Relevance of antigen presentation by T cells on T cell differentiation

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Universidad Autónoma de Madrid Faculty of Sciences

Department of Molecular Biology

Relevance of antigen

presentation by T cells on T cell differentiation

Doctoral Thesis Viola Lucrezia Boccasavia

Madrid 2018

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Doctoral Thesis

Relevance of antigen presentation by T cells on T cell differentiation

This thesis is submitted by Viola Lucrezia Boccasavia in fulfillment of the requirements for the degree of Doctor in Molecular Biology

Thesis director: Dr. Balbino Alarcón Sánchez

Research professor in Consejo Superior de Investigaciones Científicas Centro de Biología Molecular Severo Ochoa

Universidad Autónoma de Madrid

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The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under REA grant agreement n°317057 and it was entirely performed under the direction of Balbino Alarcón at the Centro de Biología Molecular Severo Ochoa (CSIS-UAM).

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Acknowledgement

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List of the abbreviations

Ags Antigens

APC Antigen presenting cell ACK Erythrocyte lysis buffer Bcl6 B cell lymphoma 6 protein BM Bone Marrow

BSA Bovine Serum Albumin CCR6 Chemokine receptor type 6 CD Cluster of differentiation

cSMAC Central supramolecular activation clusters DC Dendritic cell

DMEM Dulbecco's Modified Eagle's Medium dSMAC Distal supramolecular activation clusters EDTA Ethylene Diamine Tetra-acetic Acid FBS Fetal Bovine Serum

FcγR Fragment crystalizable region FITC Fluorescein isothiocyanate Foxp3 Forkhead box protein O GDP Guanosine diphosphate GFP Green fluorescent protein

GM-CSF Granulocyte macrophage colony-stimulating factor GTP Guanosine triphosphate

IFN Interferon IL Interleukin

JAK Janus tyrosine kinase KDa kilodalton

Lamp1 Lysosome-associated membrane protein 1 LNSC Lymph node stromal cells

MCC Moth cytochrome c 88-103 peptide MHC Major histocompatibility complex NK Natural Killer

OVA Ovalbumin peptide

PBS Phosphate-buffered solution PD-1 Programmed Death-1 PE Phycoerythrin

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PMA Phorbol myristate acetate

pSMAC Peripheral supramolecular activation clusters RAR Retinoic Acid Receptors

RORγt Retinoic acid-related orphan receptor gamma t RPMI Roswell Park Memorial Institute medium RT Room Temperature

RT-q PCR Real time quantitative polymerase chain reaction STAT Signal transducers and activators of a transcription T-Box21 T cell specific T-Box transcription factor T-Bet TCR T cell receptor

Tfh T follicular helper cell

TGFβ Transforming growth factor beta Th cells T helper cells

TNFα Tumour necrosis factor alpha Treg Regulatory T cells

WT Wild type

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List of figures

Figure 1. Acquisition of MHC-II by TCR transgenic AND and OT2 T cells……….. 68

Figure 2. Acquisition of I-E^k MHC-II by TCR transgenic AND T cell by confocal microscopy ... 69

Figure 3. Acquisition of I-E^k MHC-II from DCs to T cells is actin cytoskeleton and Src signalling dependent.. ... 70

Figure 4. Acquisition of H-2k^b(MHC-I)/OVA peptide by TCR transgenic OT1 T cells.. .... 71

Figure 6. Acquisition of I-E^k MHC-II co-localize with Lamp1 and CD63 ... 74

Figure 7. Acquisition of I-E^k MHC-II and CD80 by ELYRA super-resolution. ... 74

Figure 8. Acquisition of I-E^k MHC-II by using ImageStream X Mark II Imaging Flow Cytometer. ... 75

Figure 9. Acquisition of I-E^k MHC-II by representative transmission electron micrographs (TEM).. ... 76

Figure 10. Acquisition of I-E^k MHC-II in vivo. A. ... 77

Figure. 11. T cells stimulated by DC cells express activation markers. ... 79

Figure 12. Proliferation assay. ... 80

Figure 13. Proliferation assay. A... 81

Figure. 14. Acquisition of I-E^k MHC-II and I-A^b MHC-II by confocal microscopy.. ... 81

Fig.15. Proliferation Assay with Cell Trace staining. ... 82

. ... 83

Fig.17. Differentiation in vitro at day 6. A. ... 86

Figure 18. Differentiation in vitro at days 3 of co-culture. A. ... 89

Figure 19. Differentiation in vivo... 90

Figure 20. Differentiation in vitro at earlier time points: CCR6 and CD25 markers. ... 92

Figure 21. Differentiation in vitro at earlier time points: CD69, CD44, CD25 and PD-1.. .... 93

Figure 22. CBA Assay. ... 94

Figure 23. Comparison between T-APCs (Pres) and Pres+Resp. ... 95

Figure 24. Microarray analysis.. ... 97

Figure 25. Different mRNA expression between Presenting and Responding:. ... 99

Fig.26.Conclusive Model of the 2^nd part. ... 100

Figure 27: Preliminary experiment in vivo with L. monocytogenes. ... 102

Figure 29. EAE model in reconstituted mice. A.. ... 106

Figure 30. EAE model: analysis of extracellular markers in secondary lymphoid organs ... 107

Figure 31. Model of RhoG defect.. ... 108

Figure 32. CD4⁺ T cell differentiation relies on the number of DCs present in the culture. ... 109

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Figure 33. CD4⁺ T cell differentiation relies on the number of DCs injected in vivo. A.. 111

Figure 34. CD4⁺ T cell differentiation relies on the number of viral particles administrated in vivo. ... 113

Figure 35. T-T interactions in vivo.. ... 116

Figure 36. Analysis of intracellular markers. ... 117

Figure 37. Model of the 3^rd part. ... 118

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List of Tables

Table 1. List of media: provides an exhaustive list of all media used in this study for culture

and maintenance of cell lines and primary cells. ... 40

Table 2. Oligonucleotides. Oligonucleotide sequences for mice genotyping. ... 42

Table 3. Reagents, sources and application ... 43

Table 4. List of buffers. ... 44

Table 5: List of antibodies used in this thesis. FC: Flow Cytometry IF: Immunofluorescence. 45 Table 6: List of antibodies used in this thesis. FC: Flow Cytometry IF: Immunofluorescence. 49 Table 7. List of primers used for qRT-PCR ... 59

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ABSTRACT

T cells are known to acquire parts of the APCs through the IS by a process known as trogocytosis (from the greek trogos, to gnaw). These APC fragments include MHC complexed to antigen (pMHC) and ligands for CD28 (CD80, CD86).

