This manuscript is the culmination of three years of doctoral studies, for which acknowledgments to various people are due. I apologize in advance if I have forgotten some people, who are nonetheless appreciated.

First, I would like to thank my supervisors: Drs Loïc Dupré and Kaan Boztug, without whom none of this would have been possible. Loïc, thank you for all your advice, help, support, the long hours and fruitful discussions, and for being a humane supervisor.

Drs Joerg Menche and Audrey Ferrand for their help and input in shaping the project.

My three reviewers Drs. Christophe Le Clainche, Delphine Muriaux and Fernando Sepulveda for taking the time to read this manuscript, advise and for agreeing to partake in the thesis jury, and Dr.

Maha Ayyoub, the jury president.

A major thanks is due to Loan Vulliard, none of this would have been possible without your hard work, and the countless times you have helped me sort things out. Thank you for trying to make bioinformatics understandable for me, and for the much-appreciated occasional sarcasm.

The organoid platform of the IRSD, especially Aude Rubio, Muriel Quaranta-Nicaise and Dr. Isabelle

Fernandes, for all the long hours spent acquiring, analyzing and transferring (and re-transferring) images, and for making me fall in love with the Opera Phenix.

The ladies of both the cytometry and microscopy platforms of the CPTP, for all your help not only during experiments but also your kind and encouraging words from the start until the very end. Drs Sophie Allart and Fatima L'faquihi-Olive, you are strong, knowledgeable and amazing boss ladies.

Danièle Daviaud and Fatima, thank you for always being extremely sweet and helpful. Anne-Laure Iscache, Valérie Duplan-Eche and Lydia De La Fuente, I cannot forget to thank you for the times you have come to "rescue" me at the cytometers. "Maître" Astrid Canivet-Laffitte, I really appreciate every time you have helped me with ImageJ.

A huge thank you to all the colleagues in both Dupré /Lesourne and Boztug teams in Toulouse and Vienna: Dr. Renaud Lesourne, thank you for your feedback, advice and comprehension.

Nelly Rouquié, Cui Yang and Suzanne Mélique, you have been amazing ladies all the way. To the past members and now Drs Gaëtan Blaize and Jeremy Argenty, as well as Aurélie Mougel, it was a pleasure being in the same team with you. To Claire, our most recent newcomer, thank you for you for your company along experiments, and of course for taking care of my cells along with Hélène. Dr. Hélène Daniels, thank you for the discussions, advice and feedback.

I cannot thank each member of the big Boztug team individually, but a huge thanks to every member for their sweetness, help, and for making the 9 months I spent in Vienna so memorable. Dr Artem Kalinichenko and Jakob Huemer, thank you for always being up for discussing anything NK. Dr.

Wojciech Garncarz, you are a hero and no words can do you justice. Drs. Ana Krolo and Ozelm Yuce, you ladies are sweethearts. Ewa Lenartowicz, thank you for your kind and encouraging words, always.

Dr. Johannes Pfeifenschneider, thank you for your sweetness and positive mood.

To the Valitutti team (Dr. Salvatore Valitutti, Dr. Eric Espinosa, Dr. Marie-Pierre Puissegur, recent Drs. Roman Jugelé and Yoan Eliasse, and Liza Filali Sabina Mueller), and the previous members of the Dupré team (Drs. Javier Rey-Barrosso and Raïssa Houmadi, and Alice Munaretto) thank you for making me feel welcome among you, your help, sweetness and support, and for making the beginning of this journey a bit easier. Every single one of you has helped me throughout this experience whether it was through advice, support or even words of encouragement over a coffee. To Dr. Delphine Guipouy, thank you for helping me with the French administration, sharing your knowledge with me, and answering my endless questions. Thank you infinitely Dr. Cat.

A special thank you to my colleagues Tala Shahin, Maximilian Rau, Laurène Pfajfer and Marianne Guisset, colleagues who also became great friends. Afterall, it is friends who make some days more bearable.

The colleagues at CPTP who have given feedback and input to help better shape this project through listening to my presentations and advising. Moreover, thanks to Drs Anne Quillet-Mary, Gaël

Menasché and Abdelhadi Saoudi for their advice and feedback as part of my thesis committee.

An immense thank you to Dr. Nabila Jabrane-Ferrat for all her advice, support and help. You are the best "godmother" I could have asked for. I would also like to thank the NJF team: Drs Hicham El Costa (thank you for your encouragement through your sarcasm), Jordi Gouilly and Qian Chen, and finally the wonderful Ana Espino, also known as "my hero".

To my friends in Toulouse and away, and my family and friends back in Lebanon who have been there

for me throughout this journey. From the bottom of my heart I would like to thank Celine, Ranine and Elio, you have become a family away from home.

To my small family. Whatever I say, words will never be enough. To my adorable grandma whom I love beyond words, and who has always believed in me. To my little brother Georges, thank you for always being by my side and for all your wise words and encouragement, and for always encouraging me to have faith in myself. To my Georges (the other special Georges in my life), I could not have done this without you, without your endless support and patience. I cannot thank you enough for being there every single day on this journey.

And finally, the most important people in this journey (and my life): my parents. Without you I could not be here today. Thank you for all the sacrifices you have made for me to be who I am and where I am. Thank you for your unconditional love and for always believing in me and making sure I knew it.

I cannot possibly thank you enough for everything you have done for me. If superheroes existed, my parents would be ones. Everything I am and everything I will ever be, I owe to you. I love you beyond words and I am eternally grateful for you. Mom, dad, this (and everything I am and ever will be) is for you.

Summary

High content cell imaging reveals actin cytoskeleton-mediated control of the immunological synapse Cytotoxic lymphocytes rely on actin cytoskeleton remodeling to achieve their function. In particular cytotoxic T lymphocytes and NK cells assemble the immunological synapse (IS), a complex actin-rich structure that allows the interaction with target cells, such as infected cells or tumor cells, and permits the polarized delivery of lytic granules. Although actin cytoskeleton remodeling is known to be a driving force of IS assembly and dynamics, our understanding of the molecular control of actin remodeling sustaining IS dynamics remains fragmented. This PhD project consisted in developing a high-content imaging approach to unbiasedly define the metrics of IS from human T and NK lymphocytes and to characterize the requirements for actin cytoskeleton integrity in organizing the IS architecture.

For that purpose, the stimulation and staining of cell lines and primary cells in multiwell plates and acquisition of a unique set of >100.000 confocal images with a fully automatized high-content imager was optimized. The images were analyzed with two complementary CellProfiler analytical pipelines to characterize the morphological features associated with different treatments and disease status. We first extracted 16 morphological features pertaining to F-actin, LFA-1 or lytic molecules based on prior knowledge of IS assembly, and included features pertaining to the nucleus. We show that IS assembly in Jurkat and NK-92 cells is characterized by increased F-actin intensity and cell area. For Jurkat cells, we report an increase in LFA-1 intensity and surface area, and for NK-92 cells an increase in lytic granule detection at the IS plane. We then treated NK-92 cells with seven drugs known to affect different aspects of actin dynamics and investigated the associated effects on IS features. We report concentration dependent effects, not only on F-actin intensity, as expected, but also on lytic granule polarization. Furthermore, using a high-resolution morphological profiling based on >300 features, we

show that each drug inflicts distinct alterations of IS morphology. In a next step, we applied our experimental pipeline to primary NK cells isolated from the blood of healthy donors. Distinct morphological features were characterized among the NK cells from different donors, highlighting the sensitivity of our approach, but also revealing an unsuspected variability of immune cell morphologies among donors. We then further applied our approach to primary CD8 + T cells from patients with a rare immunodeficiency due to mutations in the gene encoding the actin regulator ARPC1B. ARPC1B

deficiency results in decreased F-actin intensity, as well as in lytic granule polarization. This prompted us to assess the ability of these cells to kill target cells, which was markedly reduced. These results illustrate how the systematic analysis of the IS might be used to assist the exploration of fonctional defects of lymphocyte populations in pathological settings.

In conclusion, our study reveals that although assembly of the IS can be characterized by a few features such as F-actin intensity and cell spreading, capturing fine alterations of that complex structure that arise from cytoskeleton dysregulation requires a high-content analysis. The pipeline we developed through this project holds promises for the morphological profiling of lymphocytes from primary immunodeficiency patients whose genetic defect has not yet been identified. Moreover, the discriminative power of our high-content approach could be exploited to characterize the response of lymphocytes to various stimuli and to monitor lymphocyte activation in multiple immune-related pathologies and treatment settings.

Résumé des travaux L'imagerie cellulaire à haut débit révèle le contrôle de la synapse immunologique par le cytosquelette d'actine

Les lymphocytes cytotoxiques dépendent du remodelage du cytosquelette d'actine pour atteindre leur fonction. En particulier, les lymphocytes T cytotoxiques et les cellules NK assemblent la synapse immunologique (SI), structure complexe riche en actine qui permet l'interaction avec des cellules cibles et la distribution polarisée de granules lytiques (GL). Bien que le remodelage du cytosquelette d'actine soit connu pour être une force motrice de l'assemblage et de la dynamique de la SI, la compréhension du contrôle moléculaire du remodelage de l'actine soutenant la dynamique de la SI reste fragmentée. Ce projet de thèse a consisté à développer une approche d'imagerie à haut débit pour définir de manière impartiale les métriques de la SI des lymphocytes T et NK et caractériser les exigences d'intégrité du cytosquelette d'actine dans l'organisation de l'architecture de la SI.

À cette fin, la stimulation et coloration des lignées cellulaires et cellules primaires dans des plaques à puits multiples et l'acquisition d'un ensemble unique d'images confocales avec un imageur à haut contenu entièrement automatisé ont été optimisées. Les images ont été analysées avec deux pipelines complémentaires avec CellProfiler pour définir les caractéristiques morphologiques associées aux différents traitements et à l'état de la maladie. Nous avons d'abord extrait 16 caractéristiques morphologiques se rapportant à la F-actine, LFA-1 ou aux GL, sur la base d'une connaissance préalable de l'assemblage de la SI, et inclus des caractéristiques relatives au noyau. Nous montrons que l'assemblage de la SI dans les lignées est caractérisé par une augmentation de l'intensité de la F-actine et la surface cellulaire. Pour les cellules Jurkat, nous rapportons une augmentation de l'intensité et de la surface LFA-1, et de la de la détection des LG sur le plan SI pour les cellules NK-92. Le traitement des cellules NK-92 avec 7 drogues affectant différents aspects de la dynamique de l'actine et l'étude des effets associés sur les caractéristiques de la SI montrent des effets dépendants de la concentration sur l'intensité de l'actine et sur la polarisation des GL. De plus, un profilage morphologique à haute résolution basé sur> 300 caractéristiques montre que chaque drogue inflige des altérations distinctes sur la morphologie de la SI. Nous avons appliqué ce pipeline à des cellules NK primaires isolées du sang de donneurs sains. Des caractéristiques morphologiques distinctes définissent les cellules NK de différents donneurs, soulignant la sensibilité de notre approche, mais révélant également une variabilité insoupçonnée des morphologies des cellules immunitaires parmi les donneurs. Nous avons appliqué notre approche aux cellules T CD8+ primaires de patients présentant une immunodéficience rare due à des mutations dans le gène codant pour le régulateur d'actine ARPC1B. La carence en ARPC1B entraîne une diminution de l'intensité de l'actine et de la polarisation des GL. Cela nous a incités à évaluer la capacité lytique de ces cellules, qui a été considérablement réduite. Ces résultats illustrent comment l'analyse systématique de la SI pourrait être utilisée pour aider à l'exploration des défauts fonctionnels des populations de lymphocytes dans des contextes pathologiques.

En conclusion, notre étude révèle que, bien que l'assemblage de la SI puisse être caractérisé par quelques caractéristiques telles que l'intensité de l'actine et la propagation cellulaire, la capture de fines altérations résultant de la dérégulation du cytosquelette nécessite une analyse à haut débit. Le pipeline développé est prometteur pour le profilage morphologique des lymphocytes de patients atteints d'immunodéficiences primaires dont le défaut génétique n'a pas encore été identifié. De plus, le pouvoir discriminant de notre approche pourrait être exploité pour caractériser la réponse des lymphocytes à divers stimuli et surveiller leur activation dans de multiples pathologies et traitements.

various people are due. I apologize in advance if I have forgotten some people, who are nonetheless appreciated.

First, I would like to thank my supervisors: Drs Loïc Dupré and Kaan Boztug, without whom none of this would have been possible. Loïc, thank you for all your advice, help, support, the long hours and fruitful discussions, and for being a humane supervisor.

Drs Joerg Menche and Audrey Ferrand for their help and input in shaping the project.

My three reviewers Drs. Christophe Le Clainche, Delphine Muriaux and Fernando Sepulveda for taking the time to read this manuscript, advise and for agreeing to partake in the thesis jury.

A major thanks is due to Loan Vulliard, none of this would have been possible without your hard work, and the countless times you have helped me sort things out. Thank you for trying to make bioinformatics understandable for me, and for the much-appreciated occasional sarcasm.

The organoid platform of the IRSD, especially Aude Rubio, Muriel Quaranta-Nicaise and Dr. Isabelle

Fernandes, for all the long hours spent acquiring, analyzing and transferring (and re-transferring) images, and for making me fall in love with the Opera Phenix.

The ladies of both the cytometry and microscopy platforms of the CPTP, for all your help not only during experiments but also your kind and encouraging words from the start until the very end. Drs Sophie Allart and Fatima L'faqihi-Olive, you are strong, knowledgeable and amazing boss ladies.

Danièle Daviaud and Fatima, thank you for always being extremely sweet and helpful. Anne-Laure Iscache, Valerie Duplan-Eche and Lydia De La Fuente, I cannot forget to thank you for the times you have come to "rescue" me at the cytometers. "Maître" Astrid Canivet-Laffitte, I really appreciate every time you have helped me with ImageJ.

A huge thank you to all the colleagues in both Dupré /Lesourne and Boztug teams in Toulouse and Vienna: Dr. Renaud Lesourne, thank you for your feedback, advice and comprehension.

Nelly Rouquié, Cui Yang and Suzanne Mélique, you have been amazing ladies all the way. To the past members and now Drs Gaëtan Blaize and Jeremy Argenty, it was a pleasure being in the same team with you. To Claire, our most recent newcomer, thank you for you for your company along experiments, and of course for taking care of my cells along with Hélène. Dr. Hélène Daniels, thank you for the discussions, advice and feedback.

I cannot thank each member of the big Boztug team individually, but a huge thanks to every member for their sweetness, help, and for making the 9 months I spent in Vienna so memorable. Dr Artem Kalinichenko and Jakob Huemer, thank you for always being up for discussing anything NK. Dr.

Wojciech Garncarz, you are a hero and no words can do you justice. Drs. Ana Krolo and Ozelm Yuce, you ladies are sweethearts. Ewa Lenartowicz, thank you for your kind and encouraging words, always.

Dr. Johannes Pfeifenschneider, thank you for your sweetness and positive mood.

To the Valitutti team (Dr. Salvatore Valitutti, Dr. Eric Espinosa, Dr. Marie-Pierre Puissegur, recent Drs. Roman Jugelé and Yoan Eliasse, and Liza Filali Sabina Mueller), and the previous members of the Dupré team (Drs. Javier Rey-Barrosso and Raïssa Houmadi, and Alice Munaretto) thank you for making me feel welcome among you, your help, sweetness and support, and for making the beginning of this journey a bit easier. Every single one of you has helped me throughout this experience whether it was through advice, support or even words of encouragement over a coffee. To Dr. Delphine Guipouy, thank you for helping me with the French administration, sharing your knowledge with me, and answering my endless questions. Thank you infinitely Dr. Cat.

A special thank you to my colleagues Tala Shahin, Maximilian Rau, Laurène Pfajfer and Marianne Guisset, colleagues who also became great friends. Afterall, it is friends who make some days more bearable.

The colleagues at CPTP who have given feedback and input to help better shape this project through listening to my presentations and advising. Moreover, thanks to Drs Anne Quillet-Mary, Gaël

Menasché and Abdelhadi Saoudi for their advice and feedback as part of my thesis committee.

An immense thank you to Dr. Nabila Jabrane-Ferrat for all her advice, support and help. You are the best "godmother" I could have asked for. I would also like to thank the NJF team: Drs Hicham El Costa (thank you for your encouragement through your sarcasm), Jordi Gouilly and Qian Chen, and finally the wonderful Ana Espino, also known as "my hero".

To my friends in Toulouse and away, and my family and friends back in Lebanon who have been there

for me throughout this journey. From the bottom of my heart I would like to thank Celine, Ranine and Elio, you have become a family away from home.

To my small family. Whatever I say, words will never be enough.

Preface

Having come from a background in microbiology and virology, immunology was not always the most evident of topics for me, as I had done my masters in a lab that worked on mice, where I had mostly done Western blots and Real-time PCR. The decision to embark on a PhD in immunology was quite the challenge for me, but the idea of putting in place a screen of cytotoxic cells by high content imaging sure was appealing. I began my PhD in the Dupré lab in January 2017, in the Valitutti team.

The team is an expert on cytotoxic cells, and the Dupré team is an expert on the immunological synapse of T cells, especially in the context of primary immunodeficiencies. At the time I joined, the Dupré team was composed of a postdoc: Javier Rey-Barrosso, three PhD students: Delphine Guipouy, Raissa Houmadi and Laurène Pfajfer, and an engineer: Alice Munaretto.

Javier Rey-Barrosso was passionate about motility and was studying the different migration patterns of B cell lines on Collagen and Fibronectin, as well as their ability to remain and individual cells or cluster based on the matrix. He did so by seeding these cells in 96 well plates and imaging them by TIRF microscopy. He also studied the roles of actin branching and actoMyosin contractility by using two drugs: the ROCK1 inhibitor Y-27632 and the ARP2/3 inhibitor CK-869. He also did live imaging of chemotaxis of B cells over a CCL19 gradient. Javier loved to perform his experiments and allowed me to observe, while giving me thorough explanations. Indeed, he was my first exposure to chemotaxis, as well as the potential that multiwell plates offer in terms of efficiently imaging cells under different conditions.

Alice Munaretto was also working on migration, but as opposed to Javier who was working on a B cell line, she was working on cells from CLL patients, to assess the migratory behavior of specific cell populations pre-and post-treatment. To do so, she was using a protocol of barcoding to phenotype her cells and was assessing their migration in response to chemokines. She did so by trans well assays, whose readout is by flow cytometry. Observing these experiments with Alice was my first exposure to flow cytometry. Moreover, to minimize bias in sample readout, Alice would set up an automatic sample mixing and acquisition, which was the first time I saw "automated" experiments.

Delphine Guipouy was in her last year of PhD when I met her. She was working on a specific type of cytotoxic Tregs referred to as ova-Tr1 T cells, that are capable of specific elimination myeloid target cells via their granzyme and granulysin. Delphine was highly passionate about cytotoxic cells, and she showed me how to set cytotoxicity assays in 96 well plate formats and how to analyze the results acquired by Flow cytometry. She also showed me how to perform live microscopy assays to assess cytotoxicity in real-time. Delphine also spoke to me about a killing experiment she had done in real-time, by using a high content microscope (The Operetta), where she could sort single CD8+ T cells and seed one cell per well in a 96 well plate, to observe their cytotoxicity over time. She also showed me how to perform imaging of the immunological synapse of cytotoxic cells incubated with target cells, on microscopy slides.

Raissa Houmadi was also in her 3 rd year of PhD, and she was working on developing a super-resolution microscopy approach. Indeed, she revealed how LFA-1 is spatially organized in nanoclusters and how this assembly is regulated by WASP to ensure proper granule docking in CD8+ T cells.

Laurène Pfajfer was a first year PhD student who was doing a part of her PhD at the LBI-RUD in Vienna, where she had worked on two primary immunodeficiencies: WIP and WDR1 to show how these defects lead to defective immunological synapse assembly and cytotoxicity.

Delphine, Raissa and Laurène, as well as Loic's background in WASp and other primary immunodeficiencies provided me the exposure I needed to primary immunodeficiencies, and the immunological synapse.

In the context of primary immunodeficiencies, NK cells are sometimes overlooked in cell biological assays due to their low numbers and the difficulty to expand them. I fell in love with NK cells only by working with the NK-92 cell line, but I admired these cells for their cytotoxic potential. The journey with these cells was not easy and being the first in the lab to have to grow them and use them in experiments was not an easy challenge. For that I would like to thank Delphine Guipouy, for her advice, feedback and help.

Despite the challenges that these cells presented, the difficulty to transduce the NK-92 cell line, not due to lack of trying, and their fragility and erratic behavior sometimes, I am glad have been able to set up the functional approaches routinely used in our lab on CD8+ T cells on the NK cells, as well as an approach that allows the high content imaging of the immunological synapse of these poorly adherent cells. Our lab is an expert of CD8+ T cells and their cytotoxicity, as well as the defects in the context of primary immunodeficiencies.

Taken together, the expertise of my host team has led me to optimize several assays established in the lab on the NK-92 cell line and eventually primary NK cells, as well as being the first in our lab to put in place the protocol for conjugation assay, which has later on been applied by colleagues to expanded CD8+ T cells.

Most importantly, the past three years have led me to develop the protocol of high content imaging of the immunological synapse of NK-92 and Jurkat cell lines, as well as primary NK cells and expanded CD8+ T cells from patients suffering from a primary immunodeficiency caused by mutations in the ARPC1B gene of the ARP2/3 complex.

This project would not have been possible without the help of the smart, dedicated and through bioinformatician Loan Vulliard, whose expertise has helped take this project to the next level. I was lucky to have been able to work closely with Loan during my 9-month stay at the Center for Molecular Medicine in Vienna, as a guest scientist in the Boztug team.

Prior to the results section, I will briefly introduce the immune system, with a focus on cytotoxic cells and highlighting the role of the actin cytoskeleton and the immunological synapse in achieving the cytotoxic function. The second part will address dysregulations that can affect the cytotoxic functions, while focusing particularly on primary immunodeficiencies where the cytotoxic function is perturbed.

The last part will comprise information of the various types of microscopy approaches, with a focus on high content imaging.

In the coming pages I hope to be able to highlight the importance of the cytotoxic function, as well the importance of the immunological synapse in this context. I also hope to convince the people reading this manuscript that high content imaging is the revolutionary step that they should invest in, and that, with some effort, even poorly adherent cells are amenable to being imaged with this method. Most

Figures and tables

Introduction

The following pages will comprise an introduction on the immune system with a focus on cytotoxic lymphocytes, their mechanism of action and receptors. In particular, the role of the actin cytoskeleton rearrangements and the immunological synapse in supporting cytotoxicity will be presented. Next, pathology-related defects in cytotoxicity will be addressed with a focus on primary immunodeficiencies, including a chapter on the methodologies used to assess defects in the cytotoxic function.

The second section of the introduction will focus on microscopy as a rapidly evolving collection of methodologies. In particular will be addressed how high content cell imaging applications provide novel opportunities to explore cell biology. This introduction will then provide a rationale for the implementation of high content cell imaging for the study of cytotoxic lymphocytes.

General overview of the immune system and immune cell activities

The human immune system is composed of a complex association of lymphoid organs, tissues, cells, humoral and soluble factors, which evolve simultaneously with the environment the body is exposed to, to ensure its protection against pathogens. Moreover, the immune system ensures the elimination cancer cells, as well as ensuring tissue reparation to maintain homeostasis. Also, it ensures symbiosis with the microorganisms of the microbiota.

For long, the immune system has been divided into innate and adaptive immunity, due to the types of cells involved, as well the specificity and duration of their effector function. However, research is

showing that cells of both types are highly interconnected. Therefore, an increasing number of immune cells is to be found at the interface between the innate and the adaptive immunity (Figure 1).

Innate immunity

Innate immunity is a quick reaction which follows a non-specific recognition of various microorganisms, and can take as little as a few hours to be put in place 2 . The cells implicated are tissue resident natural killer cells, macrophages, mast cells, monocytes, neutrophils, basophils, eosinophils and dendritic cells (DC). However, only natural killer (NK) cells will be detailed in a section of this manuscript.

Cells at the interphase of the innate and adaptive immune systems

Innate lymphoid cells (ILC) are a new category of cells which were discovered in the past decade.

Three groups of ILCs exist, based on the expression of transcription factors and cytokines that they produce 3 . NK cells were officially classified as the prototypical members of the group 1 innate lymphoid cells (ILC), which are defined by capacity to secrete interferon gamma (IFN-γ), but not type 2 cytokines 4 . Natural killer T (NKT) cells, which share surface markers of both natural killer and T cells. They express a semi-invariant receptor resembling that on T cells and which reacts with major histocompatibility complex (MHC) CD1d, as well as receptors present on NK cells such as CD161 5 .

NKT cells are both cytotoxic cells and capable of releasing cytokines, such as IFN-γ, IL-4, IL-10 and IL-13. Tγδ cells are CD3 + T cells that express the gamma/delta alternative T cell receptor (TCR) (as opposed to the conventional alpha/beta). They are cells that produce cytokines and exert a cytotoxic activity. They recognize bacterial phosphoantigens and non-classical MHCI molecules, and in response can either exhibit cytotoxicity or secrete cytokines such as IFN-γ [6][7][8] .

Adaptive immunity

Adaptive immunity requires a longer time to take place than innate immunity, as it requires the action of antigen-specific cells, some of which would eventually become memory cells. Such a process requires several days to be put in place 2 . The players in this immune response are T and B lymphocytes.

However, and as previously mentioned, innate and adaptive immune responses are interconnected. An adaptive immune response cannot be initiated without the dendritic cells, which are antigen presenting cells (APC). DCs will migrate into the lymphoid organs, where they will activate the T cells specific to their antigen. Through V(D)J rearrangements through their development, T cells would be endowed with unique and antigen specific TCR 9,10 .

A. B lymphocytes

B cells are CD19 + lymphocytes. B lymphocytes recognize specific antigens by B cell receptors (BCR)

or membrane bound immunoglobulins. BCRs recognize either soluble foreign antigens or ones bound to cell surface. Antigens are then internalized and processed in lysosomal/endosomal compartments, and subsequently expressed on the cell surface as antigenic peptides bound to MHC-II molecules for presentation to T helper cells (discussed in the next section). Activated B cells develop into plasma cells, which are antibody secreting cells.

B. T lymphocytes

T cells are CD3 + cells that can also be CD4 + or CD8 + . CD4 + T cells are known as helper T (Th) cells and can be further subclassified into Th1, Th2 and Th17, based on the cytokines and interleukins they produce. Special subsets include follicular helper T cells (Tfh) and regulatory T cells (Treg). Their main role is aiding in B cell activation and antibody production. Follicular helper T cells are essential for B cells to mature into plasma cells and achieve proper immune functions 11 . Regulatory T cells suppress immune reactions and maintain immune homeostasis. CD8 + T cells were thought to be the only cytotoxic cells, prior to the identification of a subpopulation of cytotoxic CD4 + T cells 12 . CD8 + T cells are cytotoxic T cells (CTLs) and will be discussed in detail in the next section.

The cytotoxic function: the weapon of defense of the immune system

Natural killer cells and CD8+ T cells are two cell types belonging to the innate and the adaptive arms of the immune system respectively. Even though they, broadly speaking, appear to serve the same function, which is to eliminate target cell, their mechanism of action, cell surface receptors and regulation are not the same, and will be elaborated in detail in this section. The cytotoxic activity of NKT, Tγδ and cytotoxic CD4+ T cells will not be addressed.

Cytotoxicity is achieved when the cytotoxic cells kill their targets by the means of releasing lytic molecules through the process of degranulation. CD8+ T cells and NK cells can kill target cells by a tightly regulated secretion of cytotoxic granules containing pore-forming proteins perforin and/or granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). However, a deep immune profiling of CTLs and NK cells has shown that NK cells have more abundant granulysin than CTLs 13 .

Lytic granules are delivered to the interface of the cytotoxic lymphocyte and target cell, and their exocytosis requires clearances in the actin cytoskeleton 14 . Released perforin monomers insert into the target cell membrane and polymerize to form a pore through which granule contents including the effector protease enzymes are delivered, and subsequently cleave caspases to initiate cell death.

Natural killer cells

Natural killer cells are cytotoxic lymphocytes of the innate immune system, and which play a role in protection against viral infections and solid and hematological malignancies. They comprise about 10-15% of circulating lymphocytes. For a long time, NK cells were simply identified as cytotoxic cells that could kill without prior activation. They specifically target cells that lack MHC-I expression, without expressing clonally-distributed antigen receptors 15 . Moreover, they release cytokines that cause an inflammatory response which mediate the response of the adaptive immune system. Further details on the cytotoxic activity of NK cells will be addressed in the next chapter, "cytotoxicity".

Two major markers to characterize natural killer cells are CD16 and CD56. That being said, it should be noted that approximately 90% of natural killer cells are CD16 bright CD56 dim and those are the ones capable of generating the cytolytic response, also known as antibody-dependent cellular cytotoxicity (ADCC) 16 . The other 10% are CD16 dim CD56 bright and are the ones that secrete cytokines. When it comes to expression of lytic molecules, among NK cells, CD56 bright and CD56 dim CD16+ NK cells showed clearly different cytotoxic molecule expression patterns: CD56 bright NK cells had low Gzm B, but high Gzm K expression, whereas the CD56 dim CD16+ NK cell subset expressed high Gzm B but lacked Gzm K 13 .

The cytotoxic activity of NK cells is tightly regulated by a complex interplay between stimulatory and inhibitory receptors and their ligands expressed on the target cells, which will be discussed in the coming section 17,18 .

