selection in V(D)J recombination: Accessibility and beyond[END_REF][START_REF] Dik | New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling[END_REF]..........

Figure 10 .

 10 Figure 10. T cell development during DP stage: TCR V(D)J rearrangement, surface markers and transcription factors. Adapted from (Verschuren et al., 1997; Dik et al., 2005)..................................................................................................................................

  Figure 10. T cell development during DP stage: TCR V(D)J rearrangement, surface markers and transcription factors. Adapted from (Verschuren et al., 1997; Dik et al., 2005)..................................................................................................................................

Figure 11 .

 11 Figure 11. Schematic of TCRA-TCRD locus in (A) murine and (B) humans on chromosome 14. Adapted from (Glusman et al., 2001; Krangel, 2003; Zhao et al., 2016)..................................................................................................................................

  Figure 11. Schematic of TCRA-TCRD locus in (A) murine and (B) humans on chromosome 14. Adapted from (Glusman et al., 2001; Krangel, 2003; Zhao et al., 2016)..................................................................................................................................

Figure 12 .

 12 Figure 12. CTCF. Chromatin loop organization of the TCRA-TCRD locus in DP thymocytes as defined by 3C. Adapted from (Carico and Krangel, 2015)........................

Figure 13 .

 13 Figure 13. Detection of cj δREC-ΨJα and sj δREC-ΨJα. Adapted from[START_REF] Okazaki | T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining[END_REF][START_REF] Hazenberg | T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation[END_REF] ............................................................

  Figure 13. Detection of cj δREC-ΨJα and sj δREC-ΨJα. Adapted from[START_REF] Okazaki | T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining[END_REF][START_REF] Hazenberg | T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation[END_REF] ............................................................

  Figure 16. Diagram of thymic egress and phenotypic maturation of mouse and human RTEs in secondary lymphoid organs. Adapted from (Fink, 2013)....................................

Figure 17 .

 17 Figure 17. V(D)J recombination. Adapted from (Krangel, 2003)....................................

Figure 18 .

 18 Figure 18. TCRδ rearrangement redirects a processive TCRα recombination program to expand the TCRα repertoire. Adapted from (Carico et al., 2017).................................

Figure 19 .

 19 Figure 19. Junctional diversity. Adapted from (Mak, Saunders and Jett, 2014)..............

Figure 20 .

 20 Figure 20. TCR-antigen-peptide-MHC interaction and TCR gene recombination. Adapted from (Woodsworth, Castellarin and Holt, 2013).................................................

Figure 21 .

 21 Figure 21. Thymic epithelial space (TES) decreases with age in normal human thymuses. Adapted from (Sempowski et al., 2000)...........................................................

Figure 22 .

 22 Figure 22. Milieu Intérieur network.................................................................................

Figure 23 .

 23 Figure 23. Effect of the TCRA-TCRD genetic variation on sjTRECs in human thymocytes.........................................................................................................................

Figure 24 .

 24 Figure 24. Effect of the TCRA-TCRD genetic variation on sjTRECs in females and males..................................................................................................................................

Figure 25 .

 25 Figure 25. Chromatin modifications at the target TCRA-TCRD region in human total thymocytes.........................................................................................................................

Figure 4 .

 4 Figure 4. Overview of regulated migration events during T cell development. Adapted from (Love and Bhandoola, 2011).

Figure 6 .

 6 Figure 6. Cortical TECs play a critical role in multiple stages of T cell development, including homing of T cell progenitors into the thymus via secretion of various cytokines, T cell lineage commitment via Dll4-dependent activation of Notch, T cell expansion and development via secretion of various cytokines (IL-7, SCF), and positive selection of αβT cells. Adapted from[START_REF] Abramson | Thymic Epithelial Cells[END_REF].

Figure 11 .

 11 Figure 11. Schematic of TCRA-TCRD locus in (A) murine and (B) humans on chromosome 14. Dark red triangles represent position and orientation of CBEs (INT1-2 and TEA CBEs are labeled). Gene segments are indicated by rectangles, enhancers by ovals and promoters by arrows. Eδ (orange oval) activates a region at the double-negative thymocyte stage (orange bar), whereas Eα (blue oval) activates a region at the double-positive thymocyte stage (blue bar). The region of the Jα array that depends on TEA is indicated by the green bar. Cen.,

  program of iNKT cells occurs by the downstream recruitment of the adaptor signaling lymphocytic-activation molecule (SLAM)-associated protein (SAP) and the Src kinase Fyn, which were previously recognized as essential for the expansion and differentiation of the iNKT cell lineage[START_REF] Godfrey | Control points in NKT-cell development[END_REF]. INKT cells use a semi-invariant TCR made up mostly of a single invariant TCRα chain (Vα14-Jα18 in mice, Vα24-Jα18 in humans) with certain TCRβ chains (Vβ8.2, Vβ7, or Vβ2 in mice, Vβ11 in humans) to engage CD1d. In the thymus, iNKT cells develop into three major terminally differentiated and functionally distinct iNKT cell subsets[START_REF] Lee | Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells[END_REF][START_REF] Gapin | Development of invariant natural killer T cells[END_REF]. INKT-1 cells express the transcription factor T-BET and secrete predominantly IFNγ; iNKT-2 cells express high levels of the GATA3 and promyelocytic leukemia zinc finger (PLZF) transcription factors and secrete IL-4 that affect the homeostasis of thymic cell populations and helps CD8 + T cells to become memory-like (Carty, Koretzky and Jordan, 2014); and also drives the acquisition of an activated/memory-like phenotype by Foxp3 + regulatory T cells; iNKT-17 have intermediate levels of PLZF, are positive for RORγt expression, and secrete IL-17 (Havenar-Daughton et al., 2012). In mouse, RANKL expressing CD44 -thymic iNKT cells regulate the differentiation of Aire MHC class-II mTECs that are involved in clonal deletion of self-reactive T cells and Treg cell maturation

Figure 16 .

