In some experiments, antibodies blocking immune checkpoint molecules; humanised anti-PD-1 (nivolumab, kind gift from Bristol Myers Squibb, New York, NY), humanised anti-CTLA-4 (ipilimumab, kind gift from Bristol Myers Squibb), m-a-hTIM-3 (clone F38C2E2, BioLegend), m-a-hTIGIT (clone MBSA43, eBiosciences), or isotype controls; hIgG4 (clone ET904, BioLegend), hIgG1 (clone ET901, BioLegend), mIgG1 (clone MOPC-21, BioLegend), were added to activate latent infection

In some experiments, antibodies blocking immune checkpoint molecules; humanised anti-PD-1 (nivolumab, kind gift from Bristol Myers Squibb, New York, NY), humanised anti-CTLA-4 (ipilimumab, kind gift from Bristol Myers Squibb), m-a-hTIM-3 (clone F38C2E2, BioLegend), m-a-hTIGIT (clone MBSA43, eBiosciences), or isotype controls; hIgG4 (clone ET904, BioLegend), hIgG1 (clone ET901, BioLegend), mIgG1 (clone MOPC-21, BioLegend), were added to activate latent infection. CTLA-4 and PD-1 reversed HIV latency in proliferating and non-proliferating CD4+ T cells respectively. In the absence of SEB, only the combination of antibodies to PD-1, CTLA-4, TIM-3 and TIGIT reversed latency. The potency of latency reversal was significantly higher following combination IC blockade compared to other latency reversing agents, including vorinostat and bryostatin. Combination IC blockade should be further explored as a strategy to reverse HIV latency. Introduction Antiretroviral therapy (ART) has revolutionized the treatment of human immunodeficiency virus (HIV) infection and has dramatically reduced mortality and morbidity. However, ART is lifelong, expensive, and often has side effects so there is an urgent need to identify strategies to cure HIV or induce remission to avoid lifelong treatment [1]. The major barrier to a cure for HIV infection is the persistence of latent infection in long lived resting and proliferating CD4+ T cells [2C5] that are more frequently detected in lymphoid tissue and the gastrointestinal tract VHL [6C8]. It is highly likely that the mechanisms maintaining HIV latency differ in non-proliferating and proliferating T cells, suggesting that multiple interventions may be needed to eliminate latency. One approach being tested to eliminate latently infected cells in people living with HIV (PLWH) on ART is to activate latent virus and thereby induce death of the infected cell through immune clearance or virus induced cytolysis [9, 10]. To date, clinical trials that have examined Hydroxyfasudil latency reversing agents (LRA), such as histone deacetylase inhibitors (HDACi), disulfiram or Toll-like receptor (TLR) agonists have shown modest latency reversal but without clearance of infected cells [11C15]. Furthermore, HDACi have been shown to induce adverse effects on adaptive immune function and have multiple off target effects [11, 16C21]. Therefore, alternative LRAs that are more potent and have a beneficial effect on adaptive immune function, to enhance immune-mediated clearance of Hydroxyfasudil infected cells, are needed. Latent infection is enriched in CD4+ T cells expressing immune checkpoint (IC) molecules, first described for programmed death-1 (PD-1) in circulating CD4+ T cells in blood [22, 23], and more recently in T follicular helper cells in lymphoid tissue [7]. In simian immunodeficiency virus (SIV)-infected macaques on ART, there is also enrichment of virus in CD4+ T cells expressing cytotoxic T lymphocyte-4 (CTLA-4) and PD-1 in the extra-follicular and follicular lymphoid compartments, respectively [24]. We previously demonstrated that co-expression of multiple IC molecules including PD-1, lymphocyte activation gene 3 (LAG-3) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on CD4+ T cells from blood from PLWH on ART were highly enriched for HIV infection compared to cells that expressed fewer than three IC markers [23]. HIV transcription and virus production is largely dependent on host transcription factors that increase and localise Hydroxyfasudil to the nucleus following Hydroxyfasudil T cell activation (reviewed in [1]). Ligation of IC molecules can actively suppress these pathways [25]. We recently demonstrated that engagement of PD-1 in vitro inhibits the establishment of HIV latency in resting CD4+ T cells [26] and in latently infected cells isolated from PLWH on ART, programmed death ligand 1 (PD-L1) can block viral production at the transcriptional level by abrogating T cell receptor (TCR)-induced HIV reactivation [27]. Conversely, PD-1 blockade with the monoclonal antibody pembrolizumab Hydroxyfasudil in combination with the LRA bryostatin, enhances.