Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

Impaired cell mediated immunity is the clinical hallmark of HIV infection yet the manner in which CD8 T-cells are disabled is not yet fully understood. IL-7 signalling is essential for normal CD8 T-cell development and function. Our lab has previously shown decreased expression of the IL-7 receptor a-chain (CD127) on circulating CD8 T-cells in HIV+ patients is mediated by the HIV Tat protein which results in poor CD8 T-cell function. Soluble Tat protein is secreted by infected CD4 T-cells and taken up by neighbouring uninfected CD8 T-cells through endocytosis. Once in the cytoplasm, Tat translocates to the inner leaflet of the cell membrane where it binds directly to the cytoplasmic tail of CD127 inducing receptor aggregation, internalization, and degradation by the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is able to reduce IL-7 signaling and impair CD8 T-cell proliferation and function. To determine which domain(s) in the cytoplamic tail of CD127 are required for interaction with Tat, a series of plasmids encoding for CD127 deletion mutants were successfully created. These series of mutant CD127 coding sequences were transfected into a eukaryotic expression system, the Jurakt cell line, where CD127 mutants were successfully expressed. Before determine which region on CD127 is required for Tat binding, an optimized Ni-NTA column system was used to successfully isolate histidine-tagged HIV-1 Tat at a high yield and purity from E. coli. This HIV Tat protein was used to treat the lysates of the Jurakt cells transfected with the panel of CD127 mutants. CD127 was then immunoprecipitated, followed by Western analysis of the immune complexes to detect Tat protein. Tat was immunoprecipitated with all CD127 mutants suggests neither tyrosine 449, box 1, the acidic region, serine region nor C-tail are specifically required for Tat binding to CD127.

Interleukin (IL) -7 is an essential non-redundant cytokine that influences T-cell differentiation, proliferation, homeostasis and T-cell functions. In T-cells, IL-7 signals are transduced via IL-7's heterodimeric receptor composed of a common, γ chain (CD132) and an IL-7 specific, α chain (CD127). In light of the many roles that IL-7 plays in T-cell biology, it is no surprise that CD127 expression is tightly regulated in T-cells. In this study, I explore the effects that disease specific mutations in CD127 have on CD127 expression, regulation and signal transduction using an in vitro T-cell model. Here I specifically examined four disease associated mutations of CD127: P132S associated with severe combined immunodeficiency; L242_L243insNPC associated with T-cell acute lymphoblastic leukemia; I356V & T244I associated with autoimmune diseases like multiple sclerosis, rheumatoid arthritis and type 1 diabetes. In developing my model, I decided to use Jurkat cells because they expressed high endogenous surface levels of CD132, low endogenous surface levels of CD127 and endogenous STAT5. Jurkat cells were transduced with lentiviruses that induced expression of either WT or one of the four mutant CD127. I found that transduced Jurkat cells produced the WT and all four mutant CD127 proteins. I also found that wild type CD127, I356V, L242_L243insNPC and T244I mutant CD127 proteins were all expressed at the same level on the cell surface. However, I could not detect P132S mutated CD127 protein in its native state on the surface or intracellularly. I also found no differences between the mutant CD127 and wild type CD127 with regards to the level of soluble CD127 transcripts. I found that cell lines expressing L242_L243insNPC, I356V and T244I mutant CD127 protein, down-regulated surface CD127 at high IL-7 doses (25ng/mL) to the same extent as in the cell line expressing wild type CD127 protein. Interestingly, at the low IL-7 dose (1ng/mL) these mutant CD127 cell lines down-regulated surface CD127 to a lesser degree the wild type CD127 cell line. Further studies are required to elucidate whether P132S mutated CD127 is expressed on the surface and if T224I and I356V mutations in CD127 enhance signaling. By understanding CD127 dysregulation and dysfunction in disease states, we can potentially develop therapeutics that can return the function of CD127 to normalcy.

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.

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.

Le sepsis, cause majeure de décès en réanimation, entraîne des altérations immunitaires associées à un risque augmenté de décès et d'infections secondaires. En particulier, les lymphocytes T (LT) de patients présentent des altérations phénotypiques et fonctionnelles caractéristiques d'un état d'épuisement. Pour améliorer leur réponse, l'IL-7 est actuellement proposée comme immunostimulant. Son récepteur existe sous différentes formes: protéines membranaire (CD127) et soluble (sCD127) et différents transcrits. Leur expression n'a été que peu étudiée dans le sepsis. De plus, dans d'autres contextes cliniques, le phénotype CD127lowPD1high est proposé comme marqueur d'épuisement des LT mais n'a jamais été évalué dans le sepsis. L'objectif de ce projet était d'évaluer l'expression du récepteur de l'IL-7 comme biomarqueur dans le sepsis, d'étudier sa régulation chez les patients et la présence du phénotype CD127lowPD-1high en lien avec les altérations des LT. L'objectif était également de mettre au point un modèle ex vivo reproduisant les altérations intrinsèques aux LT de patients. Nous avons confirmé l'intérêt de la concentration plasmatique de sCD127 comme marqueur de mortalité chez les patients de réanimation. Nous avons mis en évidence l'association de l'expression des transcrits IL7R correspondant à des formes solubles avec la mortalité, ainsi que leur régulation intrinsèque dans les LT. Enfin, la proportion augmentée de LT CD127lowPD-1high chez les patients en choc septique en fait également un candidat potentiel comme marqueur spécifique des altérations des LT. Dans un deuxième temps, nous avons développé un modèle d'altérations induites par l'activation à partir de LT purifiés de volontaires sains reproduisant les altérations des LT de patients en choc septique. Ce modèle suggère un rôle de l'activation des LT dans le développement de leurs altérations et pourrait permettre d'étudier de nouveaux aspects de la physiopathologie des LT dans le sepsis / Sepsis is the leading cause of death in intensive care units (ICU). Septic patients develop immune dysfunctions that are associated with an increased risk of death and secondary infections. In particular, patients’ T lymphocytes present phenotypic and functional alterations characteristic of exhaustion. To restore these alterations, IL-7 is currently proposed as an immunostimulant. Its receptor exists in different forms: a membrane (CD127) and a soluble (sCD127) proteins and different transcripts. The IL-7 receptor expression has not been studied in sepsis. Moreover, the CD127lowPD-1high phenotype is proposed as a T cell exhaustion marker in other clinical contexts but has never been explored in sepsis.In this context, the aim of this project was first to evaluate the expression of the IL-7 receptor as a biomarker in sepsis, to study its regulation in patients and the CD127lowPD-1high phenotype in parallel with T cell alterations in septic shock patients. Secondly, the objective was to develop an ex vivo model reproducing phenotypic and functional T cell alterations observed in patients.We confirmed the role of sCD127 plasmatic concentration as a marker of mortality in septic shock and more generally in ICU patients. In addition, we demonstrated the association of the expression of the IL7R transcripts, corresponding to soluble forms with mortality as well as the intrinsic regulation of their expressions in T lymphocytes. Finally, we found an increased proportion of CD127lowPD-1high T cells in septic shock patients. This population could also be evaluate as a potential specific marker of T lymphocyte alterations. Secondly, we developed an ex vivo model of alterations induced by activation of purified T cell from healthy volunteers reproducing alterations observed in patients’ T cells. This model suggests a role of T cell activation in the development of their alterations and could be used to explore new aspects of T cell alterations pathophysiology in sepsis