 16 Figure 16. Diagram of thymic egress and phenotypic maturation of mouse and human recent thymic emigrants (RTEs) in secondary lymphoid organs; RTEs (green cells) exit the blood into secondary lymphoid organs and begin a 3-week-long maturation process that is facilitated by dendritic cells, eventually becoming peripheral naïve T cells (blue cell).

  16, green cells) exit the blood into secondary lymphoid organs and it takes about 3-week-long maturation process, which is facilitated by dendritic cells, to eventually acquire the features of standard peripheral naïve T cells (Fig.16, blue cells). Throughout the lifetime of the individual, RTEs are essential for the maintenance of a diverse population of naïve peripheral T cells, ready to further differentiate into appropriate effector T cells upon encounter with foreign antigen. RTEs are both phenotypically and functionally distinct from their peripheral naïve T cell counterparts. The pool of mature TCRαβ T cells in the secondary lymphoid organs arises through slow release of young cells from the thymus[START_REF] Takada | Naive T cell homeostasis: from awareness of space to a sense of place[END_REF]. Formation of the T cell pool occurs largely in young life but continues into old age.Maintenance of the naïve T cell pool is not a default state but is dependent on constant stimulation of naïve T cells by extrinsic factors. Measurement of the proliferative capacity and life span of defined cell types is fundamental for understanding population dynamics and homeostasis. Homeostasis of T cells is essential to the maintenance of the T cell pool and TCR diversity. Two homeostatic signals are crucial to maintain naïve T cells alive: (II.2.5.2.) TCR-self-pMHC interactions and (II.2.5.3.) Interleukin 7.

Figure 18 .

 18 Figure 18. TCRδ rearrangement redirects a processive TCRα recombination program to expand the TCRα repertoire. Adapted from[START_REF] Carico | Tcrd Rearrangement Redirects a Processive Tcra Recombination Program to Expand the Tcra Repertoire[END_REF].

Figure 19 .

 19 Figure 19. Junctional diversity. If the D and J segments in this example are joined precisely

Figure 20 .

 20 Figure 20. TCR-antigen-peptide-MHC interaction and TCR gene recombination. (a) A T cell(pink) encountering an antigen-presenting cell (APC; blue). The APC presents peptide antigen (Ag; yellow) in complex with the larger major histocompatibility complex (MHC; turquoise). (b) A TCR (multi-colored) heterodimer composed of an α and a β chains, engaging peptide-MHC (pMHC). Moving outward from the T cell, the constant region (green) of the TCR is anchored to the cell membrane, followed by the J region (red). In TCRα chain the J region is followed by the V region (orange), whereas in TCRβ chain, a D region is located between the V and J regions. The complementarity determining region 3β (CDR3β; color gradients at junctions) represent the regions encoded by arbitrary, untemplated nucleotides introduced during somatic recombination. (c) Simplified representation of TCRβ VDJ gene recombination resulting in TCR diversity. Adapted from[START_REF] Woodsworth | Sequence analysis of T-cell repertoires in health and disease[END_REF].

  infection. While naïve CD8 + T cell numbers hardly changed during follow-up, naïve CD4 + T cell counts continually declined. The observed longitudinal changes in TRECs and naïve T cell numbers in HIV-infected individuals are most likely explained by a tight balance between increased T cell division and death, suggesting that these changes are intrinsically linked in HIV infection[START_REF] Drylewicz | Reconciling Longitudinal Naive T-Cell and TREC Dynamics during HIV-1 Infection[END_REF]. IL-7 clinical trial in HIV patients demonstrated an increased thymic output and tented to improve the TCR repertoire in persons with low TCR diversity. IL-7 lead to a dose-dependent CD4 T cell increase and the broadering of TCR diversity in some subjects[START_REF] Lévy | Effects of recombinant human interleukin 7 on T-cell recovery and thymic output in HIV-infected patients receiving antiretroviral therapy: results of a phase I/IIa randomized, placebo-controlled, multicenter study[END_REF]. Another recent study stablished the relevance of thymic function measured by sjTREC/ βTREC ratio in HIV disease progression by analyzing a large number of patients (n = 774) in 3 cohorts with different HIV disease progression phenotypes[START_REF] Ferrando-Martinez | Thymic function failure is associated with human immunodeficiency virus disease progression[END_REF].

  but few have focused on a deep analysis of healthy immune responses. Knowledge of baseline responses in healthy persons is crucial for the understanding of the pathologic context as it can be used as a reference. This has been achieved through the French scientific initiative, financed by the Investissement d'Avenir Laboratoire d'Excellence (LabEx) research program, the Milieu Intérieur (MI) consortium, driven by Dr. Matthew Albert and Dr. Luis Quitana-Murci, with the objective to define the determinants of human immune variance. The MI project aims to determine what are the genetic and environmental factors that contribute to the observed heterogeneity in the healthy human immune responses and to provide a large dataset

Fig. 22 .

