Unequal distribution of genetically-intact HIV-1 proviruses in cells expressing the immune checkpoint markers PD-1 and/or CTLA-4

INTRODUCTION: HIV-1 persists in resting CD4(+) T-cells despite antiretroviral therapy (ART). Determining the cell surface markers that enrich for genetically-intact HIV-1 genomes is vital in developing targeted curative strategies. Previous studies have found that HIV-1 proviral DNA is enriched in CD4(+) T-cells expressing the immune checkpoint markers programmed cell death protein-1 (PD-1) or cytotoxic T-lymphocyte associated protein-4 (CTLA-4). There has also been some success in blocking these markers in an effort to reverse HIV-1 latency. However, it remains unclear whether cells expressing PD-1 and/or CTLA-4 are enriched for genetically-intact, and potentially replication-competent, HIV-1 genomes. METHODS: We obtained peripheral blood from 16 HIV-1-infected participants, and paired lymph node from four of these participants, during effective ART. Memory CD4(+) T-cells from either site were sorted into four populations: PD-1(-)CTLA-4(-) (double negative, DN), PD-1(+)CTLA-4(-) (PD-1(+)), PD-1(-)CTLA-4(+) (CTLA-4(+)) and PD-1(+)CTLA-4(+) (double positive, DP). We performed an exploratory study using the full-length individual proviral sequencing (FLIPS) assay to identify genetically-intact and defective genomes from each subset, as well as HIV-1 genomes with specific intact open reading frames (ORFs). RESULTS AND DISCUSSION: In peripheral blood, we observed that proviruses found within PD-1(+) cells are more likely to have intact ORFs for genes such as tat, rev and nef compared to DN, CTLA-4(+) and DP cells, all of which may contribute to HIV-1 persistence. Conversely, we observed that CTLA-4 expression is a marker for cells harbouring HIV-1 provirus that is more likely to be defective, containing low levels of these intact ORFs. In the lymph node, we found evidence that CTLA-4(+) cells contain lower levels of HIV-1 provirus compared to the other cell subsets. Importantly, however, we observed significant participant variation in the enrichment of HIV-1 proviruses with intact genomes or specific intact ORFs across these memory CD4(+) T-cell subsets, and therefore consideration of additional cellular markers will likely be needed to consistently identify cells harbouring latent, and potentially replication-competent, HIV-1.