Distinct phenotypes of antigen-selected CD8 T cells emerge at different stages of an in vivo immune response.

We have previously described a unique system for identifying Ag-selected CD8 T cells during an in vivo response in normal mice. In this system, lymphocytes isolated from DBA/2 mice injected i.p. with HLA-CW3 transfected syngeneic (H-2d) P815 cells show a remarkable expansion of CD8 cells that utilize TCR expressing the V beta 10 gene segment and additional structural features characteristic of Kd-restricted CW3-specific CTL clones. We have now taken advantage of this system to characterize the surface phenotype of CD8 cells selected by Ag in vivo. We observed several distinct phenotypes at different stages of the response. At the peak of the response, Ag-selected cells were low in CD62L and CD45RB expression but displayed high levels of CD44. In addition, there was a partial down-regulation of CD8 and TCR. Cells of this phenotype were present in lymphoid tissues for several mo after immunization. Much later in the response, Ag-selected cells expressed higher levels of CD8 and TCR. Moreover, a distinct subset of these long-term immune cells emerged that now expressed CD62L and CD45RB. Analysis of CD8 cells from different tissues also revealed certain differences, particularly in TCR and co-receptor levels from liver-derived cells compared with circulating cells at the peak of the response. Our findings suggest that the function of Ag-selected CD8 cells may be regulated over time and according to location by subtle changes in cell-surface phenotype.

Processing of tumor-associated antigen by the proteasomes of dendritic cells controls in vivo T-cell responses.

Dendritic cells are unique in their capacity to process antigens and prime naive CD8(+) T cells. Contrary to most cells, which express the standard proteasomes, dendritic cells express immunoproteasomes constitutively. The melanoma-associated protein Melan-A(MART1) contains an HLA-A2-restricted peptide that is poorly processed by melanoma cells expressing immunoproteasomes in vitro. Here, we show that the expression of Melan-A in dendritic cells fails to elicit T-cell responses in vitro and in vivo because it is not processed by the proteasomes of dendritic cells. In contrast, dendritic cells lacking immunoproteasomes induce strong anti-Melan-A T-cell responses in vitro and in vivo. These results suggest that the inefficient processing of self-antigens, such as Melan-A, by the immunoproteasomes of professional antigen-presenting cells prevents the induction of antitumor T-cell responses in vivo.