Trogocytic T cells not only acquire pMHC from the APC but also display it on their own membrane. Expression of the acquired pMHC by the T cell has been given either a negative regulatory role during the immune response or on the contrary, a positive activation role, indicating that T cells can become efficient APCs. Our own experiments in vitro indicate that T cells can take up pMHC complexes as well as co-stimulatory molecules such as CD80 and are able to express them on their membrane surface, forming large clusters. T cells can also acquire bystander pMHC and present it to naïve T cells of the bystander specificity.

Most interesting, we have been able to characterize the effector antigen presentation by T cells to other T cells of the same antigen specificity. We found that T Responding (Tresp) proliferate as a consequence of antigen presentation by T Presenting (Tpres). However, T Presenting cells proliferate much more vigorously than T Responding cells and express FOXP3 and other markers of regulatory T cells. Conversely, T Responding CD4⁺T cells more frequently become IFNγ or IL-17A producing cells. In fact, using microarray gene expression and qPCR analysis we show that T Presenting become preferably Tregs whereas T responding polarise towards Th17.

Our results suggest that T-T antigen presentation after trogocytosis process may have an impact on the pro-inflammatory versus the pro-proliferative and anti-inflammatory differentiation response and therefore, condition the adaptive immune response. Finally, we used mouse models to test the nature of these findings in vivo to assess how important is antigen presentation by T cells in response to pathogens and autoimmune disease.

The results presented in this thesis pave the way for exploring a novel mechanism of cellular communication for T cells as APCs that might be relevant in conditions of scarcity of pathogen-infected cells.

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PRESENTACIÓN

Se sabe que las células T adquieren partes de las APC a través de la IS mediante un proceso conocido como trogocitosis (del griego trogos, para roer).

Estos fragmentos de APC incluyen MHC complejado con antígeno (pMHC) y ligandos para CD28 (CD80, CD86). Las células T trogocíticas no solo adquieren pMHC del APC sino que también lo muestran en su propia membrana. La expresión de pMHC adquirida por las células T se ha asignado ya sea a un papel regulador negativo durante la respuesta inmune o, por el contrario, a un papel de activación positivo, lo que indica que las células T pueden convertirse en APC eficaces. Nuestros propios experimentos in vitro indican que las células T pueden tomar complejos pMHC, así como moléculas coestimuladoras como CD80 y son capaces de expresarlas en su superficie de membrana, formando grandes grupos. Las células T también pueden adquirir al espectador pMHC y presentarlo a las células T vírgenes de la especificidad del espectador.

Lo más interesante es que hemos podido caracterizar la presentación del antígeno efector por las células T a otras células T de la misma especificidad de antígeno. Encontramos que T Responding (Tresp) prolifera como consecuencia de la presentación del antígeno por T Presenting (Tpres). Sin embargo, las células presentadoras de T proliferan mucho más vigorosamente que las células de respuesta T y expresan FOXP3 y otros marcadores de células T reguladoras.

Por el contrario, las células T CD4 + que responden T se convierten con mayor frecuencia en células productoras de IFNγ o IL-17A. De hecho, usando la expresión del gen de microarrays y el análisis de qPCR, mostramos que la presentación de T se convierte preferiblemente en Treg mientras que la respuesta de T se polariza hacia Th17.

Nuestros resultados sugieren que la presentación del antígeno T-T después del proceso de trogocitosis puede tener un impacto en la respuesta de diferenciación proinflamatoria versus proproliferativa y antiinflamatoria y, por lo tanto, condicionar la respuesta inmune adaptativa. Finalmente, utilizamos modelos de ratón para probar la naturaleza de estos hallazgos in vivo para evaluar cuán importante es la presentación del antígeno por las células T en respuesta a patógenos y enfermedades autoinmunes. Los resultados

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presentados en esta tesis allanan el camino para explorar un nuevo mecanismo de comunicación celular para las células T como APC que podría ser relevante en condiciones de escasez de células infectadas por patógenos.

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INTRODUCTION

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Introduction

1. The immune cell network at a glance

We live surrounded by microorganisms present in our everyday environment, many of which cause disease. Yet despite this continual exposure we become ill only rarely. How does the body defend itself? The term

"Immunology" comes from the latin word immunitas and is the study of the body´s defence against infection.

One feature of the immune system is that it can form long-lasting memories of the pathogens it has previously encountered by creating cells called memory cells. Immunological memory, the ability of the body to

“remember” and respond rapidly and more vigorously to a pathogen upon subsequent encounters, has long been recognized in human history. The first documentation of immunological memory came from the Greek historian Thucydides, who vividly described the plague that struck the city of Athens at the beginning of the Peloponnesian war in 430 B.C., recounting that “this disease never took any man the second time”; it took us more than two millennia to understand that immunological memory is a fundamental feature of the adaptive immunity conveyed by B and T lymphocytes and forms the basis of vaccination; by exposing the immune system to a pathogen in a controlled, safe way, memory cells form and can efficiently fight off a future infection.

The most important organs of the immune system are the bone marrow and the thymus, considered as the two primary lymphatic organs where lymphocytes are formed and mature. Lymph nodes, tonsils, the spleen, Peyer’s patches, the mucosa- and gut-associated lymphoid tissues are the secondary lymphoid tissues where lymphocytes are activated. Importantly, the cells of the immune system are all derived from specialized hematopoietic stem cells (HSC) in the bone marrow from where they either migrate towards the thymus for development or circulate in the blood or lymph to detect pathogenic or malignant threats to the body. In order to do so, all immune cells rely on a

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distinct set of receptors with which they discriminate self/healthy from non- self/diseased tissues to ensure that their effector potential is only released in dangerous situations. In vertebrates, the cells of the immune system are classically assigned to either the innate or the adaptive immune system. This is done according to the type of recognition receptors used and the timing of the subsequent immune reaction but also by the ability of the cell to exert immunological memory.

