Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.

Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.

Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.

Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.

The ability of our immune system to mount a response against non-self-antigens legitimates the semi-allogenic fetus as a target for maternal immune attack. Yet, in a normal pregnancy the fetus stays well protected due to the concerted action of several diverse mechanisms which either suppress the fetal allogenicity or spatio-temporally inhibit maternal immune cells’ growth and functions. One such factor which aids in the establishment, progression and maintenance of pregnancy is the 28 kDa dimeric sialylated glycoprotein Glycodelin-A (GdA). Synthesized by the endometrium and decidua, this protein has myriad functions, the most important being that of immunosuppression. GdA is inhibitory to all hematopoietic cells and also induces programmed cell death in activated T cells and monocytes via the intrinsic mitochondrial pathway. In the Introductory chapter of this thesis, details about GdA and the other isoforms of the glycodelin family of proteins have been presented which highlight the involvement of glycodelins in primate pregnancy, with emphasis on GdA and its pleiotropic functions associated with reproduction in females. Activated T-lymphocytes against paternal antigens are found in the uterine compartment and in the maternal circulation throughout pregnancy. Activated CD8+ T-lymphocytes have been reported to pre-dominate the uterine T-lymphocyte population during pregnancy and unlike the CD4+ T cells, are retained until term. Studies show that activated CD8+ T-lymphocytes are necessary for the establishment and progression of early pregnancy. However, how these lymphocytes harbouring cytotoxic activity are regulated at the later stages of pregnancy is poorly defined. We attempted to uncover a possible mechanism of regulation of CTL (cytotoxic T lymphocyte) activity (if any) during primate pregnancy by GdA. In the absence of established human CD8+ T cell lines, we first standardized the generation of CTLs in-vitro from hPBMCs (human peripheral blood mononuclear cells) by alloactivating them with an ovarian carcinoma cell line OVCAR-3 utilized as a mimic of an allograft. The details of the rationale behind using this method for generating CTLs and the alloactivation methodology have been put together in the Chapter 1 of this thesis. The activation of hPBMCs was confirmed by the surface expression of an early activation marker CD69 and tritiated thymidine incorporation. Differentiation of CD8+ T cells into effector cells was confirmed by the upregulation of perforin and granzyme transcripts by real time RT-PCR analysis. Target-cell specific cytolytic activity of the CTLs was assessed by using a cytotoxicity measurement assay- JAM test, details of which also form a part of chapter 1. Having generated effective CTLs in vitro, we tested the effect of GdA on CTL activity. Our findings, on the effect of GdA on CTLs have also been discussed in the Chapter 1. We observed that the cytolytic activity of CTLs was significantly reduced by GdA treatment albeit at a dose three to four times higher than that required for inhibiting CD8+ T cell proliferation, implying that a mechanism of temporal regulation of CTL activity operated at the feto-maternal interface, thereby contributing to the establishment and progression of pregnancy. Interestingly, in our quest to uncover the mechanism of inhibition of CTL activity by GdA, we found that the inhibition of proliferation was comparable in both CD4+ and CD8+ T-lymphocytes at all dosages of GdA, but unlike CD4 + T cells CD8 + T cells were resistant to GdA-induced apoptosis even at high dosage of GdA. Hence we could rule out that the loss of CTL activity upon GdA treatment was due to CD8+ T cell death. Further, we assessed the functional competence of alloactivated CTLs by quantitating the mRNA transcripts of key cytolytic molecules; perforin and granzyme B, in GdA treated alloactivated hPBMCs and found that there was a significant reduction in the mRNA of these cytolytic molecules. Additionally, we also found that GdA treated CD8+ T cells exhibited impaired release of the cytolytic molecules by the process of degranulation, measured by the surface exposure of LAMPs (Lysosome associated membrane proteins) on the surface of cells by flow cytometry and as seen by the retention of perforin protein in them assessed by intracellular staining and flow cytometry. Intrigued