Study of the proteolytic activity of the paracaspase MALT1 in T cell activation

Summary : Several signalling cascades are initiated through the triggering of the T cell receptor (TCR) by an antigenic peptide expressed at the surface of an antigen presenting cell. These pathways lead to morphological changes controlling T cell adhesiveness and migration to the site of infection, and to the activation of transcription factors that regulate key genes for the proper development of the immune response. Amongst them, the nuclear factor xB (NF-κB) is the subject of intense research since more than twenty years because deregulated NF-κB signalling in lymphocytes can lead to immunodeficiency, autoimmunity or lymphomas. Therefore, the understanding of the molecular mechanisms regulating NF-κB activation is important for the development of new therapeutics aimed at treating various diseases. In T lymphocytes, a complex composed of CARMAI, BCL10 and MALT1 relays signals from TCR proximal events to NF-κB activation. Gene translocations of the BCL10 or MALTI genes or oncogenic mutations affecting CARNA 1 result in constitutive NF-κB activation and are related to the development of certain forms of lymphomas. MALT1 contains acaspase-like domain, but it is unknown whether this domain is proteolytically active. In this study, we found that MALT1 has arginine-directed proteolytic activity. We showed that the proteolytic activity of MALT 1 is key to TCR-induced NF-κB activation and production of interleukin 2. We identified BCL 10 as a MALT 1 substrate, and we showed that its cleavage regulates T cell adhesion to the extracellular matrix protein fibronectin. Furthermore, we identified caspase 10 as another substrate of MALT1. caspase 10 is a close homologue of caspase 8 and is known to be involved in the induction of apoptosis upon Fast or TRAIL stimulation. We showed that caspase 10 is important for TCR-induced NF-κB activation and interleukin 2 production, identifying for the first time a non apoptotic function for caspase 10. These data provide evidence for previously uncharacterized roles of MALT 1 and BCL 10 in the regulation of T cell adhesion and of caspase 10 in the activation of lymphocytes, and allow a better understanding of the molecular mechanisms of T lymphocyte activation. Since the proteolytic activity of MALT1 is essential to T cell activation, it suggests that the targeting of this activity may be relevant for the development of immunomodulatory or anticancer drugs. Résumé : De nombreuses voies de signalisation sont initiées via la stimulation des récepteurs des cellules T (TCR) par un peptide antigénique exprimé à la surface d'une cellule présentatrice d'antigènes. Ces cascades de signalisation produisent des changements morphologiques qui contrôlent l'adhésion des cellules T et leur migration vers le site d'infection. Elles contrôlent également l'activation de facteurs de transcription qui régulent la transcription de gènes importants pour la réponse immunitaire. Parmi ces derniers, le facteur nucléaire KB (NF-κB) joue un rôle essentiel, puisqu'une régulation aberrante de son activité dans les lymphocytes peut causer des immunodéficiences, des maladies autoimmunes ou des lymphomes. C'est pour cela que la compréhension des mécanismes moléculaires qui contrôlent l'activation de NF-κB est donc importante pour le développement de nouvelles thérapies. Un complexe contenant les protéines CAIZMAI, BCL10 et MALT1 transmet, dans les lymphocytes T, le signal du TCR vers l'activation de NF-κB. Des translocations des gènes qui codent pour BCL10 et MALTI et des mutations affectant la fonction de CARNAI ont été liées au développement de certaines formes de lymphomes. MALTI contient un domaine qui ressemble au domaine catalytique présent dans les caspases, mais il n'est pas connu si ce domaine a une activité protéolytique. Dans cette étude, nous avons découvert que MALTI est une protéase qui a une spécificité pour les acides aminés basiques comme l'arginine. Nous montrons que l'activité protéolytique de MALTI est importante pour l'activation de NF-κB et la production d'interleukine 2 après stimulation du TCR. Nous avons observé que BCL10 est clivé par MALTI pendant l'activation des lymphocytes T, et que ce clivage est impliqué dans la régulation de l'adhésion des lymphocytes T à la fibronectin, une protéine de la matrice extracellulaire. De plus, nous avons identifié que la caspase 10, qui a une grande homologie avec la caspase 8 et qui jusqu'à maintenant est connue pour son rôle dans l'induction de la mort cellulaire en réponse à une stimulation par Fast ou par TRAIL, est également un substrat de MALT 1. En montrant que la caspase 10 est nécessaire à l' activation de NF-icB et à la production de l'interleukine 2 après stimulation du TCR, nous décrivons pour la première fois une fonction non apoptotique de la caspase 10. Ces résultats décrivent de nouveaux rôles pour MALT1 et BCL10 dans le contrôle de l'adhésion des lymphocytes T et de la caspase 10 pour l'activation des lymphocytes T. Puisque l'activité protéolytique de MALT1 est essentielle pour l'activation des lymphocytes T, nous suggérons que cibler cette activité protéolytique de MALT 1 pourrait amener de nouvelles possibilités de traitement de maladies où une activation aberrante des lymphocytes est impliquée.

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

Inhibitory Receptor Expression Depends More Dominantly on Differentiation and Activation than "Exhaustion" of Human CD8 T Cells.

