CD32+CD4+ T cells are enriched in HIV-1 DNA

Background: CD32 was reported to mark the HIV-1 reservoir harboring replication-competentproviruses, but several recent reports challenged this finding. We aimed to confirm or deny theusefulness of CD32 as a marker of the latent reservoir and to further characterize the phenotype of theseCD32+CD4+ T cells, as well as the transcriptional activity of HIV-1 residing in this reservoir.Methods: CD32 expression was measured by flow cytometry on PBMCs from ART-treated HIV-1 infectedpatients and uninfected controls. Co-expression of HLA-DR, immune checkpoint receptors (PD-1, TIGIT,LAG-3) and CD2 was measured by flow cytometry. HIV-1 DNA and unspliced RNA were quantified in bulkPBMC samples and in CD32+ and CD32- fractions of CD4+ T cells sorted with magnetic beads.Results: The median frequency of CD32+CD4+ T cells in HIV-infected individuals (n=18) was 0.07% whichwas significantly higher than in the controls (0.01%, p=0.016). We found a positive correlation betweenthe percentage of CD32+CD4+ T cells and total HIV-1 DNA load in PBMCs (rho=0.58; p=0.012).CD32+CD4+ T cells demonstrated increased expression of LAG-3 (p=0.016), TIGIT (p=0.016) and HLA-DR(p< 0.0001) compared with CD32-CD4+ T cells in HIV-infected patients. In the full sample, CD32+CD4+ Tcells were not enriched for HIV-1 DNA or RNA compared with CD32-CD4+ cells. However, in a subgroupof patients with smaller (and presumably purer) CD32+CD4+ T-cell fractions (n=9), we observed asignificant enrichment for HIV-1 DNA in this fraction (average of 6-fold, p=0.012). We thereforeoptimized our assay to isolate a purer fraction of CD32+CD4+ T cells and found a positive enrichment forHIV-1 DNA in the CD32+CD4+ fraction in all the additional patients (n=7) tested (average of 14-fold,p=0.016).Conclusions: We confirmed that CD32+CD4+ T cells are enriched for HIV-1 DNA, although the level ofenrichment was less pronounced than previously reported. Our results also highlight the importance ofgetting a sufficiently pure CD32+CD4+ T cells fraction for analysis and might explain the negative resultsobtained by others. Our data further indicate that these CD32+CD4+ T cells are activated cells, and thatthey often co-express the immune checkpoint receptors TIGIT and LAG-3.