by the observations, we probed for the regulators of perforin and granzymes in CTLs. EOMES (Eomesodermin) and T- Bet are well known transcription factors which control the differentiation of CD8+ T cells into effector and memory cell CD8+ T cell type. Interestingly we found that the expression of EOMES was significantly reduced in activated GdA treated hPBMCs, both at the transcriptional and translational level, however T-Bet did not show any variation in expression upon GdA treatment. All the above findings have been compiled in Chapter 2 along with our studies on the possibility of GdA to induce a tolerogenic phenotype in T cells. We found there was no difference in the mRNA level and surface expression of CD103 and CD28 in alloactivated PBMCs, while FOXP3 mRNA did not show any variation upon GdA treatment, indicating that GdA does not induce a tolerogenic phenotype in T-lymphocytes, further confirming our data that the decreased cytolytic activity of CTLs upon GdA treatment was not due to tolerance but due to impaired function Interestingly, IL-2/IL-2R signaling is known to directly regulate perforin and granzyme expression as well as it plays a role in the expression of T-Bet and EOMES. Therefore, as a read out of IL-2 signaling we checked for the surface expression of the high affinity IL-2R subunit, CD25. As expected, CD25 expression was more pronounced in CD4+ T cells and consistent with published reports in literature that GdA suppresses IL-2 synthesis, we also observed a significant reduction in the CD25bright population in both the T cell subsets (CD4+ and CD8+) upon GdA treatment (addressed in Chapter 3). This finding supports a mechanism of action of GdA, wherein the cytolytic activity of CTLs is compromised by the downregulation of EOMES, triggered by the low IL-2 levels. This translates to aberrant synthesis of key cytolytic molecules perforin and granzyme B, leading to low efficiency CTLs, which are further disabled by defective degranulation machinery induced by GdA. We did not look into the mechanistic aspects of how GdA suppresses degranulation, which can be addressed later as a part of another study. Building up on our observations, and taking cues from existing literature, that IL-2 regulates the expression of pro and anti-apoptotic protein levels within activated cells, we looked at the expression profile of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) in activated PBMCs upon GdA treatment. There was a significant reduction in the total mRNA and protein level of Bcl-2, while a very significant increase in Bax mRNA and protein was observed. Chapter 3 of the thesis also presents this data and explains a plausible mechanism of the inhibitory effect of GdA on T-lymphocytes. In Chapter 2, we have also addressed the probable reasons for the differences in the responses of CD4+ and CD8+ T-lymphocytes to GdA. Interestingly, surface glycan profile of CD4+ and CD8+ T-lymphocytes upon activation and the surface expression of the most probable receptor for GdA i.e. CD7 was comparable in both the T cell subsets, indicating that possibly the downstream signaling events leading to GdA-induced apoptosis and not the surface binding of GdA may vary in CD4+ and CD8+ T-lymphocytes, due to which we observed a difference in the extent of apoptosis induced in these cell types by GdA although the inhibition of proliferation in both the subsets was comparable. In summary, this study is the first to provide evidence for a possible mechanism of temporal regulation of CTL activity at the feto-maternal interface, where activated CD8+ T cells are abundantly present. We can say with much confidence that binding of GdA to T-lymphocytes causes sub-optimal IL-2 signaling which translates into reduced expression of EOMES and hence downregulation of perforin and granzyme B, leading to impaired CTL activity in CD8+ T-lymphocytes, which is further weakened by the impaired release of the cytolytic molecules from them. Insufficient IL-2 signaling in the presence of GdA can also be a cause of inhibition of proliferation in T-lymphocytes, while the resulting decrease in anti-apoptotic protein Bcl-2 and increase in pro-apoptotic protein Bax seem to contribute to the induction of apoptosis in CD4+ T cell. It will be interesting to explore the mediators involved in the IL-2 signaling pathway that are differentially regulated in CD4+ and CD8+ T cells which confer resistance in CD8+ T cells to GdA-induced apoptosis and also the mechanism by which GdA regulates the degranulation of cytolytic vesicles in CTLs needs to be worked out.

Résumé : L’IL-7 et l’IL-15 sont des cytokines impliquées dans l’homéostasie des lymphocytes T CD8 naïfs et mémoires respectivement. Lors d’une réponse immunitaire, certaines cytokines pro-inflammatoires, comme l’IL-6 et l’IL-21, sont produites par les cellules du système immunitaire inné. Nous avons observé que certaines cytokines de ces deux groupes (homéostasie et pro-inflammatoires), peuvent avoir un effet synergique sur la fonction des lymphocytes T CD8. Spécifiquement, l’incubation des lymphocytes T CD8 naïfs avec l’IL-6 ou l’IL-21, en présence d’IL-7 ou d’IL-15 cause une forte prolifération qui est indépendante de l’antigène. De plus, la combinaison d’IL-15 avec l’IL-6 ou l’IL-21 entraîne une prolifération préférentielle des lymphocytes T mémoires, tandis que la combinaison avec l’IL-7 entraîne une prolifération des lymphocytes T naïfs. La stimulation des lymphocytes T CD8 avec l’IL-6 ou l’IL-21, en présence d’IL-7 ou d’IL-15, entraîne une augmentation de la phosphorylation en tyrosine de STAT5 ainsi qu’une augmentation de liaison à l’ADN. Nous avons étudié l’effet d’une pré-stimulation des cellules T CD8 naïves par les cytokines synergiques sur leur réponse subséquente à un antigène. Nous avons observé qu’une pré-stimulation avec l’IL-6 ou l’IL-21, en présence d’IL-7 ou d’IL-15, même pour une courte durée de 24 heures, augmente leur sensibilité aux antigènes, entraînant une robuste prolifération et une forte augmentation de cytotoxité spécifique à l’antigène gp33. Nous avons observé que les cytokines pro-inflammatoires en combinaison avec l’IL-7 induisent une augmentation accrue de la prolifération chez les lymphocytes T CD8 exprimant un TCR transgénique de forte affinité (P14), ainsi que les cellules exprimant un TCR de faible affinité (H-Y). De plus, la combinaison synergique de cytokines entraîne une forte expression du récepteur de l’IL-2R[gamma] (CD132), ainsi qu’une augmentation de la production d’IL-2 après stimulation antigénique. Une forte augmentation de l’expression de CD8 et de CD45, ainsi qu’une diminution drastique de l’expression de CD5 peut expliquer l’augmentation de l’avidité fonctionnelle du TCR suite à une stimulation avec les combinaisons de cytokines synergiques. La stimulation des lymphocytes T CD8 avec les combinaisons de cytokines, induit une augmentation de la phosphorylation de LAT ainsi qu‘AKT. Cependant, la stimulation subséquente du CD3 n’entraîne pas d’augmentation de la phosphorylation de LAT ainsi qu’AKT chez les lymphocytes T CD8 pré-stimulés avec les combinaisons de cytokines. Nous avons aussi observé que les lymphocytes T CD8 stimulés avec les combinaisons de cytokines augmentent l’expression de CD62L, ce qui peut favoriser leur migration vers les ganglions lymphatiques. En conclusion, la production de cytokines pro-inflammatoires (IL-6, IL-15, IL-21) par les cellules du système immunitaire inné lors d’une infection ou d’une inflammation, ainsi que la présence constitutive d’IL-7, peuvent stimuler la prolifération et l’activation des lymphocytes T CD8 de façon non spécifique à l’antigène. Cette stimulation entraîne une augmentation de l’avidité fonctionnelle de leur TCR causant ainsi une forte prolifération ainsi que l’acquisition de fonctions effectrices spécifiques. Cette liaison entre le système immunitaire inné et adaptatif, médiée par les cytokines pro-inflammatoires et les cytokines homéostatiques joue un rôle très important dans l’élimination des pathogènes ainsi que dans le développement de maladies auto-immunitaires. / Abstract : Homeostasis of naive and memory CD8[superscript +] T lymphocytes is dependent on two cytokines IL-7 and IL-15, respectively. During an immune response to an infection, cells of the innate immune system produce several pro-inflammatory cytokines. We have observed that these two groups of cytokines, namely proinflammatory and homeostatic, can have a synergistic effect on CD8 T lymphocytes. Specifically, incubation of naive CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 induced strong proliferation in an antigen independent manner. While the combination of IL-6 or IL-21 with IL-15 induced strong proliferation of memory CD8 T cells, naïve CD8 T cells responded better to the combination with IL-7. These stimulatory cytokine combinations elicited strong STAT5 phosphorylation and it’s binding to DNA in CD8 T cells. We investigated the effect of priming CD8 T cells with the synergistic combination of IL-6 or IL-21 and IL-7 on their subsequent response to antigen. We observed that cytokine priming for only 24 hours enhanced their sensitivity to antigen, resulting in strong proliferation, effectors functions and cytotoxicity. These effects were observed with CD8 T cells expressing transgenic TCR with strong (P14) or weak (H-Y) affinity towards cognate peptide antigens. Priming CD8 T cells with the synergistic combination of cytokines increased the expression of IL-2 receptor gamma (CD132) and augmented the production of IL-2 when stimulated with antigen. These cells also expressed elevated levels of CD8 and CD45, as well as down modulate CD5, and these events may underlie the increased TCR avidity. Stimulation of CD8 T cells with the synergistic combination of cytokines induced phosphorylation of LAT and AKT. However, subsequent TCR stimulation did not further increase these phosphorylation events. We have observed that C D8 T cells primed with the synergistic combinations of cytokines up regulated CD62L, which could promote their migration through lymph nodes. In conclusion, inflammatory cytokines such as (IL-6, IL-15, IL-21) secreted by cells of the innate immune system during an infection or non-infectious inflammation, and basal levels of the homeostatic cytokine IL-7 can act in synergy with inflammatory cytokines to activate CD8 T lymphocytes in an antigen independent manner. This stimulation also results in an increase in the functional avidity of their TCR, as indicated by strong antigen responsiveness with increased proliferation and display of effectors functions. This connection between the innate and adaptive system mediated by inflammatory cytokines may play an important role in pathogen clearance and possibly in the development of autoimmune diseases.

BIM é uma proteína pro-apoptótica membro da família Bcl-2. No sistema imunológico, BIM foi descrita como reguladora da homeostase de linfócitos. Porém, ainda não foi estudado o papel do BIM no estabelecimento da resposta imune de linfócitos T CD8+. Sendo que os vetores adenovirais fortes ativadores da resposta, neste trabalho investigamos o papel de BIM na qualidade e frequência de linfócitos T CD8+ estimulados com Ad.cOVA. Camundongos C57Bl/6 selvagens, bim+/- e bim-/- foram imunizados com 2x106 PFU/100μl. Assim, observou-se uma redução da lise especifica e menor freqüência de linfócitos CD8+ produtores de IFNγ em camundongos bim-/-. Em paralelo, foi avaliada a resposta imune anti-tumoral destes camundongos sem encontrar diferencias significativas. A cinética da resposta efetora de linfócitos T CD8+ de camundongos bim-/- mostrou escassa perda das capacidades efetoras destes linfócitos, sendo o possível mecanismo para controlar a progressão tumoral. Em conclusão camundongos bim-/- apresentam uma menor freqüência de células efetoras, sugerindo um importante papel de BIM na produção de linfócitos T CD8+ antígeno-específicos trás a vacinação com Ad.cOVA. / BIM is a pro-apoptotic member of the Bcl-2 protein family. In the immune system, BIM has been described as lymphocyte homeostasis regulator. However, the role of BIM in the establishment of the immune response of CD8+ T lymphocytes has not yet been studied. As the strong adenoviral vectors activating the response, we investigated the role of BIM in the quality and frequency of Ad.cOVA-stimulated CD8+ T lymphocytes. Wild C57Bl/6 mice, bim+/- and bim-/- were immunized with 2x106 PFU/100μl. Thus, a reduction of the specific lysis and decreased frequency of IFNγ producing CD8+ lymphocytes in bim-/- mice was observed. In parallel, the anti-tumor immune response of these mice was evaluated without finding significant differences. The kinetics of the effector response of CD8+ T lymphocytes from bim-/- mice showed little loss of the effector capacities of these lymphocytes, being the possible mechanism to control tumor progression. In conclusion, bim-/- mice show a lower frequency of effector cells, suggesting an important role of BIM in the production of antigen-specific CD8+ T lymphocytes after vaccination with Ad.cOVA.