Under conditions of chronic antigen stimulation, such as persistent viral infection and cancer, CD8 T cells may diminish effector function, which has been termed "exhaustion." Expression of inhibitory Receptors (iRs) is often regarded as a hallmark of "exhaustion." Here we studied the expression of eight different iRs by CD8 T cells of healthy humans, including CTLA-4, PD1, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1. We show that many iRs are expressed upon activation, and with progressive differentiation to effector cells, even in absence of long-term ("chronic") antigenic stimulation. In particular, we evaluated the direct relationship between iR expression and functionality in CD8 T cells by using anti-CD3 and anti-CD28 stimulation to stimulate all cells and differentiation subsets. We observed a striking up-regulation of certain iRs following the cytokine production wave, in agreement with the notion that iRs function as a negative feedback mechanism. Intriguingly, we found no major impairment of cytokine production in cells positive for a broad array of iRs, as previously shown for PD1 in healthy donors. Rather, the expression of the various iRs strongly correlated with T cell differentiation or activation states, or both. Furthermore, we analyzed CD8 T cells from lymph nodes (LNs) of melanoma patients. Interestingly, we found altered iR expression and lower cytokine production by T cells from metastatic LNs, but also from non-metastatic LNs, likely due to mechanisms which are not related to exhaustion. Together, our data shows that expression of iRs per se does not mark dysfunctional cells, but is rather tightly linked to activation and differentiation. This study highlights the importance of considering the status of activation and differentiation for the study and the clinical monitoring of CD8 T cells.

IL-2/anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor alpha subunit CD25.

IL-2 is crucial to T cell homeostasis, especially of CD4(+) T regulatory cells and memory CD8(+) cells, as evidenced by vigorous proliferation of these cells in vivo following injections of superagonist IL-2/anti-IL-2 antibody complexes. The mechanism of IL-2/anti-IL-2 antibody complexes is unknown owing to a lack of understanding of IL-2 homeostasis. We show that IL-2 receptor alpha (CD25) plays a crucial role in IL-2 homeostasis. Thus, prolongation of IL-2 half-life and blocking of CD25 using antibodies or CD25-deficient mice led in combination, but not alone, to vigorous IL-2-mediated T cell proliferation, similar to IL-2/anti-IL-2 antibody complexes. These data suggest an unpredicted role for CD25 in IL-2 homeostasis.

Natural killer cell receptor-repertoire and functions after induction therapy by polyclonal rabbit anti-thymocyte globulin in unsensitized kidney transplant recipients.

Polyclonal rabbit anti-thymocyte globulin (rATG) is widely used in solid organ transplantation (SOT) as induction therapy or to treat corticosteroid-resistant rejection. In vivo, the effect of rATG on natural killer (NK) cells has not been studied. These cells are of particular relevance after SOT because classical immunosuppressive drugs do not inhibit or even can activate NK cells. A cohort of 20 recipients at low immunological risk, that had been receiving rATG as induction therapy, was analyzed for receptor repertoire, cytotoxicity and capacity of NK cells to secrete IFN-γ before kidney transplantation and at different time points thereafter. NK cells expressed fewer killer-cell immunoglobulin-like receptors (KIR), fewer activating receptors NKG2D, but more inhibitory receptor NKG2A compatible with an immature phenotype in the first 6 months post transplantation. Both cytotoxicity of NK cells and the secretion of IFN-γ were preserved over time after transplantation.

Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition.

CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.

BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

Neutrophil-derived APRIL concentrated in tumor lesions by proteoglycans correlates with human B-cell lymphoma aggressiveness.

A PRoliferation-Inducing TNF Ligand (APRIL) costimulates B-cell activation. When overexpressed in mice, APRIL induces B-cell neoplasia, reminiscent of human B-cell chronic lymphoid leukemia (B-CLL). We analyzed APRIL expression in situ in human non-Hodgkin lymphomas. APRIL up-regulation was only observed in high-grade B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma (BL). Up-regulation was seen in 46% and 20% of DLBCL and BL, respectively. In DLBCL, neutrophils, constitutively producing APRIL and infiltrating the tumor tissue, were the main cellular source of APRIL. Rare DLBCL cases showed a predominance of histiocytes or mesenchymal cells as APRIL source. APRIL secreted by neutrophils accumulated on tumor cells via proteoglycan binding. In addition to proteoglycans, DLBCL tumor cells expressed the APRIL signaling receptor, TACI and/or BCMA, indicating that these tumor cells are fully equipped to respond to APRIL. A retrospective clinical analysis revealed a significant correlation between high expression of APRIL in tumor lesions and decreased overall patient survival rate. Hence, APRIL produced by inflammatory cells infiltrating lymphoma lesions may increase tumor aggressiveness and affect disease outcome.

