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.

The cytokine, interleukin-7 (IL-7), has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in ATP levels that were attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three important regulators of glucose metabolism, the glucose transporter, GLUT-1, and two glycolytic enzymes, Hexokinase II (HXKII) and phosphofructokinase-1 (PFK1), using an IL-7-dependent T-cell line and primary lymphocytes. We found that in lymphocytes deprived of IL-7 loss of glucose uptake correlated with decreased expression of HXKII. Re-addition of IL-7 to cytokine deprived lymphocytes restored the transcription of the HXKII gene within 2 hours, but not that of GLUT-1 or PFK1. IL-7-mediated increases in HXKII, but not GLUT-1 or PFK-1, were also observed at the protein level. Inhibition of HXKII with 3-Bromopyruvate or specific siRNA decreased glucose utilization, as well as ATP levels, in the presence of IL-7, while over-expression of HXKII, but not GLUT-1, restored glucose retention and increased ATP levels in the absence of IL-7. This IL-7 mediated HXKII gene expression was abrogated with inhibition of JNK pathway. IL-7 also increased activation of AP-1 complex and DNA binding of JunD, a transcriptional complex thought to be negative regulator of proliferation. We found that over expression of HXKII caused cell cycle arrest and cell death, indicating that a potent IL-7 signal could produce negative growth signals. We conclude that IL-7 controls glucose utilization by regulating the gene expression of HXKII through activation of JNK-JunD pathway, suggesting a mechanism by which IL-7 supports bioenergetics that control cell fate decisions in lymphocytes.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Malgré leur efficacité pour réduire la réplication du VIH, les traitements antirétroviraux ne s’accompagnent pas systématiquement de la restauration du compartiment des lymphocytes T CD4+ périphériques. L’espérance et la qualité de vie des individus en échec immunologique sont grandement impactées. Concomitante avec les anomalies périphériques, une atteinte des progéniteurs hématopoïétiques rend compte des fréquentes cytopénies observées au cours des stades tardifs de l’infection. Si l’infection directe des progéniteurs CD34+ reste marginale, les études qualitatives menées in vitro évoquent la perturbation du potentiel de différenciation de ces cellules.Nous avons sélectionné et apparié les patients infectés par le VIH à chaque extrême quand au profil de leur restauration immunitaire et un groupe de donneurs non infectés. Les répondeurs (IR) et non répondeurs immunologiques (INR) traités depuis plus de 8 ans présentaient les caractéristiques similaires pour chaque paramètre pouvant impacter la magnitude de la reconstitution du système immunitaire. Les INR montraient l’activation chronique du système immunitaire, l’inflammation persistante et les signes de l’atteinte de la thymopoïèse. La fréquence des progéniteurs hématopoïétiques circulants n’étant pas différente entre les deux groupes de patients, nous avons analysé le potentiel de ces cellules aux stades pré-thymiques de la différenciation.En utilisant un système de co-culture des progéniteurs hématopoïétiques avec une lignée stromale OP9-DL1 (différenciation T) ou MS5 (différenciation B et NK) avec un cocktail cytokinique approprié, nous avons mis en évidence l’altération du potentiel de différenciation T des cellules issues de patients INR impactant leur restauration périphérique. Ce n’était pas le cas chez les patients IR qui étaient similaires aux donneurs non-VIH. En revanche, le potentiel NK était impacté chez tous les patients infectés en comparaison aux donneurs. Finalement aucune anomalie de potentiel B n’était révélée.En étudiant les voies moléculaires de l’engagement des précurseurs T (Notch), de leur prolifération (IL-7/IL7R) et leur survie (Fas/FasL, TNFR, caspase-1, P2X7) nous avons constaté une diminution de la viabilité des progéniteurs hématopoïétiques chez les patients VIH+ qui présentaient d’avantage d’activation de la caspase-1 qui orchestrait la mort cellulaire par pyroptose. De plus, l’expression de certains gènes-cibles de Notch était clairement Notch-indépendante. Néanmoins, les différences dans le profil transcriptionnel de BCL11B entre les patients IR et INR nous ont permis de proposer un modèle selon lequel la différenciation T était promue chez les patients IR au dépit de celle des précurseurs NK. Enfin, les progéniteurs CD34+ de patients INR présentait la surexpression du P2X7 (récepteur à l’ATP extracellulaire) et l’absence de l’ectonucléotidase CD73 (hydrolyse de l’ATP) ce qui suggérait leur susceptibilité accrue aux nucléotides extracellulaires.L’ensemble de données nous permet de postuler qu’il existe un microenvironnement hautement inflammatoire dans la niche médullaire des progéniteurs hématopoïétiques chez les patients VIH+ qui perturbe leur survie et différenciation. Cette mortalité accrue des cellules CD34+ et probablement des cellules voisines amplifie l’inflammation locale. Ce processus est compensé chez certains patients par la meilleure différenciation T des progéniteurs CD34+ et la réponse immunologique qui s’en suit. Quand ce n’est pas le cas l’atteinte de la lymphopoïèse est importante et l’absence de la reconstitution de la population des lymphocytes T CD4+ périphériques est observée. Ainsi, nous pensons avoir identifié une population de patients infectés par le VIH pour qui les interventions ponctuelles avec les médicaments anti-inflammatoires (par exemple, les antagonistes du P2X7) peuvent s’avérer