Orientador: Lenice do Rosário de Souza / Resumo: Segundo dados do Programa Conjunto das Nações Unidas sobre HIV/Aids (UNAIDS), existem atualmente 36,9 milhões de pessoas vivendo com HIV no mundo. A disponibilização e eficácia da terapia antirretroviral (TARV) permitiu uma mudança no cenário desta pandemia, reduzindo consideravelmente a mortalidade associada à aids. Devido à redução da carga viral plasmática do HIV (CV) para níveis indetectáveis, a TARV permite que ocorra a recuperação de células T CD4+, principal alvo do vírus, e melhora no prognóstico das pessoas que vivem com o HIV/aids (PVHA). Entretanto, as mudanças no compartimento de linfócitos T CD8+, responsáveis pelo controle da replicação viral, são relativamente pouco entendidas durante os vários estágios da infecção. Desta forma, o presente estudo, de caráter retrospectivo, teve o objetivo de avaliar, a partir do levantamento das contagens de células T CD8+, as possíveis correlações entre esta variável e a evolução clínica da infecção, considerando as contagens de células T CD4+, quantificações da CV, tempo de TARV, classe terapêutica utilizada, aparecimento de infecções oportunistas e desfecho clínico (paciente assintomático, progressão para aids ou morte). Para isso, foram analisados 200 prontuários eletrônicos de PVHA acompanhadas no Serviço de Ambulatórios Especializados de Infectologia “Domingos Alves Meira” (SAEI-DAM) e diagnosticadas a partir de 2012. Para a análise dos dados, foram realizados os testes, binomial negativa, correlação de Pearson e analisad... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Deux grandes classes de cellules composent le pool de lymphocytes T (LT) CD8 mémoires. D'une part, les LT CD8 mémoires conventionnels sont générés via la reconnaissance spécifique d'antigènes dérivés de pathogènes ou de tumeurs. D'autre part, les LT CD8 mémoires innés sont générés via différents mécanismes impliquant de fortes stimulations par des cytokines γc indépendamment de la reconnaissance d'antigènes du non soi. Le phénotype extrêmement similaire de ces deux populations cellulaires ne permet pas de les distinguer in vivo. En conséquence, la population de LT CD8 mémoires innés est relativement peu caractérisée. Mon travail de thèse comportait donc deux objectifs majeurs : 1 / Identifier des marqueurs permettant de distinguer in vivo ces deux classes de LT CD8 mémoires. 2/ Caractériser la population de LT CD8 mémoires innés. Dans cette étude, nous démontrons qu'au sein du pool de LT CD8 mémoires, seules les cellules conventionnelles expriment la chimiokine CCL5 et le récepteur NKG2D. Ces deux biomarqueurs permettent ainsi pour la première fois de distinguer les LT CD8 mémoires innés et conventionnels in vivo, à la fois chez la souris et chez l'homme. Grâce à l'expression de NKG2D, nous démontrons que ces LT CD8 mémoires innés possèdent des caractéristiques typiques de cellules mémoires, notamment une réactivité augmentée ainsi qu'un programme génétique comparable à celui des LT CD8 mémoires conventionnels. Néanmoins, cette population cellulaire conserve certaines caractéristiques de cellules naïves. Ainsi, le répertoire TCR diversifié de cette population cellulaire permet à ces cellules de participer à des réponses immunitaires primaires contre différents pathogènes. Enfin, dans un contexte inflammatoire, les LT CD8 mémoires innés présentent un défaut d'accès au tissu pulmonaire comparé aux LT CD8 mémoires conventionnels. Ceci corrèle avec un déficit d'expression de certaines intégrines par les LT CD8 mémoires innés. L'ensemble de nos résultats démontre que les LT CD8 mémoires innés, caractérisés par l'absence d'expression de CCL5 et NKG2D, constituent une population cellulaire hybride, à la frontière entre cellules naïves et cellules mémoires conventionnelles / The pool of memory CD8 T cells is composed of two major cell classes. On one hand, conventional memory CD8 T cells are generated consequently to the specific recognition of pathogen or tumor derived antigens. On the other hand, innate memory CD8 T cells are generated through several mechanisms involving strong yc cytokine stimulation in the absence of cognate antigen recognition. However, these cell classes harbor a very similar phenotype. As a consequence, innate memory CD8 T cell population remains poorly characterized. This PhD has two main objectives : 1 / Identify new biomarkers that enable the discrimination between memory CD8 T cell classes 2/ Characterize the