HIV infection leads to a phenomenon of inflammaging, in which chronic inflammation induces an immune aged phenotype, even in individuals on combined antiretroviral therapy (cART) with undetectable viremia. In this study, we investigated T cell homeostasis and telomeric DNA damage and repair machineries in cART-controlled HIV patients at risk for inflammaging. We found a significant depletion of CD4 T cells, which was inversely correlated with the cell apoptosis in virus-suppressed HIV subjects compared to age-matched healthy subjects (HS). In addition, HIV CD4 T cells were prone to DNA damage that extended to chromosome ends—telomeres, leading to accelerated telomere erosion—a hallmark of cell senescence. Mechanistically, the DNA double-strand break (DSB) sensors MRE11, RAD50, and NBS1 (MRN complex) remained intact, but both expression and activity of the DNA damage checkpoint kinase ataxia-telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in HIV CD4 T cells. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in healthy CD4 T cells following ATM knockdown or exposure to the ATM inhibitor KU60019 in vitro, recapitulating the biological effects observed in HIV-derived CD4 T cells in vivo. Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, apoptosis, and cellular dysfunction in HIV-derived CD4 T cells. These results demonstrate that failure of DSB repair due to ATM deficiency leads to increased DNA damage and renders CD4 T cells prone to senescence and apoptotic death, contributing to CD4 T cell depletion or dysfunction in cART-controlled, latent HIV infection.

No description available.

Hepatitis C virus (HCV) or human immunodeficiency virus (HIV) infection leads to a phenomenon of inflammaging, in which chronic infection or inflammation induces an immune aged phenotype with T cell dysfunction. Thus, HCV or HIV infection has been deemed as a model to study the mechanisms of T cell infammaging and viral persistence in humans. In this dissertation, T cell homeostasis, DNA damage and repair machineries were investigated in patients with chronic HCV or HIV infection at risk for inflammaging. We found a significant depletion in CD4 T cells, which was correlated with their apoptosis in chronically HCV/HIVinfected patients, compared to age-matched healthy subjects. In addition, virus-infected patients’ CD4 T cells were prone to DNA damage that extended to chromosome ends (telomeres), leading to accelerated telomere erosion - a hallmark of senescence. Mechanistically, the DNA doublestrand break (DSB) sensor MRE11, RAD50, and NBS1 (MRN) remained intact, but the DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in T cells from HCV/HIV-infected individuals. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in primary CD4 T cells following ATM knockdown, or exposure to the ATM inhibitor (KU60019), as well as in CD4 T cells co-cultured with HCV-infected hepatocytes, or a T cell line infected with HIV-1 in the presence of raltegravir in vitro, which recapitulates the biological effects observed in T cells in the setting of HCV/HIV infection in vivo. Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, survival deficit, and cellular dysfunction in T cells from both HCV and HIV-infected individuals. These results demonstrate that failure of DSB repair due to ATM deficiency leads to unrepaired DNA damage and renders virally infected patients’ T cells prone to senescence and apoptosis, thus contributing to CD4 T cell loss or dysfunction during chronic HCV or HIV infection. This study reveals a novel mechanism by which ATM deficiency promotes telomeric DNA damage and premature T cell aging, and provides a new therapeutic target for inflammaging-induced immune dysfunction during chronic viral infection.

In this thesis, we investigated T cell homeostasis and DNA damage repair machineries in HIV infection. We found that the frequencies of CD4T cells were low, which is associated with cell apoptosis in HIV patients compared to healthy subjects. Importantly, these events were closely correlated to the increase in T cell exhaustion, senescence, DNA damage, and telomere attrition. Mechanistically, while the DNA damage sensors Mer11, Rad50, and NBS1 (MRN) complexes remained intact, the ataxia-telangiectasia mutated (ATM) kinase and its downstream checkpoint kinase 2 (CHK2) were significantly inhibited during HIV infection. Additionally, telomeric repeat-binding factor 2 (TRF2) that functions to protect telomeres from unwanted DNA damage was also suppressed by HIV infection. These findings revealed an important mechanism by which telomeres undergo DNA damage that remained unrepaired due to ATM deficiency and TRF2 deprotection - a process that could promote T cell apoptosis, senescence, and cellular dysfunction in HIV infection.