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1.

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

Synthesis of a radioiodinated photoreactive MAGE-1 peptide derivative and photoaffinity labeling of cell-associated human leukocyte antigen-A1 molecules.

The synthesis of a photoreactive derivative of the human leukocyte antigen-A1 (HLA-A1)-restricted MAGE-1 peptide 161-169 (EADPTGHSY) is described. Using conventional automated solid-phase peptide synthesis, a photoreactive derivative of this peptide was synthesized by replacing histidine-167 with photo-reactive N-beta-4-azidosalicyloyl-L-2,3-diaminopropionic acid. The C-terminal tyrosine was incorporated as phosphotyrosine. This peptide derivative was radioiodinated in the presence of chloramine T. This iodination took place selectively at the photoreactive group, because the phosphate ester prevented tyrosine iodination. Following dephosphorylation with alkaline phosphatase and chromatographic purification, the radiolabeled peptide derivative was incubated with cells expressing HLA-A1 or other HLA molecules. Photoactivation resulted in efficient photoaffinity labeling of HLA-A1. Other HLA molecules or other cellular components were not detectably labeled. This labeling was inhibited by HLA-A1 but not by HLA-A2-binding peptides. This synthesis is generally applicable and can also be adapted to the synthesis of well-defined radiolabeled nonphotoreactive peptide derivatives.

Dimeric recombinant IgA directed against carcino-embryonic antigen, a novel tool for carcinoma localization.

Carcinoembryonic antigen (CEA) has been shown to be one of the best markers for in vivo tumor targeting of radiolabeled antibodies, despite the fact that it is localized predominantly at the apical side of human colon carcinoma cells within the fairly closed pseudolumen structures formed by these tumors. Due to this particular histological localization, a large proportion of the CEA molecules may remain inaccessible to the intravenously injected radiolabeled anti-CEA antibodies of IgG isotype, which are widely used in the clinic. In order to improve targeting, we made a recombinant dimeric IgA, which should have the capacity to translocate from the basolateral to the apical side of the pseudolumen formed by colon carcinoma cells after binding to the polyIg receptor (pIgR). A genomic chimeric mouse-human IgA2 construct was made using one of our most specific anti-CEA hybridomas, CE-25. The chimeric IgA (chIgA) was expressed in the Sp2/0 myeloma cell line. The secreted recombinant antibody was found to consist mostly of a dimeric form of IgA with a molecular weight of about 350 kDa. The dimeric chIgA was shown to translocate efficiently in vitro across a monolayer of epithelial cells expressing the pIgR and to retain full CEA binding activity.

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen.

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX? vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

Autoreactive T cells that bypass negative selection are not tolerant and respond to self-antigen stimulation during infection