 22 Fig. 22. Milieu Intérieur network.

  Leston Araujo, Cécile Alanio, Etienne Patin, Jacob Bergstedt, Alejandra Urrutia, Silvia Lopez-Lastra, Yan Li, Bruno Charbit, Cameron Ross MacPherson, Milena Hasan, Breno Luiz Melo-Lima, Corinne Douay, Noémie Saut, Marine Germain, David Alexandre Trégouët, Pierre-Emmanuel Morange, Magnus Fontes, Darragh Duffy, James P. Di Santo, Lluis Quintana-Murci, Matthew L. Albert, Antoine Toubert, The Milieu Intérieur Consortium

1 Université

 1 Paris Diderot, Sorbonne Paris Cité, Institut Universitaire d'Hématologie, 75010 Paris, France. 2 INSERM, UMRS-1160, Alloimmunity-Autoimmunity-Transplantation, 75010 Paris, France. 3 Center for Translational Research, Technology Core, Institut Pasteur, 75015 Paris, France. 4 Dendritic Cell Immunobiology, Department of Immunology, Institut Pasteur, 75015 Paris, France. 5 INSERM, UMRS-1223, 75015 Paris, France.Clave et al., Sci. Transl. Med. 10, eaao2966 (2018) 5 September 2018 SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE 2 of 11

Fig. 1 .

 1 Fig. 1. Thymic function associates with naïve T cell immune phenotypes. (A) bTRECs (blue) are episomal DNA generated during the TCRB recombination. sjTRECs (purple) derive from the deletion of the TCRD locus during TCRA locus recombination (shown in fig. S1B). DN, double negative; ISP, immature single positive; DP, double positive; SP, single positive. (B) Effect sizes of significant associations [adjusted P values (adj. P) < 0.05] between sjTRECs and immune cells and parameters measured by flow cytometry in 969 healthy individuals from the MI cohort. Effect sizes were estimated in a mixed model (see Supplementary Materials and Methods). MFI, mean fluorescence intensity. (C) Relationships between sjTRECs and the log 10 -transformed number of naïve CD4 + and CD8 + T cells, naïve T reg , and iNKT cells. Regression lines were fitted using linear regression. Adjusted P values were obtained using the mixed model and based on the Kenward-Rogers F test.

15 Fig. 2 .

 152 Fig. 2. Age and sex strongly affect thymic function in healthy donors. (A) sjTRECs as a function of age in 487 women (red) and 492 men (blue). (B) bTRECs as a function of age, in 264 women and 242 men donors with detectable amounts. (C) Number of intrathymic divisions as a function of age, in donors with detectable bTRECs. (D) sjTRECs as a function of sex in 487 women and 492 men. Regression lines were fitted using linear regression. P values were adjusted to control the false discovery rate at 5% and estimated on the basis of the Kenward-Rogers approximate F tests.

Fig. 3 .

 3 Fig. 3. Genome-wide association study reveals an impact of TCRA-TCRD genetic variation on thymic function. (A) Manhattan plot for genetic association with sjTRECs in the 969 donors of the MI cohort. Light and dark gray lines indicate the threshold for suggestive association (P = 1.0 × 10 -5 ) and genome-wide significant association (P = 5.0 × 10 -8 ), respectively. (B) Detailed view of the TCRA-TCRD locus. Primers (sjTREC-F/R) and probe (sjTREC-P) used to quantify sjTRECs are shown in red and cyan, respectively. (C) Fine mapping of the genetic association between the TCRA-TCRD locus and sjTRECs. Meta-analysis P values were obtained by combining arraybased, probe-based, and imputed genotypes of the MI and MARTHA cohorts (tableS2). Variants that are significantly associated at the genome-wide level are indicated in red. (D) Physical position of the four most strongly associated variants, relative to active transcription activity[START_REF]An integrated encyclopedia of DNA elements in the human genome[END_REF]. The position of Dd3 is indicated.

Fig. 4 .

 4 Fig. 4. Effect of TCRA-TCRD human genetic variation on thymic function in humanized immunodeficient mice. (A) Immunodeficient Balb/c Rag2 -/-Il2rg -/-Sirpa NOD (BRGS) mice were reconstituted with human CD34 + hematopoietic progenitors harvested from fetal livers with rs2204985 genotype AA (orange), GA (brown), or GG (purple). (B) Effects of rs2204985 genotypes on sjTRECs in all mice (AA, n = 19; GA, n = 58; GG, n = 15). (C) Effects of rs2204985 genotypes in immunophenotyped mice (AA, n = 5; GA, n = 31; GG, n = 13) on number of CD3 + thymocytes and (D) on thymocyte subsets at different developmental stages. Indicated P values correspond to the genotype effect in a linear model including genotype and mouse recipient sex.

Fig. 6 .

 6 Fig.6. Combined effects of sex, age, and TCRA-TCRD genetic variation on human thymic function. sjTRECs as a function of age and rs2204985 genotypes in (A) the MI cohort (n = 969) and (B) the replication MARTHA cohort (n = 612). Regression lines were fitted using linear regression. P values were obtained with a mixed model of log 10 (sjTRECs), including rs2204985 genotypes as predictor; covariates were selected using a data-driven variable selection scheme; and correcting for population stratification was performed using the genetic relatedness matrix (GRM) as a random effect. Orange, brown, and purple indicate AA, GA, and GG genotype, respectively. (C) Proportions of variance of sjTRECs explained by age, sex, and TCRA-TCRD genetic variation in MI (left) and MARTHA (right) cohorts. The surface area and color of subrectangles indicate proportions attributed to specific predictors, as measured by the R 2 of the regression model. (D) Difference between actual age and thymic age as a function of sex and rs2204985 variant. Thymic age is predicted from a regression model, where AA men are assumed as the baseline.