A. NK cell receptors

Natural killer cells receive signals through different receptors on target cells and use a multiple receptor recognition strategy and will kill cells that have an increased or a decreased expression of "self" proteins, and cells that express foreign proteins.

a. Natural cytotoxicity receptors

Natural cytotoxicity receptors (NCR) are key in recognizing virus infected and tumor cells and their clearance. The expression of these NCRs has been shown to correlate with the magnitude of the cytolytic activity of the NK cells.

i. CD16

CD16 is an Fc receptor (FcγIII) present on the CD56 dim population of the natural killer cells. Signaling through CD16 delivers a powerful signal that enables the natural killer cells to kill target cells mainly by cytolysis. CD16 binds to the IgG portion of antibodies. In NK cells, CD16A is expressed and colocalizes with CD3ζ and Fc-εRI-γ, thus inducing the stimulatory signals. CD16 activation following binding to its ligand induces of the transcription of the genes encoding interleukin-2 receptor (IL-2R)

and cytokines, and even though binding to anti-CD16 induces a strong cytolytic reaction, it does not lead to NK cell proliferation 19,20 . Moreover, it has been shown that the levels of expressions of CD16 may vary, and this variation correlates with the strength of serial killing 21 .

ii. NKp46

NKp46 (also known as CD335) is a member of the natural cytotoxicity receptor family, and is, along with NKG2D, one of the main receptors. In1997, a 46 kDa molecule was shown to play a role in NK mediated cytolytic activity, cytokine secretion as well as Calcium ion mobilization 22 . NKp46 is encoded in the leukocyte receptor complex on chromosome 19 23,24 . NKp46 has been shown to be expressed on subsets of natural killer cells, while it was not expressed in T and B cells 25 . NKp46

expression was shown to be decreased in post-transplant lymphoproliferative disease in all natural killer subsets, accompanied by an increase in programmed cell death protein 1 (PD-1), which may give a hint on the dysregulation in natural killer functions 26 . Cross-linking of NKp46 with a monoclonal antibody (mAb) resulted in an increase in cytolytic activity, cytokine secretion and Calcium ion mobilization. NKp46 molecules are coupled to intracytoplasmic transduction machinery through associating with CD3ζ and Fc-εRI-γ adaptor proteins that contain immune tyrosine-based activating motifs (ITAM).

iii. NKp30

NKp30 is encoded in the class III region of the MHC locus on chromosome 6 24 . It has been demonstrated that NKp30 expression varies depending on the organ in which NK cells are examined:

it is highest in spleen, lowest in liver and intermediate in blood, despite the absence of a general receptor down-regulation, thus suggesting either an organ-specific regulation of NKp30 receptors, or a preferential homing of NKp30+ NK positive cells 27 . Expression of the ligands of NKp30 on DCs is the main regulator of the supply of dendritic cells, as NKp30 can mediate the promotion or termination of dendritic cell maturation. One of the ligands of NKp30 is BAG6. Soluble BAG6 has been shown to be present in elevated levels in patients with hematological diseases 28,29 . Galectin-3 has also been shown to be a soluble NKp30 target, and to play a role in tumor evasion from NK cells 30 . B7-H6, another soluble NKp30 ligand was shown to be associated with a down-regulation of NK cells in neuroblastoma patients 31 . Another NKp30 ligand is BAT6, a nuclear factor also released by tumor cells in exosomes rather than a soluble protein, which triggers cytokine release (tumor necrosis factor alpha (TNF-α), IFN-γ) and enhances NK-cell mediated killing 27,32 . The cytoplasmic region of NKp30 lacks ITAM motifs.

iv. NKG2D

NK receptor member D of the lectinlike receptor family (NKG2D) is a natural killer receptor that binds to cells which have an upregulated expression of self-proteins. It is also expressed on CD8+ T cells, γδ T cells and Natural Killer T cells 33,34 . NKG2D binds to NKG2D-L on target cells, thus activating an effective anti-tumor response in early stages 35 . However, a sustained NKG2D-L expression and shedding of soluble ligands counteracts NKG2D-dependent NK cell activity in later stages. Contrary to NKG2A, NKG2D does not bind to CD94. The ligand for NKG2D is stress-inducible MICA, a distant homolog MHC I, and the expression of NKG2D on NK cells is increased by IL-15 34,36 . The structures of NKG2D-ligand complexes indicate that NKG2D binds diagonally over the α1 and α2 helices of the ligands, which bears a striking resemblance to the way T-cell receptors bind over MHC molecules. DAP10 is the membrane-bound signal transducing subunit of the NKG2D receptor 37 . It has been shown that one tumor evasion mechanism is the release of soluble NKp30 and NKG2D ligands, as well as a down-regulation of the expression of NCR ligands 38,39 .

v. NKp44

NKp44 is another member of the NCR family, and is about 44 kDa in size. The gene encoding NKp44 is encoded on chromosome 6. While NKp30 and NKp46 are expressed on resting and activated NK cells, NKp44 is exclusively expressed on activated NK cells in the presence of IL-2 40 . NKp44 ligands include glycoproteins and proteoglycans expressed on the surface of target cells, nuclear proteins that can be exposed outside the cell, and molecules that can be either soluble or in vesicles 41 . In the same fashion as NKp46, monoclonal antibody cross-linking of NKp44 enhances the cytolytic activity of NK cells. NKp44, which does not contain any ITIM motif in the cytoplasmic region, associates with the adaptor molecules DAP12 and KARAP, which contain an ITAM motif in the cytoplasmic region 40,42 . E molecule (a non-classical MHC-Ib) and subsequently protect the target cells from cytolysis 43 . KIRs include but are not limited the p49, p50, p58, p70 and p140 17,38 . However, each KIR is expressed on a fraction of NK cells, thus allowing the NK cells to recognize a larger pool of MHC-I deficient cells. It has been shown that once an NK cell is in contact with an MHC-I expressing cell, KIRs accumulate at the contact area in a central area surrounded by LFA-1 and ICAM-1 to drive NK cell migration 44 . Factin is a somewhat expendable player in this process, whereby it can accelerate KIR recruitment to the synapse but is not crucial for this process 44 .

c. Costimulatory molecules

2B4 (CD44) which binds to CD48, and NTBA are two members of the CD2 family which can play either an activating or inhibitory function, depending on the availability of downstream regulating element of the signaling pathway termed signaling lymphocyte activation molecule-associated protein (SAP). Both 2B4 and NTBA trigger NK cytolytic activity in cells that express high levels of NCRs 45,46 .

NTBA promotes a strong production of IFN-γ and TNF-α 46,47 . 2B4 may also enhance the cytolytic activity of NK cells 48 . 2B4 does not associate with ITAM-bearing polypeptides but has functional signaling motifs in the intracellular domain. The 2B4-CD48 interactions appear to take place at the center of the synapse 44 . Sivori and colleagues have shown that 2B4 can initiate cytolysis in the subset of NK cells that have a high level of NKp46 expression, without altering the expression of 2B4, but also that 2B4 may function as an inhibitory molecule at early stages of NK cell differentiation 49,50 .

Other co-stimulatory molecules include CD80 and CD86, which have been shown to enhance the cytotoxic activity of NK cells 51 .

B. NK cell cytotoxicity: from target binding to target lysis

Natural killers were long compared to cytotoxic T cells. However, they are unique in the sense that they use specific NK receptors, and that their response is faster than that of CTLs. Being part of the innate immune system, natural killer cells do not need prior activation to achieve their cytotoxic function. Therefore, they do not express antigen-specific receptors, but rather the activation and inhibition receptors discussed in the previous chapter "NK cell receptors". The cytotoxicity of NK cells can be broken down into several steps, leading to the formation of the immunological synapse (IS) and achievement of the cytotoxic function by release of lytic molecule content, and then detachment of scanning for another target cell.

a. NK receptors involved in immunological synapse formation

NK cell receptors can associate with ITAM bearing molecules. FcRγ and TCR ζ chains can form either homodimers or heterodimers which interact with CD16. NKp30 and NKp46 associate with FcRγ and TCR ζ, while NKG2D associates with DAP10 52 . After patrolling their environment, NK cells attach to a target cell through adhesion receptors, which include CD2, NKG2D and DNAM-1. By using tweezers and confocal microscopy, it has been demonstrated that upon NK cell contact with its target, NKG2D is recruited to the IS and forms a ring type of structure which marks a border in which lytic granules are secreted 53 . Moreover, stimulation through NKG2D was shown to lead to the formation of an F-actin ring at the periphery of the IS, and that NKG2D leads to a more stable synapse than CD16 21,53 . NKp44 associated with DAP12, a homodimer with a single ITAM motif. While signaling through CD16 is sufficient to cause NK cell -target conjugates and degranulation, stimulation of other receptors alone does not cause NK degranulation 21,54,55 . Other receptors only stimulate degranulation in pairs, suggesting a synergistic activation. CD16 binds to the Fc portions of antibodies and NK cells can therefore kill target cells coated with antibodies and eliminate them by ADCC. NKG2D signaling leads to adhesion, granule polarization and degranulation 56 . DNAM-1, NKp80 and NKp65 also have activating potential. However, there is limited information of their signaling properties. The immunological synapse of NK cells will be touched upon further in the section pertaining to the steps of cytotoxicity.

b. NK cell degranulation

Degranulation of NK cells is not random process, and it has been shown that NK cells must converge their lytic granules to prevent bystander killing and achieve a more efficient elimination of target cells 55 . Moreover, it has been shown that receptor activation must occur in a specific sequence, as initial engagement of CD16 did not affect subsequent stimulation via NKG2D, whereas stimulation through CD16 was impaired after activation via NKG2D 21 . Once firmly tethered to its target, the NK cell undergoes a change in shape and becomes more flattened, and a lytic cleft is formed 57 . Following actin rearrangement as well as de novo actin synthesis, the MTOC undergoes reorientation to allow polarization of the lytic granules towards the synapse, and this is mediated by the formin homolog hDia, which in NK cells is not essential for actin branching 58 .

DNAM-1 has been found to physically associate with lymphocyte function associated antigen 1 (LFA-1), thus indicating that it plays a role in LFA-1 mediated signaling 59 . Indeed, LFA-1 engagement was shown to mediate granule convergence, and to induce more targeted degranulation at the IS, along with CD16 than CD16 alone 55 . Therefore, the co-engagement of LFA-1 and CD16 leads to a monodirectional granule convergence, thereby reducing bystander killing rates and increasing the rates of specific killing. only a small fraction express the open conformation of LFA-1, and LFA-1 in NK cells requires activation to through the aforementioned adhesion receptors, which can induce insideout signaling 60 . LFA-1 induces a downstream signaling cascade, changes in F-actin reorganization, as well as an arrest signal and symmetrical spreading 61 . Talin is also recruited to the sites of LFA-1 prior to granule polarization, and, via constitutive association of vinculin (an actin-binding protein that was shown to act as "leaky capper" of the barbed end of the actin filament) to talin and Arp2/3, recruits ARP2/3 [62][63][64] . Moreover, talin is essential for lytic granule polarization [START_REF] Mace | A Dual Role for Talin in NK Cell Cytotoxicity: Activation of LFA-1-Mediated Cell Adhesion and Polarization of NK Cells[END_REF] . Furthermore, LFA-1 also leads to WASp recruitment, which in turn promotes Arp2/3 mediated actin polymerization 62 . Lytic granules must converge long the MTOC, and this step is independent of actin dynamics and of CD107a, and is rather mediated by dynein and does not imply a commitment for neither granule polarization nor for cytotoxicity [START_REF] Mace | Cell biological steps and checkpoints in accessing NK cell cytotoxicity[END_REF] . However, formation of an organized synapse where LAMP-1 is retrieved at the center depends on the interaction of LFA-1 with the lipid bilayer 44 . Following lytic granule polarization, they dock at the IS membrane and fuse with the plasma membrane. The docking is primarily mediated by Rab27a and Munc13-4, followed by priming, which occurs as a result of Munc13-4-mediated activation of Syntaxin 11 or bridging of the plasma membrane with lytic granule [START_REF] Mace | Cell biological steps and checkpoints in accessing NK cell cytotoxicity[END_REF][START_REF] Wood | Different NK cell-activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity[END_REF][START_REF] Kurowska | Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex[END_REF] . Fusion occurs as a result of the formation of a complex whose components are not all known, but include syntaxin11, VAMP 4 and VAMP 7 [START_REF] Mace | Cell biological steps and checkpoints in accessing NK cell cytotoxicity[END_REF] .

Following lytic granule delivery to the IS, it has been shown that only a fraction of the lytic granules are exocytosed, and that granules are highly mobile, independently of F-actin dynamics, but may depend on Myosin II A(MyH9) to generate the necessary forces 14,[START_REF] Mace | NK Cell Lytic Granules Are Highly Motile at the Immunological Synapse and Require F-Actin for Post-Degranulation Persistence[END_REF] . Indeed, MyH9 heavy chain knock-out impaired cytotoxicity, membrane fusion of lytic granules, and granzyme secretion [START_REF] Andzelm | Myosin IIA is required for cytolytic granule exocytosis in human NK cells[END_REF] . It has also been shown that MyH9 constitutively physically interact with lytic granules, and lytic granule secretion was shown to be impaired in patients suffering from MyH9 mutations [START_REF] Sanborn | Myosin IIA Associates with NK Cell Lytic Granules to Enable Their Interaction with F-Actin and Function at the Immunological Synapse[END_REF] . While Myosin IIa is not needed for the formation of an organized IS in NK cells, it is crucial for the final exocytosis step 44 . Following degranulation, the cell must undergo a termination process, followed by detachment.

These steps are elaborated in details in [START_REF] Mace | Cell biological steps and checkpoints in accessing NK cell cytotoxicity[END_REF] . Moreover, it has been shown that lytic granules require a pervasive actin meshwork of hypodense F-actin (which can be achieved through NKG2D and LFA-1 co-ligation) to pass through and that the actin clearances in which they pass are granule-sized 53,[START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] .

However, actin dynamics are independent of the presence of granules, since actin clearances are observed prior to granule arrival. It has also been demonstrated by super resolution microscopy that the center of the NK IS contains thin F-actin filaments, as opposed to the previous belief that the IS formation leads to F-actin clearance from the center 53,[START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] .

Cytotoxic T cells: the killers of the adaptive immune system

CD8+ T cells have a classical αβ T-cell receptor TCR. Through a random process of genetic rearrangements, lymphocytes express on their surface unique TCR that have single antigen specificity, which is their capacity to recognize one type of peptide-MHC complex.

Progenitor lymphoid cells leave the bone marrow and reach the thymus to proceed to differentiate, where they undergo a negative and a positive selection. Following this selection, the remaining cells will be capable of recognizing APC, and not react against self-molecules. Naïve CD8+ T cells must be activated by CD4+ cells in order to produce lytic and effector molecules [START_REF] Wang | Multiple Paths for Activation of Naive CD8 + T Cells: CD4-Independent Help[END_REF] . CD8+ lymphocytes recognize MHC I molecules presented on the surface of APCs, therefore most cells can be APC-as opposed to CD4+ T cells which recognize MHC II molecules on "professional" APC such as DC and B cells. Naïve T cells will give rise to several subpopulations, culminating in effector cells. Effector CD8+ T cells express lytic molecules and have high migratory capacity. Following stimulation of their TCR, they undergo reorganization of their actin cytoskeleton, slow down their motility and establish firm adhesions with their targets leading to the assembly of the IS [START_REF] Zhang | CD8+ T Cells: Foot Soldiers of the Immune System[END_REF] .

The delivery of lytic granules from CD8+ T cells is a highly regulated process, and the signaling and cytotoxic activity of CD8+ T cells will be elaborated in detail in the section "CD8+ T cell Cytotoxicity".

A. Receptors, adhesion and co-stimulatory and inhibitory molecules involved in CD8+ T cell cytotoxicity

The activity of cytotoxic T cells is governed by the interplay between several receptors and costimulatory molecules, which will be elaborated in the following pages.

a. The T-cell receptor

The αβ TCR on CTLs is composed of two transmembrane glycosylated polypeptide chains belonging to the immunoglobulin superfamily and linked through a disulfide bond. These glycoproteins contain a short intracytoplasmic tail composed of 4-12 amino acids and a hydrophobic transmembrane domain of 20 positively charged amino acids, thus allowing the TCR to achieve a stable association with the negatively charged transmembrane domain of the CD3 molecules (Figure 4) [START_REF] Rudolph | PEPTIDES, AND CORECEPTORS[END_REF] .

Figure 4: The TCR and its association with CD3. The TCR is composed of the α and β chains and forms a complex with CD3. From the thesis manuscript of Roxana El Khazen, 2016.

T cell activation requires sustained signaling by the pMHC specific to the TCR. However, the duration of the TCR-pMHC contact has been shown to be very short [START_REF] Matsui | Low affinity interaction of peptide-MHC complexes with T cell receptors[END_REF][START_REF] Weber | Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor[END_REF] . Therefore, it was proven that despite the rapidity of TCR-MHC interaction, sustained TCR signaling occurs via multiple rounds of TCR-MHC binding [START_REF] Huang | The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness[END_REF][START_REF] Huppa | TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity[END_REF] . Interestingly, as little as 1 MHC molecule can induce calcium ion increase in T cells, and that T cell activation can be achieved by TCR recognizing as little as 10-15 MHC molecules, and CTLs can kill target cells expressing 1-10 MHC molecules [START_REF] Irvine | Direct observation of ligand recognition by T cells[END_REF][START_REF] Purbhoo | T cell killing does not require the formation of a stable mature immunological synapse[END_REF][START_REF] Huse | Spatial and Temporal Dynamics of T Cell Receptor Signaling with a Photoactivatable Agonist[END_REF][START_REF] Jiang | Two-Stage Cooperative T Cell Receptor-Peptide Major Histocompatibility Complex-CD8 Trimolecular Interactions Amplify Antigen Discrimination[END_REF] . This can be explained by the proposed model of "serial TCR engagement", which suggests that one MHC molecule could trigger and internalize approximately 180 TCRs, thus leading to a sustained TCR signaling despite the small number of MHC molecules via a sequential high TCR occupancy [START_REF] Valitutti | Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton[END_REF] . However, another model referred to as "kinetic proofreading" suggests a prolonged TCR-MCH interaction, leading to modifications which will be amplified only by highly specific interactions [START_REF] Mckeithan | Kinetic proofreading in T-cell receptor signal transduction[END_REF] . However, it is most likely that these two types of interactions are not mutually exclusive and may be dependent on the number of MHC molecules on the APC surface.

Briefly, the steps of TCR signaling include the translocation of LCK and Fyn, two members of the Src tyrosine kinase family, into the membrane following TCR engagement, which eases the phosphorylation of ITAM motifs. ZAP-70 also associates to ITAM motifs and activates Linker for activation of T cells (LAT) protein which will allow more signaling molecules to bind (Figure 5).

However, TCR engagement is not enough and T cell activation requires signaling from co-stimulatory molecules. These include CD8 for CTLs, CD28 and the adhesion molecules CD2 and LFA-1 [START_REF] Dustin | A Novel Adaptor Protein Orchestrates Receptor Patterning and Cytoskeletal Polarity in T-Cell Contacts[END_REF] .

Figure 5: Pathways of TCR signal transduction [START_REF] Brownlie | T cell receptor signalling networks: branched, diversified and bounded[END_REF] .

b. The role of the integrin LFA-1 in cytotoxicity

Integrins are heterodimeric adhesion proteins, which allow leukocyte adhesion and play a role in their migration. Integrins play an essential role in infection control, where their absence leads to recurrent infections. They also have a role in immune homeostasis, and perturbations can lead to cancers and autoimmunity. Integrins have a crucial role in the recruitment of lymphocytes from the blood into infection sites, and for lymphocyte adhesion onto their target cells. Moreover, integrins make intracellular adhesion therefore playing a role in the activation of several signaling pathways.

Being heterodimeric proteins, they are formed of one of 18 α and one of 8 β subunits. They can be one of 24 different combinations 88,[START_REF] Takada | The integrins[END_REF] . The very late after activation (VLA) family of integrins which comprises VLA-1 to 5 has also been identified. Of the LFA-1 family, three members have been identified: LFA-1, 2 and 3 [START_REF] Campana | Human leukocyte function-associated antigens on lympho-hemopoietic precursor cells[END_REF] . In this manuscript, only the integrin LFA-1 will be addressed.

LFA-1 is required for the migration of T and NK lymphocytes. It is composed of the CD11a (αL) and the CD18 (β2) subunits. LFA-1 plays a role in immunity, as its deficiency is characterized by recurrent bacterial infection [START_REF] Anderson | Leukocyte Adhesion Deficiency: An Inherited Defect in the Mac-1, LFA-1, and p150,95 Glycoproteins[END_REF] . LFA-1 is a transmembrane protein comprised of an extracellular domain, a transmembrane domain and a cytoplasmic domain. LFA-1 binds to intracellular adhesion molecule ICAM-1, among other ICAMs (ICAM-2 and 3, as well as JAM-1). In this manuscript, only the LFA-1 -ICAM-1 interaction will be addressed. LFA-1 binding to ICAM-1 is a temperature-dependent energy requiring process, which also requires the presence of magnesium ion and an intact cytoskeleton [START_REF] Marlin | Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1)[END_REF] .

ICAM-1 (CD54) is a transmembrane glycoprotein expressed on the surface of leukocytes and endothelial cells. ICAM-1 is required for the interaction of LFA-1 on immune cells with their target cells or DCs, or between the immune cells and their environment [START_REF] Marlin | Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1)[END_REF][START_REF] Rothlein | A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1[END_REF][START_REF] Long | ICAM-1: Getting a Grip on Leukocyte Adhesion[END_REF] . Moreover, full LFA-1 activation requires ICAM-1 on the target cell to provide a physical resistance to allow for F-actin flow [START_REF] Comrie | F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse[END_REF] .

i. LFA-1 activation

Integrins in circulating lymphocytes are inactive and get activated through binding to chemokine receptors. LFA-1 activation is characterized by both affinity to ICAM-1, and avidity.

Affinity is defined as the capacity of a ligand to bind to its receptor, depending on the available number of molecules. FRET experiments have revealed that the constant of dissociation between LFA-1 and ICAM-1 to be 17.93±1.34 nM [START_REF] Chakraborty | FRET Based Quantification and Screening Technology Platform for the Interactions of Leukocyte Function-Associated Antigen-1 (LFA-1) with InterCellular Adhesion Molecule-1 (ICAM-1)[END_REF] .

Avidity is defined as the strength of interaction between two molecules. The increased probability of an LFA-1 -ICAM-1 interaction due to the aggregation of LFA-1 molecules indirectly leads to an increased adhesion strength [START_REF] Carman | Integrin avidity regulation: are changes in affinity and conformation underemphasized?[END_REF] . Avidity varies based on the conformation (openness) of the LFA-1 molecule. LFA-1 has been described to have three conformations: the closed conformation corresponds to a low affinity and thus an inactive LFA-1 (Figure 6). LFA-1 activation is modulated by bidirectional signaling: inside-out and outside-in signaling.

ii. Inside-out signaling

Inside-out signaling is mediated by intracellular signals, resulting from the activation of other receptors such as the TCR or chemokine receptors, to mediate extracellular changes to the adhesive state of LFA-1 [START_REF] Verma | Not Just an Adhesion Molecule: LFA-1 Contact Tunes the T Lymphocyte Program[END_REF] . Chemokines such as CCL19 and CCL21 only activate the LFA-1 to its intermediate iii.

Outside-in signaling

Outside-in signaling refers to the final stages of LFA-1 adhesion to its ICAM-1, which initiates an integrin-proximal tyrosine kinase cascade of signaling events. It is caused by extracellular changes which regulate the adhesiveness of LFA-1 [START_REF] Verma | Not Just an Adhesion Molecule: LFA-1 Contact Tunes the T Lymphocyte Program[END_REF] . Vav1, talin, kindlin3 and ZAP70, all proteins who are bound to the cytoplasmic tail of LFA-1 play a role in outside-in signaling, where the changes they undergo lead to a signaling cascade. Extended LFA-1 is bound with a "closed" head to ICAM-1, but following the signaling cascade, the headpiece changes shape to allow high affinity binding to LFA-1. This consequently increases the avidity and affinity of LFA-1 binding to ICAM-1 [START_REF] Verma | Not Just an Adhesion Molecule: LFA-1 Contact Tunes the T Lymphocyte Program[END_REF] .

c. Costimulatory molecules

The critical costimulatory receptor CD28, when engaged by its primary ligand CD80 becomes enriched in TCR MCs. The CD28-CD80 complex is transported to the central supramolecular activation cluster of the immunological synapse , where a CD28-PKCθ arrays around this structure 102 .

The adhesion molecule CD2 (LFA-2) not only facilitates T cell adhesion but also functions as a costimulatory molecule, by lowering the threshold for T cell activation. Following TCR activation, the avidity of the interaction between CD2 and CD58 is increased 103 . The engagement of CD2 induces phosphorylation of PLCγ1 via Fyn kinase thus potentiating intracellular calcium levels above those achieved in response to TCR triggering alone 102 .

d. Inhibitory molecules

Cytotoxic T lymphocyte antigen-4 (CTLA-4) can outcompete CD28 for binding to CD80 and CD86.

CTLA-4 co-ligation leads to an increased threshold of

B. CD8+ T cell cytotoxicity: from naïve cells to professional killers

When compared, the cytotoxicity of NK and CTLs differ in the migration dynamics, the duration of contact with their targets, whereby it was shown that CTLs decrease their migration speed when patrolling the tumor environment, and establish longer contacts with their targets, while NK cells establish more dynamic contacts 104 . The contact with an antigen presenting cell is what triggers TCR mediated signaling which, along with other costimulatory signals leads to the differentiation into CTLs in the primary lymphoid organs.

Following the first stimulation with antigen, most effector CD8+ T cells will die, and only a fraction will become memory cells (Figure 7).

a. Differentiation into professional killers

First, naïve CD8+ T cells must scan their environment and interact with antigen presenting cells, to become activated via their TCR. Such CD8/APC interactions are possible thanks to the motility of both the CD8 and the dendritic cells. This capacity to interact with APCs and high motility allows CD8+ T cells to quickly scan through DCs, wherein it is supposed that in one hour, a single DC can be probed by about 500 CD8+ T cells 106 . However, CD8+ T cells must reduce their motility and come to a halt in order to establish an interaction with the APC. This process is enabled by the interaction of their TCR with MHC molecules, where an adhesion ring is formed due to centripetal actin flow 107 .

The IS of a T cell was first defined in the late 1990s, and was defined as "a molecular machine controlling T cell activation" 108 .

CD8-APC interactions were initially thought to be a timely process, where activation of the T cell was thought to require long hours of CTL-APC contact, where the T cell remains in contact with one APC for a time ranging between 6 and 30 hours 108,109 . However, evidence in later years supported a more dynamic type of interactions, in which the CTL will have several brief and short-lived interactions with multiple DCs 110 . This has suggested the presence of a short-term memory that allows T cells to perform a "signal summation" from these intermittent interactions, which were termed "kinapses" 111- 113 . The activation signal that is subsequently generated from these interactions was shown to last for several hours 112 . However, the immunological synapse is a productive and more stable interaction between the T cell and the APC 113 . It has been shown that for T cells to be arrested and primed, they must interact with a DC that expresses high levels of adhesion molecules, a high quantity of costimulatory molecules, as well as a high quantity of high affinity MHC-I molecules 114 .

CTL-APC interaction leads to a fast tyrosine phosphorylation cascade, thus triggering several signaling pathways and a Ca ++ induction of the transcription of genes involved not only in lymphocyte activation, but also proliferation 115,116 . The calcium signaling depends on a stable IS, therefore on the actin cytoskeleton. Indeed, inhibition of actin filaments by Latrunculin B which binds G-actin reduces the number of TCR that can be stimulated, subsequently rendering the cell unable to maintain calcium ion release 117 .

CD8+ T cell cytotoxicity

CTL-mediated cytotoxicity requires a direct contact between the CTL and the antigenic peptides However, not all granules that are polarized will fuse with the synapse, as determined by total internal reflection microscopy 122 . Moreover, granule secretion relies heavily on the actin cytoskeleton remodeling, which will be addressed in detail in the next chapter.

Remodeling of the actin cytoskeleton governs the cytotoxicity of NK and CD8+ T cell cytotoxicity

Cytotoxicity is a dynamic process which involves several steps, in which the actin cytoskeleton and the rearrangements it undergoes play a pivotal role.

The actin cytoskeleton is a filamentous network composed of globular actin (G-actin) monomers, which polymerize to form filamentous actin (F-actin), and then these filaments may be severed. This involves the interplay of several actin binding proteins. This is a highly dynamic process involving several players. Nucleation involves the formation of actin dimers followed by actin trimers, which allows the start of the elongation through the process of polymerization. The next step involves adenosine triphosphate (ATP) hydrolysis.

To further elongate the actin filaments, formins such as mDia in mice (hDia in humans), and FMNL1

must interact with the barbed wire to allow monomers to be added to it, thus creating parallel elongated bundles 123,124 . Formins also acts as "leaky cappers", whereby they slow down the elongation and dissociation rates of the actin filament 125 .

Arp2/3 binds to the "mother" actin filament to allow the formation of a "daughter" filament in a dendritic fashion at a 70-degree angle. Arp2/3 is composed of seven subunits of which five unique components: ARP2, ARP3, ARPC2, ARPC3 and ARPC4, together with one molecule each of the isoform pairs ARPC1A and ARPC1B, and ARPC5A and ARPC5B. Arp2/3 generally requires activation by a nucleation-promoting factor from the Wiskott-Aldrich Syndrome protein (WASp)

family, the WASp family-Verprolin homologous protein (WAVE), and the cortactin family. WASp bound to ARP2/3 will bind to profilin bound G-actin to perform actin nucleation, until the process is stopped by capping proteins 126 .

On the other hand, proteins that prevent actin polymerization include cofilin and profilin. Cofilinmediated actin severing has been shown to be necessary in achieving T cell spreading and the formation of a proper immunological synapse, as well as cell motility 127,128 . Profilin is a protein that binds to the barbed end G-actin and prevents its addition to the nascent actin filament 129 .

Some major players in this process can be summarized in Figure 8.

Dynamic steps of cytotoxicity

A. Cell migration

Chemokine receptors allow lymphocytes to sense the chemokine gradients and migrate to where their targets cells are. Migrating cells transform from round cells to cells with a leading edge at the front, and a uropod at the back. The leading edge, also named the lamellipodium, is a thin and spread structure, composed of highly branched actin fibers. Also in the leading edge are filipodia, which are protrusions with an exploratory function, and made up cross-linked actin fibers arranged in a parallel fashion 130 . The uropod is composed of actin filaments coupled to Myosin, which allows the cell to generate the tension necessary to migrate forward. To achieve cytotoxicity, the cytotoxic T or NK lymphocyte must bind to different targets and scan them. In order to do so, lymphocytes must halt their motility upon contact with the target 106,109 . Upon binding to the potential target cell, the cell's machinery is shifted from locomotion towards immunological synapse formation, where actin polymerization continues to take place without the cell moving. The lymphocyte spreads a lamellar sheet over the target, and outward radial actin polymerization takes place.

More information on the mobility of T lymphocytes can be found in 131 .

B. The immunological synapse: the platform for cytotoxicity

Once the lymphocytes reach their target, they must establish a stable contact, which is the immunological synapse (discussed in the next section). Following the establishment of the immunological synapse, cytotoxic lymphocytes must polarize their lytic vesicles and their MTOC towards the area of contact in order to release perforin and create pores in the target, followed by granzyme release into the target 64 . The MTOC is polarized in a way to deliver the lytic granules in the open areas of the actin meshwork 53 .

The immunological synapse in the cytotoxic context, is defined as the interaction between a cytotoxic lymphocyte and a target cell. As inferred from the previous sections, the immunological synapse assembly plays a crucial role in cytotoxicity and will only be discussed in that context.

IS formation takes place when the lymphocyte halts its movement and establishes a transient but controlled contact with the target cell. However, the immunological synapse is far from being a static process, it is highly dynamic and tightly regulated and involves reorganization of cellular components as well as signaling proteins.

a. Immunological synapse of CD8+ T cells

Analysis of the structure of the mature immunological synapse of T cells has revealed a "bullseye" structure with radially distributed and spatially segregated rings which Kupfer has named supramolecular activation clusters (SMAC), where the TCR-MHC cluster in the center, surrounded by a ring of LFA-1-ICAM-1, which is itself surrounded by a distal ring which comprises CD45, a transmembrane tyrosine phosphatase [132][133][134] . These clusters are referred to, from the inside-out as:

central, peripheral and distal SMAC (cSMAC, pSMAC and dSMAC) (Figure 10), and the bullseye pattern has been suggested to be a mechanism to confine lytic granules and prevent bystander killing 44,135 . The cSMAC, as its name indicates, is the center of the synapse and the area of TCR aggregation, and was postulated to be formed of microclusters of about 100 TCR 136,137 . The pSMAC directly surrounds the cSMAC and is composed of proteins such as talin and LFA-1. The dSMAC is the most distal area, and is also referred to as radial lamellipodium that allows the cell to sense chemical and physical properties of the APC 134,138 . Methods based on fluid supported lipid bilayers showed that in the case of CD8+ T cells, TCR-MC form at the dSMAC and move centripetally through the pSMAC to the cSMAC, initially aided by actin filaments undergoing retrograde flow and subsequently through dynein-mediated movement along the microtubules toward the centrosome that translocates beneath the cSMAC following TCR stimulation [139][140][141] . LCK is, however, found at the dSMAC, and the cSMAC can be further divided into the endo-cSMAC, a poor actin region where the TCR are located, and the exo-cSMAC, a TCR poor region where CD28 molecules are located 142,143 . TCR signaling following TCR-peptide-MHC-I engagement drives the ARP2/3 dependent polymerization of branched actin filaments at the edge of the site of contact between the T cell and the APC. This drives initial T cell spreading and the centripetal flow of the acto-Myosin network, and aids in the retrograde flow towards the cSMAC. This retrograde flow may be inhibited by using Jasplakinolide, a drug that directly binds the actin filament and leads to the depletion of the G-actin pool. Jasplakinolide addition leads to the formation of a dense actin ring between the pSMAC and the cSMAC 144 . Centripetal actin flow also plays a role in the conformational change of integrins, thereby causing the T cell to adhere to its APC and plays a role in the outside-in signaling. Indeed, removing the synapse symmetry leads to the restoration of the lymphocyte motility by creating a retrograde actin flow, which in turn creates an LFA-1 focal zone 44 .