CD4 T-cell depletion is a hallmark of HIV/AIDS, but the underlying mechanism is still unclear. We have recently shown that ataxia-telangiectasia-mutated (ATM) deficiency in CD4 T cells accelerates DNA damage, telomere erosion, and cell apoptosis in HIV-infected individuals on antiretroviral therapy (ART). Whether these alterations in ART-treated HIV subjects occur in vitro in HIV-infected CD4 T cells remains unknown. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the telomeric DNA damage response (DDR) and cellular apoptosis in highly permissive SupT1 cells, followed by the validation of our observations in primary CD4 T cells with active or drug-suppressed HIV infection. Specifically, we established an in vitro HIV T-cell culture system with viral replication and raltegravir (RAL; an integrase inhibitor) suppression, mimicking active and ART-controlled HIV infection in vivo. We demonstrated that HIV-induced, telomeric DDR plays a pivotal role in triggering telomere erosion, premature T-cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This in vitro model provides a new tool to investigate HIV pathogenesis, and our results shed new light on the molecular mechanisms of telomeric DDR and CD4 T-cell homeostasis during HIV infection. IMPORTANCE The hallmark of HIV infection is a gradual depletion of CD4 T cells, with a progressive decline of host immunity. How CD4 T cells are depleted in individuals with active and virus-suppressed HIV infection remains unclear. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the chromosome end (telomere) DNA damage response (DDR) and cellular apoptosis in a T-cell line (highly permissive SupT1 cells), as well as in primary CD4 T cells with active or drug-suppressed HIV infection. We demonstrated that HIV-induced telomeric DDR plays a critical role in inducing telomere loss, premature cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This study sheds new light on the molecular mechanisms of telomeric DDR and its role in CD4 T-cell homeostasis during HIV infection.

T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation.

No description available.

L’atteinte de l’âge sur les populations lymphocytaires T conventionnelles CD4 et CD8 avec l’avancée en âge est relativement bien décrite en périphérie lymphoïde secondaire chez la souris, et dans le sang périphérique chez l’homme. Deux paramètres sont observés : réduction du nombre de ces cellules et altération du ratio naïve/mémoire. À l’inverse, l’évaluation des tissus lymphoïdes tertiaires et des tissus extra lymphoïdes dans les réponses immunes, reste à affiner. Notre étude au cours du vieillissement physiologique du compartiment T fut menée dans des tissus lymphoïdes et non lymphoïdes de souris C57BL/6 wild-type, âgées entre 2 et 6 mois, entre 10 et 14 mois et entre 22 et 26 mois. Nous avons démontré que la lymphopénie T classiquement décrite liée au vieillissement dans les organes lymphoïdes secondaires ne s’applique pas à tout l’organisme : les compartiments intestinaux étudiés présentent une accumulation de cellules TCRαβ+ CD4+ (TCD4) et CD8+ (TCD8). Nos résultats dévoilent un impact différentiel du vieillissement sur le nombre absolu des différents compartiments cellulaires TCRαβ+ dans les organes lymphoïdes et la muqueuse intestinale. Ces résultats suggèrent donc que la lymphopénie T décrite dans les organes lymphoïdes s’établissant au cours du vieillissement pourrait être essentiellement liée à une redistribution des lymphocytes. A l’inverse, la persistance des cellules T régulatrices dans les organes lymphoïdes secondaires pourrait être liée à une production locale dans la muqueuse intestinale. Il semble donc que l’équilibre TCD8/TCD4 peut être différemment affecté selon le site considéré et cette observation peut fournir une justification pour la plus grande susceptibilité aux infections observée avec l’âge. / Consequences of ageing on conventional CD4 and CD8 T lymphocytes populations is relatively well described in murine secondary lymphoid organs and in human peripheral blood: reduction the number of these cells and alteration of naïve/effector-memory ratio in favour of effector-memory cells. Conversely, evaluation in tertiary lymphoid tissues and non-lymphoid tissues remains to be refined. We conducted an exhaustive analysis of T cell compartments during physiological aging in lymphoid and non-lymphoid tissues isolated from wild-type C57BL/6 mice aged of 2 to 6 months, 10 to 14 months and 22 to 26 months. We demonstrated that T lymphopenia described classically associated with aging in the secondary lymphoid organs does not apply to the whole organism: intestinal compartments studied show an accumulation of TCRαβ+ CD4+ cells (TCD4) and CD8+ (TCD8). Our results reveal a differential impact of aging on the absolute number of different TCRαβ+ cellular compartments in lymphoid organs and intestinal mucosa. T cell lymphopenia in secondary lymphoid organs currently associated to ageing may essentially reflect T cell redistribution. TCD8/TCD4 balance may be affected differently depending on the site considered and this observation may provide a rationale for the greater susceptibility to infection observed with age.

T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation.