Objective In HIV-infected individuals on antiretroviral therapy (ART), latent HIV is enriched in CD4+ T-cells expressing immune checkpoint molecules (ICs), in particular programmed cell death-1 (PD-1). We therefore assessed the effect of blocking PD-1 on latency, both in vitro and in vivo. Methods HIV latency was established in vitro following co-culture of resting CD4+ T-cells with myeloid dendritic cells. Expression of PD-1 was quantified by flow cytometry, and latency assessed in sorted PD-1high and PD-1low/− non-proliferating CD4+ memory T-cells. The role of PD-1 in the establishment of latency was determined by adding anti-PD-1 (pembrolizumab) to co-cultures before and after infection. Additionally, a single infusion of anti-PD-1 (nivolumab) was administered to an HIV-infected individual on ART with metastatic melanoma, and cell-associated (CA) HIV DNA and RNA, and plasma HIV RNA were quantified. Results HIV latency was significantly enriched in PD-1high compared to PD-1low/− nonproliferating, CD4+ memory T-cells. Sorting for an additional IC, T-cell immunoglobulin domain and mucin domain-3 (Tim-3), in combination with PD-1 further enriched for latency. Blocking PD-1 prior to HIV infection, in vitro, resulted in a modest but significant decrease in latently infected cells in all donors (n=6). The administration of anti-PD-1 to an HIV-infected individual on ART, resulted in a significant increase in CA HIV RNA in CD4+ T-cells, without significant changes in HIV DNA or plasma HIV RNA, consistent with reversal of HIV latency. Conclusions PD-1 contributes to the establishment and maintenance of HIV latency and should be explored as a target, in combination with other ICs, to reverse latency.

Background Antibodies to PD-1 and CTLA-4 may perturb HIV persistence during antiretroviral therapy (ART) by reversing HIV-latency and/or boosting HIV-specific immunity leading to clearance of infected cells. We tested this hypothesis in a clinical trial of anti-PD-1 alone or in combination with anti-CTLA-4 in people living with HIV (PLWH) and cancer. Methods This was a substudy of the AIDS Malignancy Consortium-095 Study. ART-suppressed PLWH with advanced malignancies were assigned to nivolumab (anti-PD-1) with or without ipilimumab (anti-CTLA-4). In samples obtained pre-infusion and one and seven days after the first and fourth dose of immune checkpoint blockade (ICB), we quantified cell-associated unspliced (CA-US) HIV-RNA and HIV-DNA. Plasma HIV-RNA was quantified during the first treatment cycle. Quantitative viral outgrowth assay (QVOA) to estimate the frequency of replication-competent HIV was performed before and after ICB for participants with samples available. Results Of forty participants, 33 received nivolumab and seven nivolumab plus ipilimumab. Whereas CA-US HIV RNA did not change with nivolumab monotherapy, we detected a median 1.44 fold-increase (IQR 1.16–1.89) after the first dose of nivolumab and ipilimumab combination therapy (P=0.031). There was no decrease in the frequency of cells containing replication-competent HIV, but in the two individuals on combination ICB for whom we had longitudinal QVOA, we detected decreases of 97% and 64% compared to baseline. Conclusion Anti-PD-1 alone showed no effect on HIV-latency or the latent HIV-reservoir, but the combination of anti-PD-1 and anti-CTL-4 induced a modest increase in CA-US HIV RNA and may potentially eliminate cells containing replication-competent HIV.