The pSMAC is made up of a lamella-like actin arc network that outlines the radial symmetric contractile ability of this part of the IS. It has been shown that the assembly of the pSMAC actin arc is mediated by formins and organized by Myosin II into antiparallel concentric arcs [143][144][145][146] . Indeed, the role of formins validated by the fact that mDia depletion leads to failure in the formation of the linear actin filaments needed generate the typical concentric Myosin IIA-rich arcs, as well as the treatment with the pan-formin inhibitor SMIFH2, which decreases the presence of actin arcs within the pSMAC 146 .

b. Immunological synapse of NK cells

Most of the available information on the IS comes from the study of T cells. However, some groups have been interested in the study of the IS on NK cells. In NK cells, distinct IS properties have been identified.

For NK cells, much like T cells, cytotoxicity is mediated by the NK cell emptying its granule content into the target cell. In addition to the receptor interplay mentioned above, the actin cytoskeleton plays a crucial role in the NK cell synapse. In NK cells, cytotoxicity requires the formation of a mature immunological synapse 147 Moreover, the fact that lytic granules exist in NK cells even prior to activation, the accidental release of these granules must be prevented, and the directed release of lytic granules must be ensured. The contact between the NK cell and its target is followed by adhesion, While lytic granules aggregate around the MTOC, the MTOC also begins to polarize towards the immunological synapse, and the signals to cause this polarization include ERK, VAV1 and PYK2 147 .

Lytic granules appear to be accumulated at the cSMAC, and adhesion molecules in the pSMAC 57 . As in T cells, reorganization of the actin cytoskeleton must take place for MTOC and lytic granule polarization to the IS, and discrete actin hypodense regions in the cytoskeleton must exist to allow lytic granule access to the plasma membrane 14,[START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF]147 . However, these clearances are not mediated by a unidirectional or radial actin flow, but rather by a local nanoscale dynamism where these clearances are opening and closing [START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] . ARP2/3 and Myosin IIA also play a role in the structuring of these clearances, where ARP2/3 depletion leads to the formation of long bundled actin filaments at the IS, and MyosinII A impairment decreases the number of permissive clearances [START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] . Interestingly, these clearances were observed prior to granule arrival [START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] . Docking of the granules requires members of the Rab family of GTPases, such as Rab27a, and the SNARE family member Munc13-4 which interacts with Rab27a, as well as LFA-1 engagement [START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF]147 . The ARP2/3 complex, as well as MyosinIIA have also been shown to play a role in granule secretion, where their absence appears to impair exocytosis [START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] .

Indeed, MyosinII A not only plays a role in the actin clearances, but also in lytic granule movement towards the secretory domain of the cSMAC [START_REF] Sanborn | Myosin IIA Associates with NK Cell Lytic Granules to Enable Their Interaction with F-Actin and Function at the Immunological Synapse[END_REF] . In NK cells, it has been demonstrated that the granulesized clearances are maintained after degranulation, and so were local filament dynamics, suggesting that the formation of these clearances is independent from granule presence, despite the necessity for these clearances for a successful degranulation [START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] .

Cytotoxicity resolves by the detachment of cytotoxic lymphocyte from its target. These steps can be summarized in the figure 9 below.

Cancers

Cancer is defined as a disease of immune dysregulation, characterized by a failed immune recognition leading to the proliferation of malignant cells 148 .

The infiltration of a tumor by activated CD8+ T cells is considered a good prognosis and can be predictive of a better response to chemotherapy and immune checkpoint blockade therapy. However, in several cancers, CD8+ T cells are unable to access the tumor and will accumulate outside the tumor cell cluster. However, some CD8+ T cells can infiltrate tumors and it is speculated that the dense fiber region surrounding the tumor might not be able to protect against this infiltration 149 . However, Li et al. speculate that in the case of tumors that are indeed being infiltrated, it is due to CD8+ T cells that slow down then regain cell motility and chemotaxis 149 . NK cells were also demonstrated to play a role in cancer immunosurveilance 150 . Due to their potency to eliminate cancer cells, T and NK cell research aiming to use them as cancer immunotherapy is currently booming.

Autoimmunity

Autoimmune diseases are defined as aberrant responses towards "self". They have a high prevalence of 7-9% in the population and affect women more than men. It is thought that women are more predisposed to autoimmune diseases due to hormones as well as the direct effects of X chromosomes as well a sex-specific microbiota 151 . Autoimmune diseases can be mediated by autoantibodies or CD8+

T cells, but Th cells are always implicated. Autoimmune diseases are usually caused by a combination of genetic and environmental factors (such as infections, microbiome and UV light) which culminate in an imbalance between the effector and the regulatory responses [152][153][154][155][156][157][158][159] . They include type I diabetes, multiple sclerosis, rheumatoid arthritis and inflammatory bowel disease. Despite the large spectrum of organs they affect and their different clinical manifestations, autoimmune diseases usually undergo an initiation phase, often asymptomatic, followed by a propagation phase characterized by inflammation and tissue damaged caused by cytokine production and the imbalance between the effector and regulatory functions of T cells 160 . The last stage is the resolution, in which patients have some restored balanced between the regulatory and effector functions, but often relapse. Interestingly, and contrary to a previous belief, a larger portion than previously thought of self-reactive CD8+ T cells manage to escape the process of clonal deletion 161,162 . Therapies for autoimmune diseases are mainly cytokine antagonists, which aim at establishing a long-term resolution by increasing regulatory mechanisms 163,164 .

Primary immunodeficiencies

Primary immunodeficiencies (PID) are a heterogeneous group of approximately 400 rare genetic diseases characterized by severe defects in one or several components of the innate and/or the adaptive immune system. The clinical presentations of PIDs are variable, but PID patients usually suffer from recurrent infections, cancers and autoimmunity, which may be life-threatening. Characterized PIDs are monogenic disorders, but more complex polygenic disorders certainly remain to be discovered. In this section, only an overview on PIDs will be presented, and the focus on PIDs where defects in the cytotoxicity or the actin cytoskeleton/immunological synapse were reported will be discussed in a later

section. An exhaustive list of PIDs can be found in 165 .

A. Hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a severe condition characterized by a heightened inflammatory response and an expansion of polyclonal CD8+ T cells, most likely due to defects in the cytotoxic machinery of T and NK cells, despite their normal numbers 166 . HLH patients present with mutations is the perforin, MUNC13/4, RAB27a, LYST, Syntaxin11 genes, all of which are associated with the perforin-containing lytic granules in cytotoxic cells (Reviewed in 167 ). These defects are comprised in the category of primary HLH, while secondary HLH occurs as a complication of infection, cancer or autoimmunity. The fact that several of the mutations is some of these genes such as perforin were found in families has led to terming some HLH as familial hemophagocytic lymphohistiocytosis (FHL). These defects, summarized in Table 1, will be elaborated in further details in the coming pages, as they entail a defect in cytotoxicity.

a. Perforin deficiency (FHL2)

The for granule polarization and docking at the IS membrane, but is required through its association with Rab27a for the regulated secretion of cytotoxic granules at the priming stage of the exocytic pathway 176,177 . Therefore, Munc13-4 deficient CD8+T and NK cells exhibit defective cytotoxicity and express more perforin than healthy controls 175,176,178 .

c. STX11 deficiency (FHL4)

The syntaxin11 (STX11) protein is encoded by a gene on chromosome 6. Patients with defective STX11 present with HLH symptoms. STX11-causes defects in cytotoxicity and degranulation, and patients have a higher perforin expression levels than healthy controls. However, confocal microscopy imaging reveals that STX11 deficient cells can efficiently polarize their perforin granules but fail to degranulate, due to defective granule fusion with the membrane 178 (Figure 11). These defects may be reversed by IL-2 administration 179 .

e. Rab27a deficiency (Griscelli syndrome)

Rab27a is a member of a large family of Ras-related small GTPases, and the gene encoding it is located on chromosome 15. Loss-of-function mutations in the Rab27A gene result in Griscelli syndrome, a rare autosomal disorder in which patients suffer partial cutaneous albinism where a pigmentary dilution of the skin and the hair s observed, as well as clumps of pigment in hair shafts and an accumulation of melanosomes in melanocytes. Patients often develop hemophagocytic syndrome, which is the uncontrollable activation of lymphocytes and macrophages. Rab27a is required for granule secretion, as Rab27a-deficient T cells exhibited reduced cytotoxicity and cytolytic granule exocytosis despite the presence of a normal amount of lytic granules of the proper size [182][183][184] . Confocal microscopy images revealed that Rab27a co-localizes with Gzm B-containing granules 183 .

f. LYST deficiency (Chediak-Higashi syndrome)

The LYST gene is located on chromosome 1. Patients with LYST deficiency suffer from Chediak-Higashi syndrome, a rare autosomal recessive disease characterized by partial oculocutaneous albinism, recurrent infections, a progressive primary neurological disease, and often causing HLH 185,186 . NK cells form LYST deficient patients show no cytotoxicity and impaired degranulation, similarly to T cells 185,[187][188][189] . Lytic granules from LYST deficient patients show an aberrant morphology. There are distinct granules in patient NK cells, and not a giant granule as previously thought. However, they are larger than granules in normal NK cells, and they do not converge at the MTOC, as revealed by confocal microscopy imaging 189 . The granules containing cytokines, and cytokine release are not affected by LYST mutation 189 .

B. Severe combined immunodeficiency

Severe combined immunodeficiency (SCID) are a group of genetic diseases caused by mutations in

b. Innate immunodeficiencies

Innate immunodeficiencies are usually associated with defects in phagocytes, Toll-like receptor mediated signaling and complements. Clinical manifestations comprise bacterial and fungal infections.

Innate immunodeficiencies include severe congenital neutropenia, presented as an extremely low neutrophil count. Phagocyte defects are characterized by chronic granulomatous disease, which often has an X-linked inheritance. Innate immunodeficiencies also include defects in the IL-2/IFN-γ signaling pathway. PIDs associated with complement defects are the rarest category of PIDs, comprising less than 1% of all diagnosed cases, and are often associated with autoimmunity.

C. PIDs related to defects in the actin cytoskeleton

Even though primary immunodeficiencies with a defective actin cytoskeleton are not a category per se, we must focus on them as a group in this manuscript, since its emphasis is cytotoxic cells and several of these defects are causative of impaired cytotoxicity. As previously mentioned, much of the information available to us today on the role of the actin cytoskeleton and its regulation, especially in the control of the immunological synapse, comes from the study of PID patients. In the context of my thesis project, we are particularly interested in the regulation of the immunological synapse, and the interplay between the actin cytoskeleton and the lytic granules. The genes which cause such defects appear to belong to same signaling pathway in several instances, as seen by similar symptoms in the patients and similar functional defects. Some of these defects may also be accompanied by a defective cytotoxic activity.

a. Wiskott Aldrich protein deficiency

The Wiskott Aldrich syndrome (WAS) was first described in 1953 by Wiskott, then by Aldrich in 1954 194 . Both have identified that patients suffering from a defect linked to the X chromone present with microthrombocytopenia, eczema and recurrent otitis among other infections. In certain cases, the pathology of WAS would evolve to include tumors such as B cell lymphomas, and autoimmunity, and treatment often requires stem cell transplantation 195,196 . The gene behind WAS is located on the short arm of the X chromosome, specifically at Xp11. 22-p11.23, and WASp is a multidomain protein comprised, from the N to the C terminus, of a WH1 domain, followed by a basic domain, a G protein binding domain (GBD), a proline-rich region and a VCA domain.

WASp is autoinhibited by a set of intramolecular interactions, mainly between the basic and GBD domains and the VCA domain, therefore preventing binding of the Arp2/3 to the VCA domain and initiation of actin polymerization 197 . Indeed, WASp is involved in actin polymerization through the nucleation of nascent actin filaments in response to the TCR or chemokine receptors (Figure 13). In fact, WASp, (as well as the WAVE complex) controls the activation of the ARP2/3 complex. For more information on the detailed activation of WASp, I invite you to read 197 . WASp is not only recruited to lipid rafts following TCR activation, but is essential for lipid raft clustering 199 . WASp deficiency also causes a decreased F-actin accumulation at the IS, as well as a decreased density of LFA-1 clusters at the IS 57,101,200 .

Moreover, WASp deficiency was shown to cause a mild defect in the cytotoxicity of CTLs and an even more severe defect in NK cells. Indeed, WASp deficient cells, despite a normal expression level of lytic molecules, show an aberrant synapse morphology as well as a defect in the recruitment of the lytic molecules to the synapse, as well as the exocytosis of these molecules 57,201,202 . That is because lytic granule polarization relies on the WASp-dependent actin polarization. Interestingly, treatment with IL-2 corrects the defect in NK cells from WAS patients and restores their normal cytotoxic function, by activating WAVE2 They also showed that IL-2 treatment is effective in restoring F-actin content and cytotoxicity in WAS patients.

b. WASp Interacting protein deficiency

WASp is found in association with WASp interacting protein (WIP), which plays a role in the control of WASp activation, by easing its activation by Cdc-42 and WASp recruitment to the IS 205 . WIP was shown to be recruited to the IS in NK cells 200 . WIP is activated by phosphorylation but interestingly, this phosphorylation is independent of the WIP-WASp interaction, thus suggesting an additional role for WIP 200 . It has been shown by triple-color FRET that both the activation and degradation of WASp are dependent on the phosphorylation state of WIP 206

The mutation in the WIPF1 gene located on chromosome 2 causes a decrease in the expression levels of the WIP, which consequently leads to the absence of WASp, despite the absence of mutations in the WAS gene. Moreover, expression of WIP in the cells of the patient restores the expression of WAS to normal levels 207 .

The clinical presentations of WIP deficiency resemble those of WAS, which include a failure to thrive, eczema, thrombocytopenia, ulcerative skin lesions, pneumonia and cytomegalovirus infection 207,208 .

The patient suffered from a reduced number of CD3+ cells, with a more notable decrease in CD8+ than CD4+ cells, as well as a reduction in B cells and an increase in NK cells. WIP was also shown to negatively impact the migration of T cells towards CXCL12 and CCL19, and the cells displayed a failure to elongate 208 .

Despite the normal expression of perforin in WIP patient CD8+T cells, the cells displayed a reduced ability to degranulate, which translates into a defect in cytotoxicity and elimination of target cells 208 .

That can be explained by the fact that the patient cells were shown to be incapable of assembling the IS, rendering them incapable of polarizing their lytic granules. Moreover, patient T exposed an aberrant actin organization, where the dominant actin structures were elongated and filamentous, and oriented in one direction. This evidence supports the role of WIP in the control of the actin cytoskeleton and lamellipodia formation, therefore an essential role in the proper assembly of the immunological synapse.

c. ARHGEF1 deficiency

ARHGEF1 is an intracellular protein that has been shown to regulate migration and adhesion through its regulation of G protein-coupled receptor signaling and RhoA activation. It acts by stimulating the release of guanine diphosphate (GDP) to allow the binding of guanine triphosphate (GTP) 209 . The gene encoding ARHGEF1 is located on chromosome 19. ARHGEF1 deficiency symptoms include respiratory tract infections including pneumonia. They also showed defective antibody production 210 .

One patient presented with mucoepidermoid carcinoma.

Both patients had low B cell counts with an elevated frequency of transitional B cells, with almost no memory B cells. NK, CD4+ and CD8+ T cells were within the normal range, with one patient showing an increase in naïve CD8+ T cells and a decrease in memory CD8+ T cells. The cells from these patients displayed normal levels of RhoA, but reduced RhoA activity, suggesting that the defect could be due to a Rho regulator. F-actin levels were lower in the patients than the healthy controls. Moreover, treatment of patient and normal donor T cells with the ROCK inhibitor Y-27632 caused no effect on F-actin levels in the patients but reduced their levels in normal donors to those comparable in the patients, indicating a role of ARHGEF1 in the RhoA-ROCK mediated actin polymerization.

ARHGEF1 was also shown to play a role in mediating the LFA-1 dependent T cell adhesion to ICAM-1, but this was confirmed by siRNA not in patient cells 211 .

d. WDR1 deficiency

The deficiency in the WD repeat 1 gene, located on chromosome 4, leads to a deficiency in the actin interacting protein 1 (Aip1). WDR1 is composed of 9 repeats, which are 30-40 amino acids long. The patients present with mucosal and skin ulcerations, respiratory tract infections, stomatitis, periodic fevers and one patient died of sepsis. Patients present with mild to severe lymphopenia and suffer from recurrent infections. It has been shown that patients with WDR1 deficiency have neutrophil adhesion and migration defects, but such findings are beyond the scope of this manuscript 212,213 .

WDR1 promotes actin filament disassembly through cofilin, and with the involvement of coronin.

Indeed, WDR1 binds to cofilin-loaded F-actin to induce their rapid severing. THP1 cells transduced with shRNA targeting WDR1 do not show a defect in actin polymerization when assessed by flow cytometry, most likely because of mechanisms enabling the cells to compensate for the defects caused by WDR1 214 . However, T cells from WDR1 patients left to adhere on slides coated with anti-CD3 Ab show increased spreading, as well as aberrant actin structures such as arcs, filopodia and spikes 213 .

Increased filopodia suggest an involvement of formins.

Even tough B cells from patients migrate normally towards a CCL19 gradient, they display an increased adhesion and thin protrusions. They also display an increased accumulation of polymerized actin in the ventral plane of the cell, as shown by TIRF microscopy 213 . This suggests that a WDR1 mutation affects BCR functions.

T cells from WRD1 deficient patients show a diminished to normal response to stimulation with anti-CD3 antibody, which may be explained by the need for the forces generation by the actin cytoskeleton to activate T cells 213,214 . However, the fact that WDR1 deficiency does not hinder the ability of patient T cells to migrate in response to CXCL12 or accomplish normal cytotoxicity suggest that WDR1 is not crucial in these aspects relying on TCR activation 213 .

e. Coronin1A deficiency

The Coronin family is a family of highly conserved actin regulatory proteins. They bind to the Arp2/3 complex, but unlike other proteins, their role the inhibition of action nucleation 215 . They contain 5

WD40 repeats, and their structure is reviewed in more details in 215,216 . The gene encoding coronin 1A (Coro1A) is located on chromosome 16, and the Coro1A protein is a 57kDa protein that has the conserved WD repeats, and a leucine zipper motif 217

Coro1A, which is the member of the coronin family expressed mostly on hemopoietic cells, was shown to cause lymphopenia in mice, especially in T cells. SCID patients with coro1A deficiency were identified and exhibited T-B+ (NK+) SCID phenotype, as well as increased double-negative ϒδ Tcells 216,[218][219][220] . The absence of naïve CD4+ T despite the presence of a thymus cells is attributed to the fact that Coro1A deficiency leads to distorted mitochondria, therefore leading to apoptosis 221 . Coro1A deficient patients also suffered from upper respiratory tract infections; ear and sinus infections and inability to control EBV, associated with fatal lymphoproliferative syndrome and lymphoma at a particularly young age 216,218 . In one patient, this was due to a mutation in one copy of Coro1A coding region, as well as a heterozygous deletion of p11.2 on chromosome 16 218 . In this patient, this deletion is suggested to have predisposed her to attention deficit hyperactivity disorder.

Early is recruited to the synapse along with F-actin, and that it remains there for the duration of the mature synapse. Knock-down (KD) of Coro1A by shRNA in an NK cell line leads to a defective killing in these cells, due to a defect in degranulation (granule exocytosis), as shown by the decreased levels of CD107a expression 168,224 . However, confocal microscopy experiments show that Coro1A deficient cells can form conjugates with their targets, with no alteration of the size of the IS, the accumulation of LFA-1 at the IS and effective granule and centrosome polarization, all of which are actin-dependent processes (Figure12). STED microscopy revealed that Coro1A KD does not hinder ARP2/3 recruitment but causes the accumulation of a greater density of F-actin in smaller area. This dense Factin accumulation at the IS may be the reason the exocytosis of lytic granules is hindered, since degranulation requires a pervasive actin meshwork through which the lytic granules can be released, and since STED microscopy reveals that Coro1A KD leads to smaller clearances, and the association of the lytic granules with more dense actin, making Coro1A a probable key player in the residence of lytic molecules in the actin hypodense regions but not their movement 224 . NK cells from a patient presenting with a Coro1A deficiency show that, similarly to what is observed in the shRNA KD, the actin presents a smaller area, as well as smaller and fewer clearances, leading to a defect in granule exocytosis (Figure 14). The patient also displays a defective cytotoxic activity.

f. Ccd42 deficiency

Cdc42 is a RhoGTPase that acts upstream of WASp to activate it (when in its GTP bound state) and recruit protein kinase Cθ (PKCθ) and talin to the cSMAC and pSMAC, respectively. Indeed, GTP bound Cdc42 binds to WASp, thus releasing the VCA region and allowing WASp binding to ARP2/3 225 . Cdc42 is activated more effectively by the β1 integrin than by CD3, and the absence of the β1 integrin can be partially compensated by LFA-1 226 . Ezrin has been shown to play a role in its activation. It has been shown that following TCR engagement, Cdc42, along with WASp, is recruited to the IS 226,227 . T cells defective in Cdc42 fail to polarize F-actin at the IS when in contact with target cells, but this did not affect IL-2 production 228 . Therefore, Cdc42 has a role in multiple cellular processes involving the actin cytoskeleton, such as motility and endocytosis.

Patients with mutations in Cdc42 appear to have mid frontal bossing, failure to thrive, hepatosplenomegaly, transaminitis, recurrent febrile episodes, urticaria-like rashes, and significant cytopenia 229 . It has also been shown that when the Cdc42 defect is in the GTPase, the defect triggers the formation of broad lamellipodia and stress fiber assembly, while a defect where the rate of GDP/GTP exchange is elevated triggers filopodia formation and stress fiber dissolution 230 .

g. RhoA deficiency

RhoA, a 23kDA protein which is active when bound to GTP, plays a role in BCR signaling and induces

h. RhoH deficiency

The RHOH gene located on chromosome 4 encodes an atypical, hematopoietic cell-specific member of the Rho GTPase family, which function as intracellular switches to transduce signals from several membrane receptors including the TCR and BCR. RHOH is constantly in its active GTP-bound active conformation because it lacks a GTPase activity 234,235 . Moreover, it is essential for positive thymocyte selection and for pre-TCR and TCR signaling because it allows the efficient interaction of ZAP70 with the LAT signalosome, thus regulating thymocyte development 236,237 . Its interaction with ZAP70 is also essential for ZAP70 translocation to the IS 237,238 . RhoH also plays a role in reducing the migration in response to chemokines and maintaining the contact with APC , by increasing both Rap1 activity and LFA-1 adhesion 239 . Indeed, cells that lack RhoH migrate faster in response to chemokines, while RhoH relocates along with LFA-1 to the IS upon T cell:target conjugate formation 239 . The patients suffered from Epidermodysplasia verruciformis and a human betapapilloma virus (EP-HPV).Consistent with findings in mice, RhoH deficient patients lack naïve T cells, and ZAP70 phosphorylation in response to stimulation with anti-CD3 Ab is defective, further enforcing the role of RhoH in TCR signaling 240 .

It is most likely that these patient cells would display a defect in there is, given the role of RhoH in TCR signaling and IS stimulation.

i. DOCK2 deficiency

The DOCK180 related superfamily includes conserved genes which are guanine exchange factors, and that were shown to play a role in phagocytosis, cell migration and immune homeostasis. These proteins harbor a DOCK Homology Region-2 (DHR-2) domain that binds and activates Cdc42, Rac or another Rho family member to exchange GDP for GTP 241 . Therefore, the DOCK family of proteins may be considered as guanine exchange factors (GEF).

j. DOCK8 deficiency

Director of cytokinesis (DOCK 8) is a direct interactor GEF of Cdc42 (with little activity towards Rac1

and none with RhoA), and has been shown to mediate Cdc42 activation via its DHR-2, and was shown to be crucial for Cdc42 activation at the leading edge, therefore important in ameboid migration 245,246 .

DOCK8 patients were reported to have SCID and be susceptible to viral (most common is herpes simplex virus) and cutaneous infections, therefore suggesting a defect of cytotoxic lymphocytes.

Patients also presented with high levels of serum IgE and hypereosinophilia, low numbers of T cells and B cells, low serum IgM levels [246][247][248] . DOCK8 patient CD8 T cells show a decrease in naïve cells, and the circulating cells display an exhausted phenotype and become unresponsive to TCR stimulation with anti-CD3 249 . Moreover, cells from these patients show a defect in proliferation, which is not overcome by the addition of exogenous IL-2 249 . Clonal expansion of CD4 T cells was also diminished.

In cell lines, DOCK8 was shown to be expressed in NK cell lines, but not the Jurkat T cell line,

rendering it an incompatible model for such studies, even though Jurkat cell line is the most commonly DOCK8 was also shown to be a direct interactor of WASp and talin, and to mediate their polarization to the IS in NK cells, therefore indicating a role for DOCK8 in actin reorganization and IS maturation 246 . However, the DOCK8 deficiency remains less severe than the DOCK2 deficiency.

k. PSTPIP1 deficiency

Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene located on chromosome 15, also known as CD2-binding protein 1 (CD2BP1) encodes for a cytosolic adaptor protein involved with activation, differentiation, and migration of T cells 251 . PSTPIP1 also acts as a scaffolding protein for signal transduction towards the nucleus 252 . PSTPIP1 has been identified in patients with pyogenic sterile arthritis with PG and acne syndrome, a rare autoinflammatory disorder with autosomal dominant inheritance 251,253,254 Patients with PSTPIP1 deficiency presented with an immunodeficiency phenotype, but no symptoms of autoinflammation. They also presented with recurrent viral and bacterial respiratory tract infections and low IgG and IgA levels (low IgM for one patient). Their T cells were mostly naïve T cells, there was a reduction of CD4+ T cells in one patient, and there was a reduction in B cell numbers 252 . PSTPIP1 acts a scaffold protein to allow protein tyrosine phosphatase (PTP-PEST) to dephosphorylate WASp 255 . Therefore, through its interactions with CD2 and WASp through PTP-PEST, PSTPIP1 plays a role in F-actin dynamics and the establishment of a stable IS. In one patient, CD4+ T cells showed a pre-activated F-actin status, therefore failing to modulate their actin cytoskeleton in response to stimulation with anti-CD3 Ab, as well as higher baseline levels of calcium ion than normal controls 252 . CD4+ T cells from patients were also more motile, which may explain their failure to respond to TCR/CD2 stimulation. Jurkat T cells transfected with plasmids encoding PSTPIP1 mutation and either left unstimulated or stimulated with antiCD3/anti-CD28 beads and imaged with confocal microscopy showed more baseline F-actin levels when unstimulated, but a failure to recruit F-actin to the IS upon stimulation 252 .

l. STK4 deficiency

Serine threonine kinase 4 (STK4), previously named Mammalian sterile-like protein 1 (MST1), encoded by a gene on chromosome 3, is a ubiquitous protein that plays a dual proapoptotic and antiapoptotic role. Its proapoptotic role is suggested by the fact that in resting conditions it is a 63kDa cytosolic protein which becomes cleaved by caspases 256 . STK4 deficient patients presented with neutropenia, recurrent fever and recurrent respiratory tract infections, mouth ulcers, sinusitis, eczemalike rashes and skin abcesses [256][257][258] . Patients also presented lower levels of CD4+ and low to normal CD8+ T cells, as well as slightly lower B cell count, and a normal NK cell count 256,257 . T cells from MST1 deficient patients exhibit a defect in proliferation, and higher apoptosis rates than healthy controls 256,257 . Recurrent episodes of neutropenia also suggest a role for STK4 in neutrophils.

Unfortunately, the data on patients deficient in MST1 is limited. proliferation following stimulation with anti-CD3/CD28 Ab was impaired. IS was not impaired in MSN deficient cells and neither was F-actin content, but migration and adhesion were. However, cytotoxicity was not assessed, but it may be affected given that MSN is a regulator of the actin cytoskeleton.

p. Rac2 deficiency

Ras-related C3 botulinum toxin substrate 2 (RAC2) is a hematopoietic-specific member of the Rho family of guanosine triphosphatases (Rho GTPase) and encoded on chromosome 22. Rac2GDP can be activated by a GEF and RAC2-GTP drives several cellular functions through association and activation of downstream effector proteins. Activation of these downstream targets leads, among other, to actin cytoskeleton rearrangement and thymic T-cell selection. Indeed, RAC2 is involved in the cycling of actin between G-actin and F-actin states through interactions with cofilin and the ARP2/3 complex 270 .

The patients presented with failure to thrive, recurrent pneumonia, followed by edema, proteinuria, urinary tract infections, cellulitis and membranous glomerulonephritis, undetectable IgA and increased IgG, a normal T cell count and a slightly decreased B cell count in one patient and a reversed CD4+:CD8+ T cell ratio and severe B cell lymphopenia in the other 271,272 . Rac2 is mainly studied in neutrophils, and patient neutrophils from Rac2 deficient patients exhibit an abnormal chemotaxis, and constantly high levels of F-actin, suggesting that the role of Rac2 in actin cycling between its two forms is impaired 272,273 .

q. Myosin deficiency

Myosins

r. ARPC1B deficiency

The actin nucleator ARP2/3 is composed of 7 subunits (Figure 17) The Arp2 and Arp3 subunits act as monomers from which new actin filaments elongate in a 70-degree orientation and recruit and hydrolyze ATP 285 . The remaining subunits (the isoforms ARPC1A, ARPC1B (encoded by a gene on chromosome 7), ARPC5, and ARPC5L, and ARP4) organize to form the structural core of the complex, providing an interface for binding to preexisting actin filaments and regulatory cofactors.

Indeed, activated WASp binds to ARP2/3 and delivers the first G-actin via its VCA domain, and WAVE binds to the complex as well 286,287 . Patients with ARPC1B deficiency present with a very early onset of 2 months on average. The first patients with an ARPC1B mutation were reported in 2017, and more have been reported since then.

ARPC1B deficient patients presented with failure to thrive (due a partial growth hormone impairment), recurrent bacterial and viral infections and hemorrhages, platelet abnormalities, eosinophilia, enterocolitis, small vessel vasculitis, eczema and other indicators of inflammatory/immune disease, and such a phenotype was described as "WAS-like" [288][289][290][291] . The patients presented with an increased amount of B cells, and decreased CD4+ and CD8+ T cells, including a reduction of naïve T cells 289 .

ARP2/3 was recently shown to colocalize with actin at the IS and at lamellipodia, suggesting that the defects observed in ARPC1B deficiency may be attributed to defects in cytoskeletal organization, especially that ARPC1B cells display a defect in actin polymerization 288,291,292 . Moreover, in patients with ARPC1B deficiency, levels of ARPC1B in PBMCs are reduced and the distribution is, as revealed by confocal microscopy, in large patches instead of an even distribution along the actin cortex 288 .

APRC1B deficient patient T cells fail to spread radially on an ICAM-1/Anti-CD3 Ab coated surface, and they emit thin filopodia, which are under the control of formins, as can be inferred from their abolishment upon treatment with SMIFH2 274,288,292 . Moreover, they require more time to form weak conjugates and a very narrow, F-actin poor contact area with target cells, due to the failure to accumulate or deplete F-actin 288,292 . Treatment of T cells with the ARP2/3 inhibitor CK-666 -which like CK-869maintains the ARP2/3 complex in an inactive state blocking formation of the short pitch Arp2-Arp3 dimer -was shown to reduce T cell cytotoxicity, as well as reduced actin accumulation as compared to the inactive CK-689 292,293 . Inhibition with CK-666 also abolished lamellipodia (which were replaced by small blebs), and treated cells showed a slower migratory speed 292 . Therefore, the ARP2/3 complex is involved in CTL migration and cytotoxicity.