Acknowledgments:

  We thank J. Fellay, P. Scepanovic, and C. A. W. Thorball for support with genetic analysis and J.-M. Doisne, G. M. Ranson, and H. Strick-Marchand for support with humanized mouse experiments. We thank V. Asnafi, E. Macintyre, A. Cieslak, and I. André-Schmutz for helpful discussions. We also thank the Centre d'Immunologie Humaine (Institut Pasteur, Paris, France) for support. Funding: This work was supported by the French government's Invest in the Future Program, managed by the Agence Nationale de la Recherche (ANR; 10-LABX-69-01). I.L.A. was a recipient of the Science without Borders PhD program from Brazil Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). J.B. is a member of the Lund Center for Control of Complex Engineering Systems (LCCC) Linnaeus Center and the Excellence Center at Linköping-Lund in Information Technology (ELLIT) Excellence Center at Lund University and is supported by the ELLIIT Excellence Center. J.P.D.S., Y.L., and S.L.-L. received funding from Institut Pasteur, INSERM, the Laboratoire d'Excellence REVIVE. A.T. and E.C. received funding from ANR grant PriCelAge (ANR-14-CE14-0030-01). Genetic investigations in the MARTHA study were supported by the GENMED Laboratory of Excellence on Medical Genomics (ANR-10-LABX-0013). Author contributions: A.T. and M.L.A. conceived the project. E.C., I.L.A., and C.A. contributed to the design of experiments, supervised and conducted the experiments, generated the manuscript figures, and wrote the manuscript. E.P. and J.B. supervised and conducted statistical analyses, generated the manuscript figures, and wrote the manuscript. A.U., S.L.-L., Y.L., B.C., C.R.M., M.H., B.L.M.-L., and C.D. assisted in conducting experiments. N.S., M.G., D.-A.T., and P.-E.M. provided materials and data. M.F., D.D., and J.P.D.S. contributed to the supervision of the project and data analyses. A.T. and M.L.A. supervised the project, contributed to the design of the experiments and their analyses, and wrote the manuscript. M.L.A., L.Q.-M., and A.T. secured the funding. All authors reviewed and accepted the manuscript. Competing interests: E.C., I.L.A., C.A., L.Q.-M., M.L.A., and A.

  , and Serge Hercberg (Université Paris 13, Paris); Olivier Lantz (Institut Curie); Claude Leclerc, Hugo Mouquet, Etienne Patin, Sandra Pellegrini, and Stanislas Pol (Hôpital Cochin); Antonio Rausell (INSERM UMR 1163 -Institut Imagine); Lars Rogge, Anavaj Sakuntabhai, Olivier Schwartz, Benno Schwikowski, Spencer Shorte, and Vassili Soumelis (Institut Curie); Frédéric Tangy and Eric Tartour (Hôpital Européen George Pompidou, Paris); Antoine Toubert (Hôpital Saint-Louis, Paris); Mathilde Touvier (Université Paris 13); Marie-Noëlle Ungeheuer; Lluis Quintana-Murci; and Matthew L. Albert. Additional information can be found at http://www.milieuinterieur.fr/en. . Clave, I. L. Araujo, C. Alanio, E. Patin, J. Bergstedt, A. Urrutia, S. Lopez-Lastra, Y. Li, B. Charbit, C. R. MacPherson, M. Hasan, B. L. Melo-Lima, C. Douay, N. Saut, M. Germain, D.-A. Trégouët, P.-E. Morange, M. Fontes, D. Duffy, J. P. Di Santo, L. Quintana-Murci, M. L. Albert, A. Toubert, The Milieu Intérieur Consortium, Human thymopoiesis is influenced by a common genetic variant within the TCRA-TCRD locus. Sci. Transl. Med. 10, eaao2966 (2018).

  Human thymopoiesis is influenced by a common genetic variant within the Consortium Darragh Duffy, James P. Di Santo, Lluis Quintana-Murci, Matthew L. Albert, Antoine Toubert and The Milieu Intérieur Douay, Noémie Saut, Marine Germain, David-Alexandre Trégouët, Pierre-Emmanuel Morange, Magnus Fontes, Lopez-Lastra, Yan Li, Bruno Charbit, Cameron Ross MacPherson, Milena Hasan, Breno Luiz Melo-Lima, Corinne Emmanuel Clave, Itauá Leston Araujo, Cécile Alanio, Etienne Patin, Jacob Bergstedt, Alejandra Urrutia, Silvia DOI: 10.1126/scitranslmed.aao2966 , eaao2966. 10 Sci Transl Med investigators to examine the Milieu Intérieur data. in an independent cohort and mouse models. The authors have also developed a Web application for other a genome-wide association study revealed variants that were associated with thymic output, which was confirmed output, known to decrease over time, is affected by other factors. In addition to seeing sex-dependent differences, . leveraged samples from this cohort to see how thymic et al environment influence the immune system. Clave overt disease. The Milieu Intérieur Consortium enrolled 1000 healthy adults to study how genetics and the Personalized medicine partly depends on understanding what causes variance even outside the context of Mining the Milieu Intérieur

  200 µM of each dNTP, 2.5 mM MgSO4 and 1.25 unit of Platinum Taq DNA pol High Fidelity (ThermoFisher Scientific, Courtaboeuf, France) in 1x buffer. Columns of 48.48 Dynamic array IFCs were loaded with 5 µl containing 2.25 µl of a 1/400 th dilution of preamplified DNA, 2.5 µl of 2x Takyon Low Rox Probe MM (Eurogentec) and 0.25 µl of sample Loading Reagent and raws with an equal mixture of 2x Assay loading Reagent and 2x

7

 7

2 ,

 2 ∈ { , … , 6} and ~ , 2 , and where

  , ~ , , ∈ { , … , }, ~ , , and = , … 9 9.

  is the batch variable for TREC processing (what plate was used for sample i), ~ , and = , … 9 . The model was fitted, and profile likelihood based confidence intervals was constructed, using the lme4 package. Testing 14 was done using Kenward-Rogers approximated F-tests implemented in the pbkrtest package in R.Meta-analysis of the MI and MARTHA cohorts was conducted with the rna.mi function in the metafor R package (60), with the random effects approach based on the DerSimonian and Laird estimator.Thymic age estimationTo illustrate how thymic function is impacted by non-genetic and genetic factors, we sought to estimate the effect of an individual's genotype at rs2204985 and sex in terms of a quantity we call "thymic age", which we define for an individual as the age of the average male with the AA rs2204985 genotype with the same number of predicted sjTRECs as this individual. Genotypes in this context are considered a categorical variable that take values in {AA, AG, GG}. Consider the regression model: indicators that individual i is of genotype AG or GG. Because we pooled data of the MI and MARTHA cohort, we adjusted for cohort differences by including the MARTHA term, which equals 1 if individual i is part of the MARTHA cohort.The previous equation gives the following relationship for AA males: {log ( )| } -= +

  log10 sjTREC values was estimated by the fitting linear regression model,

Fig. S1 .

 S1 Fig. S1. Technical workflow of the study and validation of the TREC assay in the MI cohort.

r 2

 2 =0.99). Finally, the PCR1 technical replicates were compared to the values obtained for the same 43 donors tested on different plates during the course of the study (PCR1 and MI; r 2 =0.99). (F) To assess the biological reproducibility of the TREC assay, we quantified sjTREC levels on biological replicates collected for 29 same individuals at 2 to 3 weeks interval. A high correlation was found between the two measures (r 2 =0.99). (G) Impact of the rs76132819 SNP on probe binding and assay efficiency. This SNP was found to locate inside the sjTREC probe (position 26 C/A), and was in moderate linkage disequilibrium (D'=1.0, r²=0.13) with the SNPs identified by genome wide association study (table

Fig. S2 .

 S2 Fig. S2. Association of sjTRECs with immune cell counts and parameters.(A) Significance of the effect of sjTRECs on immunophenotypes, as estimated by the -log10(adjusted P-values) of the Kenward-Rogers approximated F tests from mixed models including age, sex, CMV serostatus and smoking status as fixed effect covariates together with day of blood draw as a random effect. Normalized log-scale effect sizes were colorcoded. The vertical black line indicates the -log10 of chosen threshold for statistical significance (-log10(adj. P=0.05)=1.30103). (B) Significant associations between sjTRECs and the numbers of circulating T-cells, CD4 + and CD8 + T-cells, and neutrophils (n=969).

Fig. S3 .

 S3 Fig. S3. Association of TRECs with immune cell counts and parameters. (A) Significance of the effect of βTRECs on immunophenotypes, as estimated by the -log10(adjusted P-values) of the Kenward-Rogers approximated F tests from mixed models including age, sex, CMV serostatus and smoking status as fixed effect covariates together with day of blood draw as a random effect. Log-scale normalized effect sizes were colorcoded. The vertical black line indicates the -log10 of chosen threshold for statistical significance (-log10(adj. P=0.05)=1.30103). Significant association between βTRECs and (B) the number of circulating naive CD8 + T-cells, (C) the number of circulating naïve CD4 + T-cells and (D) the number of circulating naïve Treg cells (n=832). Regression lines were fitted using simple linear regression. Adjusted P-values were obtained using the mixed model described in (A).

Fig. S4 .

 S4 Fig. S4. Association of sjTRECs with non-heritable factors. Significance of the effect of demographic, medical and lifestyle variables on sjTRECs, as estimated by the -log10(adjusted P-values) of the Kenward-Rogers approximated F tests

Fig. S5 .

 S5 Fig. S5. Association of TRECs with non-heritable factors.

Fig. S6 .

 S6 Fig. S6. Association of intrathymic division number with non-heritable factors.(A) Significance of the effect of demographic, medical and lifestyle variables (tableS2) on the number of intrathymic divisions, as estimated by the -log10(adjusted P-values) of the Kenward-Rogers approximated F tests from mixed models including the tested treatment variable, age, sex, (unless these were the treatment) as fixed effects covariates and TREC processing plates as random effects. Log-scale normalized effect sizes were color-coded.

Fig. S7 .

 S7 Fig. S7. Association of thymic function parameters with specific non-heritable factors. Effects of cytomegalovirus (CMV) serostatus, influenza A virus (IVA) serostatus and metabolic score (10) on (A) sjTRECs (n=969), (B) βTRECs (n=506), and (C) number of intrathymic divisions (n=506). Effects of body mass index (BMI) and log10 of C reactive protein (CRP) on (D) sjTRECs (n=969), (E) βTRECs (n=506) and (F) number of intrathymic divisions (n=506).

Fig. S8 .

 S8 Fig. S8. Age and sex impact on sjTRECs in the MARTHA cohort.Effects of (A) age and (B) sex on sjTRECs in the independent MARTHA cohort (n=612).Adjusted P-values are the FDR-adjusted P-values for the Kenward-Rogers approximated F test of a age (or sex) effect obtained using a mixed model for the response variable log10 sjTRECs including sex (or age) and TREC processing plate as fixed effects, and additional batch variables as random effects. Regression lines were fitted using linear regression. Blue indicates men (n=160) and red indicates women (n=452).

Fig. S9 . 35 Figure S11 Fig.

 S935S11 Fig. S9. Human immune system mice flow cytometry gating strategy.

Fig. S12 .

 S12 Fig. S12. Annotated screenshot of Shiny webapp showing interface for visualization and prediction.