d’un bénéfice clinique irréfutable. / Despite the efficient reduction of the HIV replication, the administration of combination antiretroviral therapy (c-ART) is not systematically accompanied by the restoration of the peripheral T CD4+ lymphocyte compartment. The life expectancy and quality are severely impacted in individuals with immunological failure. Together with peripheral abnormalities, an alteration of CD34+ hematopoietic progenitor may explain the frequency of the cytopenia observed in the latest stages of the disease. While a direct infection of CD34+ progenitors is thought to be extremely rare, quantitative studies performed in vitro have highlighted the impairment of the differentiation potential of these cells.We selected and matched individuals infected with HIV presenting extremely opposite immunological profile in response to c-ART as well as non-infected donors. The Immune Responders (IR) and Immune Non Responders (INR) treated since more than 8 years, presented similar characteristics for each parameter known to be involved in poor reconstitution of immune system. INR patients showed chronic immune activation, persistent inflammation and thymic regeneration failure. The frequency of circulating CD34 hematopoietic progenitors being not different between both groups of patients, we analyzed the differentiation potential of these cells at pre-thymic stages of lymphopoiesis.Using a co-culture system of hematopoietic progenitors with stromal cell lines OP9-DL1 (T-cell assay) or MS5 (B- and NK-cells assay) with appropriate cytokines, we highlighted an alteration of T-cell differentiation potential in INRs impacting their peripheral restoration. This was not observed in IRs who were similar to non-HIV donors. On the other hand, NK-cell differentiation potential was impaired in both groups of patients in comparison to non-HIV donors. Lastly, no abnormalities in B-cell potential were revealed.Studying molecular pathways involved in T-cell specification (Notch), proliferation (IL-7/IL7R) and survival (Fas/FasL, TNFR, caspase-1, P2X7) we observed the decreased viability of hematopoietic progenitors in HIV patients with increased caspase-1 activation involved in cellular death by pyroptosis. Moreover, expression of some Notch target genes was clearly Notch-independent. However, differences in transcriptional profile of BCL11B between IRs and INRs allowed us to postulate that T-cell differentiation is promoted over NK-cell differentiation in IR patients. Finally, CD34+ cells from INRs presented P2X7 overexpression (extracellular ATP receptor) and absence of CD73 ectonucleotidase (ATP hydrolysis) pointing out their increased susceptibility to extracellular nucleotides.Taken together our data, we postulate that highly inflammatory microenvironment of hematopoietic progenitor’s bone marrow niche disturbs their survival and differentiation in HIV patients. Thus, increased cellular death of CD34+ cells and probably neighboring cells amplifies the local inflammation. This is compensated in some patients by enhanced T-cell differentiation of CD34+ progenitors and results in immunological success. When it is not the case, the alteration of lymphopoiesis is important and the absence of reconstitution of peripheral T CD4+ lymphocyte compartment is noted. We believe have identified the population of HIV-infected individuals who will benefit from occasional administration of anti-inflammatory drugs (such as P2X7 antagonists).

B cell development is a carefully orchestrated process involving many transcription factors acting in concert with cytokine signals, particularly IL-7. The transcription factor c-Myb has long been implicated in B cell development, however surprisingly little is known about the function of c-Myb in B cell progenitors. I have used several mouse models of c-Myb deficiency to investigate the role of c-Myb in the B cell lineage. Conditional deletion of c-Myb in early B cell progenitors using mb-1Cre (c MybΔmb1/Δmb1) leads to a striking lack of B cells from the pre-pro-B cell stage onwards, demonstrating that c-Myb is absolutely required for B cell development. Mice homozygous for a hypomorphic allele of c-Myb (c MybPlt4/Plt4) also display a severe reduction in B cells; in these mice, defects in lymphoid development can be detected within the multipotent progenitor compartment of bone marrow. c-Myb activates transcription via coactivator proteins, particularly CBP and p300. Mice bearing a point mutation in p300 (p300Plt6/Plt6) that inhibits the interaction of p300 with c Myb display a partial block in B cell development, highlighting the importance of the c Myb-p300 complex for B cell development. Together, these mice demonstrate that c-Myb regulates B cell development by functioning both in multipotent progenitor cells and directly in B cell progenitors. In addition, I show that the B-lymphopenia in c-Myb deficient mice is related to a profound defect in IL-7 signalling. IL-7 normally stimulates the proliferation, survival and differentiation of B cell progenitors, however pro-B cells from c-MybPlt4/Plt4 and c MybΔmb1/Δmb1 mice fail to respond to IL 7. Expression of the IL-7Rα chain is reduced on pro-B cells from c MybPlt4/Plt4 and c-MybΔmb1/Δmb1 mice, suggesting that Il7r may be a c-Myb target gene in B cells. Reporter gene assays show that c-Myb can activate the Il7r promoter in synergy with the transcription factor Pu.1. Overall, this work demonstrates that c-Myb is essential for early B cell development and plays a critical role in linking cytokine signals to the transcription factor networks in B cell progenitors.