ARPC1B deficient T cells show a reduced migration in response to CXCL12, reduced degranulation, reduced centrosome polarization and a reduced ability to lyse targets, which are due to defective TCR signaling, as well as a reduced Gzm B content 292 . Of note, even though ARPC1B deficient cells could secrete granules, the defect in degranulation can be traced back to the fact that only a minority of lytic granules are polarized to the IS. Some ARPC1B deficient CTLs (perforin positive) showed a loss of CD8 as well. ARPC1B deficient CD16 neg CD56 bright NK cells also show defects of degranulation and cytotoxicity, which may be partially restored with IL-2 289 .

The study of patients with several immunodeficiencies related to the cytotoxic function lead to the observation that ARP2/3 is a key player in the regulation of the cytotoxic function in lymphocytes.

Therefore, it would be interesting to study the response of the ARPC1B deficient cells to different levels of activation by anti-CD3 and assess their cytotoxic potential under these different levels of activation.

Figure 18: PID-associated actin regulators

Methodologies to assess defects in cytotoxicity.

To assess the defects in cytotoxic lymphocytes in patients or mouse models, different methodologies exist. In this chapter, the methods employed to assess functional defects in the PIDs mentioned in this chapter will be elaborated, following the logical order of the steps of cytotoxicity.

A defect in actin content or polymerization may be assessed by flow-cytometry, as done for WDR1, DOCK2, DOCK8 , Rac2 and ARPC1B 214,244,250,273,291 .

Defects in lytic granule content are generally assessed by flow cytometry, as was done for RASGRP1 262 .

Conjugate formation capacity can be assessed by flow cytometry or immunofluorescence, as was done for DOCK8 and Coro1A respectively 224,250 .

The immunological synapse is typically monitored by confocal microscopy. It has been shown that WIP deficient cells display actin organization and form improper IS 208 . WDR1 deficient cells are more spread and form actin arcs 213 . Coro1A cells form a synapse of normal size while Myh9 deficient cells appear to spread more than control cells and ARPC1B deficient cells display a failure to spread radially 224,278,292 . DOCK8, PSTPIP1, RASGRP1 fail to recruit actin to the IS 246,252,262 . Coro1A deficient cells can successfully recruit LFA-1 to the IS, which is not the case for DOCK8 deficient cells 224,246 . Granule convergence at the MTOC appears defective in Lyst deficient cells, and the granules appear larger in size that controls 294 .

Defective degranulation can be monitored by flow cytometry, by monitoring the levels of CD107a exposure by flow cytometry, as was done for WIP, Coro1a, Rab27a and Lyst 183,185,208,224 . It can also be monitored by observing the recruitment of lytic granules to the IS, where it has been shown that while Munc13-4, Munc18-2, Coro1A can usefully polarize their granules to the IS, DOCK8 and STX11 and MyH9 deficient cells fail to do so [176][177][178]181,224,246,249,250,281 .

Finally, the ability of cytotoxic cells to eliminate target cells can be assessed by flow cytometry, where it has been shown that WASp, WIP, Coro1A, DOCK8, Rab27a, Munc18-2, Lyst and ARPC1B have impaired ability to eliminate target cells with respect to healthy controls 180,182,202,208,224,246,250,292 . This shows that there is currently no systematic and unified process of detecting defects in the cytotoxic activity of lymphocytes, and that several tests are usually performed to assess it. Therefore, there is a need for an approach which can predict a functional defect in the cytotoxic activity of NK and CD8+

T cells.

Microscopy imaging of biological objects

In the previous section, I have tried to highlight the importance of the cytotoxic function of T and NK cells, as well as the important role of the immunological synapse in the context of cytotoxicity. In the next section, I will highlight the importance of microscopy in immunology, as well as the advantages of high content imaging pipelines as opposed to traditional low throughput microscopy.

The human eye is highly capable of viewing different objects of varying sizes. However, the smallest it can see is an object of around 100µM, when that object is about 25 cm away.

The human eye is thus incapable of seeing infectious organisms such as bacteria, without an object that would enhance their size. For that reason, the microscope, which was invented by Antoni Van Leeuwenhoek and Robert Hooke, has proven to be a vastly useful tool. A microscope is therefore a tool that allows one to see samples while enhancing their size through one or multiple lenses. Any sample that allows light to pass through it may be viewed with a microscope.

The basics of microscopy

A. Light sources to illuminate a sample

Every microscope essentially needs a light source, and the light is composed of an electromagnetic wave having a specific wavelength, and photons. Lights can illuminate a sample either directly

B. From an object to an image

When light passes through lenses, the image can be enlarged to different sizes, depending on the magnification of a given lens. Light exits a lens and rays parallel to the optical axis may only be converged onto one focal point if the lens is spherical. The objective lens typically determines the magnification and resolution of an image, as well as the sensitivity of the microscope with regards to how much light will be collected from the sample. Magnification refers to how large the final collected image will be with respect to the sample.

The object being detected by a microscope is referred to as a sample, and it should be placed on a support in order to be illuminated. Therefore, an essential characteristic of a sample is that it should allow the passage of light.

The human eye is the structure that detects the image in the context of a simple light microscope.

However, most commonly used nowadays is a camera, which also allows saving images. However, noises may occur and interfere with proper image detection. Notably we name the black noise generated by the camera, the reading noise caused by the electronic signal amplification and the photonic noise due to the random nature of the light.

The term "resolutive" is employed to refer to a microscope that can detect two close points as separate.

In other terms, the resolution of a microscope is defined as the smallest distance between two points of a sample at which they can still de distinguished as two separate points. This distance, as demonstrated by Abbe in 1873, has been shown to depend on the light diffraction from the sample through the lens. The formula to determine resolution is:

d = λ / NA,
where λ is the wavelength of light and NA is the numerical aperture of the objective lens.

Technological advancements in the field of microscopy are always aiming to increase the resolution.

The timeline of microscopy in biology

The discovery of microorganisms through microscopes began in the 17 th century with the Dutch scientist Antony Van Leeuwenhoek, who created lenses with 300x magnification, and used single lenses to view his samples, and referred to what he saw as "animalcules". Hooke, a contemporary of Van Leeuwenhoek, also created settings that allowed object magnification and imaged seeds, the eyes of a fly and the structure of a cork. He however used compound lenses, which resulted in more aberrations due to the lenses themselves at the time.

In the nineteenth century, the theoretical and technical aspects of the modern light microscope were developed. The most notable are diffraction-limit theory, aberration-corrected lenses and an optimized illumination mode called Köhler illumination.

In 1868, Abbe invented the apochromatic lens, using more fused lenses, which better corrected chromatic and spherical aberrations. In 1883, he collaborated with Schott, a glass chemist, to produce the first lenses that were engineered with sufficiently high quality to produce diffraction-limited microscopes.

Fritz To improve contrast in biological samples, stains have proved to be quite a useful tool, especially stains that allow staining of specific parts of a sample.

The first reported staining of a biological sample was reported by von Gerlash in 1858, who managed to differentially stain the cytoplasm and the nucleus of brain tissue with carmine. Golgi introduced silver staining in 1873, through which he visualized the nervous system. In 1884, Gram introduced the Gram staining, which is until now still used to stain different bacteria. However, the revolution in microscopy staining of biological samples occurred upon the introduction of fluorescent stains. Stokes was the first to define fluorescence in 1852, as the emission of light at a wavelength different from that of excitation. In 1941, Albert Coons published the first work on immunofluorescence, where he used a fluorescein-derivative-labelled antibody and showed that it could still bind to its antigen 300 . This was the first steps towards the use of fluorescent antibodies as highly specific stains.

The first natural fluorescent stain used was green fluorescent protein (GFP), isolated from jellyfish in 1962 301 . However, it was not expressed outside of jellyfish until 1994, when it was incorporated into a promoter gene for β-tubulin and shown to be useful in determining expression levels 302 . A real breakthrough was optimizing a method to fuse the genes of a protein of interest with a GFP and express this in a cell while also leaving it relatively unperturbed. This was performed in 1994 by Wang and colleagues by fusing green fluorescent protein GFP to the bcd transcription factor in Drosophila 303 .

Blue and yellow fluorescent proteins were produced by mutating GFP, while red fluorescent protein was produced from Anthzoa, a species of coral, in 1999 304,305 . In 1980, Roger Tsien introduced the first calcium probe, capable of detecting cell membrane voltages and ion concentration 306 .

In To overcome this limitation, systems which scan multiple focus spots across the sample simultaneously and image the resulting emission on a camera have been designed. The spinning disk confocal is the most common, and it uses a disk of pinholes which sweep across the sample in a way that a revolution of the disk scans over every point in the sample during a single exposure 314 . Spinning disk confocal microscopes have gained popularity given their high speed and sensitivity.

Even though the imaging of biological samples had come a long way, the size of the smallest object that could be imaged was still a limiting factor. Two objects could be separated (or resolvable) as long as the nearest-neighbor separation of the dye is greater than the limit of the optical resolution. In other words, the wavelength of the light used, as well as the objective used were the barrier to imaging single molecules. Hell and Wichmann were the first to show that it was possible to break the diffraction limit with a technique he called stimulated emission depletion microscopy (STED), and Hell and Klar implemented it in 1999 315,316 . Alexa dyes also came to use in the late 1990s and are more fluorescent than the dyes previously used. Alexa derivatives coupled to phalloidin can be used to visualize F-actin, and may also be used to detect antibodies 317 . Currently, a common approach to stain cells is indirect immunofluorescence, where the cells are fixed, labeled with a primary antibody specific to the protein to be detected, followed by staining with secondary antibodies, which generally have a broad specificity and can be coupled to Alexa dyes.

In 2006, two new techniques were introduced. Photo-activated-localization microscopy (PALM) was used by Hess and Betzig, in which photoactivable proteins which change colors when exposed to UV light change colors 318,319 . The main difference is that Hess and colleagues used TIRF while Betzig and colleagues used confocal microscopy. In 2006 as well, Rust and colleagues showed that by using a technique called stochastic optical reconstruction microscopy (STORM), they could generate a super resolution by using a Cy5/Cy3 switchable probe 320 . When the probe is illuminated by a red laser, Cy5 is kept in a dark state, while the pair is brought back to an exited state by a green laser.

Advancements have also been made in 3D imaging microscopy which allow the reconstitution of cells and even organoids. However, they will not be addressed in this manuscript.

The timeline of microscopy is summarized in Figure 19 below.

High content imaging: A game changer

One problem microscopy has faced is the time image acquisition and processing can take. This is true especially in a time where screens have become highly popular as an approach, and the fact that phenotypic analysis can be a powerful technique to reveal information. Indeed, phenotypic analysis has been employed by Brenner who performed a screen on Caenorhabditis elegans to assess how different mutations could impact behavior, and by Nüsslein-Volhard and Wieschaus who performed a screen on the Drosophila melanogaster embryos to assess the effect of different mutations on the number and the polarity of the segmental patterns of the larva 321,322 . However, these screens were manual and consequently laborious, which explains the need for an automated approach. Screens may be conducted in several different ways and may include biochemical assays, neurite growth assays, as well as translocation and differentiation assays (reviewed in 323 ).

Screens can also include specific inhibitions with small interfering RNA (siRNA), short hairpin RNA (shRNA) and CRISPR cas9 screens, and include genome wide screens [324][325][326] . Indeed, Carlin and colleagues have designed an siRNA screen and images samples using fluorescence lifetime imaging microscopy (FLIM), which is the optimized FRET technique to image protein interactions, and monitored the activity of Cdc42 and its interactors 327 . However, CRISPR cas9 screens are growing in popularity since shRNA and siRNA may generate off-target effects and incomplete inhibition 328

High content screens are a useful tool in the identification of small molecules that may be used for drug development, as well as determining interactions between different proteins or compounds.

A. High content imaging: microscopy at a large scale

High content imaging (HCI), a term which began to be employed in the early 2000s, combines approaches of automated imaging and image analysis of cells and cellular organelles, to quantify phenotypic and functional parameters, usually linked to genetic or pharmacological perturbations.

High content imaging, with its corresponding analysis methods, has become increasingly popular in the fields of drug or chemical compound discovery, as well as identification of gene functions, especially because it allows for quick, efficient and lower cost screens [329][330][331][332][333][334][335][336] .

Some low content imaging approaches may be amenable to screening by high content imaging.

Sieprath and colleagues have optimized an HCI protocol on fibroblasts seeded in 96 well plates, where they use live imaging to assess the levels of reactive oxygen species as well as mitochondrial membrane potential and mitochondrial morphology 337 . Szalai and Engedal have optimized a protocol for the assessment of real-time cell proliferation and death in a prostate carcinoma cell line 338 . Massey has developed an HCI approach to measure target engagement in living adherent cells based on the principle of altered protein thermal stabilization or destabilization in response to ligand binding 339 .

HCI has several biological applications and may be used to answer different biological questions. The following pages will focus on the questions that may be answered with HCI, and the tools needed for such screens.

a. HCI screens may be used to define gene function.

Bai et al. used an siRNA HCI screen to knock-down 26 genes whose function was not previously assessed, in a prostate carcinoma cell line and showed that 25 of these genes affected cytoskeletal organization or cell shape 340 . Indeed, they used 14 morphological including parameters pertaining to F-actin, cell shape and area. Chia et al. showed, by using an HCI on a genome-wide siRNA screen in human embryonic stem cells (hESC), that PRDM14 is a crucial transcription factor required for the maintenance of hESC identity and the re-acquisition of pluripotency in human somatic cells 341 . It has been shown that an HCI RNA screens can be useful to determine genes involved in mitotic spindle formation in yeast 342 . Indeed, an HCI screen coupled to gene knock-down has served to identify mutants with aberrant spindle morphology, by using 78 spindle-specific features for the analysis, as well as to identify new candidate genes that may play a role in spindle disassembly.

Billmann et al. have used an

RNAi screen in Drosophila, to screen for modulators of the cell cycle.

They started with a genome-wide RNAi screen where, following image analysis for nuclear and mitotic parameters, they selected 300 candidates to connect novel genes to known regulators of the cell cycle 343 .

b. HCI screens can be effective in determining gene interactions.

Genetic interactions can be assessed by using pairwise gene perturbations to generate interaction

profiles. An image-based genome-wise RNAi screen in Drosophila cells has identified 168 genes whose phenotypic profiles are sensitive to MEK, involved in the MAP/ERK pathway 344 . Genes may also be co-depleted by RNAi and imaged by HCI to determine genetic interactions and generate correlation networks. Using this technique, it was determined that there is a link between Ras signaling and chromatin remodeling 345 .

Chong et al. have applied an HCI screen to budding yeast open reading frame (ORF) -GFP collection,

where each of the ∼4,100 strains carries a unique fusion gene construct in which an ORF is fused to the GFP gene, generating a full-length protein with a COOH-terminus GFP fusion, whose expression is driven by the endogenous ORF promoter 346 . They also introduced a cytosolic RFP, to create an automated platform to quantify the abundance and localization of these fusion genes, instead of the manual quantification that was applied 347 . Their results led to the generation of the abundance localization map with 2834 proteins connected to at least one of the 16 quantitative localization scores that they defined for each protein and which reflect 16 defined subcellular compartments.

c. HCI screening is a useful tool to assess alterations to cell morphology in response to chemical compounds.

Coupling treatment with compounds to genetic perturbations may reveal how genes are sensitive to specific inhibitions 344 .

Bray and colleagues showed that it is possible to use six fluorescent dyes to perform what they describe as "Cell Painting", in which they can reveal eight cellular components and organelles 331 . Subsequently, they could extract roughly 1500 morphological features to describe their cells, and profile them accordingly. They then show that it is possible to profile cells treated with different chemical compounds and detect even subtle morphological differences.

A recent study by Caldera et al. has shown that it is possible to benefit from the interactome of the physical interactions of more than 16000 proteins and perturb them with chemical compounds, which they summarized in a 3D perturbation space 348,349 . For that, they applied an HCI approach in the adherent MCF 10A cell line and showed that it is possible to use cell morphology to evaluate the interaction between different perturbations in high dimensionality space.

HCI may be used to determine levels of protein interactions in response to different chemical compounds. Preistly et al. have developed an immunocytochemical protocol in which they use HCI to determine the specific binding of Dickkopf-related protein 1, whose elevated levels have been linked to Alzheimer's disease 350 . Using HCI they measured the specific binding of the protein to its receptor in response to treatment with different inhibitors.

d. HCI screens are a powerful tool to image disease models.

Schulte and colleagues show that high content imaging screens can be used to build a Huntington's disease model and identify compounds which can rescue dystrophic neurite formation 351 . Being a spinning disk confocal microscope, it has an array of illuminated pinholes equally paced to illuminate the whole field of view and positioned at one of the microscope's intermediate image planes will create a de-magnified array of tiny excitation focal volumes at the nearest objective lens object plane. The spinning disk optics and precise excitation laser and camera synchronization lead to minimal phototoxicity and photobleaching. This minimalized spectral cross-talk allows for high quality images and a reduced acquisition time (Figure 21). This microscope also offers the advantage of water immersion objectives, which capture more photons, thus increasing image resolution. Moreover, the system has a fast laser-based autofocus, thus ensuring the maintain of focus throughout different fields. The system also benefits from a CO2 chamber and temperature control, to allow live cell imaging. From: The Perkin Elmer website

C. High content imaging analysis requires specialized software

Companies developing and selling HCI microscopes usually develop in parallel compatible analysis software that may be purchased in parallel. Such software are specifically designed for the analysis of data from HCI screens. They are generally user-friendly and easy to navigate and may include pre-designed analysis pipelines. They may be used to detect specific phenotypes of alterations to cell morphology or intensities of a fluorophore. However, such software may not be adapted for more complex questions which go beyond basic biology and require knowledge of bioinformatics to a certain extent. Therefore, there is the need for software that are more adapted to deal with such demands.

a. CellProfiler

CellProfiler is an open source image analysis software, developed by the Carpenter lab at the Broad Institute of Harvard and MIT. It allows the user to measure, simultaneously, several parameters related to cell size, shape, intensity and texture. Moreover, it is specifically designed to perform the analysis of data incoming from screens, in a high throughput and automated manner [355][356][357][358] . CellProfiler is an easy to use software which can be installed on a personal computer to analyze a small number of images, while a larger number of images will have to be processed on a cluster. The details on how the software works, its advantages and its applications can be found in 355

b. EBImage

EBImage is an image processing toolbox for R whose features include multi-dimensional image processing, a range of fast image processing functions, support of more than 80 image formats, fast interactive image display. It allows for automated phenotyping of images by performing the steps of segmentation, feature extraction and statistical analysis, as used in the screens by Breinig et al,and Heigwer et al. 344,359,360 .

High content imaging screens have come a long way in recent years. The first screens were 2D, but the need to mimic in-vivo conditions has led to the development of microscopes and image analysis software capable of handling 3D structures. The advantage that 2D screens offer are the simplicity and the lower cost, but cells cultured and subsequently imaged in 2D layers lose some of their morphological properties, giving 3D screens an advantage in terms of physiological value [361][362][363] . One limitation of high content screens is the difficulty to apply them to cells in suspension, thus hindering the study of several diseases such as lymphomas or primary immunodeficiencies.

3D screens will not be addressed in this manuscript.

D. Image analysis pipeline in high content imaging screens

As previously mentioned, high content imaging screens generate a significant bulk of data to be processed, which require analysis to quantify the phenotypes of interest. ImageJ and Metamorph, can be a tedious tool to analyze many images, even with the use of macros. That is because any change that needs to be made to the analysis pipeline will require a direct intervention with the code (Matlab, Java), which may be prone to errors. Therefore, they will not be discussed in this manuscript. However, they have been reviewed in 355 .

There are however specialized tools for HCI data analysis and image-based cell profiling. In imagebased profiling, hundreds of morphological features are measured, and the morphological profile refers to this collection of measurements 364 . The typical analysis work flow for image based profiling can be divided into eight main steps 364 . Assuming the cellular model, fluorescent probes and image acquisition have been performed successfully, the transition from images to phenotypes can take place 365

The typical workflow of image-based cell profiling is represented in figure 22 below and will be elaborated in the following pages.

Figure 22: Representative workflow for image-based cell profiling. Image-based cell profiling involves eight main steps which lead to the transformation of images into quantitative information

from which conclusions can be drawn 364 .

a. Image analysis

First, image analysis must transform images into measurements (features) describing the state of each cell. Every image must undergo field of view illumination correction, as intensities can vary between the center and the edges of a field of view, due to optic artifacts. This process recovers the true image to avoid corruption of an accurate segmentation and intensity measurements 366 . Illumination correction can be achieved by building corrections from dark and bright images with no sample acquired along with the imaged samples, in a process termed "prospective methods" 367 . However, such an approach often yields an incomplete correction. Alternatively, retrospective single-image methods can correct illumination by calculating corrections on a per image basis, which can alter relative intensities 364 .

Retrospective multi-image methods correct illumination by using the totality of the images acquired in an experiment 364,367,368 . This approach usually produces more robust results. Next, the images must undergo segmentation, which allows the distinction between pixels that belong to the object of interest and background pixels. This approach is typically used in CellProfiler analyses and requires

b. Image quality control

The second step is image quality control, and an automated approach must be put in place to remove defective images. It has to be done a field of view level to account for blurring, saturation and the presence of debris. Cell-level quality control must also be performed to remove cells that exhibit a highly unusual phenotype. Outliers can be detected in a model-free fashion by the use of robust statistics such as the mean and media, or multivariate methods such as principal component analysis (PCA) and Mahalanobis-based outlier detection. The latter is based on robust estimation of mean and covariance matrix to detect outliers 364,369 . Model-based outlier detection is based on training a model to detect outliers.

c. Processing the extracted features

The third step is the processing of extracted features, which must consider several factors in order to yield the proper conclusions. Features extracted may present with non-finite values, which can be problematic for statistics and machine learning approaches. Therefore, cells with such values may be removed if they are a minority, or the feature itself may be removed if it appears to be missing in a large proportion of the sample. A third way, which should generally be avoided as it may create a bias and false conclusion, is the imputation of missing values. The effect of the plate layout must also be corrected, to account for artifacts that may be present in a plate or across a batch. Ideally, samples should be placed in random positions in the plate, and in different positions in multiple replicated.

However, this is not a practical approach in the absence of robotic sample dstribution. Caicedo et al.

recommend using a two-way median polish to correct for these effects when the samples are randomized, via iterative median smoothing of rows and columns to get rid of positional effects, followed by the division of each well value by the plate median absolute deviation to generate a B score 364 . Local smoothing may also be applied via 2D polynomial regression or running averages.

Batch effect must also be corrected via standardization and quantile normalization to plates if the samples are placed in a random order in each experiment. Otherwise, canonical correlation analysis could be applied. Feature transformation and normalization must be applied to features that display varying shapes of statistical distribution. These include logarithmic transformations used to obtain approximate normal distributions for features that have highly skewed values or require range correction either by the logarithmic functions or one of its adaptations, or relative normalization whereby the population is scaled and centered with respect to one part of the population in which statistics have been computed. Cofounding effects must also be removed for subtle phenotypes, to avoid masking a phenotype of interest 368 .

d. Dimensionality reduction

The fourth step involves dimensionality reduction, which aims to remove related, redundant or uninformative features. Feature selection includes finding correlated features by computing the feature-feature correlation matrix and keeping only the features with the largest mean absolute correlation 364 . Replicate correlation filtering keeps only the features that provide the highest additional information content. Minimum redundancy-maximum relevance adds a constraint based on mutual information to the selection algorithm. The resulting selected features have high replicate correlation while preserving a diverse set of measurements. Support-vector-machine-based recursive-feature elimination involves a support vector trained machine that is trained to weigh feature usefulness.

Linear transformation methods can be performed to achieve lower-dimensional subspaces of higherdimensional data that maintain information content. An additional step of decorrelation must be implemented in the case of a technical bias, such as the choice of features to measure. In that instance, the output space becomes biased as well, as should be accounted for.

From the fifth until the eighth step, the pipeline becomes less straightforward, as steps may be skipped or reordered, based on the experimental setup and the answers sought.

e. Single-cell data aggregation

The fifth step is single-cell data aggregation, from which population-level morphological states can be concluded. Aggregation can rely on the mean profile which assumes a normal feature distribution and where a profile is built from the means of each feature for the entire population. It can also rely on the median profile, which can provide more robustness for non-normal distributions and minimize outlier effect. It appears to perform better than other profiling strategies 364 . KS profile compares the probability distribution of a feature in a sample with respect to negative controls by using the KS nonparametric statistical test and yields a profile where the KS statistics reveal how the sample behaves differently with respect to the control. Next is subpopulation identification and aggregation, where cells are first identified and clustered according to their morphological phenotypes from single cell profiles. This can be either supervised or unsupervised. Single cell data points are then classified to one of the identified subpopulations and aggregated to calculate the number of cells within each of the identified subpopulations.

f. Profile similarity measurement

The sixth step is measuring profile similarity, to compare experimental conditions. Similarity metric calculation involves three main types of metrics which allow a simpler data visualization and analysis. They include distance measures to calculate how far apart two features are in the feature space and quantify the magnitude between two given profiles. They include Euclidian, Mahalanobis and Manhattan distances. Similarity measures compute a statistical estimation of how likely two profiles are to be related and to find groups that share common properties. They include Pearson's correlation, cosine similarity and Spearman's and Kendall's rank correlations. Machine learning-based learned similarity measures involve training models with prior knowledge about the samples to highlight patterns that may not be discriminated by regular metrics. Concentration-effect handling must be taken into consideration to identify similarities among different compounds that may occur at different concentrations. Titration-invariant similarity score computes a score between each dose and the negative control, and the scores are sorted in a concentration-dependent fashion. Maximum correlation is practical when a small number of concentrations is used, and the correlation matrix is computed between all concentration pairs 370 . The maximum value is used as the dose-independent similarity score.

g. Assay quality assessment

The seventh step is the assessment of the assay quality. Comparison to ground-truth relies on the use of known similarities between pairs of treatments. Classification accuracy may be applied if treatment results are known to fall into specific classes, and concentrations of the same treatment are expected to cluster together. Replicate reproducibility should also be achieved, where the similarity between replicates should be higher than the similarity to profiles of other treatments or conditions 364 .

Effect size computes the difference between the positive and negative controls but may lead to a bias as some phenotypes which do not particularly distinguish a control may be ignored.

h. Downstream analysis and result interpretation

The final step is downstream analysis, which allows the interpretation of the results of morphological profiling. Clustering is an effective way to extract meaningful relationships between different morphological profiles. Hierarchical clustering is the most commonly used strategy to identify correlation as well as positive and negative connections, and is computed by a similarity matrix which can reveal patterns in the data 371,372 . The data from the heat map is structured as a dendrogram to link features together based on proximity in the feature space. Next comes data visualization, which usually takes the form of a 2D (sometimes 3D) map to reduce dimensionality and allow for an easier interpretation and less crowding. While tools to represent data in more than two dimensions have been used, which include the Chernoff faces and the pixel-based techniques, they are a mere visual tool where the user is let to interpret the data. Visualization can include data projections or hierarchical visualizations. Data projection includes the following:

i. Linear dimensionality reduction: Principle component analysis

PCA is a linear dimensionality reduction technique which has long been the most employed tool, as it relies on simple linear algebra and is faster than most other methods. It consists in finding a lowdimensional embedding of the data points in a way to best preserve their variance, as it was measured in the high-dimensional space, and representing them as a new set of orthogonal and uncorrelated variables called the principal components 373,374 .A comprehensive and recent review on PCA was published in 2016 by Jolliffe and Cadima 374 .

ii. Non-linear dimensionality reduction

The need and usefulness of new tools is being recognized, in order to extract not only linear relationships between input dimensions (as in PCA), but any kinds of relationships. Such non-linear dimensionality reductions include but are not limited to the following:

c) t-distributed stochastic neighborhood embedding

t-distributed stochastic neighborhood embedding (t-SNE) has stochastic neighbor embedding (SNE) as its basis, which converts the high-dimensional Euclidean distances between datapoints into conditional probabilities (pj|i) that represent similarities. The mathematical aspect of SNE is elaborated in 377 . The limitation of SNE is the crowdedness of the data points and a suboptimal cost function, whereas t-SNE is an improvement since it uses a symmetrized thus simpler cost function and a studentt rather than a Gaussian distribution. t-SNE is elaborated in further detail in 377 .

Figure23: Visualization of 6000 handwritten digits from the MNIST data set by t-SNE 377

However, the disadvantage of tSNEs is that such a representation causes the loss of large scale information such as the inter-cluster relationships, while it is precise for local structures rather thatn global trends 378 . Therefore, a tSNE might not be the best adapted method to extract meaningful relationships between large-scale clusters

d) Uniform manifold approximation and projection

When compared to other tools, the uniform manifold approximation and projection (UMAP) proved Unsupervised methods aim to find internal structure or relationships in a data set instead of trying to determine how best to predict a "correct" answer and reveal clusters and associations within the data set without any prior teaching model. They are useful especially when class data labels are unavailable.

They include hierarchical clustering on principal components, because PCA allows the reduction of the number of variables without losing important information and is a very stable method compared to other unsupervised machine learning techniques 387,388 . Unsupervised clustering finds clusters without a pre-determined number of clusters and the data is divided into clusters in a way where the data within one cluster bares high similarity among themselves, and dissimilarity to data in other clusters 389 .

The performance of a classifier can be determined by comparing the prediction of the classifier to the expected outcome. These include binary classifications and multilabel classifications. The F1 score is a measure that can express the counts of true positives, false positives and false negatives 390 .

E. Statistics

The performance of a high content screen is usually quantified with statistical parameters. When performing a high content screen, it is assumed that the data is normally distributed, and logarithmic transformations (usually log2) are often applied in the pre-processing stage, to generate data that is more symmetrically distributed around the mean, to facilitate the understanding between positive and negative values 391,392 The next step involves dealing with outliers and can be achieved in two ways: classical and robust.

Instead of eliminating outliers, Robust statistics, which have gained in popularity in high content screens, may be applied. Robust statistics are less sensitive to outliers as they use the median and median absolute deviation instead of the mean and standard deviation. This diminished the effect of outliers on the final results 392 . The robustness of a technique can be determined by a breakdown point, which is the number of outliers allowed before the parameters become no longer representative of the original dataset. The median and median absolute deviation have a breakdown point of 50, while the mean and standard deviation have a breakdown point of 0%, implying that robust statistics can successfully handle a data set where 50% of the data points are outliers 392 .

Data must be normalized to account for and reduce assay variability, as well as to eliminate false positives and negatives which may be caused by plate patterns 391 . Data normalization can be controlbased, where the positive and negative controls are used as the 100% and 0% bounds respectively, and the features of the other data points are calculated with respect to these values. The activity of the sample can be calculated as percent of control or normalized percent inhibition.

Non-control-based data normalization is favored in high content screens, as it assumes that most samples are inactive and serve as their own control. IT can be calculated as percent of samples, robust percent of samples, Z-score or robust Z-score. The Z-score accounts for signal variability in the samples. This method is usually favored as it leads to a better understanding of the overall activity but can be quite misleading in screens where the basal activity shifts upwards.

Data analysis must also consider variability detected in experiment-wise patterns. Median polish accounts for row and column effect (the spatial trends in a plate), and the B-score utilized to account for inter-plate variability uses the residual values calculated in median polish and the mean absolute deviation 368,391,392 . Background correction must also be applied by calculating the average activities within each well and generating a background surface for each experiment. Well correction is performed in a similar manner to background correction, but the fitting is performed for each individual well, proving to be successful for hit selection 392 . Diffusion-state model corrects variability in environmental factors such as temperature or humidity distribution across well and is generated from a control column.

Quality control statistics include signal/background which is the ratio of the mean of the positive control to the mean of the background signal, signal/noise which is similar to signal/background byt takes into account signal variability, signal window which measures the data range in an assay, assay variability ratio which measures the data variability in the positive and negative controls, the Z'-factor which assumes a normal distribution to account for variability in the control wells, the Z-factor which is a modification of the Z'-score where the sample mean and standard deviation replace that of the negative control, and the SSMD, which relies on probability and statistics and has been shown to be more effective that Z' and Z scores in handling controls with different effects 392,393 .

To assess HCI results, specific statistical analyses must be employed to analyze and interpret the large amount of data generated. The different analysis methods and requirements include normalization, logarithmic transformation and quality control.

To compare any two treatments, a multidimensional perturbation value (mp-value) may be calculated. The difference between a treated condition and a control is considered as activity, while no difference is considered inactivity. An mp-value to the Mahalanobis distance is assigned, and permutation tests are used to determine statistical significance. This is done by a permutation approach, where under the null hypothesis, shuffling the replicate labels would not result in a drastically different Mahalanobis distance between the two groups, while if the treatment replicates are significantly different, the Mahalanobis distance would likely be smaller after randomly shuffling the replicate labels 120 . Significance values are interpreted in the same way as significance values from parametric statistical analyses, where an mp-value < 0.05 is considered significant.

Implementation of a high content imaging pipeline to study the immunological synapse of cytotoxic lymphocytes

Rationale

Despite the great advancements made in the field of high content imaging in terms of techniques and analysis, these assays lack standardized protocols. The work towards standardization is however in progress.

The cytotoxic function is highly important, and a tightly regulated process. Moreover, the immunological synapse involves a complex interplay between a plethora of molecules, in order to drive the successful delivery of lytic molecules into the target cells. All the aforementioned research suggests a crucial role for the actin cytoskeleton and the process of polymerization and depolymerization in maintaining immune homeostasis, and that cytoskeletal defects such as defective immunological synapse and cytotoxicity may lead to several life-threatening illnesses. In the context of primary immunodeficiencies, the data is not systematic, and the analysis methods vary. Current advancements in the field of imaging and image analysis have made high content imaging of a large number of samples feasible, with a good image resolution that allows the detection of subtle morphologies, and robust statistics specially designed for such approaches. However, microscopy is not widely used among immunologists, but the advancements made in that field have undeniably helped our understanding of the mechanisms governing the cytotoxic activity, actin dynamics and the assembly of the immunological synapse.

Taking together the crucial role of the immunological synapse in cytotoxicity, and the findings that perturbations of the cells can alter morphology, the following could be hypothesized the following:

• The immunological synapse of CD8+ T cells and NK cells should present different morphological features

• Different perturbations to the actin cytoskeleton of cytotoxic lymphocytes should yield distinct immunological synapse morphologies.

• The immunological synapse of ARPC1B deficient patients should have a morphology that is distinct from that of normal donors

Driven by the importance of the role of the immunological synapse in cytotoxicity and the need for a systematic method to screen for PIDs that may display a defective cytotoxic activity, I have elaborated a new high content imaging and analysis pipeline of the immunological synapse. This pipeline could be a first step to assess defects in the immunological synapse assembly, from which functional defects may be inferred. Such a pipeline, which overcomes the bottleneck of low throughput microscopy typically used to image the immunological synapse, could be systematically applied to screen cytotoxic lymphocytes from PID patients and adapted to screen other types of immune cells.

Objectives

Therefore, the objectives of my thesis project were the following:

1. Set up a high content imaging and analysis pipeline of the immunological synapse of cytotoxic

Results

I first started by optimizing assays on the NK-92 cell line and primary NK cells isolated from the PBMCs of normal donors.

Indeed, the review by Emily Mace entitled: "Cell biological steps and checkpoints in accessing NK cell cytotoxicity" [START_REF] Mace | Cell biological steps and checkpoints in accessing NK cell cytotoxicity[END_REF] , as well as the expertise of our lab on lymphocytes inspired me to set up functional assays to assess the main steps of cytotoxicity in NK cells. During the first year of my PhD, I learned a lot about the cytotoxic function, and had the chance to establish assays on a new cell type that was not used in our lab.

Indeed, I have established a methodology where the functional steps of cytotoxicity can be assessed in parallel. Migration, conjugation and cytotoxicity were amenable to assessment high throughput cytometry readings in 96 well plates. However, the step of imaging the immunological synapse which is done by low throughput confocal microscopy revealed to be tedious in terms of the time required for image acquisition, the analysis approach to adopt and the parameters to analyze. I was therefore driven to test the feasibility of staining the immunological synapse in a high content format, to match this assay format to that of the other steps. It was also interesting to benefit from imaging the cells in a high content format and apply a compatible analysis. For that reason, I imaged the IS using the Opera Phenix microscope, and its compatible analysis software Harmony, to analyze the individual wells.

However, the number of parameters that may be analyzed by such approaches remain limited, and the analysis on a per well basis can hide some interesting phenotypic features. It was evident that a more powerful bioinformatics approach was needed to extract hundreds of relevant IS features on a per cell basis, and with statistics on hundreds of cells. With my thesis supervisor, we therefore established a close collaboration with the Menche lab at the CeMM in Vienna, to benefit from their expertise in morphological profiling in high content imaging screens 348 .

I will first start by showing the results of experiments that I set up and optimized on the NK-92 cell line and primary NK cells isolated from PBMCs, followed by the setup of a high content imaging approach of the IS. This approach should be overcome the technical bottleneck of low throughput imaging, and its power in terms of the number of cells and the parameters to be analyzed, and resolutive power should lead to identifying meaningful biological morphological features of the immunological synapse.

Setting up assays in the NK-92 cell line

Cell migration is a crucial step in the function of cytotoxic cells. Indeed, cytotoxic cells migrate along a chemokine gradient.

Our results show that NK-92 cells migrate in response to CXCL12, even at low chemokine concentrations (Figure 24a). However, the response to chemokines appears to stop beyond a certain threshold. Indeed, cells appear to stop responding to the two highest CXCL12 concentrations tested.

Alternatively, cells may undergo chemorepulsion, as shown in other cell models at high chemokine concentrations, which has been previously described for mature T cells 394

In order to achieve their cytotoxic function, NK cells must establish conjugates with their targets.

Conjugate formation is assessed by flow cytometry by staining NK cells and K562 target cells with two different stains and assessing the percentage of NK cells bound to K562 cells.

Our results show that NK-92 cells establish conjugates with their targets after 15 minutes of incubation, and conjugate formation seems to plateau at 45 minutes (Figure 24b).

Our lab has quite an expertise in confocal microscopy imaging of cytotoxic lymphocytes. Therefore, I had the opportunity to perform staining of NK-92 cells incubated with their targets to visualize the formation of the immunological synapse. Our results show that upon formation of the immunological synapse, NK-92 cells polarize their lytic granules towards the area of contact with the target cell (Figure 24c). After 1hour of incubation, the mean distance between the lytic granules and the synaptic area is about 4.3μm, providing support to the visual observation that NK-92 cells do not polarize all their lytic granule content.

Also benefitting from the expertise of our lab in performing cytotoxic assays, I could perform killing assays where NK-92 cells and stained K562 cells are incubated for 4 and 24 hours and evaluated by flow cytometry. Cells are also stained with 7AAD which only stains dead cells and allows the discrimination of alive cells. The percentage of lysis is determined by evaluating the residual number of 7AAD-/stain+ K562 cells. Our results indicate that NK-92 cells are efficient at eliminating target cells starting 4 hours, and it appears that to achieve maximum cytotoxicity an excess of effector NK-92 cells is needed with respect to target cells (Figure 24d). Indeed, we observe a better killing efficiency at a 5:1 effector to target ratio than 1:1. I was also the first in the lab to set up IRM and TIRF microscopy imaging of NK-92 cells in 8-well chambers for a collaboration, with the help of Javier Rey-Barrosso and Astrid Canivet-Laffitte from the microscopy platform. Our results show that most WT NK-92 cells spread efficiently over a coating of ICAM-1 and anti-NKp30 Ab and form an IS where they display a large peripheral belt of tight adhesion and a less adherent central patches, and that lytic granules dock in these loose adhesion patches (Figure 24e). We report a defect in synapse formation and granule docking in NK-92 cells knocked down for a yet unpublished gene, so these results may not be disclosed. The manuscript revealing the role of that gene has been submitted. Representative image of an NK-92 cell forming a synapse imaged by IRM and lytic granules (red) imaged by TIRF microcopy using lysotracker.

Setting up assays in primary NK cells.

Once assays were set up and optimized in the NK-92 cell line, the next logical step was to apply them to primary NK cells.

For that, I tried to optimize the expansion on primary NK cells from PBMCs by using a feeder system, like the expansion of CD8+ T cells using a feeder system routinely performed in our team. PLH cells, which are EBV transformed lymphoblastoid cell line, kindly provided by DR. Martin Villalba were used as feeder cells.

The expansion process was not optimal, as it does not yield large numbers of NK cells, and the cells are only viable for two weeks and needed stimulation with feeder every 2-3 days. Moreover, the protocol is optimized for NK cells isolated from umbilical cord blood which yields a much higher starting number of PBMCs than a 10ml blood tube. Moreover, there are other expansion protocols using another type of irradiated feeder referred to as NK cell feeder (NKF) 395,396 , which I could not try.

However, I could manage to set up functional assays on the primary NK cells.

Even though primary NK cells appear to be spontaneous migrators, they are also capable of a specific migratory response towards both CXCL10 and CXCL12 (Figure 25a). They are also capable of forming conjugates with target K562 cells, and similarly to NK-92, the % of primary NK cells forming conjugates appears to increase over time (Figure 25b). The expanded primary NK cells appear to be cytotoxic and capable of efficient ADCC, even at 4h and in a surplus of target cells (Figure 25c). Our results also show that an excess of primary NK cells is not needed to efficiently eliminate most target cells, and a ratio of 2 NK cells for 1 K562 cell efficiently removes all target cells at 24 hours. Primary NK cells could also be assessed by confocal microscopy for the formation of the immunological synapse when conjugated with target K562 cells, and to polarize their lytic granules (Figure 25d).

However, the % of granules polarized and the mean distance to the IS was not assessed in these cells.

I also performed some microscopy experiments on NK cells isolated from frozen PBMCs of normal donors and a PID patient for a collaboration where a reduced lytic granule content and a defect in granule polarization were observed, but these have not yet been published and the gene name and specific results shall not be disclosed in this manuscript.

In conclusion, I was the first to set up the functional assays routinely performed on CD8+ T cells in our lab on an NK cell line and primary NK cells, and add the conjugation assay. However, if our lab is to start performing experiments on primary NK cells (especially from patient material), the expansion of primary NK cells should be tested using the feeder system developed by Bae an Lee 396 . Seeing how important the immunological synapse is for the cytotoxicity process, we posited that imaging the immunological synapse under different conditions can be predictive of the cytotoxic potential of CD8+ T cells and NK cells. However, traditional confocal microscopy revealed to be limited in terms of the conditions that were amenable to imaging, and the limitation of the parameters that can be assessed, as well as the time required for image analysis.

These optimizations led to the development of my main project, which is the establishment of a high content imaging approach of the immunological synapse under different conditions, to determine the role of the actin cytoskeleton in regulating cytotoxic activity.

We established a close collaboration with the Institut de Recherche en Santé Digestive (IRSD), who had acquired the Opera Phenix, to set up the high content imaging on the IS. This collaboration, as well as a close collaboration with the Menche team at the Center for Molecular Medicine (CeMM) in Vienna made it possible for me to conclude my PhD work in a scientific paper that is soon to be submitted (to Nature Communications), and of which I will share co-first authorship with Loan Vulliard, a PhD student in bioinformatics in the Menche lab.

High content imaging of the immunological synapse

Morphological profiling of human T and NK lymphocytes identifies actin-mediated control of the immunological synapse Yolla German, 1,2,3,4+ Loan Vulliard, 5+ Aude Rubio, 2,6 Kaan Boztug, 4,5,7,8 Audrey Ferrand, 2,6 Jörg Menche 5 and Loïc Dupré* 1,2,3,4 The detection and neutralization of infected cells and tumors by cytotoxic lymphocytes is a vital immune defense mechanism. The immunological synapse orchestrates the target recognition process and the subsequent cytotoxic activity. Here, we present an integrated experimental and computational strategy to systematically characterize the morphological properties of the immunological synapse of human cytotoxic lymphocytes.

Our approach combines high-content imaging with an unbiased, datadriven identification of high-resolution morphological profiles. Such profiling discriminates with high accuracy immunological synapse perturbations induced by an array of actin drugs in both model cell lines and primary lymphocytes. It reveals inter-individual heterogeneity in lymphocyte morphological traits. Furthermore, it uncovers immunological synapse alterations in functionally defective CD8 + T cells from immunodeficient patients carrying ARPC1B mutations. Our study thus provides a foundation for the application of morphological profiling as a powerful and scalable approach to monitor lymphocyte activation status in experimental and disease settings.

The immunological synapse (IS) is a complex cellular structure that sets lymphocyte activation and function during encounter with antigenpresenting cells and target cells. The canonical IS is characterized by a symmetrical architecture consisting of concentric rings of F-actin and integrins, while the antigen receptors occupy a central position 1,2 . The lymphocyte spreading associated with IS assembly, as well as the molecular organization defining IS architecture, rely on actin cytoskeleton dynamics. In cytotoxic lymphocytes, including CD8+ T cells and NK cells, the IS is particularly important because it sustains the polarized delivery of cytolytic molecules such as perforin and granzymes towards target cells 3 . Indeed, activation of the integrin LFA-1 via an inside-out signaling from the T-cell receptor (TCR) in T cells, and several stimulatory receptors in NK cells [4][5][6][7] , leads to the formation of a tight adhesive ring allowing confinement of the degranulation process. Additional layers of control of lytic granule delivery at the IS are their polarization via the orientation of the microtubule organizing center 8,9 and their restricted passage through pervasive actin cytoskeleton clearances 10 . Given the key events occurring at the IS, this structure is a window of choice to monitor lymphocyte activation and function. Indeed, the positioning and dynamic behavior of multiple receptors and signaling molecules have been characterized within the IS 11 , and alterations of the its architecture have been reported in multiple disease settings 12,13 . However, the various microscopy approaches employed so far to characterize spatial organization of the IS have remained low throughput and have been restricted to the analysis of a limited number of morphological features. A more systematic indepth assessment of the IS would better exploit this structure as a pivotal read-out for the characterization of lymphocyte activation and function.

Recent advances in high content imaging (HCI) now allow for the profiling of cells at a much richer level of detail and in an unbiased fashion. It has therefore been widely employed in cancer and toxicology research, in particular for screening drug effects on adherent cell lines and implementing genetic screens based on the siRNA, shRNA and CRISPR technologies 14-17. However, HCI has not yet been applied to the study of leukocytes because of the difficulty to overcome the relatively poor adherence of these cells.

In this study, we report the implementation of an HCI approach that allows the high-resolution confocal imaging of T and NK cells stimulated over 2D surfaces functionalized with ICAM-1 and stimulatory antibodies, and the effect of pharmaceutical and genetic perturbations on the IS morphology. In addition to extracting a previously studied features related to staining of F-actin, LFA-1 and perforin, we develop an unbiased analytical approach allowing highdimensional profiling and clustering of IS morphologies. Our data shows non-identical perturbations caused by drugs affecting different facets of actin cytoskeleton remodeling and highlights that actin cytoskeleton integrity is required not only for lymphocyte spreading but also for lytic granule polarization and LFA-1 distribution. Application of our HCI pipeline to lymphocytes isolated from human blood reveals distinct morphological profiles in individual healthy donors. Furthermore, our method allows characterizing synapse defects in untransformed CD8 + T cells from ARPC1B-deficient patients, illustrating its potential to identify disease-related synapse alterations and to predict functional defects, such as cytotoxicity.

Results

Morphological profiles of T cell and NK cell immunological synapses.

In order to systematically analyze the morphological profile of lymphocyte populations, we here sought to develop an adapted HCI workflow. It consisted in seeding cells of interest on stimulatory surfaces in microwells of 96-or 384-well plates, fixation and staining with combinations of fluorescent dyes and antibodies. Confocal images were acquired on an Opera Phenix high-content screening system and analyzed with CellProfiler 18 to automatically segment individual cells and extract features pertaining to cell morphology and each of the fluorescent markers (Fig. 1a). As proof of concept, we first applied our approach to NK-92 and Jurkat cells, two human cell lines commonly used as models for NK cells and T cells, respectively. Cell morphologies were compared upon interaction with a neutral poly-Llysine (PLL) surface or co-stimulation with the LFA-1 ligand ICAM-1 and stimulatory antibodies (Ab) in order to evoke IS assembly. Upon co-stimulation with ICAM-1 and anti-NKp30 / NKp46 Ab, NK-92 cells spread, emitted F-actin-rich peripheral pseudopodia and polarized perforin-containing granules towards the center of the cell to substrate interface (Fig. 1b and Fig. S1a). These observations are in line with the characteristics of the IS from cytotoxic lymphocytes 11,19 , therefore validating our highthroughput stimulation and staining procedure. Based on literature describing the IS and reporting a polarization of F-actin and lytic granules in NK cells [20][21][22] , we first selected quantitative features pertaining to the F-actin and perforin stainings and extracted them as mean values per field of view averaged across 3 experiments. We also included features related to the nucleus, available since the DAPI staining was used in a primary nucleus segmentation step before the identification of the cytoplasms around the nuclei (Fig. 1c and Table S1). Increase in Factin intensity and cell area were prominent features of the stimulation, as compared to the PLL condition. Furthermore, the number of perforin-containing granules detected at the cell to substrate interface increased upon stimulation, which is indicative of their polarization towards the IS. Interestingly, this polarization process was associated with a relative spreading of the area covered by lytic granules, supporting the notion of multiple docking domains at the synapse 23 . Our analysis also highlights that increase of nucleus area is a typical feature of the IS in the NK-92 cells. Interestingly, nucleus area appears to increase along with F-actin intensity when assessed across 3 experiments (Fig. 1d), suggesting that nucleus flattening and F-actin polymerization are related events, probably as components of the cell spreading mechanism. Of note, the absolute values for F-actin intensity were higher in one of the 3 experiments, possibly resulting from differences in staining quality. This indicates that absolute value for staining intensities across experiments should be considered with caution. To further estimate morphological heterogeneity in individual cells, F-actin intensity was assessed on a per-cell basis (Fig. 1e), rather than on a per field of view basis. The unimodal increase of F-actin intensity driven by the stimulation of NK-92 cells indicates a relatively homogenous activation and IS assembly in these cells. It also validates our approach to consider mean cell measurements on a per field of view basis.

We then applied our HCI workflow to Jurkat cells, which were co-stimulated with ICAM-1 and anti-CD3 Ab. We selected 12 features pertaining to the F-actin, LFA-1 and DAPI stainings to monitor hallmarks of the T cell IS 11 . As compared to the neutral PLL stimulation, LFA-1/CD3 costimulation led to cell spreading, assembly of a peripheral F-actin ring-like structure and redistribution of the integrin LFA-1 as an inner belt at the cell to substrate interface (Fig. 1f and Fig. S1b), which are characteristic for the IS 11,24 . Our quantification over multiple fields showed that similarly to NK-92 cells, F-actin intensity, cell area, LFA-1 intensity and LFA-1 area are prominent features of the Jurkat cell IS (Fig. 1g and Table S2). Likewise, F-actin and LFA-1 intensities correlated in individual fields of view with a Pearson correlation coefficient of 0.50 (Fig. 1h). At the single cell level, F-actin clearly increased in response to the ICAM-1 and anti-CD3 Ab stimulation, despite noticeable heterogeneity in both stimulated and unstimulated cells (Fig. 1i). When taken together, the data collected on the assembly of the IS in NK-92 and Jurkat cells highlight F-actin intensity rise and cell spreading as common traits. However, in line with the distinct appearance of their actin rich peripheral protrusions, Jurkat cells, but not NK-92 cells, became rounder upon activation. Furthermore, while NK-92 flattened their nucleus, the effect was not marked in Jurkat cells, indicating a distinct cell spreading behavior. Overall, our data validate the reliability and power of HCI with high spatial resolution to unbiasedly define the morphological profiles of lymphocytes in response to stimulatory regimens.

Cytoskeleton drugs induce non-identical alterations of the NK cell IS.

Given the prominent actin remodeling activity sustaining IS assembly, we next exploited our HCI approach to monitor how pharmacological modulation of cytoskeletal dynamics would affect IS architecture. NK-92 cells were treated with seven drugs known to target distinct aspects of actin and acto-Myosin dynamics, which were used at 3 concentrations in order to detect possible dose-dependent effects. NK-92 cells were pre-treated with drugs for 30 minutes before seeding on ICAM-1 and anti-NKp30 / NKp46 Ab. In a first step, the effects of these drugs on the previously selected quantitative morphological features of the IS were assessed.

Upon treatment with Latrunculin B, which binds actin monomers and inhibits actin polymerization, a concentration-dependent decrease in F-actin intensity was detected as compared to the untreated control (Fig. 2a), in concordance with an expected reduction in actin polymerization. However, IS assembly was not fully impeded as revealed by a minor reduction of cell area. Remarkably, Latrunculin B treatment elicited an increase of the number of perforin granules and the area they occupied at the cell to substrate interface. This might reflect impaired exocytosis and accumulation of aberrantly spread lytic granules at the IS. Such explanation is in agreement with the role of actin dynamics in facilitating the docking and exocytosis of lytic granules 21,25 . Upon treatment with Jasplakinolide, which stabilizes actin, a mild decrease in F-actin intensity was detected at 1 µM, supporting the notion that actin turnover is required to fuel polymerization 26,27 (Fig. 2b). A higher concentration of 2.5 µM was tested but could not be exploited because of its apparent detrimental effect on cell viability. In comparison to Latrunculin B, Jasplakinolide treatment elicited an increase in the perforin-related features, confirming that actin turnover is required for lytic granule exocytosis 25,27 . Treatment with the Myosin inhibitor Blebbistatin induced a slight increase in F-actin intensity at 5 and 10 µM (Fig. 2c). More strikingly it increased the number of granules detected at the synapse and the area they occupied, consistent with previous findings that Blebbistatin hinders granule exocytosis without affecting their polarization 28,29 . Treatment with the ROCK inhibitor Y-27632 affected F-actin intensity, cell area and lytic granule features similar to those elicited by Blebbistatin treatment (Fig. 2d), in agreement with the activity of ROCK as an upstream regulator of acto-Myosin contractility. Upon CK-869 treatment, a concentration-dependent decrease in F-actin intensity was detected (Fig. 2e), showing, as expected, that ARP2/3 complex inhibition reduced actin polymerization 30 . Moreover, CK-869 treated cells displayed reduced radial spreading, as shown by decreased area and increased cell width to length ratio, indicative of a severe impairment of IS assembly. A distinct property of CK-869 treatment was a reduction in the number of and area covered by perforin granules, possibly reflecting the inability of CK-869 treated cells to polarize lytic granules towards the stimulatory surface because of defective IS assembly. Upon treatment with Wiskostatin, an inhibitor of WASP, which drives ARP2/3dependent actin branching, a slight increase in Factin intensity was measured for the two lowest concentrations (Fig. 2f). In comparison with CK-869, Wiskostatin displayed minor effects on perforin features. This suggests that the ARP2/3 activator WASP plays a limited role in the overall actin polymerization rate at the IS and in lytic granule polarization and secretion 24,31 . Treatment with the pan-formin blocker SMIFH2 led to a concentration-dependent increase in F-actin intensity (Fig. 2g). Low-concentration SMIFH2 treatment resulted in an increase in perforin intensity and area. Collectively, these data indicate that drugs affecting different facets of actin remodeling differentially altered the assembly of the NK cell IS.

To further assess whether the morphological alterations inflicted by the drugs could be distinguished one from another, we trained a random forest classifier based on the 13 selected features. The image set was split to carry out a parameter optimization and to validate the performance of the model. The obtained overall accuracy of 69% and 𝐹 1 score of 0.7 confirmed that our method performed relatively well at distinguishing the morphological effects of the actin drugs. The confusion matrix shows that most drugs were predicted with high accuracy based on the corresponding image features, while the morphological effects of Blebbistatin and Y-27632 could not be easily distinguished, in line with their highly related mechanism of action (Fig. S2a). It also confirmed F-actin intensity as a major discriminating feature and identified cell eccentricity and roundness as key features to account for the morphological alterations induced by the drugs (Fig. S2b).

Overall, our comparative image-based analysis of the effects of different drugs affecting actin and acto-Myosin dynamics reveals that distinct effects on actin turnover and polymerization yield distinguishable IS morphologies but converge in affecting lytic granule positioning. This supports the notion that multiple actin-dependent steps control lytic granule docking and exocytosis 32 .

High-resolution morphology profiling of NK cells upon drug treatment.

To further enrich our morphological analysis of the IS in the context of drug treatment, we considered additional morphological features beyond the previously analyzed quantitative features. From 1898 measured features, a set of 383 features was retained following filtering of non-informative and redundant features. In order to visualize the information contained in this large feature set, as well as to quantify the significance of morphological changes upon drug treatment as compared to untreated cells, we applied a UMAP dimensionality reduction. This allows the visualization of all cell images, recapitulating in a 'morphological space' the relation between the morphology they display, by summarizing the variation of the 383 features into two dimensions (Fig. S2c). By examining the relation between these features, we saw that both the different types of measurements acquired, and the different biological objects studied provided complementary and non-redundant information about the global changes occurring between images and between treatments (Fig. S2d). This also showed that none of these morphological features were repeating technical confounders, such as experimental plate position effect or cell count. As clearly visible in the morphological space, images of cells treated with Latrunculin B, Jasplakinolide and CK-869 clustered away from the untreated cells and from one another, most likely owing to these drugs having prominent and distinct 27632, e. CK-968, f. Wiskostatin, and g. SMIFH2. h. Confusion matrix and class-wise performance on held-out data of a random forest model trained to predict drug treatment based on the morphology of NK-92 cells seeded on ICAM-1, anti-NKp30 and anti-NKp46. i-j Total and average importance for the prediction of morphological features per measurement type and biological object described of NK-92 cells seeded on ICAM-1, anti-NKp30 and anti-NKp46 and treated with i. CK-869 or j. SMIFH2. effects on the ability of NK-92 cells to assemble the IS (Fig. 3a, b ande). In comparison, morphologies of cells treated with Blebbistatin, Y-27632, Wiskostatin and SMIFH2 appeared to be less distinct from the untreated condition and to cluster at close vicinity to one another (Fig. 3c, d, f andg). The three concentrations assayed per treatment fell into distinct sub-clusters, clearly indicating dose-dependent effects, as detailed for CK-869 and SMIFH2 (Fig. S2e andf). All drugevoked morphological profiles were found to be significantly distant from the untreated state. Indeed, the median robust Mahalanobis distances between the fields of view per treatment and their matching negative controls are larger than expected at random (Fig. S2g) 33,34 . To get insight into the nature of the changes that are causative of the observed clusters on the UMAP representation, we trained a random forest classifier on the set of 383 features. This achieved a satisfactory performance, as shown on the confusion matrix (Fig. 3h) with an 𝐹 1 score and an accuracy of 0.89 and 89%, respectively. The importance of each feature for the classification was proxied by the average increase in accuracy obtained by including the given feature in a decision tree. In particular, our analysis shows that CK-869 treatment mostly affected nucleus and cytoplasm shape descriptors (Fig. 3i), while SMIFH2 treatment altered radial intensity distributions in the cytoplasm (Fig. 3j). Only four features described intensities in the cytoplasm within our feature set. Interestingly, those few features were in average increasing the model accuracy the most, strengthening the necessity, but not sufficiency, of actin intensity measurements to profile the IS. Features pertaining to lytic granules also played a determinant role in reinforcing model accuracy, providing further evidence of a tight regulation of lytic granule distribution at the IS. Our data therefore demonstrates the ability of the unbiased profiling to identify relevant spatially localized events and characterize perturbed cell states with high-resolution power.

Morphological profiling of primary human NK cells upon drug treatment.

We next applied our HCI approach to assess the susceptibility of primary human lymphocytes from different donors to cytoskeletal drugs. For that purpose, NK cells were purified from the peripheral blood of three normal donors, treated with four concentrations of either CK-869 or SMIFH2, and stimulated with ICAM-1 and anti-NKp30 / NKp46 Ab (Fig. 4a). Although the untreated cells from the three different healthy donors displayed variation in morphology, an actin-rich IS with the lytic granules concentrated in one area towards the cell periphery was observed upon stimulation. The four tested concentrations of CK-869 caused a marked decrease in F-actin intensity in the NK cells from the three donors, demonstrating the capacity of our approach to detect actin cytoskeleton alterations in primary lymphocytes. Notably, the area covered by the perforin granules, taken as an absolute value or divided by the cell area, was increased in the CK-869 treated NK cells from the three donors, showing a clear dose-dependent response (Fig. 4b). This effect is opposite to what was measured in the NK-92 cells, highlighting contrasting responses of model cell lines and primary cells. Moreover, the four tested concentrations of SMIFH2 also caused a decrease in F-actin intensity in the NK cells from the three donors, thereby highlighting the importance of formins for actin remodeling at the IS (Fig. 4c). SMIFH2 treatment also strongly affected the distribution of perforin granules. Interestingly, in NK cells from donors 1 and 2, a dose dependent reduction of both perforin granule number and covered area was observed, a response opposite to that to CK-869. In contrast with donors 1 and 2 , low concentrations of SMIFH2 resulted in an increase of perforin granule number and covered area in NK cells from donor 3. It should be noted that lower number of perforin granules were detected in the untreated cells from this donor, possibly influencing the response to the tested drugs. Those observations highlight the potential of HCI to identify features underlying inter-donor variability upon stimulation and treatment of lymphocytes populations, which may be explained by the phenotypic variation of each donor's NK cells 35 . Together, the dataset collected on primary NK cells demonstrates that HCI is amenable to the morphological profiling of primary human lymphocytes in the context of drug treatments and that it can discriminate specific responses from individual donors.

Immunological synapse defect is associated with impaired cytotoxicity in CD8 + T cells from ARPC1B deficient patients.

We then reasoned that the HCI approach might be adapted to characterizing lymphocyte impairments in the context of pathology. For that purpose, we implemented a morphological profiling of CD8 + T cells isolated from two patients suffering from a primary immunodeficiency caused by mutations in ARPC1B, which encodes one subunit of the ARP2/3 complex. Cells from the two patients and three normal donors were stimulated with ICAM-1 and either 1 or 10 µg/ml anti-CD3 Ab, and stained for perforin and LFA-1, F-actin and the nucleus.  images suggests that the CD8 + T cells from both patients spread less than control cells and failed to assemble a typical IS (Fig. 5a). Analysis of 16 selected morphological features highlighted that F-actin intensity was decreased in T cells from the two patients, as compared to the cells from the normal donors, following stimulation with anti-CD3 Ab at both concentrations (Fig. 5b and Fig. S3a). This is comparable to the data collected above in cell lines and primary cells upon treatment with the ARP2/3 inhibitor CK-869. Cells from the two patients however displayed distinct morphological aberrations. While cell area was mostly affected in cells from patient 1, implying an impaired spreading ability, cell roundness was prominently decreased in patient 2, most likely resulting from aberrant peripheral actin spikes. T cells from patient 2 displayed an increased number and dispersion of perforin granules, similarly with what we observed in primary NK cells treated with CK-869. However, fewer perforin granules were detected in T cells from patient 1. Their dispersion was reduced in absolute terms but increased when normalized for the cell area, the latter being reduced. LFA-1 intensity was increased in the T cells from both patients and LFA-1 was localized at the cell rim rather than at the cell to substrate contact area, suggesting abnormal distribution of adhesive forces. Our morphological profiling clearly establishes that CD8 + T cells from the two considered ARPC1B-deficient patients have severe impairments in IS assembly. The distinct synaptic alterations revealed by our approach in the two patients could not be explained by differences in the phenotype of the cells, which showed similar expression of CD8, perforin, LFA-1, and granzyme B (Fig. S3 f-i). To further enrich our analysis, we applied once more a UMAP approach to explore the morphological space, which evidenced a marked segregation of the two patients from the control donors, but also among each other (Fig. 5c and Fig. S3b). This analysis therefore reinforces the finding that patient T cells have aberrant synaptic traits and that the nature of these aberrations is distinct between the two patients. Interestingly, we noticed that even though the normal donors clustered closely to each other, donor 3 did not overlap with the other two at either anti-CD3 Ab concentration, confirming that heterogeneity in IS morphology exists among normal donors, as observed above for NK cells. A first random forest model showed that we could determine the concentration of anti-CD3 Ab used to stimulate the cells with an accuracy of 95% and 𝐹 1 score of 0.95 (Fig. S3c), and indicated that IS assembly varied according to the concentration of anti-CD3 Ab (Fig. S3d). This was associated with changes in shape and radial distribution in the cytoplasm, while cytoplasm intensities were the most discriminative feature category in average, fitting a scenario in which TCR stimulation strength would differentially remodel the actin cytoskeleton and associated synapse morphology.

A second model showed that our approach is powerful enough to distinguish patient cells from normal donor cells seeded on ICAM-1 and 10 µg/ml anti-CD3 Ab by achieving a perfect classification on a validation set (Fig. 5d), distinguishing ARPC1B deficient cells mostly on the basis of textural and intensity distribution changes within the cytoplasm (Fig. 5e). Moreover, some features changed not only between ARPC1B patients and normal donors but were as well discriminating between patient 1 and patient 2 (Fig. S3e), further reinforcing that the two patients have distinct IS architectures. The aberrant IS characterized in the patients through the morphological profiling approach alluded to a possible functional defect. We therefore assessed the cytotoxic activity of CD8 + T cells towards anti-CD3 Ab-coated P815 target cells. Whereas, normal CD8 + T cells started to kill target cells in four hours, CD8 + T cells from the ARPC1B deficient patients failed to do so. The patient derived CD8 + T cells remained defective at killing target cells over a prolonged 24-hour incubation (Fig. 5f). This indicates that the T cells from the patients most likely fail to secrete lytic molecules, despite a normal content in perforin and granzyme B (Fig. S3g andi). Our results therefore indicate that the defects in IS organization characterized in both patients are leading to a severe impairment of the cytotoxic activity.

Discussion

By combining automated cell imaging with computational image analysis pipelines, HCI provides novel opportunities to systematically analyze cellular mechanisms 15,36,37 . Howeverm, the potential of such approach has not yet been explored for the study of immune cells. We here tailor a HCI approach for the high-resolution morphological profiling of various human cytotoxic lymphocyte population, and focus on the imaging of the IS as a mean to capture the activation state and effector potential of these cells. We validate our HCI approach by identifying distinct morphological signatures evoked by a panel of actin-targeting drugs. We further reveal the power of our HCI approach to discriminate individual donors on the basis of immune cell morphological traits. We also exemplify the clinical applicability of such approach by identifying cytotoxic lymphocyte aberrations in patients with a severe congenital immunodeficiency.

Although we use a minimalistic 2D static approach based on the adsorption of stimulatory molecules on the surface microwells, it proves to robustly stimulate the assembly of morphological structures qualifying as IS. We show that various human lymphocyte populations, including model cell lines, cells freshly isolated from the blood, as well as expanded primary cells can be stained and imaged with an automated confocal microscope at high resolutive power in a 384-well format, allowing the analysis of several samples, activation conditions and perturbations in parallel. Computationally, we use robust statistics and work at an image-level resolution, typically gathering a few dozens of cells imaged over four z planes representing the 2-µm section of the cells most proximal to the stimulatory substrate. While most morphological profiling studies have been limited to average profiles over wells or replicates 14,37,38 , a few approaches have defined profiles based on single cells 39,40 . We here rather consider the variability in morphology displayed in each image by including measures of dispersion that are proven to be beneficial for morphological profiles and could potentially be further improved by adding a higher order joint statistical moment 41 . From an analytical point of view, we elaborate two complementary methods. First, we focus on a pre-defined set of morphological features based on prior knowledge and including cell and nucleus shape parameters as well as intensities of F-actin, LFA-1 and perforin at the synaptic plane. We show that such method can be applied to relatively low numbers of images and provides meaningful identification of discriminative features when comparing experimental conditions. Second, we implement a high-resolution and unbiased morphological profiling pipeline, from which novel relevant features can be identified and from which highperformance classifiers can be trained to discriminate cell states corresponding to different stimulations, drug treatments or genetic defects.

Beyond the methodological advance provided in this study, we present data relevant to the understanding of lymphocyte activation in both a fundamental and medically relevant perspective. Among the pre-defined set of morphological features, we identify increase of F-actin as a hallmark of T and NK lymphocyte stimulation by combinations of ICAM-1 and antibodies directed against CD3 or NK receptors, respectively. This is in line with the previously established role of the actin cytoskeleton in driving the cell spreading behavior supporting IS assembly 42,43 . The further investigation of the role of actin cytoskeleton remodeling by the treatment of NK cells with a drug array reveals distinct morphological alterations upon targeting actin polymerization, depolymerization and Myosin II. Our data also point to converging morphologies induced by some of the drugs with distinct modes of action, possibly related to a limited number of configurations of the cytoskeleton, as recently described in an adherent neuroblastoma cell line 36,44 . Strikingly, most tested drugs yield prominent alteration of the distribution of perforin-containing granules, indicating that the different facets of actin cytoskeleton dynamics are all important to regulate the polarized delivery of lytic granules at the IS 25,45 .

Owing to the distinct morphological profiles observed for each drug, and the detection of dosedependent effects, both in cell lines and primary cells, such an approach could be applied in the context of immunotherapeutic drugs. A striking finding of the application of morphological profiling to lymphocyte populations is that it reveals a previously unappreciated level of heterogeneity in cellular morphological traits among individuals. When considering the data pertaining to the NK cells freshly isolated from the blood, we cannot rule out that morphological differences arise from distinct activation states of the cells from different donors. However, in vitro stimulation and expansion of T lymphocytes, which is expected to robustly drive cells towards a differentiated phenotype 46 , was also associated with distinct morphological traits. Further analysis of larger cohorts of donors and sorted subpopulations of lymphocytes will be required to precisely appreciate the degree of morphological heterogeneity among individuals and lymphocyte subsets. The detection of distinct morphological profiles among healthy individuals certainly highlights the extreme sensitivity of the HCI approach to characterize and compare cell populations. A further illustration of this property is provided by the characterization of IS alterations in T lymphocyte populations isolated from 2 patients with ARPC1B deficiency. Interestingly, again, our approach points to distinct morphological alterations in the cells from the 2 patients considered. Such differences might be inherent to the severity of the ARPC1B genetic defect. 47,48 The study of larger cohorts of patients, which would be compatible with the herein developed approach, would be required to answer such question.

CD8 + T cells from ARPC1B patients display an aberrant IS morphology including defects pertaining to the distribution of perforin granules and LFA-1. Comparably to other studies, we show a reduced cell area and a failure to spread radially and emit lamellipodia upon TCR stimulation 47 . The lack of lamellipodia formation was also observed upon NK-92 and primary NK cell treatment with CK-869. Our data reveals increased accumulation of lytic granules at the IS for one patient, which could indicate a defect in granule exocytosis, opposed to a decrease in perforin related parameters for the other patient, most likely indicative of failed lytic granule polarization. These observations are in agreement with a recent study showing defective lytic granule polarization and degranulation in ARPC1B deficient CD8 + T cells 49 . The detection of such IS defects is suggestive of a possible alteration of the cytotoxic activity. Our data shows that ARPC1B deficient cells fail to eliminate target cells, as recently reported 49 . This illustrates the potential of HCI to provide guidance for the implantation of complementary low throughput assays to assess defects at the functional and molecular level. At this stage, we cannot generalize the case of the ARPC1B deficiency in establishing a systematic relationship between IS alteration and functional defect. However, it is interesting to mention that multiple primary immunodeficiencies have been found by us and others to associate IS defects and functional impairments [50][51][52][53] . Previous reports have also shown that PIDs where the IS is defective fail to eliminate target cells 51,53,54 The systematic analysis of multiple such pathologies and corresponding cellular models would certainly provide a unique opportunity to establish rules linking morphology to function.

Overall, we provide here an innovative HCI approach to unbiasedly interrogate the biology of lymphocyte populations. It provides a rich way to identify and interpret details of the IS architecture and surpass current approaches in detecting morphological traits of specific lymphocyte populations, as illustrated by the distinct morphological profiles identified among the primary lymphocytes of individual donors. This hold promises to stratify patients based on specific morphotypes of lymphocytes or other leukocytes. Therefore, we thoroughly report both the experimental and computational methods employed and provide all scripts used in the analysis to maximize the reproducibility of the approach developed herein. We hope this encourages further research leveraging the application of HCI to blood-derived cell subsets, for potential translation in the field of cancer therapy and personalized medicine.

Materials and methods

Cell lines and primary cells

Jurkat cells were cultured in RPMI (Gibco) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% non-essential amino acids and 1% HEPES (all from Thermo Fisher Scientific). NK-92 cells were cultured according to the recommendations from ATCC. Primary NK cells were purified from freshly isolated PBMCs using the MagniSort Human NK enrichment kit (Invitrogen) and maintained in RPMI supplemented with 5% human serum, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% non-essential amino acids and 1% HEPES. Primary CD8 + T cells were purified from frozen PBMCs of 3 healthy donors and 2 ARPC1B deficient patients by negative selection using the EasySep Human CD8 + T cell enrichment kit. CD8 + T cells were stimulated in RPMI supplemented with 5% human serum, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% non-essential amino acids, 1% HEPES 1 µg/ml PHA and 100 IU/ml IL-2. CD8 + T cells were expanded for further rounds every 2 weeks with a mixture of irradiated PBMCs from 3 normal donors. Peripheral blood from healthy donors and patients was obtained in accordance with the 1964 Helsinki declaration and its later amendments or ethical standards. Informed consents were approved by the relevant local Institutional Ethical Committees.

Culture and staining conditions used for High content imaging

CellCarrier Ultra tissue culture treated plates (Perkin Elmer) were coated with either 0.01% PLL (Merck) or a combination of 2 µg/ml ICAM-1 (R&D Systems), 1 µg/ml NKp30 (R&D systems, MAB18491) and 1 µg/ml NKp46 (BD Biosciences, 557487). NK-92 cells were cultured in IL-2 free medium overnight. 15000 NK-92 and 5000 primary NK cells were seeded per well and left for 30 min at 37°C to adhere and form the synapse. Cells were fixed with 3% paraformaldehyde (Merck) and stained with antiperforin Ab and phalloidin-AF 488. Goat antimouse AF 555 was used to reveal perforin staining. Nuclei were stained with DAPI. NK-92 were treated with 5, 10 and 50 µM Blebbistatin, 10, 25 and 50 µM CK-869 (Merck), 0.1, 1 and 2.5 µM Jasplakinolide (Merck), 0.1, 0.25 and 0.5 µM Latrunculin B (Merck), 50, 100 and 250 µM SMIFH2 (Merck), 10 50 and 100 µM Wiskostatin (Merck) and 5, 10 and 25 µM Y-27632 (Merck) for 30 min at 37°C and washed twice in PBS before seeding onto the plates and letting them adhere for 30 min. The same procedure was applied to primary NK cells treated with 5, 10, 25 and 50 µM CK-869 and 25 50, 100 and 250 µM SMIFH2.

CellCarrier Ultra multiwell tissue culture treated plates were coated with either 0.01% poly-Llysine or a combination of 2 µg/ml ICAM-1 and 10 µg/ml anti-CD3 (eBioscience). 10000 Jurkat or 5000 CD8 + T cells were seeded per well and left for 15 min at 37°C to adhere and form the synapse. Cells were fixed with 3% paraformaldehyde and stained with anti-LFA-1 (BioLegend, 301202) and phalloidin-AF 488 (Thermo Fisher Scientific) in permeabilization buffer (eBioscience). Goat anti-mouse AF 647 antibody (Thermo Fisher Scientific, A-21240) was used to reveal LFA-1 staining. CD8 + T cells were in addition stained with anti-perforin and goat anti-mouse AF 555 (Life technologies) was used to reveal perforin staining. Nuclei were stained with DAPI (Thermo Fisher Scientific). Stained cells were kept in PBS at 4°C until imaging.

Image acquisition and processing

Images were acquired on an automated spinning disk confocal HCS device (Opera Phenix, Perkin Elmer) equipped with a 40x 1.1 NA Plan Apochromat water immersion objective and a sCMOS camera. For each well, 40 randomly selected fields and 8 stacks per field (0.5 µm step) were acquired. Stacks of images were combined with maximum projection for four focal slices (z from 1 to 4 with a 0.5 µm step), then assembled in sets of images per field of view corresponding to DAPI, phalloidin and LFA-1 or perforin depending on the cell type imaged. These datasets were processed, and measurements were made using CellProfiler 3.0 18 (see Supplementary Files [CellProfiler pipeline]). In brief, we assess the image quality, log-transform the intensities for experiments with high background noise, correct the illumination on each image based on background intensities, avoid DNA precipitations by multiplying intensities on DAPI channel by phalloidin intensities before segmenting cell nuclei using global minimum cross entropy thresholding. We perform a secondary segmentation of the cytoplasms using the watershed method 55 and global minimum cross entropy thresholding on the phalloidin channel. Image sets with low maximal DNA intensity or showing no nucleus were discarded. Cells having more than 30% of their cytoplasm surface at less than 5 pixels of another cell were removed, in order to ignore clusters of cells and to focus on single cells displaying an IS. We segmented small actin speckles in the cytoplasm at more than 3 pixels from the membrane as well as speckles of perforin and secondary objects spanned around the nuclei by LFA-1 staining. Additionally, primary NK and expanded CD8 + T cells associated with less than 2 perforin granules were excluded from the analysis. Finally, we measured colocalization of these objects, intensities in the nuclei and cytoplasms, granularity on all channels, textural and shape features, intensity distributions, distance and overlap between objects and counted speckles neighbors less than 10 pixels away. We then kept the average and the standard deviation of these features per field of view. This led to 1898 and 2076 morphological features in NK92 and Jurkat cells respectively. For primary NK cells and expanded CD8 + T cells, features related to actin speckles were excluded, as they were not found to be informative, resulting in 2386 and 1517 features, respectively. Stacks of images were combined with maximum projection for four focal slices (z from 1 to 4 with a 0.5 µm step), then assembled in sets of images per field of view corresponding to DAPI, phalloidin and LFA-1 or perforin depending on the cell type imaged. These datasets were processed, and measurements were made using CellProfiler 3.0 18 (see the pipelines provided).

In brief, we assess the image quality, logtransform the intensities for experiments with high background noise, correct the illumination on each image based on background intensities, avoid DNA precipitations by multiplying intensities on DAPI channel by phalloidin intensities before segmenting cell nuclei using global minimum cross entropy thresholding. We perform a secondary segmentation of the cytoplasms using the watershed method 55 and global minimum cross entropy thresholding on the phalloidin channel. Image sets with low maximal DNA intensity or showing no nucleus were discarded. Cells having more than 30% of their cytoplasm surface at less than 5 pixels of another cell were removed, in order to ignore clusters of cells and to focus on single cells displaying an IS. We segmented small actin speckles in the cytoplasm at more than 3 pixels from the membrane as well as speckles of perforin and secondary objects spanned around the nuclei by LFA-1 staining. Additionally, primary NK and expanded CD8 + T cells associated with less than 2 perforin granules were excluded from the analysis. Finally, we measured colocalization of these objects, intensities in the nuclei and cytoplasms, granularity on all channels, textural and shape features, intensity distributions, distance and overlap between objects and counted speckles neighbors less than 10 pixels away. We then kept the average and the standard deviation of these features per field of view. This led to 1898 and 2076 morphological features in NK92 and Jurkat cells respectively. For primary NK cells and expanded CD8 + T cells, features related to actin speckles were excluded, as they were not found to be informative, resulting in 2386 and 1517 features, respectively.

Data processing and visualization

We subsequently conducted analyses in R 3.5.1 with the data visualization package ggplot2 3.1.1 and Microsoft Excel (Version 1902). We further selected a smaller set of informative morphological features and checked the quality of processed images by (i) removing wells with low maximal DNA intensity and cell count, (ii) removing features and images generating missing values and (iii) removing constant features in the study dataset or the subset of negative controls used as reference.

From these images passing our quality checks, up to 16 raw summary variables were extracted based on their interpretability and on their known relevance to describe ISs. The fold changes compared to unstimulated or untreated controls were further reported and displayed in the form or radar charts. On the other hand, for all features, per-image values 𝑋 were transformed successively with the following functions 𝑓 1 and 𝑓 2 , with 𝑋 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 the negative controls in 𝑋 on which the data is normalized:

𝑓 1 (𝑋) = log (𝑋 + 1 -min(𝑋)) 𝑓 2 (𝑋) = 𝑋 - median(𝑋 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 ) mad(𝑋 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 )
To remove redundancy in the set of features used for downstream analyses, we ensured that the selected variables were not excessively linearly correlated. To do so, all features were ordered from highest to lowest median absolute deviation (hence by variation in the experiment compared to negative controls). Starting from the top of this list, all other features linearly correlated to the first feature with a Pearson's coefficient higher than 0.6 were excluded. We sequentially went on with the next remaining feature in the list and iterated until the acquisition of a small and informative set of uncorrelated features.

This set of features was used for visualization and quantification of the overall morphological changes induced by perturbations. We then reduced the dimensionality of the data using the UMAP algorithm 56 to 2 dimensions for visualizations and 3 dimensions for computation of the statistical significance of morphological effects in the drug screen on NK92. This pipeline succeeded in selecting a wide range of features that were not excessively biased by confounders (Fig. S2d).

Robust Morphological Perturbation Value

To quantify the significance of overall changes in morphology between a perturbed state and a reference state (healthy or untreated cells), we define the Robust Morphological Perturbation Value. This extends the concept of Multidimensional Perturbation Value 33 , which defines a single value summarizing the statistical significance of morphological changes in multidimensional spaces, by using robust statistics and the minimum covariance determinant 34 decreasing the sensitivity to technical (unfiltered artifacts) and biological outliers (images displaying extreme morphologies or uncommon cell states). In brief, the RMPV is obtained for 𝑋 the set of all filtered and uncorrelated features and 𝑋 𝑊𝑇 the subset of the data corresponding to images of the reference population in five steps. First, the minimum covariance determinant estimator 𝑀(𝑋 𝑊𝑇 ) is calculated to describe the variation of morphologies observed in the reference set, using its implementation in the R package robustBase version 0.93. Second, this value is used to determine 𝑅, the robust Mahalanobis distance of each images of 𝑋 to 𝑋 𝑊𝑇 [arXiv:1904.02596 [stat.ME]]. Third, the median value 𝑅 ̃= median(𝑅) was obtained for each drug tested. Fourth, for 2000 iterations the labels of the condition and the reference were randomly permuted to obtain an empirical distribution of 𝑅 ̃ under the assumption that there was no difference between the multivariate location and scatter of the morphological parameters of the perturbation and the reference. Finally, the RMPV is defined as the empirical p-value obtained from these distributions after FDR adjustment for testing changes in multiple conditions and indicates the probability of observing at least half of the images displaying morphological changes of a similar intensity if the perturbation was similar to the reference.

Random forest classification and interpretation

Using the set of informative and uncorrelated morphological features -previously used for dimensionality reduction, we trained a random forest classifier 57 using the R package randomForest version 4.6.

Using the set of informative and uncorrelated morphological features -previously used for dimensionality reduction, we trained a random forest classifier 57 using the R package randomForest version 4.6. Each forest included 1000 decision trees. The data was split in 6 folds of equal size, each containing all possible classification label. To select the optimal number mtry of variables selected at each split, we incremented the parameter value from 20 to 90 by steps of 10 and assessed the performance using the macro 𝐹 1 score as defined below in a 5-fold cross-validation scheme. One extra fold was used as validation set to estimate the performance of the model after selection of the optimal parameters and retraining on all of the 5 folds used for cross-validation. In the case of the drug screen on the NK-92 cell line, we used a similar approach using the 13 features of known relevance in describing the IS as input, and testing mtry values from 1 to 13 with steps of 3. Overall the performance was evaluated using the macro 𝐹 1 score:

𝐹 1 = 1 𝑛 ∑ 2 × 𝑇𝑃 𝑖 2 × 𝑇𝑃 𝑖 + 𝐹𝑃 𝑖 + 𝐹𝑁 𝑖 𝑛 𝑖=1
where 𝑛 is the number of categories in the classification, and 𝑇𝑃 𝑖 , 𝐹𝑃 𝑖 and 𝐹𝑁 𝑖 are respectively the number of true positives, false positives and false negatives for category 𝑖 in the validation set. To interpret the feature importance in the prediction, we extracted the mean decrease in accuracy obtained when including each feature, either for the prediction of a given class or overall using micro averaging. The total and average importance of features split in distinct groups based on the type of measurements and biological object described were calculated as well. These feature groups were defined based on the corresponding CellProfiler measurement types and biological objects. Features that did not describe the cytoplasm, nucleus, perforin granules or actin granules were counted in the "Other" biological object category. Similarly, features that did not correspond to the "Texture", "AreaShape", "RadialDistribution", "Granularity" or "Intensity" measurements were grouped under the term "Other".

Cytotoxicity assay

Target P815 cells were stained for 30 min with Cell Tracker green CMFDA (Thermo Fisher Scientific) and coated with 10 µg/ml anti-CD3 (eBiosciences, 16-0037-81) for one hour at 37°C. They were also treated with 0.2 µg/ml of aphidicolin to prevent their proliferation. P815 were incubated with effector CD8 + T cells in Ubottom 96 well plates at an effector: target ratio of 1:1 for 4 and 24 hours 58 . Cells were then stained with Amino-Actinomycin D (7-AAD) (BD Biosciences) to discriminate dead and alive cells using the MacsQuant VYB (Miltenyi) and analyzed with FlowJo. The number of residual alive CMFDA + / 7-AAD -cells was assessed to calculate cytotoxicity. Student's t-test was used to calculate significance.

Phenotypic analysis

Expanded CD8 + T cells from normal donors and ARPC1B-deficient patients were stained with fluorochrome-coupled antibodies recognizing the extracellular markers CD8 (BioLegend, 344718) and LFA-1 (BioLegend, 363404) for 30 min at 4°C. Intracellular staining was performed following fixation and permeabilization, with the following antibodies perforin (BioLegend, 308110) and granzyme B (BDPharmigen, 561142) for 45 min at 4°C. The data were acquired on MacsQuant Q10 (Miltenyi) and analyzed with FlowJo. Student's t-test was used to calculate significance.

Materials and methods: optimizations on NK-92 and primary NK cells.

NK-92 cells 1.1 Migration

NK-92 cells were resuspended at 0.2, 0.4 and 1*10 6 /ml in alpha-MEM + IL-2. 50μL of cell suspension was added on top of the 5 μM polycarbonate transwell membrane. 100 μLof 1, 10, 25, 50, 100, 500 and 1000 ng/ml CXCL12 were added to the bottom wells. Negative and positive controls were added.

The cells migrated for 4 hours at 37°C. Following migration, cells in the bottom wells were corrected and the number of cells that migrated was assessed using the MacsQaunt VYB. The percentage of migration in response to CXCL12 was calculated with respect to the negative control.

Student's t-test was used for statistical significance.

Conjugate formation kinetics

NK-92 cells were stained with CTV and K-562 cells were stained with CMFDA. 100μL of NK-92 cells and 100μL of K-562 cells at 1*10 6 cells/ml for 15, 30, 45 and 60 minutes at 37°C. Cells were fixed with 1% PFA and results were read at the MacsQaunt VYB. Gentle automated sample mixing was performed, to ensure minimal conjugate disruption. The percentage of NK-92 cells involved in conjugates was calculated as the fraction of double positive cells with respect to the total CTV+ population.

Student's t-test was used for statistical significance.

NK-92 -K562 conjugate assessment by microscopy

Microscope slides were coated with Poly-L-Lysine overnight. NK-92 and K562 cells at 0.1*10 6 /ml were incubated for 30 minutes at 37°C in U-bottom 96 well plates. The cells were gently mixed, and a 50 μL was deposited on each well. The cells were left to adhere for 5 minutes on the wells at 37°C. The cells were subsequently fixed with 3% PFA, permeabilized with 0.1% saponin and stained with mouse anti-human anti-perforin Ab for 1 hour at RT, followed by staining with phalloidin (AF-488) to reveal F-actin, goat-anti mouse IgG2b Ab (AF-647) to reveal perforin and DAPI to reveal nuclei.

The sealed slides were images with the 63x oil-immersion objective at the LSM710, and the images were analyzed with ImageJ to calculate the mean distance of lytic granules to the IS.

Student's t-test was used for statistical significance.

NK-92 cytotoxicity

NK-92 cell suspensions were prepared at 0.25 and 1.25*10 6 cells/ml. K562 cells stained with CMFDA were prepared at 0.25*10 6 cells/ml. NK-92 cells were incubated with K562 cells at a 1:1 and a 5:1 effector: target ratio. Aphidicolin was added to prevent cell proliferation, and the cells were incubated at 37°C for 4 and 24h. 7AAD was added to the cell suspension prior to cytometry reading. Using the MacsQuant VYB, the number of residual alive target cells was assed to calculate lysis, by assessing the number of CMFDA+ 7AAD-cells.

Student's t-test was used for statistical significance

TIRF microscopy

NK-92 cells were loaded with LysoTracker Red for 30 min at 37 °C. Cells were then washed and transferred to µ-Slide 8-well chambers pre-coated with ICAM-1 and anti-NKp30Ab, and rapidly transferred to a pre-warmed microscope stage. TIRFM experiments were performed using a 100 × 1.45 numerical aperture TIRF objective on a Nikon TE2000U microscope custom modified with a TIRF illumination module. The images were recorded at 1-sec intervals. Images were analyzed using ImageJ to quantify lytic granule position and movement within the TIRF plane.

Primary NK cells

Primary NK cell expansion

Primary NK cells were isolated from blood PBMCs of two normal donors using a CD3 negative selection kit. The negative fraction was stimulated with irradiated PLH cells at a ratio of 4 PLH for 1 NK cell, in RPMI 10% FCS containing 100U/ml IL-2 and 5ng/ml IL-15. The first cycle of stimulation was 5-7 days long, during which the cells were not used for experiment. The following cycles were 2-3 days long and involved stimulation with 1PLH cell for each NK RPMI 10% FCS containing 100U/ml IL-2 and 5ng/ml IL-15. The number of NK cells was calculated by using CD16 and CD56Ab, and 7AAD to exclude dead cells.

Migration

Primary NK cell cells were resuspended at 1*10 6 /ml in RPMI 10% FCS + IL-2. 50μL of cell suspension was added on top of the 5 μM polycarbonate transwell membrane. 100 μLof 50 ng/ml of either CXCL10 or CXCL12 were added to the bottom wells. Negative and positive controls were added. The cells migrated for 2 hours at 37°C. Following migration, cells in the bottom wells were corrected and the number of cells that migrated was assessed using the MacsQaunt VYB. The percentage of migration in response to CXCL10 and CXCL12 was calculated with respect to the negative control.

Student's t-test was used for statistical significance.

Conjugate formation kinetics

NK cells were stained with CTV and K-562 cells were stained with CMFDA. 100μL of NK-92 cells and 100μL of K-562 cells at 1*10 6 cells/ml for 15, 30, 45, 60 and 120 minutes at 37°C. The same protocol used for NK-92 cells was followed.

Primary NK-K562 conjugate assessment by microscopy

The same protocol used for NK-92 cells was applied. However, no calculations were performed on these cells.

Primary NK cell cytotoxicity

Primary NK cell suspensions were prepared at 0.1, 0.2, 0.4, 1 and 2*10 6 cells/ml. K562 cells stained with CMFDA were prepared at 0.2*10 6 cells/ml. NK-92 cells were incubated with K562 cells at a 1:2, 1:1, 2:1, 5:1 and 10:1 effector: target ratio. The same procedure as for NK-92 was followed.

HCI as a methodological advance in imaging the immunological synapse assembly

In our study, we provide a novel high content imaging and analysis pipeline of the immunological synapse in different cytotoxic lymphocyte subsets. We use a 2D imaging approach over 4 z planes which correspond to a 2μm slice encompassing the area of contact between the lymphocyte and a microwell surface coated with stimulatory molecules. We successfully manage to mimic the assembly of the immunological synapse, whereby the cells adhere successfully to the microwell surface, and that they are amenable to staining and imaging as such. In our study, the selection of the fields that are imaged is randomized, therefore eliminating the bias that may come into play when choosing the cells to image, which may in turn lead to the exclusion of "extreme" phenotypes that are a minority of the cells but may be of interesting biological relevance. This infers the need for an analysis pipeline which allows an extensive profiling of the IS. Coupling our imaging approach to robust statistics to evaluate the data on a per image (field of view basis) allows to account for the variability observed in each field of view. Therefore, this analysis pipeline comes as an intermediate between analyses of high content imaging screens on well/condition level and on a single cell level basis 360,364,401 .

Our pipeline was applied to study the immunological synapse assembly and the use of robust statistics allowed us to draw conclusions that are statistically significant, and less sensitive to outliers while still able to identify distinct or striking morphologies. Moreover, such a pipeline allowed us to establish a classifier, which can discriminate treatment conditions and segregate normal donor from patientderived cells.

It is to be noted that even though we imaged 8 z planes encompassing a height of 4 μm, we only opted for the assessment of the first 2 μm closest to the coated surface. Therefore, our data only captures the events at the synaptic plane and misses events occurring at higher planes. The large set of images generated might be of interest to study the 3D structure of the cells, which can provide a better understanding of granule positioning, and could potentially provide insight into granule polarization defects.

HCI validates the role of the actin cytoskeleton in governing IS assembly

As a first step, we validated our approach on the NK-92 and the Jurkat cell lines, and only considered a set of targeted morphological measurements that were compiled from previous knowledge about the IS assembly. These features include intensities of F-actin, the nucleus, perforin granules and LFA-1, as well as parameters pertaining to the size and morphology of the cell and the nucleus 44,57,402 . At the field of view and single cell levels, our results reveal a positive correlation between actin intensity and nucleus area and LFA-1 intensity. However, there amount of correlation varies, indicating that cells in a given population may behave differently in response to a certain stimulus. Therefore, despite the usefulness of population-averaged assays and their ability to report how a population can respond to perturbations, these assays assume that the entire population behaves similarly to the mean cell, which leads to the loss of valuable information. Population distributions also lead to the exclusions of extreme phenotype that represent a minority, but that may have valuable biological relevance. Since our analysis pipeline eliminates any field of view that has less than three cells, as well as cell clusters, we offer a statistical robustness that permits the study of individual cells and their behavior. Having more than three cells per field of view eliminates the doubt of poor coating conditions in that particular field which may have led to poor cellular adhesion, while removing clusters allows the study of individual rather than collective cell behavior. Therefore, combining the approach of imaging a large number of cells with robust statistics decrease the sensitivity of our approach to outliers and artifacts and allows for keeping "extreme" phenotypes if they have a biological relevance.

Our results confirm the importance of the actin cytoskeleton in the assembly of the immunological synapse, whereby we show that synapse assembly entails actin accumulation and spreading. It also confirms that lytic granules polarize to the IS, and that LFA-1 accumulates there as well. There results highlight the importance of the actin cytoskeleton dynamics in the normal context, which led us to probe whether our approach identify changes to the immunological synapse assembly upon targeting the actin cytoskeleton whether it be by drug treatment or by using cells from patients presenting a genetic defect in an action-regulating gene. For that reason, we implemented an additional analysis approach, which goes beyond the analysis of a set of pre-defined morphological features to include a high-resolution analysis approach which includes a much larger set of morphological features.

We show that affecting actin cytoskeleton dynamics with drugs targeting the processes of actin polymerization and depolymerization yields alterations on the lytic granules, which is expected, as lytic granule polarization to the IS requires rearrangements to the actin cytoskeleton 53,64 . However, the changes caused by the drugs need to be considered in the context where these drugs may not be specific to only one mechanism, but could be affecting several mechanisms, as has recently been claimed for SMIFH2. Indeed, some recent data implies that SMIFH2 is not only a formin inhibitor but could also be targeting Myosins (unpublished data). Interestingly, we note that even though the parameters pertaining to perforin appear to change upon drug treatment or in ARPC1B deficient patient cells, the morphological features pertaining to perforin in the robust morphological profiling do not appear to have a high importance in the prediction of the drug treatment or distinguishing patient cell from normal donors. This suggests that the changes underwent by perforin granules are a consequence of the defects of the actin cytoskeleton, rather than a direct consequence of the drug treatment or genetic defect. This hypothesis is backed up by previous findings that there is a specific interplay between the actin cytoskeleton and the lytic granules from their polarization until their exocytosis 44,[START_REF] Mace | NK Cell Lytic Granules Are Highly Motile at the Immunological Synapse and Require F-Actin for Post-Degranulation Persistence[END_REF]224,403 .

HCI coupled to robust statistics reveal important and sometimes unexpected IS morphological features

Coupling our HCI approach to robust statistics has allowed us to perform a thorough morphological profiling of the IS, beyond the initial set of limited features. By performing a morphological analysis based on more than 300 morphological features, our results reveal the importance of some parameters which have been so far overlooked in the study of the IS. We show that targeting the IS with drugs that affect actin dynamics and analyzing cells from ARPC1B deficient patients reveals the importance of the shape of the cell, its radial distribution, its granularity as well as the shape of the nucleus are important determinants in the morphological profile of each treatment condition or deficiency. This finding is confirmed by the fact that the classifier performs with better accuracy when the model is trained to predict the treatment condition based on the 381 morphological features rather than only 13.

Interestingly, robust morphological profiling results reveal that the nucleus increases in area upon IS assembly, indicating a role of the actin cytoskeleton dynamics in shaping the morphology of the nucleus. It has been shown that the MTOC must translocate around the nucleus to IS, and that the nucleus must form an open arch structure toward the IS to allow this translocation 404,405 . Robust morphological profiling highlights that the shape of the nucleus is indeed affected upon a defective IS assembly, and the morphological changes underwent by the nucleus upon IS formation may be linked to the formation of the open arch structure and facilitation of centrosome translocation. To explore further the effect of the changes of nuclear morphology on function, single cell RNA sequencing could be performed to check for a relationship between the nuclear morphology and transcription and try to answer whether changes in nucleus shape could relate to the changes in the IS assembly, and whether changes in the actin dynamics could impact on the functions of the nucleus. Therefore, our results show a clear need for a morphological analysis of the immunological synapse that encompasses a large set of morphological features in order to properly understand the effect and impact of a given drug treatment or a mutation. However, we also note that morphological profiling is not enough to answer questions pertaining to function.

HCI could be indicative of a defect in the cytotoxic function

Our results reveal clear differences in the immunological synapse assembly of each of the two ARPC1B deficient patients compared to that of normal donors. The role of the immunological synapse assembly in the steps leading to successful cytotoxicity has been inferred from genetic defects where a defective IS assembly impacts on the cytotoxic activity 208,224,262 . For that reason, it was evident that the cytotoxic activity of ARPC1B deficient CD8+ T cells was to be tested. We show that indeed, these cells harbor a defective cytotoxic activity at both a high and low anti-CD3 concentration. This could be due to a defect in the TCR signaling, since phenotyping analysis shows a normal expression of lytic molecules. Indeed, a recent study has confirmed that ARPC1B deficient CD8+ T cells have impaired TCR activation 292 . In agreement with our results, they also report a decreased cytotoxic activity in these cells. Our analysis of the IS in these patients reveals distinct responses to stimulation with ICAM-1 and anti-CD3 Ab for each of these patients. While both of them display a reduced F-actin accumulation concordant with defective ARP2/3 and treatment with CK-869, we show that patient 1 has decreased perforin accumulation at the IS, while patient 2 appears to have more perforin granules at the IS 292,406 . It could be inferred that patient 1 has a defect in granule polarization, which has been observed in a recent study, while for patient 2 it could be presumed that the defect lies in degranulation despite proper granule polarization, in agreement with the study by Randzavola et al. in which defects in granule polarization and degranulation were reported 292 . However, the differences between the two patients could be explained by the fact that they harbor two different mutations, indicating that nature of the mutation most likely impacts the IS assembly and therefore morphological features. For that reason, even though it might be hard in the field of rare diseases, it could be of interest to study how different mutations in one gene could affect IS morphology, and how the IS morphology could translate into functional defects.

As introduced earlier, several PIDs caused by mutations is actin regulatory genes are often accompanied by a cytotoxic defect. Therefore, such an approach as the one developed during m PhD could be useful to screen samples from these patients in parallel and perform a robust morphological profiling to potentially determine the common traits observed during an impaired IS formation that may be causative of the cytotoxic defect. Creating such a database would not only be helpful in trying to establish the morphological features of a defective IS, but such a database could also be used to compare patients with unknown PID mutations to patients in said database, in an attempt to narrow down the potential gene mutations underlying their defects. The more patients that are added to such a database, the more accurate it would become to build a network of possible gene interactions of actinregulating genes.

HCI of the immunological synapse reveals discrepancies in the regulation between cell lines and primary cells.

In our assays, we denote a difference in the response of immortalized NK-92 cell lines treated with SMIFH2 and CK-869, and primary NK cells isolated from the PBMCs of three donors to the same drugs. This raises the question of discrepancies between the responses of cell lines and primary material. The use of immortalized cell lines is more advantageous, is these cells are easily grown and expanded in culture, and may be amenable to different manipulations such as gene knock-down and knock-in. However, the fact that cell lines are grown in culture over a long period of time has been shown to cause phenotypically altered populations 407 . The question of how actin dynamics may differ between immortalized cell lines and primary cells arises due to this fact.

Our results show that NK-92 cells appear to be more affected by treatment with CK-869 than by SMIFH2 and Blebbistatin. This most likely indicates that retrograde actin flow in NK-92 cells is more dependent on the ARP2/3 complex than the formins. In primary NK cells, treatment with CK-869 and SMIFH2 lead to a decrease in actin intensity, most likely implying a control of actin dynamics by both the formins and the ARP2/3 complex. This could imply a difference in the regulation of the IS assembly between cell lines and primary cells isolated from PBMCs. A recent study has addressed this question and compared the actin dynamics between Jurkat cells and primary mouse CD4+ T cells, and they showed that Jurkat cells lack actin arcs, that primary cells are more dynamic at the lamellar leading edge, and that the cortical actin network in primary cells undergoes more undulation 146,280 . Moreover, upon treatment with CK-666 and SMIFH2, they show that the retrograde actin flow in primary cells is mainly generated by MyoII, while it is more dependent on nucleation and polymerization in Jurkat cells 280 . Therefore, it can be concluded that despite the advantages cell lines present, they may not always be the best model from which conclusions should be drawn.

HCI reveals the lack of a standard "normal" or "disease" phenotype

Our results reveal that treating primary NK cells isolated from the PBMCs of three normal donors leads to a unique response in each donor. We show that as a general response to CK-869 treatment, primary NK cells show a general increase in perforin-related parameters. However, when examined closely, we denote that in one donor, perforin-related parameters are decreased. This could imply that the regulation of the immunological synapse is fine-tuned with slight differences among healthy donors. Interestingly, our pipeline also reveals a difference is IS morphological features among the three different normal donors. This most likely indicates that the fine-tuning of the immunological synapse in normal conditions might differ among people based on differences in differentiation status, ongoing infection or treatments, as well as possibly the genetic background. Testing this hypothesis would require the analysis of much larger pools of donors. It should be noted that a solid conclusion cannot be made on inter-donor heterogeneity, and such a conclusion would require a larger number of donors to rule out the possibility that the differences observed are not due to different activation states.

Previous studies have shown that differentiated CD8+ T cells from patients deficient in ARPC1B have aberrant IS morphologies 288,292 . Indeed, they reveal a defective radial spreading and a reduced cell area, and they appear to emit filopodia in response to TCR stimulation. Our images reveal the same IS morphology shown in that study (P1 and P2 are the P1 and P7 in that study), and our analysis confirms the reduced cell area. In addition, we observe different responses to stimulation with ICAM-1 and anti-CD3 Ab for each patient, which shows in the radar plots, the UMAP and the performance of the classifier which can distinguish between the two patients. Patient one has a mutated ARPC1B, while patient 2 has no ARPC1B 288,408 . Our approach therefore confirms that the immunological synapse of two patients harboring different genetic mutations will have different morphologies. It is therefore possible that due to different expression levels or ARPC1B that different compensation mechanisms (such as by formins) acting at different levels may explain the difference in IS morphologies among the two patients.

HCI as a first step preceding functional assays

The immunological synapse has been referred to as a molecular machinery behind lymphocyte activation, as it involves a complex interplay between a myriad of signaling molecules 108 . Our results show that a defective immunological synapse assembly distinguishes normal cells from cells drug treated or patient cells. By examining ARPC1B patient CD8+ T lymphocytes and performing cytotoxicity assays, we confirm that a defective immunological synapse relates to a defect in cytotoxicity. However, we only stained F-actin, perforin, LFA-1 and the nucleus, therefore we cannot conclude the molecular mechanism behind the defective cytotoxicity and answering that question would require analysis of the degranulation process in these cells, as our observation of the IS of these patients imply. Therefore, our approach, despite its usefulness in allowing us to extrapolate a functional defect, cannot be a replacement to functional assays, and must be coupled with them for an accurate depiction of the functional defect.

Since our approach is versatile in terms of the cells that may be imaged and the molecules to be stained, one could stain for a multitude of signaling molecules and functional molecules (LFA-1, granzymes, granulysin, LAMP-1) as well as tubulin in cytotoxic cells from different PID patients in an effort to narrow down the source of the functional defect. Such an approach would be of high value if applied on patient cell material, as it has a low requirement of cell numbers. Indeed, with only half a million cells, one could potentially image a hundred different conditions.

Additionally, a gene defect is usually determined by genetic sequencing. Once a mutation is confirmed, the expression of the normal proteins is usually determined by Western Blot analyses 208,213,224,409 . One potential application of our high content imaging pipeline is to, as an alternative to NGS and Western Blot, start by imaging patient lymphocytes. Indeed, if a patient presents with symptoms that are closely related to a known PID where the IS is also defective, one could image the immunological synapse of the lymphocytes of this patient by staining for the suspected defective protein and proteins from the same family or from the same signaling pathway. Such a process could aid in directing researchers towards where the mutation could lie.

HCI of the immunological synapse as a useful tool in the field of personalized medicine

Our results show that HCI can aid in shaping a morphological profile of the IS of each donor. Such interesting findings could be applied in cases of personalized medicine, where the response to treatments can be assessed on a per patient basis. This could be promising if applied to the immunological synapse as a pharmacological target. Indeed, pharmacological compounds that target the IS have been tested and are being optimized, and understanding the mechanisms underlying IS assembly and maintenance is essential for the development of new successful immunotherapeutic strategies. Namely, treatments with specific inhibitors of perforin activity are being tested as immunosuppressive treatment in autoimuunity 410 . Blocking CD3 by compounds such as chimeric, humanized and fully human anti-CD3 mAbs is being tested as potential treatment for patients with Crohn's disease and hepatitis 411 . The blockade of co-stimulatory molecules such as binding of CTLA4-IgG to CD80 to suppress CD28 co-stimulatory signaling is being employed to treat patients with autoimmune disorders 412 .

Our HCI approach could be of value when determining responders from non-responders to these treatments, by the assessment of alterations occurring to there IS parameters upon treatment. Assessing the IS morphology in these patients could be an indicator of treatment impact on IS assembly, and examination of several patient responses in parallel could be carried out in an effort to draw conclusions.

In fact, one project we are currently setting up in our lab is the high content imaging of CLL cells from patients before and after treatment with Ibrutinib, an agent that has been shown to destabilize IS assembly, as assess whether different responses to Ibrutinib treatment may be linked to its distinct IS morphologies of leukemic cells in each patient.

HCI could be the method study different functional steps of cytotoxicity

To successfully eliminate their targets, cytotoxic cells must migrate towards the site of infection, recognize target cells, form stable conjugates with them and secrete their lytic granule content into the target cells to ensure their elimination.

Motility can be studied in several setups, including transwell assay and migration chambers where the cells migrate in response to a chemokine gradient. Such results would be assessed by flow cytometry and live microscopy respectively. The motility can also be predicted by stimulating the cells with chemokines and seeding them on a matrix, and the morphological changes can be captured by fixed confocal microscopy 208 . This has been done in a low throughput format, and our experimental approach offers the potential to assess the response to chemokines in a high content fashion, where the response to several chemokines and on different matrices can be tested in parallel. Testing several conditions in parallel while doing only one staining, imaging and analysis process not only saves time but would also allow for the parallel comparison of these different conditions, as the images would be acquired under the same conditions, decreasing variability in experimental settings. Robust morphological profiling allows to efficiently detect where the response is impaired and reveal intrinsic defects that may be due to the actin cytoskeleton.

Conjugate formation between the cytotoxic cell and the target may be assessed by flow cytometry or low throughput confocal microscopy 224,246 . However, it could be more interesting to study it by microscopy, as staining for tubulin and lytic granules could be informative of the cells' capacity to polarize their granules. Conjugate formation could be amenable to imaging by high content microscopy, and this would allow the parallel imaging of several patients and normal donors in parallel. When cells are imaged with low throughput confocal microscopy, the usual number of cells imaged per experiment is around 30, which can take around 3 hours and may be subject to bias as the cells are selected manually, and some extreme phenotypes may be ignored. HCI offers the potential to image a large number of cells in a much shorter time, and automated acquisition allows for the elimination of bias. The application of robust statistics allows a lesser sensitivity to extreme phenotypes while still revealing the different phenotypes that may exist within a population, and when comparing different conditions. Imaging of conjugates in expanded CD4 and CD8+ T cells has been implemented in our lab, and I have implemented it for primary NK cells. Therefore, one application of the pipeline set up could be the simultaneous imaging of T and NK cells from the same donors.

Coupled to approaches of flow cytometry amenable to be assessed in plate formats, this approach completes the lack that was present in assessing the function steps of cytotoxicity in a high throughput format.

Limitations of HCI of the immunological synapse

One limitation that I encountered during my PhD was the fact that coating the multiwell plates and seeding the cells had to be done manually, due to the absence of robotics. Had robotics been accessible, it would not only have saved time in coating plates, as well as seeding and staining cells, but would have most likely decreased the variation in the number of cells seeded in each well.

It is to be noted that even though we imaged 8 z planes encompassing a height of 4 μm, we only opted for the assessment of the first 2 μm closest to the coated surface. Therefore, our data only captures the events at the synaptic plane and may be missing events occurring at higher planes. Indeed, a 3D study would be more comprehensive, and would reveal more information about the state of the lymphocyte, which can provide a better understanding of granule positioning, and could potentially provide insight into granule polarization defects.

Moreover, our study captures the immunological synapse assembly in the context where the lymphocytes are seeded on an ICAM-1 and stimulatory antibody coated surface. In such a scenario, these ligands are fixed, while when expressed on a target cell, they exhibit intermediate motility, depending on the cell state 413 . For that reason, it might be of interest to optimize the HCI of the immunological synapse of lymphocytes when conjugated to target cells and assess the differences the IS morphology that may arise. Another alternative could also be the optimization of this approach on planar lipid bilayers.

In our results upon drug treatment, we observed an increase in perforin-related parameters, implying that the perforin granules were at the IS surface at the time of imaging. However, since the images we captured were of fixed cells, one question that cannot be answered accurately is whether or not this increase in granules at the IS is due to an increased granule recruitment to the IS, or to a defect in granule exocytosis. To accurately answer this question our approach would need to be coupled to a degranulation assay, or a more resolutive live imaging microscopy to be more precise in determining whether the granules fail to fuse with the membrane or cannot exit through the actin clearances. In some instances, we also observed a decrease in perforin-related parameters. However, HCI of the IS could be helpful in this case but the whole height of the cell would be imaged to determine whether the cells have degranulated too fast (by a decreased lytic granule count) or if the cells fail to polarize.

Despite the fact that we report different IS morphologies for each of the two patients, the end result for both is a defective cytotoxic activity. For that reason, it could be of interest to study the steps of cytotoxicity of these patients by live microscopy, in order to confirm at which step the defect lies, and confirm the results seen and the assumption made by examining IS morphology. Therefore, despite the usefulness of HCI screens of the immunological synapse, it is not yet able to replace live microscopy. Additionally, it would be of high informative value to apply high content imaging to decipher ongoing at the nanoscale, and even determine single molecule localization. The compromise between high throughput and high resolution is slowly beginning to become narrower, as we are seeing developments in high throughput super resolution microscopy techniques 414 .

The value of HCI is the ability to perform screens using this technology. In fact, an aspect of my PhD project that I have attempted to set up was an HCI screen of the IS of NK-92 cells that were knockeddown with an arrayed library of 85 genes known to play a role in the actin cytoskeleton regulation, and their neighbors. The aim of that screen was to build an interactome of these genes, based on the obtained IS morphologies for the generated knock-downs. However, that project was discontinued due to the difficulty in transducing NK-92 cells with the lentiviral particles. An alternative that could make mapping these interactions possible would be imaging PID patient material. Additionally, if one could image the different cell types from several patients simultaneously, it could give an idea of the variations to this interactome in different cellular subsets. I contributed to the Guipouy et al. paper by performing the cytotoxicity assays for supplementary figure 2A, which serves as proof that the ova-specific Tregs can kill in an MHC dependent fashion.

For this paper I could use the expertise in cytotoxicity that I had learned from Delphine and apply it to her own project, as well as mine.

High content cell imaging reveals actin cytoskeleton-mediated control of the immunological synapse

Cytotoxic lymphocytes rely on actin cytoskeleton remodeling to achieve their function. In particular cytotoxic T lymphocytes and NK cells assemble the immunological synapse (IS), a complex actin-rich structure that allows the interaction with target cells, such as infected cells or tumor cells, and permits the polarized delivery of lytic granules. Although actin cytoskeleton remodeling is known to be a driving force of IS assembly and dynamics, our understanding of the molecular control of actin remodeling sustaining IS dynamics remains fragmented. This PhD project consisted in developing a high-content imaging approach to unbiasedly define the metrics of IS from human T and NK lymphocytes and to characterize the requirements for actin cytoskeleton integrity in organizing the IS architecture.

For that purpose, the stimulation and staining of cell lines and primary cells in multiwell plates and acquisition of a unique set of >100.000 confocal images with a fully automatized high-content imager was optimized. The images were analyzed with two complementary CellProfiler analytical pipelines to characterize the morphological features associated with different treatments and disease status. We first extracted 16 morphological features pertaining to F-actin, LFA-1 or lytic molecules based on prior knowledge of IS assembly, and included features pertaining to the nucleus. We show that IS assembly in Jurkat and NK-92 cells is characterized by increased F-actin intensity and cell area. For Jurkat cells, we report an increase in LFA-1 intensity and surface area, and for NK-92 cells an increase in lytic granule detection at the IS plane. We then treated NK-92 cells with seven drugs known to affect different aspects of actin dynamics and investigated the associated effects on IS features. We report concentration dependent effects, not only on F-actin intensity, as expected, but also on lytic granule polarization. Furthermore, using a high-resolution morphological profiling based on >300 features, we show that each drug inflicts distinct alterations of IS morphology. In a next step, we applied our experimental pipeline to primary NK cells isolated from the blood of healthy donors. Distinct morphological features were characterized among the NK cells from different donors, highlighting the sensitivity of our approach, but also revealing an unsuspected variability of immune cell morphologies among donors. We then further applied our approach to primary CD8 + T cells from patients with a rare immunodeficiency due to mutations in the gene encoding the actin regulator ARPC1B. ARPC1B deficiency results in decreased F-actin intensity, as well as in lytic granule polarization. This prompted us to assess the ability of these cells to kill target cells, which was markedly reduced. These results illustrate how the systematic analysis of the IS might be used to assist the exploration of fonctional defects of lymphocyte populations in pathological settings.

In conclusion, our study reveals that although assembly of the IS can be characterized by a few features such as F-actin intensity and cell spreading, capturing fine alterations of that complex structure that arise from cytoskeleton dysregulation requires a high-content analysis. The pipeline we developed through this project holds promises for the morphological profiling of lymphocytes from primary immunodeficiency patients whose genetic defect has not yet been identified. Moreover, the discriminative power of our high-content approach could be exploited to characterize the response of lymphocytes to various stimuli and to monitor lymphocyte activation in multiple immune-related pathologies and treatment settings.

L'imagerie cellulaire à haut débit révèle le contrôle de la synapse immunologique par le cytosquelette d'actine

Les lymphocytes cytotoxiques dépendent du remodelage du cytosquelette d'actine pour atteindre leur fonction. En particulier, les lymphocytes T cytotoxiques et les cellules NK assemblent la synapse immunologique (SI), structure complexe riche en actine qui permet l'interaction avec des cellules cibles et la distribution polarisée de granules lytiques (GL). Bien que le remodelage du cytosquelette d'actine soit connu pour être une force motrice de l'assemblage et de la dynamique de la SI, la compréhension du contrôle moléculaire du remodelage de l'actine soutenant la dynamique de la SI reste fragmentée. Ce projet de thèse a consisté à développer une approche d'imagerie à haut débit pour définir de manière impartiale les métriques de la SI des lymphocytes T et NK et caractériser les exigences d'intégrité du cytosquelette d'actine dans l'organisation de l'architecture de la SI.

À cette fin, la stimulation et coloration des lignées cellulaires et cellules primaires dans des plaques à puits multiples et l'acquisition d'un ensemble unique d'images confocales avec un imageur à haut contenu entièrement automatisé ont été optimisées. Les images ont été analysées avec deux pipelines complémentaires avec CellProfiler pour définir les caractéristiques morphologiques associées aux différents traitements et à l'état de la maladie. Nous avons d'abord extrait 16 caractéristiques morphologiques se rapportant à la F-actine, LFA-1 ou aux GL, sur la base d'une connaissance préalable de l'assemblage de la SI, et inclus des caractéristiques relatives au noyau. Nous montrons que l'assemblage de la SI dans les lignées est caractérisé par une augmentation de l'intensité de la F-actine et la surface cellulaire. Pour les cellules Jurkat, nous rapportons une augmentation de l'intensité et de la surface LFA-1, et de la de la détection des LG sur le plan SI pour les cellules NK-92. Le traitement des cellules NK-92 avec 7 drogues affectant différents aspects de la dynamique de l'actine et l'étude des effets associés sur les caractéristiques de la SI montrent des effets dépendants de la concentration sur l'intensité de l'actine et sur la polarisation des GL. De plus, un profilage morphologique à haute résolution basé sur> 300 caractéristiques montre que chaque drogue inflige des altérations distinctes sur la morphologie de la SI. Nous avons appliqué ce pipeline à des cellules NK primaires isolées du sang de donneurs sains. Des caractéristiques morphologiques distinctes définissent les cellules NK de différents donneurs, soulignant la sensibilité de notre approche, mais révélant également une variabilité insoupçonnée des morphologies des cellules immunitaires parmi les donneurs. Nous avons appliqué notre approche aux cellules T CD8+ primaires de patients présentant une immunodéficience rare due à des mutations dans le gène codant pour le régulateur d'actine ARPC1B. La carence en ARPC1B entraîne une diminution de l'intensité de l'actine et de la polarisation des GL. Cela nous a incités à évaluer la capacité lytique de ces cellules, qui a été considérablement réduite. Ces résultats illustrent comment l'analyse systématique de la SI pourrait être utilisée pour aider à l'exploration des défauts fonctionnels des populations de lymphocytes dans des contextes pathologiques.

En conclusion, notre étude révèle que, bien que l'assemblage de la SI puisse être caractérisé par quelques caractéristiques telles que l'intensité de l'actine et la propagation cellulaire, la capture de fines altérations résultant de la dérégulation du cytosquelette nécessite une analyse à haut débit. Le pipeline développé est prometteur pour le profilage morphologique des lymphocytes de patients atteints d'immunodéficiences primaires dont le défaut génétique n'a pas encore été identifié. De plus, le pouvoir discriminant de notre approche pourrait être exploité pour caractériser la réponse des lymphocytes à divers stimuli et surveiller leur activation dans de multiples pathologies et traitements.

Figures

  Figures and tables ................................................................................................................................ Introduction .......................................................................................................................................... General overview of the immune system and immune cell activities ...................................... 1.1 Innate immunity .................................................................................................................... 1.2 Cells at the interphase of the innate and adaptive immune systems ..................................... 1.3 Adaptive immunity ................................................................................................................ A. B lymphocytes...................................................................................................................... B. T lymphocytes ...................................................................................................................... The cytotoxic function: the weapon of defense of the immune system .................................... 2.1 Natural killer cells ................................................................................................................. A. NK cell receptors ........................................................................................................... a. Natural cytotoxicity receptors .................................................................................... i. CD16 ....................................................................................................................... ii. NKp46 .................................................................................................................... iii. NKp30 .................................................................................................................... iv. NKG2D .................................................................................................................. v. NKp44 .................................................................................................................... b. Inhibitory NK receptors (iNKR) ................................................................................ c. Costimulatory molecules ............................................................................................ B. NK cell cytotoxicity: from target binding to target lysis ............................................... a. NK receptors involved in immunological synapse formation .......................................

  importantly, I hope to shed light on the importance of morphology in predicting disease.

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 13 :Figure 16 :Figure 17 :Figure 18 :Figure 19 :Figure 21 :

 1234567891011121314131617181921 Figure 1: Overview of the immune system

Figure 22 :Figure 24 :Figure 24 :Figure 25 :

 22242425 Figure 22: Representative workflow for image-based cell profiling

Figure 1 :

 1 Figure 1: Overview of the immune system. The immune system is composed of the innate arm, the adaptive arm and cells at the interface of both arms. Adapted from 1 .

Figure 2 :

 2 Figure 2: Natural killer cell surface receptors involved in cytolytic activity, and their association to signaling molecules 24 .

Figure 3 :

 3 Figure 3: Schematic representation of T cell activation and differentiation in response to APC stimulation. Adapted from the thesis manuscript of Julie De Meester (Thesis, 2011).

  The intermediate conformation corresponds to an intermediate affinity, while the open conformation corresponds to a high affinity.

Figure 6 :

 6 Figure 6: Schematic representation of the different components of the LFA-1 molecule in its different conformations. A. Resting, B. Intermediate and C. Open conformations 98 .

  conformation and lead to a binding to ICAM-1 with intermediate affinity 100 . However, to fully activate LFA-1 and cause T cell arrest, shear forces were shown to be necessary 100 Inside-out signaling mediated by the TCR occurs upon the formation of the IS. TCR activation induces Rap1 activation through ADAP and SKAP55, leading to LFA-1 inside-out signaling. As previously mentioned, PLC, among others, plays a role in LFA-1 activation. Inside-out signaling cascades increase the ligand binding affinity of LFA-1 receptors and cause their redistribution on the T cell surface into highly organized patch-like clusters. Comrie et al. have shown that LFA-1 in the extended conformation is enriched in the pSMAC, while open LFA-1 was concentrated in a smaller and more central ring of the IS, which will be elaborated further in section pertaining to the IS 95 . A recent study by Houmadi et al. in our laboratory, shows that the nanoscale topology of LFA-1 at the T cell IS consists of a belt of nanoclusters embedded in the interstices of the cortical actin cytoskeleton meshwork 101 .

Figure 7 :

 7 Figure 7: Changes undergone by CD8+ T cells before/after infection. Antigen-specific naïve CD8+T

  presented on the MHC-I molecules on the target cell. Following contact with the target an immunological synapse is formed and the lytic machinery of the CTL is quickly assembled. The formation of the CTL-target stable immunological synapse relies on the binding of the integrin LFA-1 to ICAM-1 on the target cell 118 . TCR signaling following TCR-peptide-MHC-I engagement drives the ARP2/3 dependent polymerization of branched actin filaments at the edge of the site of contact between the T cell and the target cell.The MTOC polarizes towards the target cell, causing the release of soluble lytic molecules such as perforin and granzymes and membrane-anchored effector molecules such as the Fas ligand103,119 . It has also been shown that polarization of the lytic molecules to the IS requires a strong TCR signal, whereas weak TCR signaling cannot induce granule recruitment to the IS, despite proper MTOC polarization 120 . The activity of Lck, Fyn, and ZAP-70 are all essential for promoting MTOC polarization to the lytic synapse, where Lck is essential for signal propagation through ZAP70 103 . ZAP 70 phosphorylates LAT which recruits several proteins including SLP76, PLCγ1 and Vav1 to form the LAT signalosome. SLP76 and Itk mediate PLCγ1 activation, therefore inducing MTOC polarization and calcium ion influx, as well as inducing a cascade of signaling events which contribute to MTOC polarization 103 . Ca 2+ influx into the cell is essential for degranulation, but not for MTOC polarization, reinforcing the observation that MTOC polarization is not a requirement for granule polarization and exocytosis 103 . The granules are then released from the secretory cleft and into the target cell 121 .

Figure 8 :

 8 Figure 8: Overview of families of actin-binding proteins involved in the different processes pertaining to actin 101 .

Figure 10 :

 10 Figure 10: Top view model of the spatial organization of the immunological synapse 135 . The immunological synapse is organized into a central, peripheral and distal supramolecular activation clusters (SMAC), where distinct molecules aggregate in each SMAC.

  mediated by LFA-1 among other adhesion molecules147 . In the absence of inhibitory signals, the stable IS begins to form, involving F-actin rearrangements and morphological changes to NK morphology, which occur downstream of Vav1, and is WASp dependent 147 . It has been shown that NKp46 aggregates into clusters which are enriched at the IS. It has also been shown to play a role in F-actin accumulation at the IS. Even though the NK cell synapse was thought to be static and stable at the micrometer scale, it has been shown to exhibit local nanoscale dynamism, where actin filament rearrangement appears to be mediated by ARP2/3 and MyosinIIA[START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] . As opposed to the retrograde Factin flow observed in T cell synapse, NK cells appear to only display short sequences of outward Factin polymerization[START_REF] Carisey | Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells[END_REF] .

Figure 9 :

 9 Figure 9: Dynamic steps of cytotoxicity. The main steps of cytotoxicity include migration towards the target cell, conjugate formation, lytic granule polarization followed by degranulation, and end with the detachment from the target cell

  pore forming 70 kDa perforin glycoprotein is encoded by the PRF1 gene on chromosome 22 and is stored in the lytic granules of CTLs and NK cells. Patients with complete perforin deficiency present with HLH symptoms, and present an impaired CD8+ T cell and NK cell cytotoxicity 169,170 . Partial loss of perforin leads to certain malignancies, and perforin loss may lead to certain autoimmune diseases such as a systemic lupus erythematosus, Hashimoto's disease and Grave's disease 171 . A missense mutation in the PRF1 gene has also been shown to lead to neurological manifestations 172 . In certain cases where cytotoxicity of CD8+ is impaired, some CD4+ T cells may become perforin positive 173 . Perforin polymerizes and binds target cells through membrane phospholipids, forming a channel in the target cell membrane through which Gzms may be released. T cell gene therapy has been proposed as a curative mechanisms for HLH patients with perforin defects 174 . member of Munc13 family of proteins involved in vesicle priming function, has been described as a positive regulator of secretory lysosome exocytosis. It is encoded by the UNC13D gene located on chromosome 17. Patients presented with fever, hepatosplenomegaly, pancytopenia, coagulation abnormality, liver dysfunction, and features of hemophagocytosis in the bone marrow or cerebrospinal fluid 175 . Confocal and TIRF microscopy imaging reveal that Munc13-4 is dispensable

Figure 11 :

 11 Figure 11: NK cells from STX11 deficient patients do not fail to polarize their perforin granules (red) to the IS 178 .

