Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpson??s diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into ??early?? and ??late?? differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ??-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection.

Cytotoxic T-lymphocytes are important for the control of viral replication during HIV infection, however the magnitude and breadth of HIV-specific CD8+ T-cell response does not correlate well. The purpose for this study was the examination of the HLA-B*2705-specific CD8+ T-cell response to the KRWIILGLNK (KK10) epitope as a definitive model of immune control over HIV replication. The breadth of the T-cell receptor (TCR) repertoire was determined for an association between the qualitative nature of this response and immune escape and therefore, disease progression. Methodology was developed and validated for TCR repertoire analysis in formaldehyde fixed antigen-specific CD8+ T-cells. The TCR repertoire for the KK10-specific CD8+ T-cell response was defined in cross-section and longitudinally for 6 HLA-B*2705+ patients. Comparison was made to cognate HLA-A*0201 CMV NV9 and HLA-B*2705 EBV RL9-specific CD8+ T-cell populations using the Simpson??s diversity index and the Morisita-Horn similarity index for standardized repertoire analysis. HLA-B*2705 KK10-specific TCR repertoire was not found to be a determinant of control. Greater clonotype variation was found within CMV-specific CD8+ T-cell populations, suggesting an association with reactivation of CMV and disease state. An association was found between KK10-specific population diversity and the prevalence of cognate KK10 epitope in vivo. Cross-reactivity observed for dominant KK10-specific clonotypes suggested that avidity of CD8+ T-cells was important for in vivo survival. Phenotype and function was tested through multiparameter analysis of HIV and CMV-specific CD8+ T-cells. Increased frequency of CD127 (IL-7R) and Bcl-2 expression within dominant populations was suggestive of selective advantage. Division of dominant and sub-dominant CMV-specific CD8+ T-cell populations into ??early?? and ??late?? differentiation phenotypes indicated virus-specific mechanisms of clonotype turn over. No simple association of TCR expression was found for HIV and CMV-specific CD8+ T-cells with published examples of definitive TCR bias. Over-represented TCR ??-chain families of patients were found in association with public clonotypes. Convergent recombination of TCR genes was demonstrated as a mechanism for the prevalence of shared clonotypes. Standardized assessment of T-cell repertoire successfully identified mechanisms of antigen-specific CD8+ T-cell recruitment. A substantial increase in sample numbers is required before this methodology can be used to accurately demonstrate the importance of TCR repertoire usage in the control of human viral infection.

Les chimiokines et leurs récepteurs respectifs jouent un rôle important dans l’immunité innée et adaptative. Les récepteurs de chimiokines identifient des cellules T CD4+ avec potentiel de migration dans des tissus spécifiques et à fonctionnalité distincte du point de vue de la spécificité antigénique et de la production de cytokines. L’identité de la population des cellules T CD4+ susceptibles versus résistantes à l’infection par le virus de l’immunodéficience humaine (VIH) reste mal définie. Le recrutement dans les muqueuses intestinales d’un excès de cellules T effectrices (CD8+) comparé aux cellules cibles (CD4+) représente un bon pronostic de l’infection par le virus de l’immunodéficience simienne (VIS), tandis que la déplétion des cellules Th17 dans les tissus lymphoïdes associés au tractus gastro-intestinal (GALT) est un marqueur de la progression de l’infection à VIH. L’effet régulateur des chimiokines sur l’activation de la réplication virale dans différentes sous-populations cellulaires T CD4+ reste peu étudié. Ce projet de maîtrise est divisé en 3 parties: (1) l’identification des récepteurs de chimiokines CCR4, CXCR3 et CCR6 comme marqueurs de surfaces des sous populations T CD4+ avec susceptibilité distincte à l’infection par le VIH; (2) la caractérisation phénotypique et fonctionnelle des cellules T CD4+ et T CD8+ spécifiques au VIH de sujets à progression lente vers le stade sida (LTNP); et (3) les effets des chimiokines ligands de CCR4, CXCR3 et CCR6 sur l’activation cellulaire et la réplication virale in vitro. Nos résultats démontrent que les cellules T CD4+ CCR4+CCR6+ (profile cytokinique Th17) et CXCR3+CCR6+ (profile cytokinique Th1/Th17) sont hautement permissives à l’infection par le VIH. Nous proposons également de nouveaux corrélats de protection immunitaire contre le VIH chez les sujets LTNP: (i) le potentiel de co-localisation dans les muqueuses intestinales des cellules T CD4+ et CD8+ spécifiques au VIH via l’intégrine β7, (ii) le ratio élevé entre les cellules T effectrices (CD8+) versus les cellules cibles (CD4+) spécifiques au VIH, (iii) le profil cytokinique Th17 et (iv) la capacité des cellules T CD4+ et CD8+ spécifiques au VIH à produire des ligands de CCR5 bloquant l’entrée virale. Finalement, nos résultats sur l’effet co-stimulateur des chimiokines sur les cellules T et leurs effets opposés sur la réplication virale démontrent l’implication du réseau des chimiokines dans la régulation de la pathogenèse de l’infection à VIH. / The chemokines and their counter receptors play an important role in regulating innate and adaptive immunity. The chemokine receptors serve as markers for distinct CD4+ T cell subsets with specific tissue homing potential, antigenic specificity, and polarization profile. There is limited knowledge on the identity of primary CD4+ T cell subsets selectively targeted by human immunodeficiency virus (HIV) infection. The recruitment in the intestinal mucosa of excess effector (CD8+ T) versus target (CD4+ T) cells predicts a good outcome for the simian immunodeficiency virus (SIV) infection, while the depletion of Th17 cells in the gut-associated lymphoid tissues (GALT) represents a marker for HIV disease progression. The regulatory role of chemokines on cellular activation and on HIV replication in different CD4+ T cell subsets remains poorly investigated. This M.Sc. project is dived in 3 parts: (1) the identification of chemokine receptors CCR4, CXCR3 and CCR6 as surface markers of CD4+ T cell subsets, with susceptibility to HIV replication (2); the phenotypic and functional characterization of HIV-specific CD4+ and CD8+ T cells in HIV-infected long-term-non progressor (LTNP) individuals and (3) the effect of chemokine ligands of CCR4, CXCR3 and CCR6 on cellular activation and viral replication in vitro. Our results reveal that CD4+ CCR4+CCR6+ T (Th17 cytokine profile) and CD4+ CXCR3+CCR6+ (Th1/Th17 cytokine profile) T cells are highly permissive to HIV replication. Also, our results suggest new correlates of immune protection against HIV in LTNP subjects: (i) the co-localization potential of HIV-specific CD4+ and CD8+ T cells in the intestinal mucosa via the integrin β7, (ii) the high ratio between effector (CD8+) versus target (CD4+) T cells, (iii) the Th17 cytokine profile and (iv) the ability of HIV-specific CD4+ and CD8+ T cells to produce CCR5 ligands blocking viral entry. Finally, our results on the co-stimulatory effect of chemokines on T cell activation and their opposite impact on HIV replication in CD4+ T cells demonstrate the role played by the chemokine network during HIV pathogenesis.