40

 40

  primers and probes are from Eurogentec (Paris, France) except Alb-VIC probe (ThermoFisher Scientific). Underlined nucleotides represent Locked Nucleic Acids (LNA). All primers are together in preamplification. H. = Human. Part II Intrathymic role of the polymorphism in healthy human thymus: molecular mechanisms acting during the TCRA-TCRD locus recombinations Purpose Our previous study, published in Science Translational Medicine in 2018, demonstrated that non-genetic and genetic determinants could affect the thymic function in healthy adults. It

  SNP rs2204985 could interfere in human thymic function specifically in the TCRA-TCRD locus recombination in human thymus. We aimed to identify the molecular elements and putative transcriptional factors located in the open-chromatin region that could be binding in the TCRA-TCRD region under influence of the SNP rs2204985 based on ENCODE data (ENCODE Project Consortium, 2012). However, the difficulty in accessing human thymic samples and the poor number of thymocytes in adults are limitations to this study. In collaboration with the Necker hospital for sick children (Hôpital Necker Enfants Malades) in Paris -France, we have been able to develop such human study. Thymi were obtained as surgical tissue discards from a total of 76 healthy children (50 females and 26 males) aged ~ 20 weeks undergoing cardiac surgery, with informed consent from the parents. Thymic lobes were cutting into small pieces, in house mechanically dissociated (non enzymatic) using a Cell Strainer (100µm nylon Falcon ® ) and isolated by Ficoll-Paque gradient. These cells were immediately prepared to (1) DNA genotyping and TREC quantification and (2) Chromatin ImmunoPrecipitation (ChIP) experiments.

  increasing the binding of RNAP II, located in the open-chromatin region at the TCRA-TCRD region and under influence of the SNP rs2204985, indicating a possible influence on the transcription machinery, possibly contributing positively to a greater gene transcription activity at the TCRD locus. Further studies in thymi of healthy children will allow to go deeper and investigate the molecular mechanisms that influence the TCRA-TCRD genetic variation early at the DN stages of the developing thymocytes. By providing reference values of thymic function in a large healthy population via a key genetic control, our data provide a

  The human TCRB locus has 42-50 Vβ gene segments, 2 Dβ gene segments and 12-13 Jβ gene segments, while the mouse TCRB locus has 35 Vβ, 2 Dβ and 12 Jβ functional gene segments

  5 September 2018 SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

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  Orange, brown, and purple indicate AA, GA, and GG genotype, respectively. (C) Proportions of variance of sjTRECs explained by age, sex, and TCRA-TCRD genetic variation in MI (left) and MARTHA (right) cohorts. The surface area and color of subrectangles indicate proportions attributed to specific predictors, as measured by the R 2 of the regression model. (D) Difference between actual age and thymic age as a function of sex and rs2204985 variant. Thymic age is predicted from a regression model, where AA men are assumed as the baseline.

	by guest on November 21, 2018

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  5 September 2018 SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE . Y. Benjamini, D. Yekutieli, False discovery rate-adjusted multiple confidence intervals for selected parameters. J. Am. Stat. Assoc. 100, 71-81 (2005). 58. N. Meinshausen, P. Bühlmann, Stability selection. J. R. Stat. Soc. Series B Stat. Methodol. 72, 417-473 (2010). 59. A. Ziyatdinov, M. Vázquez-Santiago, H. Brunel, A. Martinez-Perez, H. Aschard, J. M. Soria, lme4qtl: Linear mixed models with flexible covariance structure for genetic studies of related individuals. BMC Bioinformatics 19, 68 (2018). 60. W. Viechtbauer, Conducting meta-analyses in R with the metafor package. J. Stat. Softw.

	36, 1-48 (2010).

  Significance of the effect of demographic, medical and lifestyle variables (tableS2) on the probability of having detectable βTREC levels, as estimated by the -log10(adjusted P-values) of the likelihood ratio tests from logistic regression models including the tested treatment variable, age and sex (unless these were the treatment).

	(adjusted P-values) of the Kenward-Rogers
	approximated F tests from mixed models including the tested treatment variable, age, sex,
	(unless these were the treatment) as fixed effects covariates and TREC processing plates
	as random effects. Normalized log-scale effect sizes were color-coded. The vertical black
	line indicates the -log10 of chosen threshold for statistical significance (-log10(adj.
	P=0.05)=1.30103). (B)

Normalized log-scale effect sizes were color-coded. The vertical black line indicates the -log10 of chosen threshold for statistical significance (-log10(adj. P=0.05)=1.30103). (C) βTREC levels as a function of sex (n=506). Adjusted P-values were obtained using the mixed model described in (A). (D) Percentages of having detectable βTREC values as a function of age in men (blue) and women (red). P-values were obtained using a logistic regression with a likelihood ratio chi-square test, with the βTREC binary variable (i.e., 0 and 1 indicating undetectable and detectable βTREC levels) as the response, and age and sex as treatments (n=463).

Table S3 . TCRA-TCRD Next Generation Sequencing Data.

