Identifying cellular subsets that preferentially harbour HIV is important for curative strategies. We aimed to address the role of the immune checkpoints, programmed cell death 1 (PD1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4) for HIV persistence on ART.
We collected peripheral blood mononuclear cells (PBMCs) and lymph node (LN) mononuclear cells by performing leukapheresis and LN biopsies in people with HIV (PWH) on suppressive ART. Memory CD4+ T cells were sorted into four subsets based on their expression of PD1 and CTLA4 to obtain: double-positive (PD1+CTLA4+), PD1 single positive (PD1+CTLA4-), CTLA4 single positive (PD1-CTLA4+) and double-negative (PD1-CTLA4-) cells. Within each sorted subset from blood and LN we quantified total HIV DNA and cell-associated unspliced HIV RNA (CA-US HIV RNA) and also performed the tat/rev limiting dilution assay (TILDA) to quantify the frequency of cells with inducible multiply-spliced HIV RNA.
We enrolled 21 PWH with 4.2-14.1 years of ART-mediated viral suppression. We obtained paired LN biopsies and leukapheresis samples in 8 participants and leukapheresis only in 13 participants. The frequency of memory CD4+ T cells co-expressing PD1 and CTLA4 was higher in LN tissue compared to blood whereas double-negative cells were more frequent in blood. We found a significant enrichment of total HIV DNA in blood memory CD4+ T cells co-expressing PD1 and CTLA4 with a median 1.8-fold (IQR 1.1-2.5, P=0.018) higher level of HIV DNA when compared to their double-negative counterpart. This enrichment was not seen in LN cells. The frequency of cells containing HIV DNA within most PD1/CTLA4 subsets in both blood and LN correlated with higher CD8+ T cell counts and percentages at study entry. Despite their enrichment for total HIV DNA, a lower proportion of double-positive memory CD4+ T cells in blood produced multiply spliced HIV RNA upon PMA/ionomycin stimulation. There was no difference across PD1/CTLA4 subsets in the level of CA-US HIV RNA in blood or LN.
The frequency of HIV-infected cells was moderately higher in blood memory CD4+ T cells co-expressing PD1 and CTLA4 but this enrichment was not seen in LN. Double-positive memory CD4+ T cells from blood had a lower frequency of inducible virus, potentially indicating these cells are characterised by their negative signalling and a limited susceptibility to induction of latent HIV.