2. T cell immune response

2.1 The innate and adaptive immune system

The immune system is a highly developed network of cells, which recognizes and fights pathogens. This system consists out of two core groups comprising distinct features, referred to as innate and adaptive immunity. In general terms, innate immune cells express surface receptors that recognize evolutionarily conserved structures (DNA, RNA, glycoproteins etc.) mainly derived from potential pathogens; the innate system is also able to sense “danger signals”, or danger associated molecular patterns (DAMPs) through different receptors (Matzinger 2002); innate immune defences are characterized by fast assimilation and rapid responses, which are of limited duration. They include humoral factors (e.g. complement and certain cytokines) and cellular components, a broad range of differentiated cells, neutrophils, macrophages, Dendritic Cell (DCs), Natural Killer cells (NK-cell) etc. each of these cell types has a specific differentiation pathway and exert specific and sometimes overlapping function. This allows early recognition of invading pathogens and subsequently either clearance through innate immune cell effector functions (e.g. phagocytosis, degranulation) or activation of the adaptive immune system. In particular, the process of phagocytosis is initiated by the formation of a phagocytic cup, leading to internalization of very large particles of bacteria that end up with the destruction of the pathogen. If the body’s first line of defence is not successfully in destroying the pathogens, after about four to

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seven days the antigen-specific adaptive immune response sets in. In contrast to the innate immune response, the adaptive needs more time to develop, it is more specific and last longer. It is crucial for antigens to interact with the immune system for an effective activation, especially with antigen presenting cells (APC) including monocytes, macrophages and, most importantly, DCs. Adaptive immune cells globally identify potential dangers based on their ability to recognize “non-self antigens”, which are elements that are not present in the human body under normal conditions. The second major attribute of the adaptive immune system is the ability of maintaining an “immunological memory” of previously encountered antigens; the appearance of memory B and T cells share the ability to be very quickly reactivated upon secondary infection with a similar pathogen (Zinkernagel and Doherty 1997).

There are two types of adaptive immune responses: humoral immunity mediated by various substances in the blood and antibodies produced by B lymphocytes, and on the other hand cell-mediated immunity that is the one we are going to focus on in this work and is mediated by lymphocytes, T and B lymphocytes primarily, antibodies and cytokines in the blood.

An adaptive immune response can be divided into a primary response and a secondary response. During a primary response, naive T cells encounter their cognate antigen, therefore get activated, differentiate and ultimately generate memory T cells. The secondary response is orchestrated by already existing memory T cells (generated during a primary response), which recognize the previously encountered antigen upon re-infection by the same pathogen, leading to its elimination.

3. Main characters of the adaptive immune response

3.1 APCs: Dendritic cells

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The specialized components of innate immunity that play a critical role in the initiation and development of adaptive immunity are called ‘APCs’. To play such an important role and keep the balance between health and disease, they have a unique set of features that enables them to operate at the interface of host defence and tolerance. Among various APCs, Dendritic Cells (from the Greek word for tree, “dendron”) are regarded as professional APCs since they share the ability to efficiently take up and process Ags for presentation to naïve T cells. DCs represent a heterogeneous populations of cells (Banchereau and Steinman 1998); are distinct from other APCs in that they possess stellate morphology, show elevated expression of major histocompatibility complex (MHC) I and II molecules as well as co-stimulatory molecules (cluster of differentiation CD40, CD80, CD86 and CD45), exhibit motility, and most importantly, switch from an Ag-capturing status to a T-cell sensitizing status called maturation (del Rio et al. 2010). 44 years after their discovery by Ralph Steinman, it is now confirmed that DCs possess characteristic T-cell sensitizing properties and control many aspects of immunity, forming a bridge between the innate and adaptive immune responses. Their role in initiating and coordinating adaptive immune responses is a consequence of their localization within tissues and their specialized ability for migration (Hu and Pasare 2013). DCs form a physical link between skin/mucosae in the periphery and secondary lymphoid organs, for they capture harmful pathogens in the periphery and induce the immune response by activating T lymphocytes. Upon phagocytosing pathogens, DCs can secrete important cytokines and mediate antiviral defence mechanisms (Mogensen 2009). To achieve these functions, DCs undergo a definitive maturation process where, after capturing invaded pathogens, they rearrange cytoskeletal structures to downregulate their phagocytic activity, process and present antigens to T cells (Granucci et al. 2003).

3.2 T lymphocytes

In the adaptive immune system, T cells are specialized in the recognition of peptide antigens presented in the context of a major histocompatibility complex (Zinkernagel and Doherty 1997). As a result, T cells can see antigen exclusively on the surface of an antigen presenting cells, such a dendritic cells or B cells.

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T cells are responsible for the special defence in the tissue, they recognize infected cells and they eliminate them from the body. During the course of an immune reaction, T lymphocytes develop into specialized effector cells: T helper cells or CD4 which contribute to the orientation of the immune response through secretion of cytokines, and T killer or cytotoxic cells or CD8 which can induce cell death in an antigen specific manner.

3.3 CD4⁺ T cells

CD4⁺T cells are one of the most versatile immune cell types and exhibit multi- faceted roles in regulating pathogen clearance and host protection. Generally, both naïve CD4⁺and CD8⁺ T cells live for few to several months. During this period, unless they encounter foreign Ags, CD4⁺ T cells inexorably migrate through the circulation and lymphoid organs, performing extensive sampling of self-pMHC, exiting secondary lymphoid organs, and returning to circulation (Cahalan and Parker 2006).

3.4 CD8⁺ T cells

CD8⁺ T cells are primarily involved in host immune responses against intracellular pathogens, e.g. bacterial and viral infections but also in anti-tumor immunity. Depending on their differentiation state, CD8⁺ T cells may commonly be partitioned into naïve, effector, effector memory and central-memory populations. Upon activation, T cells undergo massive proliferation while upregulating surface activation markers i.e. CD25, CD69, CD44, and producing major effector molecules such as IFN-γ, granzyme-B, perforin as well as Fas- L.

Activation of T cells by APCs requires the formation of a specific and long- lasting (up to 24h) interface, a very tight adhesion between the two cells, known as the immunological synapse (Dustin et al. 1997, Stoll et al. 2002, Mempel et al. 2004).

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4. The Immunological Synapse

T cells play a pivotal role in orchestrating the immune system. T cell responses are induced by antigen recognition through the T cell receptors (TCRs), which bind antigen peptide–major histocompatibility (MHCp) complexes on antigen-presenting cells (APCs). It was known that upon interaction between the T cells and the APCs, TCRs and other accessory molecules accumulated at the interface between the two cell types (Paul et al.