 S3

	Sample Name	Mouse Recipient Sex	Human Donor Sex	rs2204985	locus	Age (weeks)	total rearrangements	productive rearrangements	productive clonality	max productive frequency	Shanon Equitability	Inverse Simpson
	45605T3RD	Female		Men		AA		TCRAD		19		2 945 927		897 631			0.0417	0.038182	0.98428115051	369 487
	45609T3RD	Female		Men		AA		TCRAD		19		216 585			76 022			0.0664	0.401470 0.970865080466	14 895
	45615T3RD	Male		Men		AA		TCRAD		19		270 333			88 760			0.0558	0.266205 0.982650106856	-30 808
	17 14 45742T3RD	PRKCA TCRD Male rs4790904 rs3811236		C C Men	T T	AA	0 0	0,229 0,505 TCRAD	0,0959 0,0737	0,0212 6,2E-06 0,0181 5,2E-05 C___2609758_10 0,506 --19 872 887	-0,075 272 706 -0,018 4,1E-05 -	0 0 0.0362	0,228 0,529 0.058768 0,0093 0,0726	0,0237 6,9E-01 0,0195 2,1E-04 0.98924927183 -0,0732	-0,0133 3,5E-08 -90 236
	14 47139T5TH	TCRD Female rs2301199		G Men	C	AA	1	0,505 TCRAD	0,0727	0,0182 6,9E-05 9	-	5 959	-	-		-2 118	-		1 0.0595	0,529 0.471729 0.995952765477 0,0726 0,0195 2,1E-04 0,0727	0,0133 4,7E-08 1 941
	14 14 47140T5TH	TCRD TCRD Female rs2301200 rs3216790		C CG Men	T C	AA	1 1	0,505 0,505 TCRAD	0,0727 0,0721	0,0182 6,9E-05 0,0182 8,1E-05 9	--68 065	--	--	--21 518	--		1 1 0.0585	0,529 0,529 0.336460 0.986729670136 0,0726 0,0195 2,1E-04 0,0727 0,0726 0,0195 2,1E-04 0,0723	0,0133 4,7E-08 0,0133 5,5E-08 11 682
	14 14 47143T5TH	TCRD TCRD Male rs62762262 TAGTC rs2331618 A Men	T G	AA	1 1	0,505 0,504 TCRAD	0,0740 0,0736	0,0182 5,2E-05 0,0183 6,0E-05 9	--243 539	--	--	--75 292	--		1 1 0.1143	0,527 0,526 0.361357 0.932140757822 0,0701 0,0195 3,4E-04 0,0722 0,0734 0,0195 1,8E-04 0,0735	0,0133 5,8E-08 0,0133 3,4E-08 6 149
	14 47145T5TH	TCRD Female rs1109130		G Men	A	AA	1	0,505 TCRAD	0,0728	0,0182 7,0E-05 9	-49 935	-	-	-22 221	-		1 0.0665	0,529 0.682569 0,0726	0,0195 2,1E-04 0.97468615593 0,0727	0,0133 4,7E-08 6 829
	14	TCRD	rs2072616		G	C		1	0,505	0,0728	0,0182 7,0E-05	-		-	-		-	-		1	0,529	0,0726	0,0195 2,1E-04	0,0727	0,0133 4,7E-08
	14 47692T13TH	TCRD Female rs8012481		G Women	A	GG	1	0,505 TCRAD	0,0728	0,0182 7,0E-05 14	-149 212	-	-	-45 726	-		1 0.0430	0,529 0.052720 0.997330235266 0,0726 0,0195 2,1E-04 0,0727	0,0133 4,7E-08 42 327
	14 14 47693T13TH	TCRD TCRD Female rs8014525 rs6572448		A T Women	G A	GG	1 1	0,456 0,506 TCRAD	0,0700 0,0727	0,0180 1,1E-04 0,0181 6,1E-05 14	--18 656	--	--		--6 186	--		1 1 0.0533	0,473 0,527 0.421263 0.989667839494 0,0493 0,0195 1,2E-02 0,0605 0,0680 0,0195 5,3E-04 0,0706	0,0132 4,9E-06 0,0133 1,0E-07 4 727
	14 14 47694T13TH	TCRD TCRD Female rs741712 rs2222940		A C Women	G T	GG	1 1	0,455 0,455 TCRAD	0,0720 0,0708	0,0179 6,1E-05 0,0179 8,2E-05 14	--224 288	--	--	--68 310	--		1 1 0.0407	0,474 0,471 0.020894 0.995161945481 0,0499 0,0195 1,1E-02 0,0619 0,0499 0,0196 1,1E-02 0,0613	0,0132 2,7E-06 0,0132 3,5E-06 59 381
	14 47695T13TH	TCRD Female rs2141988		A Women	G	GG	0	0,506 TCRAD	0,0731	0,0180 5,1E-05 C__15828563_10 0,505 14 211 684	0,074 64 792 0,018 4,3E-05	0 0.0419	0,525 0.030033 0.994933552869 0,0661 0,0195 7,6E-04 0,0699	0,0132 1,3E-07 55 786
	14 14 47697T13TH	TCRD TCRD Female rs916052 rs916053		A C Women	G A	GG	1 1	0,508 0,458 TCRAD	0,0729 0,0709	0,0180 5,4E-05 0,0179 8,3E-05 14	--327 052	--	--	--99 201	--		1 1 0.0395	0,525 0,471 0.036163 0.995302652077 0,0661 0,0195 7,6E-04 0,0698 0,0479 0,0196 1,5E-02 0,0604	0,0132 1,3E-07 0,0132 4,9E-06 85 880
	14 14 47702T13TH	TCRD TCRD Male rs8021297 rs7150030		T T Women	C C	GG	1 1	0,507 0,458 TCRAD	0,0750 0,0718	0,0180 3,3E-05 0,0179 6,7E-05 14	--372 808	--	--114 460 --	--		0 1 0.0396	0,523 0,471 0.058301 0.993467458253 0,0649 0,0195 9,4E-04 0,0703 0,0479 0,0196 1,5E-02 0,0609	0,0132 1,0E-07 0,0132 4,1E-06 91 406
	14 47707T13TH	TCRD Male rs11157596		G Women	A	GG	1	0,458 TCRAD	0,0718	0,0179 6,7E-05 14	-340 604	-	-104 208 -	-		0 0.0422	0,471 0.112624 0.990646547876 0,0479 0,0196 1,5E-02 0,0609	0,0132 4,1E-06 71 032
	14 14 62T14TH	TCRD TCRD Female rs7492759 rs2204984		G A Men	A G	GG	1 1	0,507 0,505 TCRAD	0,0735 0,0765	0,0180 4,8E-05 0,0181 2,6E-05 C__16160754_10 0,504 --16 3 548 800	-0,075 1 081 567 -0,018 3,6E-05 -	1 1 0.0394	0,526 0,522 0.025733 0.989872263019 0,0657 0,0195 8,0E-04 0,0699 0,0654 0,0196 8,7E-04 0,0714	0,0132 1,2E-07 0,0133 7,7E-08 601 867
	14 14 64T14TH	TCRD TCRD Female rs71115550 rs201497432	C T Men	CATAAA TA GG	1 1	0,505 0,521 TCRAD	0,0782 0,0726	0,0182 1,8E-05 0,0184 8,8E-05 16	--616 551	--	--191 175 --	--		1 1 0.0366	0,523 0,548 0.024099 0.994079147466 0,0646 0,0195 9,9E-04 0,0719 0,0748 0,0196 1,5E-04 0,0737	0,0133 6,3E-08 0,0134 4,3E-08 154 894
	14 65T14TH	TCRD Female rs2331620		C Men	T	GG	1	0,457 TCRAD	0,0733	0,0180 5,1E-05 16	-66 190	-	-	-20 664	-		1 0.0493	0,473 0.272425 0,0464	0,0196 1,8E-02 0.99531082054 0,0610	0,0134 5,5E-06 17 642
	14 14 68T14TH	TCRD TCRD Male rs11623632 rs11851663		A G Men	G A	GG	1 1	0,456 0,455 TCRAD	0,0733 0,0726	0,0180 5,3E-05 0,0180 6,2E-05 16	--647 721	--	--200 035 --	--		1 1 0.0365	0,471 0,471 0.027283 0.994379025079 0,0467 0,0196 1,7E-02 0,0610 0,0474 0,0196 1,6E-02 0,0610	0,0133 4,2E-06 0,0133 4,3E-06 163 730
	14 14 69T14TH	TCRD TCRD Male rs8013419 rs10873018		A G Men	G A	GG	1 0	0,490 0,488 TCRAD	0,0811 0,0803	0,0177 5,1E-06 C___2609804_10 0,490 0,0177 6,7E-06 C___2609805_20 0,488 16 435 543	0,081 0,080 135 036 0,018 5,1E-06 0,018 6,7E-06	1 1 0.0412	0,515 0,514 0.030474 0.993517174734 0,0655 0,0196 8,7E-04 0,0741 0,0668 0,0196 8,6E-05 0,0742	0,0131 1,6E-08 0,0131 1,6E-08 109 246
	14 14 Median AA	TCRD TCRD	rs10137305 rs12147006		G A	C C		1 1	0,328 0,490	0,0781 0,0811	0,0191 4,7E-05 0,0177 5,1E-06 C__11721207_10 0,490 --14 230 062	-0,080 75 657 -0,018 6,1E-06 -	1 0 0.0590	0,345 0,518 0.348908 0.983465628683 0,0503 0,0209 1,6E-02 0,0654 0,0670 0,0196 6,5E-04 0,0748	0,0141 3,4E-06 0,0131 1,2E-08 13 289
	14 Median GG	TCRD	rs2204985		G	A		1	0,489	0,0794	0,0177 8,5E-06 C___2609811_10 0,488 14 333 828	0,078 101 705 0,018 1,2E-05	0 0.0409	0,515 0.033318 0.9942290862725 0,0669 0,0195 9,7E-05 0,0737	0,0131 1,9E-08 78 456
	14	TCRD	rs11844715		C	T		1	0,485	0,0774	0,0179 1,6E-05 C___2609812_10 0,482	0,070	0,018 8,6E-05	1	0,513	0,0641	0,0196 1,1E-03	0,0714	0,0132 6,3E-08
	14 p (Mann-Whitney) TCRD	rs7144549		C	G		1	0,323	0,0765 1.000 0,0193 8,3E-05	-0.521		-	-0.624	-	-	1 0.016	0,345	0,0516 0.005	0,0208 1,3E-02 0.003	0,0649	0,0142 4,6E-06 0.069
	14	TCRD	rs1882704		G	A		0	0,260	0,0843	0,0207 5,2E-05	-		-	-		-	-		0	0,275	0,0520	0,0219 1,8E-02	0,0689	0,0161 2,0E-05

