HIV Tat protein downregulates surface expression of the interleukin-7 receptor alpha-chain (CD127) on CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. Once taken up by CD8 T cells, Tat binds directly to the cytoplasmic tail of CD127 inducing receptor internalization and degradation. Given the important roles of CD127 in proper immune function, the Tat/CD127 interactions were characterized and the mechanisms required to induce receptor loss from the surface of CD8 T cells were investigated. Tat deletion mutants were generated each sequentially lacking a region of the protein. CD8 T cells isolated from HIV negative volunteers were exposed to exogenous or intracellular Tat proteins before surface CD127 expression was analyzed by flow cytometry. To characterize Tat/CD127 physical interactions, wild type Tat and Tat mutants were incubated with lysates from a CD127+ Jurkat cell line followed by CD127/Tat co-immunoprecipitation. The effect of Tat on CD127 post-translational modifications was also investigated. Removal of the N-terminus of Tat (aa 1-10 or aa 17-21) prevented Tat from downregulating CD127 and prevented Tat from binding CD127 as assessed by co-immunoprecipitation. Deletion of the basic region (aa 48-59) also prevented Tat from downregulating CD127 but did not prevent Tat from interacting physically as demonstrated by co-immunoprecipitation. Strikingly, endogenously expressed Basic Tat acted as a dominant negative mutant, causing an accumulation of CD127 at the cell surface. These observations suggest that Tat may bind CD127 via its N-terminus to disrupt the normal recycling of the receptor, and then recruit cellular endocytic machinery to the receptor via it’s basic region, to remove the receptor from the cell surface and target it for degradation. Furthermore, Tat encourages the ubiquitination of CD127 by recruiting the cytokine-inducible SH2 containing (CIS) protein to the receptor, possibly leading to accelerated CD127 internalization and proteasomal degradation. I propose a model whereby Tat binds CD127 via its N-terminal region then recruits CIS via its basic region. CIS in turn recruits a cellular E3 ubiquitin ligase to ubiquitin tag the receptor for internalization and proteasome degradation. This research may lead to novel treatments designed to maintain IL-7 signalling and strengthen CD8 T cell function in HIV+ persons.

ABSTRACT Background Interleukin-7 (IL-7) is a cytokine crucial for T-cell development and homeostasis. IL-7 is thought to be a limited resource, and its interaction with the IL-7 receptor (IL-7R) has effects on increasing cell survival, proliferation and cytolytic function. Considering the roles of IL-7, it is no surprise that the expression of the IL-7 receptor alpha chain (CD127) is tightly regulated. Despite increased levels of soluble CD127 (sCD127) being detected in a number of disease states and being associated with disease activity, the biological function of sCD127 and its clinical relevance remains to be established. In this study, I explore the post-translational mechanisms leading to the release of the soluble form of CD127 receptor through IL-7 and αCD3/αCD28 stimulation. Here I specifically established two different mechanisms by which CD127 is processed; shedding of the receptor ectodomain and clipping. Results In CD8+ T-cells, IL-7 plus TcR stimulation resulted in an increased release of sCD127. Here I found that matrix metalloproteases (MMPs), in particular MMP-9, have a role in the proteolytic clipping of CD127 resulting in the release of sCD127. In addition, I found that IL-7 plus TcR stimulation resulted in an increase in MMP activity and this activity was particularly dampened when MMP-2 and -9 inhibitors were used. I also found that neither MMP-3 nor cysteine and serine proteases seem to be directly involved in the generation of sCD127. Using a biotinylation assay I found that CD127 is being shed from the surface of CD8+ T-cells as well as thymocytes through a MMP-independent mechanism. Conclusion These results demonstrate that MMPs (in particular MMP-9) have a role in the generation of sCD127. Further studies are required to determine the specific sheddase responsible for the ectodomain shedding of CD127, as well as the details behind the regulation of MMP-9 activity both in CD8+ T-cells and thymocytes. A thorough understanding of these mechanisms will aid in the development of alternative and more specific strategies to control IL-7 mediated processes in both normal and disease states.