1987). T cells recognize cognate antigen by interacting with APCs to form immunological synapses (Huppa and Davis 2003). The term “synapse” was first used in the immune system by Norcross in 1984 in a prescient theoretical paper (Norcross 1984) describing the accumulation and function of various molecules at the T cell–APC interface, and, ten years later, Paul revived this term (Paul and Seder 1994). Similarly to the CD4⁺ T cell–APC synapse, Kupfer noticed the reorientation of the microtubule organizing center (MTOC) and Golgi apparatus toward the cytotoxic T lymphocyte (CTL)–target cell interface as an early event in CTL killing. Later, his group reported membrane and cytoskeletal reorientation at the junction between a T cell–B cell conjugate, leading to the important discovery of the supramolecular activation cluster (SMAC), a highly patterned clustering and segregation of cell surface molecules, particularly antigen receptors and adhesion molecules (Monks et al.

1998, Monks et al. 2015). The immunological synapse has been identified not only in the T cell–APC conjugates but also at the interface between B cell–

membrane-bound antigen (Fleire et al. 2006), NK cell– target cell (Orange 2008), and NKT cell–CD1d-expressing cell (McCarthy et al. 2007).

After this historic digression, we can define the immunological synapse as a special molecular architecture for recognition and signalling, where the receptors and adhesion molecules could be structurally and kinetically organized for the initial and sustained T cell activation (Monks et al. 1998);

(Grakoui et al. 1999). The concept of the immunological synapse beautifully correlated with what was known about T cell antigen recognition and activation;

however, this model could not explain early activation events, which can occur

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within 1 min. A much smaller signalling unit was predicted to form prior to the mature immunological synapse formation. Indeed, the TCR microcluster was discovered as a signalling cluster containing receptors, accessory molecules, and downstream signalling molecules (Bunnell et al. 2006); (Campi et al. 2005), (Yokosuka and Saito 2005). Microclusters dynamically change the localization and the assembled molecules at the immunological synapse and induce initial and sustained TCR signalling as well as costimulation signals (Depoil et al.

2008); (Yokosuka et al. 2008). The centripetal motion of TCR microclusters is dependent on constant remodelling of the actin cytoskeleton (Lasserre and Alcover 2010). This model is now known to describe the signalling of other lymphocytes, including B cells, natural killer (NK) cells, and natural killer T (NKT) cells (Davis and Dustin 2004).

4.1 Architecture of the Immunological Synapse

The immunological synapse is traditionally characterized by a “bull’s eye”

structure, c-SMAC, and peripheral-SMAC (p-SMAC) (Monks et al. 1998);

(Huppa and Davis 2003); (Dustin 2009) (Fig. 1). The major components of the c-SMAC are key molecules for T cell signalling, such as TCR/CD3–MHCp, CD28 – or cytotoxic T-lymphocyte antigen-4 (CTLA-4) – CD80/CD86, and protein kinase C y (PKCy). In contrast, the p-SMAC is composed of cytoskeleton-related or adhesion molecules structurally supporting the immunological synapse, such as leukocyte function- associated antigen-1 (LFA-1)/talin – intracellular adhesion molecule-1 (ICAM-1) and CD2–

CD48/CD58. The distal-SMAC (d-SMAC) was defined later as a region enriched in molecules with long extracellular domains, such as CD45 (Freiberg et al. 2002) and CD43 (Allenspach et al. 2001); (Delon et al. 2001); (Revy et al. 2001); (Roumier et al. 2001); (Stoll et al. 2002). It was thought that the c- SMAC mediates antigen recognition and subsequent T cell activation, whereas the p-SMAC supports T cell–APC conjugation and maintains the architecture of the immunological synapse.

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Illustration 1. Architecture of the conventional immunological synapse. (a) The CD3 core is clearly identified at the stable conjugation between a T cell and an APC by fluorescence- labeled anti-CD3e antibodies (lateral view, top). (b) The alignment of the receptors and the adhesion molecules are considered to be ordered by size of ectodomain (Davis and van der Merwe 2006); T cell receptor (TCR)/CD3 complex – MHC-peptide (MHCp), CD28/protein kinase C y (PKCy) – CD80/86, cytotoxic T-lymphocyte antigen 4 (CTLA-4) – CD80/CD86, Agrin, and lysobisphosphatidic acid (LBPA) in the c-SMAC; CD2–CD48/CD58, leukocyte function-associated antigen-1 (LFA-1)/ talin–intracellular adhesion molecule 1 (ICAM-1), F- actin, and CD4/Lck in the p-SMAC; and CD43/moesin, CD45, and F-actin in the d-SMAC Adapted from (Davis and Dustin 2004).

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4.2 Localization of a APC-T cell interaction

Particulate antigens, as well as antigen-containing dendritic cells and macrophages, enter the lymph node via the lymph through afferent lymphatic vessels. Lymphocytes also arrive in the lymph node via afferent lymphatic vessels, as well as from the blood through specialized high endothelial venules.

The cortex of the lymph node consists of B cell follicles (containing B cells and follicular dendritic cells) and a T cell zone (made up of mostly T cells and dendritic cells). The inner medulla contains strings of lymphocytes and macrophages known as medullary cords, as well as medullary sinuses that drain into the efferent lymphatic vessels and help guide lymph and activated lymphocytes into the blood. Naïve T cells are in constant motion, scanning the lymph node at high rates (10-15 µm/min average, 25 µm/ min burst speeds) in search for the appropriate antigen and danger signals are capable of contacting 5000 dendritic cells in one hour (Fooksman et al. 2009).

5. The mechanisms of antigen uptake

The recognition between cells of the immune system involves the activation of pathways for receptor internalization. Antigen uptake can occur at different levels. There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells; in addition to the classical clathrin-dependent mechanism of endocytosis, several endocytic pathways that do not use clathrin have also emerged (Mayor and Pagano 2007) .

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Illustration 2. Pathways of entry into cells: Large particles can be taken up by phagocytosis, whereas fluid uptake occurs by macropinocytosis. Numerous cargos can be endocytosed by mechanisms that are independent of the coat protein clathrin and the fission GTPase, dynamin.

Most internalized cargos are delivered to the early endosome via vesicular (clathrin- or caveolin- coated vesicles). Adapted from (Mayor and Pagano., 2007).

Endocytosis is defined as the process of engulfing molecules. It has four subcategories which are clathrin-mediated endocytosis, caveolae, macropinocytosis, and phagocytosis.

Clathrin-mediated endocytosis involves molecules that must be 100 nanometers in diameter in order for them to absorb and digest. Caveolae (small invaginations of the cell’s plasma membrane, composed of lipids and caveolin), on the other hand, absorb particles that are less than 50 nanometers. Lastly, macropinocytosis engulfs particles sized 0.5-5 nanometers.

Particularly interesting is the mechanism of phagocytosis; the term comes from the Greek word “phagein” meaning “to devour,” “kytos” meaning “cell” and “- osis” meaning “process” which is, on the other hand, the process of engulfing nutrients with a particular size only which is 0.5 nanometers in diameter.

Phagocytosis is an active and highly regulated multi-step complex process that involves specific cell-surface receptors and signaling cascades. In mammals, it takes place primarily in specialized cells, such as macrophages, monocytes,

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and neutrophils, which function to clear away large pathogens such as bacteria, parasites and large cell debris.

Phagocytic uptake involves actin dynamics including polymerisation, bundling, contraction, severing and depolymerisation of actin filaments.

Lately, it has been described a new process for the incorporation of membrane lipid material, that requires an intact contact between cells, without any modifications, and it is called Trogocytosis. This is a distinguishable phenomenon in contrast to Phagocytosis, the process of engulfing whole pathogens and death-cell fragments by phagocytes.

Illustration 3. from Dynamics of macrophage trogocytosis of rituximab-coated B cells. (Pham et al. 2011).

6. Trogocytosis: phenomenology

Trogocytosis (from trogo, to nibble in ancient Greek.) corresponds to the active capture of membrane fragments by another cell. This phenomenon, the unidirectional TCR-mediated capture, seems to occur very broadly among cells of the immune system. Indeed, after the formation of an immune synapse, lymphocytes will extract a significant portion of the components of the plasma membrane of the other cell that was involved in the formation of that synapse (Joly and Hudrisier 2003). Trogocytosis has been documented in α/β T cells (Arnold and Mannie 1999), γ/δ T cells (Espinosa et al. 2002)B cells (Batista et al. 2001), natural killer cells (Carlin et al. 2001), monocytes, neutrophils APCs (Herrera et al. 2004) and tumor cells (Vanherberghen et al. 2004). By nibbling rigid areas from the surface of other cells, lymphocytes, and possibly other

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leukocyte types, may not only be surveying their neighbouring cells for the development of dangerous pathogens, but may also have an important role in the refuse disposal of membrane docks that may be unwanted at the surface of resting healthy cells. Lymphocytes inherit many different molecules from conjugating cells. Some of these molecules, which are not transcribed by lymphocytes, may directly or indirectly influence the phenotype and function of the lymphocytes. From an evolutionary perspective, trogocytosis may have developed initially as a symbiotic arrangement: leukocytes may be ‘feeding´off other cell types in return for undertaking the defence of the organism against pathogens (Joly and Hudrisier 2003).

So, it seems that trogocytosis could be a vector for intercellular communication. T cells may acquire peptide/MHC complexes at the T-APC interface, forming clusters within minutes that are subsequently acquired and internalized in T cells.

Although the physiological consequences of the intercellular transfer are still questionable, several observations suggest an active role in the immune responses. Two schools of thinking have been described: the first one shows a positive regulation by CD8⁺ T cells that acquire MHC class II molecules in vitro and in vivo in response to a viral infection, a transfer which confers them the capacity to directly activate CD4⁺ T cells. The connotation of this intercellular transfer of antigen-MHC complexes may expand the repertoire of cells that can act functionally as APCs and regulate the immune response.

Conversely, the second show that intercellular transfer may downregulate immune responses. There is some evidence that the presence of APC- derived peptide MHC complexes on T cells may render them susceptible to fratricide lysis. As a negative consequence, active lymphocytes that naturally spend time in close proximity to pathogens, could contribute to the spread of pathogens within the host either through direct capture of the pathogen or its genome.

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Illustration 4. TCR Internalization and Trogocytosis in T Cells.

Constitutive TCR turnover occurs in resting cells. Upon stimulation, TCR nanoclusters aggregate to signaling-active microclusters that can be internalized in the pSMAC in a clathrin-dependent manner. Once the microclusters have reached the cSMAC, patches of the APC containing the pMHC molecules are trogocytosed in a TC21- and RhoG-dependent mechanism. Trogocytosed membrane proteins from the APC can be re-expressed on the T cell. Adapted from (Dopfer et al.

2011).

An effective immune response is vital in the protection against invading foreign pathogens: CD4⁺ T cells play a pivotal role in host defence by secreting cytokines to drive appropriate immune responses.

7. CD4+ T cell differentiation

The differentiation of CD4⁺ T cells into various T helper subsets, in vivo, is predominantly dictated by the cytokine milieu surrounding the T cell at the time of its first encounter with an antigen.

The induction of CD4⁺ T cell differentiation is generally thought of as a two- step process: cytokine stimulation which triggers JAK/STAT signalling cascades, followed by induction of master regulator transcription factors.

( D a r n e l l e t a l . 1 9 9 4 ) . Expression of so-called “master regulators” has been identified for all of the T helper cell populations introduced in this dissertation: T helper 1 (Th1), T helper 2 (Th2), T helper 17 (Th17), T

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follicular helper (Tfh) and regulatory T cells (Treg). Those master regulators are, in fact, transcription factors defined as essential for promoting differentiation into respective T helper cell populations ( Z h u e t a l . 2 0 1 0 ) . P re se n te d b e l o w in th e Il lu st ra t io n 5 an overview of T helper cell populations, the cytokine-dependent STAT-signaling needed for their differentiation, their inhibitory function against each other and the distinct set of cytokines that different T helper cell populations.

Illustration 5. Overview of naïve CD4+ T cells differentiation into T helper subsets.

The outer circles represent naïve CD4⁺T cells and different helper T cell subsets. Cytokines depicted above arrows drive differentiation towards the respective subsets. Inner circles contain designated master-regulator (transcription factors) and outer circles show STAT- molecules associated with the respective differentiation process. Cytokines in italic are effector cytokines produced by corresponding T helper cells.