Le répertoire cellulaire Τ a pour but d'être tolérant aux antigènes du soi afin d'éviter l'induction de maladies autoimmunes. C'est pourquoi les lymphocytes Τ autoréactifs sont éliminés dans le thymus lors de leur développement par le processus de sélection négative. La plupart des recherches étudient les lymphocytes Τ de haute avidité. Ces lymphocytes Τ de haute avidité sont très sensibles et réagissent fortement à un antigène du soi. En conséquence, ces cellules induisent le développement de maladies autoimmunes lorsqu'elles ciblent des organes exprimant l'antigène du soi. Plusieurs études ont montré que les lymphocytes Τ qui réagissent faiblement aux antigènes spécifiques à un tissu, nommé lymphocytes Τ de faible avidité, peuvent contourner les mécanismes de tolérance centrale et périphérique. J'ai utilisé des souris Rip-mOva qui expriment l'Ovalbumine comme antigène du soi spécifique à un tissu. Dans ces souris transgéniques Rip-mOva, les lymphocytes Τ de faible avidité survivent à la sélection négative. Une fois stimulés à la périphérie, ces lymphocytes Τ CD8+ de faible avidité ont la capacité d'infiltrer les organes qui expriment l'antigène du soi chez les souris Rip-mOva et peuvent induire une destruction tissulaire. L'objectif principal de mon projet de thèse était de comprendre les caractéristiques phénotypiques et fonctionnelles de ces lymphocytes Τ dans un état d'équilibre et dans un contexte infectieux. Pour étudier ces cellules dans un modèle murin bien défini, nous avons généré des souris exprimant un récepteur de cellule Τ transgénique appelé OT-3. Ces souris transgéniques OT-3 ont des lymphocytes Τ CD8+ de faible avidité spécifiques à l'épitope SIINFEKL de l'antigène Ovalbumine. Nous avons démontré qu'un grand nombre de lymphocytes Τ CD8+ OT-3 ne sont pas éliminés lors de la sélection négative dans le thymus après avoir rencontré l'antigène du soi. Par conséquent, les lymphocytes Τ OT-3 de faible avidité sont présents dans une fenêtre de sélection comprise entre la sélection positive et négative. Cette limite se définie comme le seuil d'affinité et est impliquée dans l'échappement de certains lymphocytes Τ OT- 3 autoréactifs. A la périphérie, ces cellules sont capables d'induire une autoimmunité après stimulation au cours d'une infection, ce qui nous permet de les définir comme étant non tolérante et non dans un état anergique à la périphérie. Nous avons également étudié le seuil d'activation des lymphocytes Τ OT-3 à faible avidité à la périphérie et avons constaté que des ligands peptidiques plus faibles que l'épitope natif SIINFEKL sont capables de les activer au cours d'une infection ainsi que de les différencier en lymphocytes Τ effecteurs et mémoires. Les données illustrent une déficience lors de la sélection négative dans le thymus de lymphocytes Τ CD8+ autoréactifs de faible avidité contre un antigène du soi spécifique à tissu et montrent que ces cellules sont entièrement compétentes lors d'une infection. - The diverse Τ cell repertoire needs to be tolerant to self-antigen to avoid the induction of autoimmunity. This is why autoreactive developing Τ cells are deleted in the thymus. The deletion of self-reactive Τ cells occurs through the process of negative selection. Most studies investigated high avidity Τ cells. These high avidity Τ cells are very sensitive and strongly react to a self-antigen. As a consequence, these cells induce the development of autoimmunity when they target organs which express the self-antigen. High avidity autoreactive CD8+ Τ cells are deleted in the thymus. However, several studies have shown Τ cells that weakly respond to tissue-restricted antigen, referred to as low avidity Τ cells, can bypass central and peripheral tolerance mechanisms. I used Rip-mOva mice that expressed Ovalbumin as a neo self-antigen in a tissue-restricted fashion. In these transgenic Rip-mOva mice low avidity CD8+ Τ cells survive negative selection. Upon stimulation in the periphery, these low avidity CD8+ Τ cells have the ability to infiltrate organs that express the self-antigen in the Rip-mOva mice and can also induce the destruction of the tissue. The major aim of my PhD project was to understand the phenotypic and functionality characteristics of these Τ cells in a steady-state condition and in a context of an infection. To study these cells in a well-defined mouse model, we generated OT-3 Τ cell receptor transgenic mice that express low avidity CD8+ Τ cells that are specific for the SIINFEKL epitope of the Ovalbumin antigen. We have been able to demonstrate that a large number of OT-3 CD8+ Τ cells survive negative selection in the thymus after encountering the self-antigen. Thus, low avidity OT-3 Τ cells are present in a window of selection comprised between positive and negative selection. This boundary defined as the affinity threshold is involved in the escape of some autoreactive low avidity OT-3 Τ cells. Once they circulate in the periphery, they are able to induce autoimmunity after stimulation during an infection, allowing us to allocate these cells as being non-tolerant and not in an anergic state in the periphery. We have also looked at the threshold of activation of low avidity OT-3 CD8+ Τ cells in the periphery and found that peptide ligands that are weaker than the native SIINFEKL epitope are able to activate OT-3 Τ cells during an infection and to differentiate them into effector and memory Τ cells. The data illustrate the impairment of negatively selecting low avidity autoreactive CD8+ Τ cells against a tissue-restricted antigen in the thymus and shows that these cells are fully competent upon an infection.