Billions of cells die everyday as a result of normal tissue turnover, infection, trauma or injury. These dead cells are taken up, processed and presented to T cells by antigen presenting cells resulting in tolerance or immunity. Apoptotic cells induce tolerance; however, the precise mechanisms are still unknown. Previous studies have shown that direct infusion of apoptotic cells induce tolerance mediated by TRAIL-expressing CD8+ T cells. We hypothesized that immunologic tolerance induced by apoptotic cells is dependent on the activation status of apoptotic cells and mediated by direct killing of target cells by TRAIL-expressing CD8+ T cells. Three different experimental systems were used to elucidate the mechanisms by which apoptotic cells regulate immune responses. Using a classical system of tolerance induction, we examined the immunological consequence of intravenous (i.v.) delivery of ex vivo-generated naïve or activated apoptotic cells. Naïve apoptotic cells induced tolerance when injected i.v.; however, previously activated apoptotic cells induced immunity. Further analysis revealed a key role for CD154 in the tolerogenic or immunogenic nature of the naïve or activated apoptotic cells, respectively, as tolerance resulted after i.v. injection of either naïve or activated apoptotic CD154-/- cells, while co-injection of an agonistic anti-CD40 mAb with naïve apoptotic T cells induced robust immunity. The infusion of large numbers of apoptotic cells has limited physiological relevance, so the investigation of the influence of apoptotic cells on the immune system turned to another experimental tolerance model where soluble peptide antigen is injected systemically to induce the peripheral deletion of a population of antigen-specific T cells. Using this system, we investigated how apoptotic cells generated in vivo leads to T cell tolerance. Following adoptive transfer of OT-II cells, wild-type mice injected with soluble OVA323-339 became unresponsive to subsequent CFA/OVA immunization. Interestingly, Trail-/- or Dr5-/- mice developed robust immunity; even though all strains displayed peripheral deletion of OVA-specific T cells. Subsequent investigation found the mechanism of action of the CD8+ T cells was TRAIL-mediated deletion of the OVA-responsive T cells in a TCR-specific manner. The experimental systems used above have some clinical relevance but are still not physiologic. To study the impact of apoptotic cells in a physiologic setting, we took advantage of the medical condition sepsis, which is accompanied by massive apoptosis of multiple immune cell populations. Thus, the final set of experiments in this thesis examined the tolerance induced during sepsis using a clinically-relevant cecal-ligation and puncture (CLP) model that included a secondary bacterial infection. CLP-treated WT mice had a reduced ability to control the secondary bacterial infection, which was paralleled by suppressed T cell responses, compared to sham-treated WT mice. In contrast, CLP- and sham-treated Trail-/- and Dr5-/- mice were able to similarly control the bacterial infection and generated bacterial antigen-specific T cell responses. The ability of CLP-treated wild-type mice to control the secondary infection and generate T cell immunity could be restored by the administration of a blocking anti-TRAIL mAb. These results suggest the importance of TRAIL in the induction of sepsis-induced immune suppression, such that TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.

Les cytokines jouent un rôle essentiel dans la réponse immunitaire ; elles modulent les propriétés et la survie de nombreuses cellules immunitaires. Les lymphocytes T (LT) sont régulés par un large spectre de cytokines. Néanmoins certaines sous-populations de LT restent peu définies à ce jour tout comme l’impact de cytokines sur ces cellules en conditions physiologiques et pathologiques. Une population de LT caractérisée par la double expression des corécepteurs CD4 et CD8 a été identifiée en périphérie chez des donneurs sains et sa fréquence est augmentée chez les patients atteints de certaines pathologies. Cependant, peu d’études ont déterminé le phénotype et les fonctions de ces cellules. Nous avons caractérisé le phénotype ex vivo des LT CD4+CD8+ issus de sujets sains et étudié la réponse de ces cellules à plusieurs cytokines importantes dans l’homéostasie et l’activation des lymphocytes. Notre étude révèle que les LT CD4+CD8+ forment une population hétérogène présentant de nombreuses caractéristiques des cellules mémoires. De plus, notre étude montre que ces cellules ont une capacité accrue de produire des cytokines et enzymes lytiques comparée aux LT CD4 et CD8. Finalement, nous avons observé qu’une plus grande proportion de LT CD4+CD8+ répond aux cytokines IL-2, IL-15 et IL-7 comparée aux LT CD4 et CD8 simples positifs. Plusieurs études ont révélé que certaines cytokines