CD4⁺ T cells have the potential to differentiate into multiple effector T helper (Th) cells depending on TCR signal strength and on the cytokine milieu, which

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is mainly shaped by innate immune cells. In this context, dendritic cells (DCs) represent master regulators of effector T cell responses to invading pathogens. DCs can indeed instruct T cell polarization by providing proper antigen-dependent TCR stimulation via major histocompatibility complex (MHC) molecules, as well as co-stimulation through surface receptors, which are up-regulated on the DC surface following pattern recognition receptor engagement by pathogen-associated molecular patterns. In addition, according to the qualitative/cytokine model of differentiation, DCs have the potential to instruct T cell differentiation by altering the microenvironment through the release of specific cytokines, including interleukin (IL)-12, IL-4 or IL-6 and Transforming Growth Factor β (TGF-β), which are Th1-, Th2- and Th17-polarizing cytokines, respectively.

i. Th1 cells

T helper 1 cells mediate immune responses mainly against intracellular viral and bacterial pathogens. The major Th1 related cytokine is IFN-γ, and t h e designated master regulator for Th1 differentiation is transcription factor T-bet (Szabo et al. 2000).

IL-12 receptor beta (IL-12Rβ) 1 is constitutively expressed on CD4⁺ T cells (Kano et al., 2008). However, upon TCR stimulation IL-12Rβ2 is upregulated, thereby forming the IL-12 receptor complex and increasing responsiveness to IL-12 (Gadina et al. 2001) ( S za b o e t a l . 1 9 9 7 ) . Engagement of IL-12 triggers STAT-4 signalling which induces IFN-γ production, leading to an autocrine signal amplification involving IFN-γ receptor signalling via STAT-1. Subsequently, sustained STAT-1 signalling drives the expression of master regulator, T-bet, and therefore Th1 cell differentiation ( L i g h v a n i e t a l . 2 0 0 1 ) .

Macrophages and DCs are the major source of IL-12 for CD4 T cells during initiation of Th1 differentiation. Th1 cells then migrate to infected tissue through up-regulation of appropriate chemokine receptors e.g.

CXCR3 ( B o n e c c h i e t a l . 1 9 9 8 , S a l l u s t o e t a l . 1 9 9 8 ) and CCR5

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(Loetscher et al. 1998) (Loetscher et al. 1998). Even though Th1 cells have been reported to produce and secrete granzyme B (a serine protease important for contact dependent cytotoxic activity), their major role relies on the ability to produce high amounts of interferon-γ. This localized Th1- mediated change in the cytokine environment provides help in attracting and activating other immune cells such as macrophages, NK cells and cytotoxic CD8 T cells, which in turn mediate target killing through phagocytosis or degranulation of cytotoxic agents.

ii. Th2 cells

T helper 2 cells are responsible for host immunity against extracellular parasites such as helminths. Cytokines associated with Th2 responses include IL-4, IL-5, IL-13 and IL-25. Moreover, the master regulator driving Th2 differentiation is transcription factor GATA-3 ( Z h e n g a n d F l a v e l l 1 9 9 7 ) .

Activation of CD4⁺ T cells in the presence of IL-4 leads to STAT-6 activation (Takeda et al. 1996), through w h i c h chromatin signalling leads to upregulation and sustained GATA-3 expression (Onodera et al. 2010).

Moreover, IL-4 driven GATA-3 expression selectively stimulates commitment to Th2 ( Z h e n g a n d F l a v e l l 1 9 9 7 ) (, while suppressing Th1, Th17 and Tfh cell differentiation directly or through upregulation of transcriptional repressor Gfi- 1 (Zhu et al. 2002) and B – lymphocyte induced maturation protein 1 (Blimp-1) ( C i m m i n o e t a l . 2 0 0 8 ) ; ( L i n e t a l . 2 0 1 3 ) . Other transcription factors such as c-Maf, IRF4 and NFATc2 work collaboratively forming a transcriptional complex in the promoter region of the il4 gene to stimulate IL-4 production and subsequently Th2 differentiation (Rengarajan et al. 2002). Interestingly, c-Maf and Gfi-1 play a major role in regulating STAT-5 mediated IL-2 signalling (Zhu et al.

2006) (Ho et al. 1998), which cooperates with IL-4 induced STAT-6 signalling to drive Th2 polarization (Cote-Sierra et al. 2004). In vitro experiments have shown that constitutive expression of STAT -5 in CD4+ T cells allows Th2 differentiation even in the absence of IL-4 and in presence o f

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I L - 1 2 . ( Z h u e t a l . 2 0 1 0 ) . However, in the absence of GATA-3 T cells failed to commit to Th2 cells, demonstrating that although IL-4 independent Th2 polarization mechanisms exist, GATA-3 is signalling ( Z h e n g a n d F l a v e l l 1 9 9 7 ) .

iii. Th17 cells

Th17 cells play a critical role in host defence against extracellular pathogens and particularly in the gut mucosa. Effector cytokines produced by this subset are IL-17 (or IL-17A), IL-17F, IL-21 and IL-22. Moreover, the transcription factor RAR-related orphan receptor (ROR)γt has been established as master regulator of Th17 cell differentiation ( I v a n o v e t a l . 2 0 0 6 ) .

The discovery of a novel cytokine chain, IL-23p19 (Oppmann et al. 2000) was a key finding for the identification and detailed description of Th17 cells. It became evident that the heterodimeric IL-12 receptor would share the same beta subunit (IL-12p40) with what is known today as the IL-23 receptor. Until that time, self-reactive Th1 cells were thought to be the major cell type involved in experimental autoimmune encephalomyelitis (EAE). However, by comparing T cell immune responses of IL-12p35- deficient mice with IL-23p19 knockout mice, Cua and colleges managed to demonstrate that IL-23 but not IL-12 would induce EAE, through expansion of IL-17 producing T cells ( C u a e t a l . 2 0 0 3 ) . A follow- up study showed different gene expression profiles for Th1 cells compared to IL-17 producing T cells, which led to the introduction of the ThIL-17 or Th17 subset (Langrish et al. 2005).

Despite the early recognition of IL-23-driven IL-17 production in T cells, it was unclear how CD4⁺ T cells, which do not express the IL-23 receptor, would differentiate into Th17. The presence of other mediators, such as transforming growth factor-beta (TGF-β) and IL-6 (or IL-21 in human), was found to be crucial for initiating Th17 differentiation ( M a n g a n e t a l . 2 0 0 6 , V e l d h o e n e t a l . 2 0 0 6 ) . Indeed, TFG-β promotes

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RORγt expression, while at the same time repressing its function through FOX-P3.

iv. Treg cells

Tregs are indispensable for maintenance of immune homeostasis.

