HIV-1 persists after many years of suppressive antiretroviral therapy (ART). It is well established that lymphoid tissues serve as primary anatomic sites for HIV-1 replication. We have previously shown that PD-1+/Tfh cells serve as primary cell compartment for HIV-1 replication in viremic patients and for persistent HIV transcription in long-term treated aviremic individuals. Recently, CD32 has been identified as a marker of HIV reservoir in blood memory CD4 T cells. We therefore investigated the distribution of the HIV reservoir in lymph nodes (LNs) memory CD4 T cell populations defined by the expression of CD32 and PD-1.
LN biopsies were obtained from 10 HIV-1 infected viremic individuals naive to ART and 13 aviremic long-term treated individuals. Expression of CD32, PD-1 and a large panel of cell lineage, activation and migration markers was assessed in memory CD4 T cells using flow- and mass cytometry that included 30 isotope conjugated antibodies. HIV-DNA was measured in total CD32+ and PD-1+ cell populations and cell associated HIV-RNA in memory CD4 T cells isolated on the basis of PD-1 and CD32 expression (PD-1-CD32-, PD-1-CD32+, PD-1+CD32-, PD-1+CD32+).
Similar to what previously shown for PD-1+ CD4 T cells, the frequency of CD32+ CD4 T cells was increased in viremic as compared to treated individuals (3% vs 1.2% p<0.0001) and positively correlated with viremia while negatively with years of suppressive ART and CD4 cell count. CD32+ CD4 T cells were enriched for total HIV DNA as compared to CD32- cells in both viremic and treated individuals (average 210 fold, n=6, p=0.01, and 1.9 fold, n=7, p=0.03 respectively) but no difference was found as compared to PD-1+ cells. In both viremics and ART treated individuals CD32+ CD4 T cells were found predominantly within the PD-1+ and Tfh cell populations. Double positive CD32+ PD-1+ cells were phenotypically similar to CD32-PD-1+ cells and Tfh cells as indicated by the expression of ICOS, CD57, CD38, CXCR5, CD40L, CCR5 and CXCR4. CD32+PD-1+ CD4 T cells were enriched in cell associated HIV RNA as compared to CD32-PD-1- (average 7.6 fold), to CD32+ PD-1- (5.2 fold) and to CD32-PD-1+ cell populations (average 7.6 fold) (n=4 ART treated).
Co-expression of CD32 and PD-1 defines a population of Tfh cells serving as the major HIV cell reservoir in HIV long-term ART treated individuals.