participent à la pathobiologie de maladies auto-immunes notamment la sclérose en plaques (SEP). La SEP est une maladie inflammatoire chronique du système nerveux central (SNC) caractérisée par une destruction de la gaine de myéline et une perte axonale à l’origine des symptômes cliniques. L’impact des médiateurs immunitaires dans la destruction et/ou la réparation reste néanmoins à éclaircir. Plusieurs études suggèrent que l’interleukine-27 (IL-27) joue un rôle central dans la SEP : l’IL-27 diminue la sévérité de la maladie dans un modèle murin de la SEP ; la réponse des patients à certaines thérapies corrèle avec des niveaux périphériques élevés d’IL-27. Notre étude avait pour but de déterminer comment l’IL-27 module les LT des patients atteints de SEP. Nous avons observé des niveaux d’IL-27 augmentés à la fois en périphérie et dans le SNC des patients SEP. Nous avons mis en évidence que les effets induits par l’IL-27 sont altérés dans les LT des patients SEP ; notamment l’induction de PD-L1 et la réponse à l’IL-12 sont plus faibles comparées aux LT de sujets sains. La voie de signalisation STAT3 est induite plus fortement en réponse à l’IL 27 dans les cellules de patients SEP en comparaison à celles de sujets sains. Finalement, nous avons démontré que la forme soluble du récepteur à l’IL-27 est présente en plus grande quantité dans le sérum des patients SEP et ces niveaux sont suffisants pour bloquer l’effet de l’IL-27 sur ses cellules cibles. En conclusion, nos résultats permettent de mieux comprendre comment certaines cytokines participent à la régulation des populations lymphocytaires. En condition pathologique, la modulation de ces cytokines pourrait s’avérer efficace dans un but thérapeutique afin de promouvoir et/ou inhiber certaines réponses immunitaires notamment dans la SEP. / Cytokines play an essential role in modulating the immune response. They act on cells from both innate and adaptive immunity by modulating their phenotype and influencing their survival. T cells are highly regulated by a broad spectrum of cytokines. Nevertheless, some subsets of T cells remain ill-defined and a better understanding of cytokine-mediated responses in physiological and pathological conditions requires further investigation. A T cell population characterized by the dual expression of CD4 and CD8 co-receptors has been identified in the periphery of healthy donors and its frequency is notably increased in several pathologies. However, very few studies have determined the phenotype and functions of these cells under physiological conditions. We performed an ex vivo phenotypic characterization of CD4+CD8+ T cells from healthy subjects and analyzed the response of these cells to several important cytokines implicated in homeostasis and activation of immune cells. Our study reveals that CD4+CD8+ T cells are heterogeneous and exhibit many characteristics of memory cells. In addition, our study shows that these cells have an enhanced ability to produce cytokines and lytic enzymes compared to CD4 and CD8 T cells. Finally, we found that a higher proportion of CD4+CD8+ T cells respond to IL-2, IL-15 and IL-7 cytokines compared to CD4 and CD8 counterparts. Numerous studies showed that lymphocyte populations and some cytokines are involved in the development of several autoimmune diseases, including multiple sclerosis (MS). MS is a chronic inflammatory disease of the central nervous system (CNS) characterized by the destruction of the myelin sheath, axonal loss and oligodendrocytic death leading to clinical symptoms. The immune mediators involved in the destruction and/or repair remain to be clarified. Several studies suggest that interleukin-27 (IL-27) plays a central role in MS: IL-27 dampens the severity of the disease in a MS mouse model and in MS patients the response to some therapies correlates with higher levels of peripheral IL-27. The purpose of our study was to determine how IL-27 modulates T cells in MS patients. We observed that IL-27 levels are increased in both periphery and CNS of MS patients. We found that IL-27-mediated effects are impaired in T cells from MS patients in particular PD-L1 induction and IL-12 response that are lower compared to T cells from healthy subjects. IL-27-triggered STAT3 signaling pathway is also enhanced in cells from MS patients compared to healthy subjects. Finally, our study showed that the soluble form of the receptor of IL-27 is present in greater levels in the serum of MS patients and the measured doses are sufficient to block the capacity of IL-27 to act on its target cells. To conclude, our results provide a better understanding of how certain cytokines participate in the regulation of lymphocyte populations. Under pathological conditions, modulation of these cytokines may be effective for therapeutic applications in order to promote and/or inhibit immune responses, particularly in MS.