The main function of Tregs is down-modulating immune responses to prevent autoimmunity and eliminate potential auto-reactive cells. TGF-β and IL-10 are the predominant effector cytokines produced by Tregs and the master regulator of this subset is transcription factor forkhead box P3 (Fox-P3) (Fontenot et al. 2003, Hori et al. 2003); ( K h a t t r i e t a l . 2 0 0 3 ) . Of note, previous to the identification of Fox-P3, Tregs were identified by constitutive surface expression of CD25, hence, in some publications they are referred to as CD4⁺ CD25⁺ T cells.

Treg cells can be divided into thymic-derived natural regulatory T cells (nTregs) (Josefowicz and Rudensky 2009) and extrathymically-derived induced regulatory T cells (iTreg) according to CD25 expression ( B i l a t e a n d L a f a i l l e 2 0 1 2 ) .During thymus development, hyperresponsive CD4 T cells are mostly eliminated from the T cell pool. However, a small fraction of those cells differentiate into nTregs and acquire the ability to react to self- antigen inducing and regulating central tolerance, specific to self-antigens ( J o s e f o w i c z a n d R u d e n s k y 2 0 0 9 ) . In contrast, iTregs acquire their suppressive potential in the periphery and they are a natural by-product of any ongoing immune response. This process can be divided into two mechanistically distinct categories of which the first one is the better understood. 1) iTreg differentiation can be induced when T cells are activated in the absence of co-receptor stimulation e.g. under non- inflammatory conditions (Kretschmer et al. 2005). 2) iTreg differentiation is also induced in the course of an immune response, concomitant to the generation of cognate effector T cells ( G o t t s c h a l k e t a l . 2 0 1 0 ) . Thus, iTregs have the ability to introduce immunological tolerance to (foreign) elements previously unknown to the immune system.

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Tfh cells are essential in providing help for B cell maturation, class switch recombination (CSR) and somatic hypermutation (SHM) through which they modulate humoral responses. Tfh helper cells can be distinguished from other CD4⁺ T cells lineages by their low expression levels of cytokines (IFN-γ, IL-4, IL-17) and transcription factors (T-bet, GATA3, and Rorγt) characteristic of Th1, Th2, and Th17 cells, respectively. (Crotty 2014). Furthermore, Tfh cells express a unique combination of effector molecules that are critical for their development and function, including high levels of the surface receptors ICOS,CD40 ligand (CD40L), OX40, BTLA and CD84, the cytoplasmatic adaptor protein SLAM-associated protein (SAP), and the transcription factors Bcl-6 and c-Maf (Nurieva et al. 2009).

Tfh cells are CXCR5hi, CCR7lo and canonically secrete IL-21, IL-4 and CXCL13 (Kroenke et al. 2012) . CCR7 is a chemokine receptor needed for migration to the T cell zone, c-Maf is a transcription factor involved in Tfh differentiation and PD-1 is a marker associated to exhaustion in cytotoxic CD8 T cells;

8. Ras superfamily

The Ras superfamily consists of 150 Ras GTPases, known as small or monomeric G proteins with low molecular weight (20–30 kDa). The Ras superfamily is divided into five large families: Ras, Rho, Rab, Ran and Arf.

(Goitre et al. 2014).

The story of small GTPases started more than three decades ago with the discovery of the Ras oncogenes, which was soon followed by the discoveries of related proteins now forming the Ras superfamily (Bourne et al. 1990). The three human Ras proteins, H-Ras, K-Ras, and N-Ras, are the founding members of this large superfamily of small GTPases with evolutionarily conserved orthologs found in Drosophila, Caenorhabditis elegans, Saccharomyces cerevisiae, Saccharomyces pombe, Dictyostelium, and plants.

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This superfamily is divided into families and subfamilies on the basis of sequence and functional similarities.

Illustration 6. Dendrogram of the small G protein superfamily: Distribution of the members of the 5 different families of Ras superfamily: Ras, Rab, Ran, Rho and Arf. Taken from: Takai et al.

(2001) Small GTP-Binding Proteins.

They are monomeric GTP-binding and hydrolysing (GTPases) proteins that act as binary, GDP-/GTP-regulated, molecular switches in coupling extracellular signals to intracellular signalling networks that regulate a wide range of fundamental cellular processes, including proliferation, differentiation, morphology, polarity, adhesion, migration, survival, and apoptosis. When activated and bound to GTP, they interact with downstream effectors; inactive GDP-bound, Rho family G proteins are thought to be cytosolic and bound to guanine dissociation inhibitor (GDI). In order to cycle between GDP- and GTP- bound states, Ras proteins need guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP). GEF activity is required to activate Ras proteins by catalysing GDP exchange to GTP. GAPs increase the intrinsically low catalytic activity of G proteins causing GTP hydrolysis and rapid inactivation.

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The Rho (Ras homologous) family of small GTPases is closely related to the Ras family and is composed of 23 members, which are involved in signalling networks regulating actin cytoskeleton organization, cell adhesion, polarity and motility, cell-cycle progression, and gene expression (Heasman and Ridley 2008). According to their homology in the amino-acid sequence, this group is divided into 6 subfamilies: Rho, Rac, Cdc42, Rnd, RhoBTB y RhoT/MIRO (Wennerberg and Der 2004).

The first function attributed to the GTPases of the Rho family was controlling cytoskeletal polymerization; the actin cytoskeleton is a constantly evolving network formed of higly dynamic polarized actin filaments (Alberts et al., 2014). The well-studied proteins considered as prototypical of this family of Rho GTPases are RhoA, Rac and Cdc42. Each of them acts as a link between

signalling

through membrane receptors and the assembly or disassembly of the actin cytoskeleton (Bustelo et al. 2007).

RhoG

RhoG is a member of the Rho family, a classically regulated GTPase, most closely related to Rac, sharing the homology with Rac1 and Cdc42 of 72% and 62% respectively. The Rho family member RhoG was identified as a serum inducible gene in fibroblasts (Vincent et al. 1992).

