CD32 expressing doublets in HIV-infected gut-associated lymphoid tissue are associated with a T follicular helper cell phenotype

Abstract

Gut-associated lymphoid tissue (GALT) is a key location for the HIV reservoir. The observation that B-cell-T-cell doublets are enriched for CD32a (a low-affinity IgG receptor) in peripheral blood raises interesting questions, especially as these cells have been associated with HIV DNA in some studies. We sought to determine if similar doublets were present in GALT, the significance of these doublets, and their implications for the HIV reservoir. Given the importance of GALT as a reservoir for HIV, we looked for expression of CD32 on gut CD4 T cells and for evidence of doublets, and any relationship with HIV DNA in HIV + individuals initiated on antiretroviral therapy (ART) during primary HIV infection (PHI). Tonsil tissue was also available for one individual. As previously shown for blood, CD32 high CD4 cells were mainly doublets of CD4 T cells and B cells, with T-cell expression of ICOS in tonsil and gut tissue. CD4 T cells associated with CD32 (compared with 'CD32−' CD4 cells) had higher expression of follicular markers CXCR5, PD-1, ICOS, and Bcl-6 consistent with a T follicular helper (TFH) phenotype. There was a significant correlation between rectal HIV DNA levels and CD32 expression on TFH cells. Together, these data suggest that CD32 high doublets are primarily composed of TFH cells, a subset known to be preferentially infected by HIV.

The Quest for Cellular Markers of HIV Reservoirs: Any Color You Like

“…Although it has been shown previously that HIV can establish latent infection in activated CD4 + T cells (110, 111) and that the peripheral blood HIV DNA load is higher in activated compared to resting cells in ART-treated individuals (112), the existence of latently infected CD4+ cells that are activated, and therefore relatively short-lived, suggests continuous replenishment of this component of the reservoir by cellular proliferation (113). At present, however, high HLA-DR expression on CD32 + CD4 + cells should be interpreted with caution, as it cannot be ruled out that the HLA-DR signal may partly originate from the non-T cells within the contaminating CD32 + T-non-T cell doublets or conjugates, as recently demonstrated by Thornhill et al (107).…”

“…The excess of residual non-T cells in the sorted CD32 + fraction can easily obscure the enrichment for HIV DNA in CD32 + CD4 + cells. Alternatively, certain cell sorting strategies and/or settings might result in the isolation of T-B cell doublets or conjugates instead of bona fide CD32 + CD4 + T cells, as reported by two groups (105, 107). Because another CD32 isoform, CD32b, is highly expressed on the surface of B cells, and consequently on T-B cell doublets or conjugates, the latter cells may be preferentially recognized during FACS sorting for CD32 + CD4 + cells as most anti-CD32 antibodies do not discriminate between the CD32a and CD32b isoforms.…”

Current Status of Latency Reversing Agents Facing the Heterogeneity of HIV-1 Cellular and Tissue Reservoirs

“…Recently, the expression of CD32a has been reported as a potential marker of memory CD4 + T cells harboring a replication-competent latent virus in aviremic patients under ART (Descours et al, 2017;Darcis et al, 2019). The role of CD32a as a cellular marker of HIV-1 reservoirs has been the subject of several works Martin et al, 2018;Osuna et al, 2018;Thornhill et al, 2019). A complete study presented at CROI by Darcis et al (CROI 2019, Poster 346 -CD32 + CD4 + T cells are enriched in HIV-1 DNA) showed that active CD4 + T cells co-expressing HLA-DR and CD32a are highly enriched with HIV-1 DNA.…”

Editorial: Immune Surveillance of the HIV Reservoir: Mechanisms, Therapeutic Targeting and New Avenues for HIV Cure

“…Due to the importance and implications of this finding, many investigators turned to CD32, including Martin et al. Although they were able to find evidence of CD32 expression in analysis of cells that contained HIV DNA, there was no correlation with total or integrated HIV DNA levels (or time to rebound on stopping ART), consistent with subsequent findings that CD32 may in fact be a marker of T cell-B cell doublets that have survived flow cytometry gating (10), but this remains a contentious area.…”