*Effect allele frequency.

†Standard error of the effect size. ‡A selection of eight SNPs were re-genotyped on Biomark in the Milieu Intérieur primary cohort, to confirm association at imputed SNPs.

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Sexing of human donors was made by single amplification of the ZFX/ZFY genes in 25 µl PCR using of the hSex2 primer pair (tableS4), 200 µM each dNTPs, 1.5 mM MgSO4

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/10/457/eaao2966/DC1 Composition of the MI Consortium Materials and Methods Fig. S1. Technical workflow of the study and validation of the TREC assay in the MI cohort. S1. Demographic, medical, and lifestyle variables included in the MI study. Table S2. Statistics of association with sjTRECs for suggestive loci and replication at the TCRA-TCRAD locus. Table S3. TCRA-TCRD next-generation sequencing data. Table S4. Primers and probes used for sex determination and TREC quantification. Table S5. Primary human immune system mice data (Excel file). References (54)[START_REF] Stekhoven | MissForest-Non-parametric missing value imputation for mixed-type data[END_REF][START_REF] Halekoh | A Kenward-Roger approximation and parametric bootstrap methods for tests in linear mixed models-The R Package pbkrtest[END_REF](57)(58)(59)(60) 1 Table S1. Demographic, medical and lifestyle variables included in the MI study. Table S2. Statistics of association with sjTRECs for suggestive loci and replication at the TCRA-TCRAD locus Table S3. TCRA-TCRD Next Generation Sequencing Data. Table S4. Primers and probes used for sex determination and TREC quantification. Table S5. Primary human immune system mice data (Excel file).

Supplementary Materials

Materials and Methods

Composition of the MI Consortium.