population of innate memory CD8 T cells in physiological condition Our results show that among the pool of memory CD8 T cells, only the conventional ones express the chemokine CCL5 and the NK receptor NKG2D. These two biomarkers enable for the first time the discrimination of memory CD8 T cell classes in physiological settings, in both mouse and human. Thanks to these new tools, we show that innate memory CD8 T cells hold typical memory features, such as an increased reactivity compared to naïve cells and a genetic program similar to the one of conventional memory cells. Nevertheless, this cell population also retains some features typical of naïve cells. The diversified TCR repertoire of this cell population allows it to participate to primary immune responses against various intracellular pathogens. Moreover, like naïve cells, innate memory CD8 T cells fail to access peripheral tissues upon local inflammation, which correlate with an absence of expression of some integrins. Altogether, these results demonstrate that innate memory CD8 T cells, characterized by the absence of expression of CCL5 and NKG2D, represent a hybrid cell population, at the boundary between naïve cells and conventional memory cells

Les tumeurs solides emploient diverses stratégies afin de se maintenir dans l’organisme et d’échapper à l’inhibition du système immunitaire. Un des mécanismes les plus puissants est la production de la cytokine Transforming Growth Factor Beta (TGF-bêta). Cependant, cette cytokine est sécrétée dans le micro-environnement tumoral sous une forme inactive, incapable de se lier à son récepteur et donc d’exercer ses fonctions hautement immunosuppressives. Ces travaux de thèse démontrent qu’une population de lymphocytes T (LT) CD4+ dite T régulateurs (Tregs), qui exprime le facteur de transcription Forkhead box P3 (Foxp3), est responsable de l’activation du TGF-bêta au sein de la tumeur. Nous avons montré que parmi les cellules du système immunitaire, les Tregs constituent la principale population exprimant l’intégrine avb8 (Itgb8), protéine responsable de l’activation du TGF-bêta. L’absence de l’Itgb8 spécifiquement à la surface des Tregs entraîne une forte diminution de la croissance tumorale. Par conséquent, l’activation de la signalisation du TGF-bêta est réduite dans les LT CD8+ qui infiltrent la tumeur, conduisant à une exacerbation de leurs fonctions cytotoxiques et donc à une élimination accrue des cellules tumorales. La relevance de ces données obtenues chez la souris a été confirmée chez l’Homme à la fois par des approches ex vivo sur des tumeurs fraîches ainsi que par des approches bio-informatiques et biostatistiques à partir d’étude de cohortes de patients. Nous proposons donc que les Tregs et les cellules tumorales travaillent de concert pour fournir une source bio-active de TGF-bêta capable de réprimer efficacement la réponse immunitaire anti-tumorale et donc de permettre à la tumeur d’échapper au système immunitaire / Solid tumors employ diverse strategies to be maintained in the organism and escape the suppression mediated by the immune system. One of the most powerful mechanisms they use is through the production of Transforming Growth Factor Beta (TGF-beta). However, this cytokine is secreted within the tumor microenvironment in its inactive form, unable to bind to its receptor and exert its highly immunosuppressive functions. The present thesis project demonstrates that a population of CD4+ T lymphocytes called regulatory T cells (Tregs), which express the transcription factor Forkhead box P3 (Foxp3), is responsible for TGF-beta activation in tumors. We show that among the cells of the immune system, Tregs constitute the main population expressing the integrin avb8 (Itgb8) which is responsible for TGF-beta activation. The absence of Itgb8 specifically on Tregs surface leads to strong decrease of tumor growth. As a result, TGF-beta signaling pathway is impaired in tumor infiltrating CD8+ T lymphocytes leading to exacerbation of their cytotoxic and efficient elimination of tumor cells. The relevance of these data obtained in mice was confirmed in the human pathology by ex vivo approaches using fresh tumors as well as by bioinformatics and biostatistics approaches from studies on patient cohorts. We propose that Tregs and tumor cells cooperate to provide a bioactive source of TGF-beta which is able to efficiently repress the anti-tumor response and thus allowing tumors to escape the immune system