Background
Latently HIV-infected cells persist during antiretroviral therapy (ART) and these viral reservoirs inevitably trigger a viral rebound when treatment is interrupted. Identifying the mechanisms contributing to the persistence of these reservoirs is essential to the development of strategies aimed at eradicating them. In this study, we used a single-cell approach to measure multiple parameters that could contribute to the persistence of HIV-infected cells after prolonged ART.
Methods
Using a modified version of the HIV-Flow assay, we phenotypically analyzed the cells harboring inducible proviruses (p24+ cells) in blood samples from 10 participants who maintained undetectable plasma viral loads for 3-24 years. CD4+ T cells were stimulated for 24h with anti-CD3 and anti-CD28 antibodies, and cellular markers associated with T cell quiescence, senescence, cytotoxicity and immune functions were measured.
Results
The phenotype of 395 p24+ cells was determined from a total pool of 390 million CD4+ T cells. p24+ cells more frequently expressed the anti-apoptotic protein Bcl-2 (p=0.006, fold increase 1.1), the immunotoxins granzymes A and K (p<0.0001 for both, fold increase 7 and 3, respectively) and the immune checkpoint molecules PD-1 and TIGIT (p<0.0001, fold increase 1.8 and p<0.0001, fold increase 3.4). We also found that p24+ cells more frequently expressed the cytokines TNF-a, IFN-g, IL-2 and IL-17 (p<0.02 for all, fold increases 5, 9, 14 and 55 respectively). Intriguingly, simultaneous expression of multiple cytokines, a signature of senescent T cells, was much more frequent in p24+ cells than in p24- cells (p<0.0001, fold increase 10). Finally, p24+ cells co-expressed the senescence markers KLRG-1 and CD57 more frequently than p24- cells (p=0.0009, fold increase 2.5).
Conclusions
Our data indicate that immunosenescence is a signature of persistently infected cells after prolonged ART, suggesting that senolytic agents may be used to reduce the HIV reservoir.
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