Figure 12 :

 12 Figure 12: NK cells from a normal donor and an STXBP2 deficient patient stained for F-actin (red) and perforin (green) reveal a normal granule polarization in patient cells181 .

  genes regulating the development and function of T, NK and often B cells. SCID patients that lack T cells and can be sub-classified further based on the presence or absence of NK and B cells. However, since antibody production from B cells requires functional T cells, patients in which T cells are absent usually have a combinedT and B deficiency 190 . Moreover, within the same family, two siblings suffering from SCID may present with different cellular phenotypes191 . SCID patients often present with symptoms early on, in the first weeks or months of life. They fail to thrive and suffer from viral and fungal infections which include infections by rare microorganisms192 . One symptom that SCID patients present with and that physicians often overlook is lymphopenia.a. B cell immunodeficienciesB cell immunodeficiencies, also known as antibody deficiency, are the most common type of PIDs, making up for about 50% of diagnosed PIDs. Clinical manifestations include a susceptibility to bacterial respiratory tract infections. They are characterized by the reduction or absence of serum immunoglobulins (Ig), or an increased Ig level but with impaired function. More than twenty Ab deficiency disorders have been defined, and include X-linked agammaglobulinemia and common variable immunodeficiency 193 .

Figure 13 :

 13 Figure 13: Wiskott-Aldrich syndrome protein activation, molecular partners, and cytoskeleton remodeling 198 .

  assays of two-color confocal microscopy on mouse T cells and Jurkat cells showed colocalization between Coro1 and F-actin, within the T cell subcortical zone and in membrane protrusions, and that Coro1 accumulates in the protrusions caused by actin polymerization following cell contact with anti-CD3 Ab 222 . Coro1A plays its role in actin depolymerization in association to cofilin and WDR1. It performs its function mostly by redirecting the spatial organization and activity of cofilin in a way to disassemble older actin filaments 223 . Mace and Orange have shown by confocal microscopy that Coro1A accumulates at the IS of NK cells in contact with their targets (Figure 12) 224 . A FRAP experiment in the same study shows that Coro1A

Figure 14 :

 14 Figure 14: The role of Coronin1A in the immunological synapse of NK cells. A. Coro1A (red) colocalizes with F-actin (green) and polarized lytic granules (blue) at the NK cell immunological synapse. B.Coro1A KD does not alter IS size, F-actin (red), LFA-1 (green) recruitment to the IS and perforin (gray) and pericentrin (blue) polarization 224 . C. STED microscopy reveals a smaller but more intense F-actin (green) area with less pervasive areas, thereby hindering granule (red) release.

  the positive selection of CD8+ T cells. RhoA plays a role in T cell development and is important for the survival and proliferation of T cell progenitors in the thymus, as well as playing a role in thymocyte adhesion231 . RhoA inhibition also prevented Rac1 activation of integrins in thymocytes. Moreover, RhoA increases LFA-1 activation during lymphocyte migration, and most likely during the IS 232 .RhoA can bind Rho-associated, coiled-coil-containing protein kinase (ROCK) and mDia. It also plays a role in uropod retraction, and microtubule formation at the uropod, and its absence leads to elongated cell tails. It is also important for the maintenance of cell shape. RhoA is also speculated to play a role in the control of IS size. (Reviewed in 233 ). Patients with low RhoA suffer from a defective antibody production, and in T and B cells there is a defect in F-actin polymerization as indicated by low levels of F-actin upon stimulation 210

  DOCK2 has been shown to be essential for the Rac1 signaling downstream of the TCR. Mice defective in DOCK2 harbor T and B cells fail to activate via Rac in response to chemokines, thus leading o migratory defects and suggesting a role of DOCK2 in cytoskeletal organization 242,243 . DOCK2 patients presented with T and B cell lymphopenia, defective T cell proliferation, recurrent respiratory infection and pneumonia episodes 244 . One of the patients suffered multiorgan failure which caused his death. Similarly to mouse models, DOCK2deficient patient ells exhibit defective Rac1 activation, as well as defective migration and F-actin polymerization 244 . NK cell cytotoxicity and IFN-γ were reduced, as well as the number of circulating NKT cells.

  used T cell line. In NK cell lines,Ham et al. have shown that depletion of DOCK8 with siRNA leads to a dose-dependent defect in cytotoxicity, as well as a decreased redirected cytotoxicity through FcR and NKG2D receptors, implying a role for DOCK8 in the cytotoxic function of NK cells 246 .Moreover, they have shown that DOCK8 plays a role in the upstream step of conjugate formation, where primary and NK cell lines deficient in DOCK8 showed defective conjugate formation despite the normal expression levels of CD18 246 . In primary DOCK8 deficient NK cells, the defect in cytotoxicity was confirmed, and it was shown that, similarly to T cells, it cannot be rescued by exogenous IL-2250 .It has been demonstrated by confocal microscopy that the CD8 T cells of mice harboring a DOCK8 deficiency show a defect in recruitment of LFA-1 to the IS, whereby the distribution of LFA-1 is reported to be uneven. Moreover, DOCK8 deficient mice cells failed to polarize actin to the IS 249 .Consistent with these findings, NK cell lines in which DOCK8 expression is suppressed by siRNA or shRNA, as well as primary NK cells from DOCK8 deficient patients show weaker accumulation of LFA-1 at the pSMAC, as well a diminished F-actin accumulation, as well as a defective granule polarization (Figure15)246,250 . Notably, the F-actin content in DOK8 deficient cells is the same as in healthy cells, as opposed to WASp deficient cells in which the overall actin content is reduced. Taken together, these data suggest a role for DOCK in cytotoxicity.

Figure 15 :

 15 Figure 15: DOCK8 suppression leads to diminished F-actin (green) and lytic granule (red) at the IS 246 .

  Moreover, MST1 was shown to play a role in the regulation of the IS, where it plays a role in the transport of TCR to cSMAC259 . It also phosphorylates VASP, which is a player in the F-actin polymerization. More on the role of MST1 can be found in the review 259 m. RASGRP1 deficiency RASGRP1 is a guanine nucleotide exchange factor whose gene is on chromosome 15. RASGRP1 is and activator of the RAS-MAPK pathway following TCR signaling. Following TCR simulation, RASGRP1 translocates to the membrane and converts RAS, a GTPase, from an inactive GDP-bound form to an active GTP-bound form, which in turn process mediates activation of the MAP-kinase.Therefore, and as confirmed by mouse studies, RASGRP1 plays and important role in T cell proliferation and development and survival260,261 . The RASGRP1 deficient patient presented with recurrent infections including pneumonia, failure to thrive, and finger clubbing 262 . The patient presented a decreased CD4+ T cell and B cell count, increased CD8+ T cells of which Tγδ cells were increased. Moreover, Tαβ CD8+ displayed an exhausted phenotype. CD4+ and CD8+ T cells displayed a failure to proliferate upon anti-CD3/CD28 Ab stimulation. Despite the increased expression of perforin and Gzm B in patient CD8+ T and NK cells, they failed to eliminate target. In NK cells, it was shown by confocal microscopy that this defect was due to the defective IS formation, with decreased F-actin accumulation, increased distance from the MTOC to the synapse and defective granule convergence (Figure16), as well as reduced granule motility as shown by video microscopy.Defects in granule motility and convergence may be attributed to the inability of RASGRP1 to bind dynein. CD8+ T cells showed a reduced migratory speed in response to CXCL12 gradient, which due to a slower actin turnover. B cells also showed defective BCR stimulation and defective MAPK activation.

Figure 16 :

 16 Figure 16: Defective RASGRP1-deficient NK cell IS assembly. Immunological synapse from a healthy donor and RASGRP1 deficient patient shows reduction in F-actin (green) accumulation, increased MTOC distance from IS (pericentrin; yellow) and a defect in lytic granule (red) convergence 262 .

  are actin-dependent molecular motors that use the energy of ATP hydrolysis to move along actin filaments. Myosin II in non-muscle cells plays important roles in cytokinesis and cell migration, and the most abundant form is IIA (MyH9). Indeed, to migrate forward the cell must retracts its trailing edge by combining both the acto-myosin contractility and the disassembly of adhesions at the rear (uropod)125 . The actin filaments generated via ARP2/3 elongation are organized by Myosin II into antiparallel concentric arcs in the pSMAC, as revealed by SIM imaging of Jurkat cells and primary mouse T cells 274 . It has been shown that upon LFA-1 binding to ICAM-1, LFA-1 associates with MyH9 275 . Treatment with Blebbistatin, a MyH9 ATPase inhibitor, and siRNA inhibition of MyH9 both result in a polarized cell morphology, in which the uropod was extended in length as opposed to shorter uropods in untreated cells. This suggest that MyH9 binding to LFA-1 and the force it exerts is essential for the LFA-1 de-adhesion at the uropod 275 . However, MyH9 association to LFA-1 is not required for LFA-1 activation nor its recruitment to the leading edge of the cell.In 2004, Jacobelli et al. Showed that MyH9 function is essential for maintenance of the uropod and for T cell motility but is dispensable for synapse formation, even thoughIlani et al. show that it is recruited to the IS and essential for its sability276,277 . However, mouse experiments revealed that siRNA inhibition of MyH9 leads to more spread IS, suggesting that MyH9 plays a role in constricting IS size, as well as playing a role in pSMAC and cSMAC formation regardless of the concentration of MHC available, but not for TCR microcluster transport 278 . Conversely, Yu et al. demonstrate that Myosin is required in the TCR transport in the first two minutes of IS formation, and that its inhibition by Blebbistatin affects the directed TCR movement 279 . Treatment of Jurkat cells with Blebbistatin and shRNA inhibition in primary CD4+ T cells and observation with TIRF microscopy of supported planar lipid bilayers reveals, on the other hand, that MyH9 activity is required for centripetal TCR microcluster movement, but not for microcluster formation 277 . Ilani et al. also show a role for MyH9 in TCR signaling277 . Moreover treatment with Y-27632, which inhibits Myosin-II contractility by specifically targeting rho-associated protein kinase (ROCK) leads to a perturbed IS280 . In NK cells it was shown that NK MyH9 deficiency causes an inability of NK cells to properly polarize their granules to the immune synapse and subsequently degranulate and lyse target cells 281 . Moreover, it was shown to polarize at the IS but its absence does not affect Is formation. MyH9 constitutively associates with lytic granules and plays a role in their fusion with the cell membrane, as shown by siRNA inhibition or Blebbistatin treatment[START_REF] Andzelm | Myosin IIA is required for cytolytic granule exocytosis in human NK cells[END_REF] . MyH9 also plays a role in B cell migration but not BCR signaling, as shown by use of Blebbistatin and mouse models 282 . Mutations in the motor domain Myosin II heavy chain lead to MYH9-related disease, characterized by hematologic defects such as macrothrombocytopenia and in some cases cataracts, deafness, and glomerulonephritis 283 . However, mutations in the tail domain of Myosin IIA lead to no relevant clinical symptoms 284 .

Figure 17 :

 17 Figure 17: Crystal structure of the ARP2/3 complex from Baus taurus access protein data bank:1K8K 288

(

  transmission) or can be reflected through a mirror before hitting the sample (reflection). Light can be divided into two main categories: brightfield and fluorescence.Widefield microscopy consists of the microscope forming an image of the object on the camera. For widefield imaging, traditional lighting methods include xenon, mercury and halogen lamps, combined with excitation and emission filters, as well as shuttering devices which control the duration of elimination. Light emitting diodes (LEDs) are also a cheap and stable alternative. LEDs can be turned on and off rapidly, eliminating the needs to shutters 295 .Fluorescence microscopy relies on the use of fluorophores (fluorescent dyes) which absorb one wavelength of light and emit another. Techniques to introduce fluorescence to biological cells include fluorescent antibodies which bind to specific proteins (a process termed immunofluorescence), and genetic manipulation to introduce fluorescent proteins into the cell. LEDs can also be used for fluorescent microscopy due to a narrow emission spectrum, as have been designed to provide illumination at very specific wavelength bands from UV to infrared295,296 . Confocal microscopy is also used to illuminate fluorophores, where the sample is illuminated by a laser beam at a single point in the focal plane 297 .

  Zernike developed phase contrast microscopy in the 1930s to overcome the poor contrast of transparent biological samples 298 . Smith and invented Nomarski invented differential interference contrast (DIC) in 1955, as an alternative to phase contrast 299 . The images produced are exceptionally sharp in comparison with other transmission modes. DIC is still the current standard technique for imaging unstained microbiological samples in having an exceptional ability to reveal the boundaries of cells and subcellular organelles.

  1946, Förster postulated that if a donor and acceptor molecule were sufficiently close together, nonradiative transfer of energy could occur between the two, now known as Förster resonance energy transfer (FRET)307 . Fluorescence can be used as a metric of putative molecular interaction through FRET in the event that the donor and acceptor molecule are fluorescent dyes.Stryer & Haugland showed in 1967 that this phenomenon could be used as a molecular ruler over a length scale of approximately 1-10 nm 308 . Since then, FRET has been widely used to image molecular interactions and the distances between biological molecules, and also in fluorescence lifetime imaging309 .In 1964, Curtis introduced the principles of interference reflection microscopy (IRM), where the interference of reflected light waves generates images with high contrast and definition. This technique has been improved and is now used to study cell adhesion or cell mobility on a glass coverslip310 . With all these advancements came the fluorescent microscope, specifically designed for samples to be stained and imaged with fluorescent dyes or probes. Fluorescent imaging techniques include confocal microscopy, total internal reflection fluorescence (TIRF) introduced in the 1980s, fluorescence recovery after photobleaching (FRAP) introduced in the 1970s, and two-photon and light-sheet microscopy. TIRF microscopy is based on the principle that when excitation light is totally internally reflected in a transparent solid at its interface with liquid, the evanescent wave (an electromagnetic field), is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. The intensity of the evanescent wave exponentially decays with distance from the surface of the solid, and only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited 311 . Two-photon scanning laser microscopy was introduced by Denk in 1990 and has since been used to observe molecules in live tissues 312 .The confocal microscope was invented by Minsky in 1955 but the nomenclature "Confocal" was first used byBrakenhoff in 1979, to refer to a microscope where the illumination is in a diffraction-limited spot within the specimen, and where the detection is also confined by positioning an aperture in front of the detector in a position that would be optically conjugate to the focus spot 313 . Confocal microscopy aims to eliminate the signals from fields other than the imaging plane, through the use of a pinhole, which would be situated in front of the detector. Laser-scanning confocal microscopes typically use point detectors instead of cameras to record an image point by point and are therefore less sensitive.

Figure 19 :

 19 Figure 19: The timeline of microscopy

Figure 21 :

 21 Figure 21: Schematic representation of the confocal light path of the Opera Phenix high content screening system. The projection of non-overlapping excitation pinhole patterns onto the sample achieves the separation of excitation and emission wavelength in space. Excitation lasers are placed into two groups in a way that adjacent laser lines are separated, and are focused through different quadrants, to allow the emission light passage only through the pinhole disc in the respective quadrant.

  . Caldera et al. have used CellProfiler to analyze fluorescent microscopy images of cells treated with an array of drugs and extracted 78 morphological features (out of 438) to position each treatment within the 78-dimension morphological space 348 .

  adjustments for each experiment. Segmentation can be model-based and performed manually based on visual parameters, or by machine learning, where a classifier is trained to find the optimal segmentation solution. Next is feature extraction, which measures the phenotypic characteristics of each cell. It includes shape features such as boundaries on nuclei and cells, perimeter, area and roundness. Intensity-based features are computed from actual intensity values per channel, on a single cell basis and separately within each segmented compartment. They include simple statistics such as minimum, maximum and mean intensity. Texture features are used to quantify the regularities of intensities in an image and rely on mathematical equations such as cosines and correlation matrices364,368 . Microenvironment features include counts and special relationships among cells in a given field of view.

  complete isometric feature mapping, the Isomap consists of a three-step algorithm. The first step determines neighboring points, the second estimates the geodesic distances and the third applies multidimensional scaling to the distances in the matrix, thus culminating in an embedding of the data in a d-dimensional Euclidian space. This technique is well elaborated in 375 b) Diffusion map Diffusion maps were introduced by Coifman and colleagues in 2005. Diffusion maps consist in computing the data set into the often low-dimensional Euclidian space, where the Euclidian distance between two points in the embedded space would be equal to the diffusion distance between the probability distributions which are centered at those points 376 .

  to have not only the fastest run times and highest reproducibility, but also and most importantly, the most relevant organization of clusters 379 . They are a recent technique, introduced by McInnes and colleagues in 2018. The algorithm of UMAPs assumes a uniform distribution of the data in a Riemannian manifold, that the Riemannian metric is locally constant, and that the manifold is locally connected 380 . The subsequent manifold, which has a fuzzy topology, can then be made into a low dimensional projection.

Figure 24 :

 24 Figure 24: Visualization of 6000 handwritten digits from the MNIST data set by UMAP 380 .

lymphocytes 2 .

 2 Provide an understanding of the actin-driven mechanisms regulating IS architecture in cytotoxic lymphocytes 3. Apply this pipeline in the clinical context, by providing an insight as to how the IS architecture may vary in patients with actinopathies. 4. Assess how a defect in the IS morphology could imply a functional defect in cytotoxicity

Figure 24 :

 24 Figure 24: Functional assays on the NK-92 cell line. A. % migration of 10000, 20000 and 50000 NK-92 was determined in response to a CXCL12 gradient (n=2). B. % conjugate formation between NK-92 cells and target K-562 cells was determined over the course of 1 hour, at 15-minute intervals (n=3). C. Confocal microscopy images of an NK-2 forming a conjugate and an immunological synapse

Figure 25 :

 25 Figure 25: Functional assays on primary NK cells. A. % migration of 50000 primary NK cells was determined in response to CXCL10 and CXCL12 (n=2). B. % conjugate formation between NK cells and target K-562 cells was determined over the course of 2 hours (n=2). C. 4 and 24h cytotoxicity assays to determine the lytic activity of primary NK cells against target cells (n=2). Student's t-test was used to determine statistical significance. Values represent means and the error bars show SD. Significance is noted as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. D. Confocal microscopy

Fig. 1 |

 1 Fig. 1 | High content imaging of the immunological synapse in lymphocytic cell lines. a. Schematic representation of the High content imaging pipeline. b. Representative images of NK-92 cells seeded on Poly-L-Lysine (PLL) or ICAM-1 plus anti-NKp30 / NKp46 Ab, stained for F-actin (green), perforin (yellow) and the nucleus (DAPI). Scale bars: 10 μm. c. Selected IS features analysed as fold change of ICAM-1 plus anti-NKp30 / NKp46 Ab over PLL. The data represent the mean of three separate experiments (n= 933-5860 cells). d. Mean nucleus area in pixels and median F-actin intensity per image across PLL and ICAM-1 plus anti-NKp30 / NKp46 Ab conditions. e. F-actin intensity distribution per cell across PLL and ICAM-1 plus anti-NKp30 / NKp46 Ab conditions. f. Representative images of Jurkat cells on PLL or ICAM-1 plus anti-CD3 Ab, stained for F-actin (green), LFA-1 (red) and the nucleus (DAPI). Scale bars: 10 μm. g. Selected IS features analysed as fold change of ICAM-1 plus anti-CD3 Ab over PLL. The data represent the mean of triplicates (n= 125-940 cells). h. Median F-actin and LFA-1 intensity per image across PLL and ICAM-1 plus anti-CD3 Ab conditions. i. F-actin intensity distribution per cell across PLL and ICAM-1 plus anti-CD3 Ab conditions.

Fig. 2 |

 2 Fig. 2 | Drug treatments yield changes to immunological synapse morphology. Graphs representing the fold change of IS parameters and representative images of NK-92 cells seeded on ICAM-1, anti-NKp30 and anti-NKp46, stained for F-

Fig. 3 |

 3 Fig. 3 | Morphological profiling of the NK cell immunological synapse upon drug treatment. Drug-treated NK-92 cell images were analysed with CellProfiler for an array of measurements and visualized using UMAP to position drug treated cells with respect to untreated cells from the same row. a. Latrunculin b. Jasplakinolide, c. Blebbistatin, d. Y-

Fig. 4 |

 4 Fig. 4 | CK-869 and SMIFH2 treatments alter immunological synapse architecture and lytic granule polarization in primary NK cells. a. Representative images of primary NK cells isolated from PBMCs of three normal donors seeded on ICAM-1, anti-NKp30 and anti-NKp46, stained for F-actin (green), perforin granules (yellow) and nuclei (DAPI) and either untreated or treated with four concentrations of CK-869 or SMIFH2. Scale bars: 10 μm. b-c Graphs representing the fold changes of immunological synapse parameters of primary NK cells treated with (b) CK-869 and (c) SMIFH2 with respect to untreated controls. The data represent the mean of 4 replicates for each drug concentration (60-409 cells).

Fig. 5 |

 5 Fig. 5 | High content imaging of the immunological synapse in ARPC1B deficient CD8+ T cells. a. Representative images of CD8+ T cells from normal donors and ARPC1B deficient patients seeded on ICAM-1 and either 1 or 10 μg/ml anti-CD3 and stained for F-actin (green), perforin granules (yellow), LFA-1 (red) and nuclei (DAPI). Scale bars: 10 μm. b. Characteristics of the immunological synapse of CD8+ T cells of the two ARPC1B deficient patients represented as fold change with respect to the average of the three normal donors seeded on ICAM-1 and 1 μg/ml anti-CD3. The data represents the mean of 6 replicates for each donor (10687-19353 cells) c. UMAP projection of CD8+ T cells morphological profiles from the two patients and the three normal donors seeded on ICAM-1 and10 μg/ml anti-CD3. d. Confusion matrix and class-wise performance on held-out data of a random forest model trained to discriminate between patient and normal donors based on the morphology of CD8+ T cells seeded on ICAM-1 and 10 μg/ml anti-CD3. e. Total and average importance of morphological features per measurement type and biological object described for the prediction of patient and normal donor CD8+ T cells seeded on ICAM-1 and 10 μg/ml anti-CD3. f. Specific lytic activity of patient and normal donor CD8+ T cells incubated with P815 cells coated with 10 μg/ml anti-CD3 after 4 h and 24 h. Values represent the mean of triplicates and error bars show SD. Significance is noted as *(P <0.05).

  you, without your endless support and patience. I cannot thank you enough for being there every single day on this journey.And finally, the most important people in this journey (and my life): my parents. Without you I could not be here today. Thank you for all the sacrifices you have made for me to be who I am and where I am. Thank you for your unconditional love and for always believing in me and making sure I knew it. I cannot possibly thank you enough for everything you have done for me. If superheroes existed, my parents would be ones. Everything I am and everything I will ever be, I owe to you. I love you beyond words and I am eternally grateful for you. Mom, dad, this (and everything I am and ever will be) is for you.

To my adorable grandma whom I love beyond words, and who has always believed in me. To my little brother Georges, thank you for always being by my side and for all your wise words and encouragement, and for always encouraging me to have faith in myself. To my Georges (the other special Georges in my life), I could not have done this without

Table 1 :

 1 HLH classification, causes and underlying defects

Table 2 :

 2

Main criteria used in the assessment of a defective immunological synapse in cytotoxic lymphocytes by immunofluorescence.

T cells before/after infection. Antigen

	-specific naïve CD8+T
	cells undergo differentiation into effector, early memory late memory and secondary memory cells 105 .
	The pattern of expression of lytic molecules varies based on the stage of differentiation they are at 13 .
	20-60% of early differentiated T cells express all granzymes (GzmB expression was minimal) and
	intermediate levels of perforin., granulysin or high-levels of perforin. Late differentiated cells express
	high levels of perforin and GzmB. The accumulated expression of Gzm B, granulysin and perforin as
	T cells differentiate to effector memory cells is in line with greater cytotoxicity observed in late
	differentiated effectors compared to early differentiated central memory or naïve CD8 T cells.

Table 1 : HLH classification, causes and underlying defects 168 .

 1

  203,204 . This was demonstrated by blocking WAS using the inhibitor Wiskostatin, which stabilizes WASp in its autoinhibited conformation and blocks its actin branching promoting activity. Using platinum replica electron microscopy, Orange et al. showed that treatment

with Wiskostatin abolished the branched actin meshwork in treated NK cells, but IL-2 restored this branched network, suggesting that IL-2 can activate a mechanism to circumvent WASp inhibition 204 .

Table 2

 2

	below summarizes the criteria used to assess IS defects in cytotoxic cells by
	immunofluorescence.
	Defect	Assessment measurement
	Actin Organization	Actin intensity at IS

Table 2 : Main criteria used in the assessment of a defective immunological synapse in cytotoxic lymphocytes by immunofluorescence.

 2

  . It is illuminated with LEDs and has a Sony CMOS camera. It offers digital confocal deconvolution, which enhances image contrast, thus decreasing exposure time and allowing for imaging of clearer cell structures, therefore a more robust image analysis.

	From: Molecular Devices website
	Logos Biosystems offers "Celena®" X high content imaging system, which offers laser autofocus,
	live cell assay support, objectives that range from 1.25x to 100x, a vast selection of LED filter cubes
	adapted to a wide array of fluorophores. It offers fluorescence imaging in four channels, brightfield,
	color brightfield, and phase contrast imaging, and a user-friendly interphase for both image acquisition
	and data analysis. It has a monochrome CMOS and an optional dual camera module.
	From: Logos Biosystems website.
	Puls and
	colleagues have shown that it is possible to develop a 3D model of tumor invasion that is amenable to
	high content imaging screens, in which they could test potential drugs 335 .

Zahir et al.

have recently shown that it is possible to use a high content imaging screen coupled to morphological profiling to assess the response of different strains of E. coli to different antibiotics 352 . analysisGE Healthcare Life Sciences offer the IN Cell Analyzer 6500HS system which comes as a successor to previous models, and which features the IRIS confocal technology for a better visualization and segmentation of biological objects, especially in thicker samples. It is a high content screening confocal microscope adapted for fixed and live-cell imaging and is illuminated with four lasers and has a sCMOS camera. Objectives range from 2x to 100x magnification, and the autofocus is maintenance is laser-based. The acquisition software of this device is user-friendly, and image analysis is performed with the IN Carta software, which also has an intuitive interface.

From: GE Life Sciences website Another high content imaging system is the Opera Phenix™, developed by Perkin Elmer. It is a confocal microscope endowed with a Nikpow spinning disk with dual view confocal optics and up to four sCMOS cameras that allow simultaneous acquisition in up to four channels, with the advantage of a low signal: background noise ratio, a wide dynamic range and high resolution.

Acknowledgements ................................................................................................................................ Summary .............................................................................................................................................. Résumé des travaux ............................................................................................................................. Acknowledgements .............................................................................................................................. Preface.................................................................................................................................................. Abbreviations .......................................................................................................................................

Acknowledgements Acknowledgements

This manuscript is the culmination of three years of doctoral studies, for which acknowledgments to

Acknowledgements

The authors thank Fatima-Ezzahra L'Faqihi-Olive, Valérie Duplan-Eche, Anne-Laure Iscache and Lydia De La Fuente-Vizuete from the cytometry platform of the CPTP and Isabelle Fernandes from the Organoid platform of the IRSD, Muriel Quaranta-Nicaise, Laurène Pfajfer, Michael Caldera, Marianne Guisset and Anton Kamnev for discussion and technical advice, Alessandro Aiuti, Marco Gattorno and Stefano Volpi for patient-derived cell lines. Y.G. performed the experiments, analyzed the results and wrote the paper, L.V. designed and performed data analysis and wrote the paper. A.R. contributed in image acquisition. A.F., J.M. and K.B. participated in research design and scientific discussions. L.D. designed the research, supervised the experiment analysis and wrote the paper.

The authors declare no competing interests.

Funding

This work was supported by the Vienna Science and Technology Fund (WWTF-LS16-060 to K.B. J.M. and L.D.), the INSERM Plan Cancer program (C15092BS to L.D.) and the Association Laurette Fugain (to L.D.).

Data availability

All the CellProfiler pipelines and morphological measurements used in this analysis are made available on FigShare with the DOI 10.6084/m9.figshare.11619960

[already available for reviewers with the following private link: https://figshare.com/s/3c06753839d77783a899].

The advantage that high content screens offer is the large assay format, where samples may be placed in 96 or 384 well plates instead of the traditional microscopy slides. Moreover, robotics has also been developed to minimize sample processing time and human error. High content imaging relies on fluorescence. Therefore, the typical fluorescent antibodies, proteins and dyes that are used in low throughput microscopy are commonly used. HCI may be used to establish a process of machine learning, where image "fingerprints" are extracted to define a specific cellular morphology, corresponding to a specific biological state. Once these fingerprints have been established, prediction models may be used to extract features from images and predict biological states 353 . Supervised training models refers to using a part of a whole set of images in order to establish these fingerprints, and then apply these models to the set and test their predictive performance. These steps can be summarized in figure 20, and image-based profiling will be elaborated further in the HCI analysis section.

B. Instrumentation: High content imaging requires specialized devices

Several companies have now worked on building high content imaging devices, to account for the growing need for such technologies in both basic research and drug discoveries.

ThermoFisher Scientific introduced the "ArrayScan" in 1999 and have over the years continues to produce several high content analyzers. They have recently released their latest model: the Cellnsight CX7 LZR high content analysis platform, which comprises fluorescence microscopy, as well as automated image analysis tools to extract relevant information from the imaged samples. Their latest system is endowed with objectives that range from 2x to 60x magnification, a seven-color laser illumination source for confocal imaging with spinning disk technology, a 5 color LED array for brightfield imaging and HCS Studio Cell Analysis Software which allows a user-friendly image acquisition setup and result analysis.

Supplementary Table 1 | Mean values and fold increase of immunological synapse parameters in

Supplementary table 2. Mean values and fold increase of immunological synapse parameters in Jurkat cells.

Mean values of individual parameters pertaining to the immunological synapse in Jurkat cells seeded on PLL or ICAM-1 and anti-CD3, and the fold change of the ratio of each mean value on the stimulated condition with respect to PLL. Intensity is measured in arbitrary units and area in (μm 2 ).

Discussion and perspectives

The role of the cytotoxic lymphocytes in the immune response and host protection in undeniable, and their role is emphasized further by the consequences of the diseases in which they are defective 165,397,398 . Moreover, their capacities are highlighted further by the developing cancer immunotherapies 399,400 . Therefore, it is essential to understand the mechanisms governing the activation and the effector capabilities of these cells. Cytotoxicity involves several dynamic steps, of which the immunological synapse has been shown to play a crucial role [START_REF] Mace | Cell biological steps and checkpoints in accessing NK cell cytotoxicity[END_REF] . Indeed, the immunological synapse has been proposed to be the sight of lytic granule convergence and their exocytosis in order to achieve target cell lysis 44,147 . Low throughput imaging approaches are traditionally used to study the IS assembly and architecture, and these approaches have helped shaped the current knowledge into IS regulation and the role of several genes in the dynamic process that is the formation of the lytic IS 44,201,224 .

Given the crucial role that the immunological synapse plays in the process of cytotoxicity, we proposed a high content imaging approach coupled to robust morphological profiling to assess the IS architecture in various cytotoxic lymphocyte populations. We posit that such an approach could be relevant to determine the activation state of these cytotoxic lymphocytes, which may reflect on their cytotoxic functions.

Annexes

I contributed to the Rey-Barrosso et al. paper by performing the immune phenotyping of JY cells for collagen and fibronectin (supplementary figure 1), as well as well as supplementary figure 4, in which the effect of extracellular matrix on the cells' ability to cluster was assessed.

Generating supplementary figure 4 was my first exposure to microscopy in a 96 well plate instead of slides or chambers.

However, imaging 96 wells for the course of 7 hours was a tedious process, despite being able to "save" a specific location in the well to re-image at a later point. By doing this experiment I was made aware of the importance of automation when acquiring images from a large number of wells, whether it be for fixed or live microscopy assays.

Discussions with Javier also exposed me to the different inhibitors of the actin/Myosin components, which was a basis for the drug screen performed during my PhD.