Journal of Investigative Dermatology (2015) 135, 1007–1015; doi:10.1038/jid.2014.516; published online 8 January 2015 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18890号 / 医博第4001号 / 新制||医||1009(附属図書館) / 31841 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 髙折 晃史, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Enteric viruses are infectious human pathogens that initiate infection in the gastrointestinal tract. They follow a fecal-oral route of transmission and are spread by contamination of food, water, or contact between individuals. Furthermore, enteric viruses also cause significant morbidity, mortality, and economic burdens yearly. Coxsackievirus (CV) is commonly isolated among enteric viruses and is an etiological agent of hand, foot, and mouth disease, hemorrhagic conjunctivitis, and myocarditis. The virus predominantly infects infants and young children and accounts for 11% of the fatality rate in neonates. Despite CV’s impact on human health, there are no treatments or vaccines for CV infections. Using a mouse model to study a key CV, Coxsackievirus B3 (CVB3), our laboratory has found two critical factors that impact CVB3 replication and pathogenesis. First, we have demonstrated that intestinal bacteria enhance intestinal CVB3 replication. We found that certain specific bacteria (Salmonella enterica) and its cell wall components, like lipopolysaccharides (LPS), enhanced CVB3 stability and infectivity in vitro. Additionally, we found that particular constituents of LPS are required for stability to occur. These data suggest that specific bacteria may be integral in maintaining CVB3 infectivity in the intestine. Besides virus-microbiome interaction, CVB3 is also impacted by sex hormones. Using castrated mice models, we observed a sex bias to CVB3 infection, with male mice succumbing to CVB3-induced disease at an increased rate compared to female mice. Our data suggest that testosterone, a predominant male sex hormone, enhanced CVB3 intestinal replication and viral dissemination to organs in male and female mice, but lethality only in male mice. Moreover, testosterone also affected the immune response by reducing the activation of the CD8+ T cells. CD8+ T cells are required to clear the viral infection and are integral in vaccine development. In contrast, we found an enhanced CD8+ T cell response in female mice to CVB3 infection, suggesting a sex-dependent T cell response that may underlie the sex bias in disease. Overall, these data represent an essential advancement in the CV field and will help develop future therapeutics and aid in vaccine design to limit CV infections.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder, characterized by platelet destruction and/or underproduction. The pathophysiology is heterogeneous and can be mediated by autoantibodies and cytotoxic T lymphocytes (CTLs). While platelet destruction in ITP is well documented, there is little support for platelet underproduction due to the inhibition of megakaryocyte growth and considerably less support for CTL-mediated platelet underproduction. Our objective was to develop an assay that could test for CTL-mediated inhibition of megakaryocyte growth (megakaryopoiesis) in ITP, using healthy controls. Peripheral blood from healthy donors was used to prepare hematopoietic stem and progenitor cells (HSPCs). These cells were expanded with StemSpan to culture a large number of megakaryocytes for the CTL assay. Our studies show that CTLs can be stimulated in-vitro using anti-CD3 antibodies and that they can be used after freezing and thawing. We also assessed CTL stimulation via peptide presentation, using viral peptides whom almost 100% of the general population have memory CTL specificity to, in order to activate a lower frequency of CTLs and to model levels of CTL activation in autoimmune disease. Both stimulants were found to stimulate CTLs in healthy donors with donor variability in the IFN-γ ELISpot. The CTL assay was developed by co-culturing thrombopoietin (TPO) stimulated HSPCs with autologous CTLs for 7 days to observe inhibition of megakaryocyte growth. To induce CTL stimulation, CTLs were either incubated with anti-CD3 or HSPCs were incubated with viral peptides before co-culturing with CTLs. Results showed that while viral peptides can be used as an internal control for the CTL assay, it could not serve as a positive control as inhibition was donor dependent. Inhibition of megakaryocyte growth in the presence of anti-CD3 stimulated CTLs was observed in all donors, validating its use as an appropriate positive control to study CD8+ T cell pathophysiology in ITP. / Thesis / Master of Science (MSc)

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