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Illustration 7. RhoG can be located hijacked in its inactive form in the cytoplasm by a specific GDI. After appropriate receptor stimulation, some form of modification at the GDI level should favor the release of the GTPase and the action of the corresponding GEF (TRIO) for the exchange of GDP by GTP. During this process, RhoG must be located on the membrane where it performs its function forming a trimeric complex with ELMO and Dock 180, this being last GE1 of Rac1, thus promoting the activation and the induction of actin polymerization by this GTPase.

Adapted from (Elfenbein et al. 2009).

In various cell types, RhoG regulates the actin cytoskeleton and is involved in filopodia formation (Gauthier-Rouviere et al. 1998), membrane ruffling (Bellanger et al. 2000), neurite outgrowth (Katoh et al. 2000), in an evolutionarily conserved process of phagocytosis of apoptotic cells in macrophages (Nakaya et al. 2006), macropinocitosis or endocytosis mediated by caveolae(Prieto-Sanchez et al. 2006) and control of granule secretion present in neuroendocrine cells (Alabed et al. 2006) T-cell spreading (Vigorito et al. 2004), dendritic spine morphogenesis (Kim et al. 2011) and lamellipodia formation (Ho and Dagnino 2012). Its cellular localization includes plasma membrane, intracellular vesicles and Golgi apparatus (Gauthier-Rouviere et al.

1998, Prieto-Sanchez et al. 2006). It has been described that several GEFs can control the activity of RhoG, and TRIO is the most recognized. TRIO has been described as a protein with triple function due to the presence of 3 different functional domains (Debant et al. 1996): two GEF DH domains (Bellanger et al. 2000), one with specificity for activation of RhoG / Rac1 (GEF

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D1) and another for RhoA (GEF D2), and a serine-threonine protein domain kinase (PSK).

Once activated, RhoG controls the activity of Rac1 and Cdc42, through the union with ELMO (deBakker et al. 2004); (Katoh et al. 2006) (Katoh and Negishi 2003)forming a trimeric complex with Dock180 (Gumienny et al. 2001).

The functions of RhoG in lymphocytes have been investigated in cell culture systems and using constitutive RhoG ^−/− mice (Vigorito et al. 2004) where it has shown that a deficiency in RhoG does not lead a major impact upon the development of either B or T cells. However, RhoG-deficient lymphocytes show a modestly enhanced response to antigen challenge and respond better to in vitro stimulation than their wild-type counterparts(Vigorito et al. 2004).

We already know, from previous studies, that T cells are able to acquire antigens from other cells and RhoG is implicated in this process; in this thesis we investigate the meaning of this acquisition in a new context that drives T cell differentiation.

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MATERIALS AND METHODS

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Materials and Methods

1. Materials

1.1 Cell lines

DCEKs is a cellular line from fibroblasts transfected with plasmids encoding I-E^kand CD80. These cells were cultured in DMEM with 10% fetal bovine serum (FBS) supplemented with 2Mm L-Glutamine, 100 U/ml penicillin and 100 U/ml streptomycin.

Naïve T lymphocytes isolated from mouse peripheral lymphoid organs, were maintained in RPMI with 10% fetal bovin serum (FBS) supplemented with 2mM L-Glutamine, 100U/ml penicillin and 100U/ml streptomycin, 10mM Sodium pyruvate and 20µM β-Mercaptoethanol.

Dendritic cells were generate from bone marrow cultured with RPMI-1640 10% FBS, supplemented with 100U/ml penicillin, 100U/ml streptomycin, 10 Mm sodium pyruvate, 20 µM β-Mercaptoethanol and 20 ng/ml of Granulocyte macrophage colony-stimulating factor (GM-CSF).

Table 1. List of media: provides an exhaustive list of all media used in this study for culture and maintenance of cell lines and primary cells.

Product Commercial Brand

RPMI 1640 CBMSO Culture service

DMEM CBMSO Culture service

Culture plates BD-Falcon

FBS Sigma

L-Glutamine CBMSO Culture service

Penicilin CBMSO Culture service

Streptomycin CBMSO Culture service

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βMercaptoethanol Sigma

AANE CBMSO Culture service

GM-CSF Peprotech

Table 1: List of media used in this thesis

1.2 Mice

A range of lines of genetically modified animals were used in this thesis, from different

sources. Different mice models characterized by RhoG GTPases deficiency and the expression of transgenic TCR were used.

Non-transgenic C57BL/6: express the allele CD45.2 of the Ptprc gene.

Congenic C57BL/6 CD45.1: express the other allele CD45.1 of the Ptprc gene. These animals were used to carry out adoptive transfer experiments in order to differentiate the donor form the receptor cells. Those mice were kindly provided by Dr. Carlos Ardavín (CNB, Madrid).

Rhog^-/-: these mice were found in C57BL/6 background and were generated as it is described in (Vigorito et al. 2004)

Rhog^-/-OT-II: these mice were crossed with mice transgenic for the OT-II TCR (Vα2/Vβ5) specific for a peptide 323-339 of chicken ovalbumin presented by I-A^b (Hogquist et al. 1994), (Barnden et al. 1998).

Rhog^-/-AND: these mice were crossed with mice transgenic for the AND TCR (Vα11.1/Vβ3) specific for a peptide MCC presented by I-E^kMHC (Kayne et al. 1989).

Cd3ε ^-/- : these mice were obtained from Jackson Laboratories (DeJarnette et al. 1998).

Congenic C57BL/6 x B10.BR: APCs expressing both the I-E^kand I-A^b MHC class II molecules were generated from bone marrow precursors isolated from a mouse line (KB) derived from an original cross between B10.BR (H-2^k

Relevance of antigen presentation by T cells on T cell differentiation

Universidad Autónoma de Madrid Faculty of Sciences

Department of Molecular Biology

Relevance of antigen

presentation by T cells on T cell differentiation

Doctoral Thesis

Relevance of antigen presentation by T cells on T cell differentiation

Acknowledgement

List of the abbreviations

Table of contents

List of figures

List of Tables

ABSTRACT

PRESENTACIÓN

INTRODUCTION

Introduction

1. The immune cell network at a glance

2. T cell immune response

3. Main characters of the adaptive immune response

4. The Immunological Synapse

5. The mechanisms of antigen uptake

6. Trogocytosis: phenomenology

7. CD4+ T cell differentiation

8. Ras superfamily

signalling

MATERIALS AND METHODS

Materials and Methods

1. Materials

Product Commercial Brand