For apoptosis/cell loss of life analysis, cells were harvested, washed once in PBS, resuspended in annexin V binding buffer (Becton, Dickinson, NJ, USA) containing a saturating concentration of an APC-conjugated annexin V antibody and Zombie NIR (BioLegend), and incubated for 15 min at room temperature (RT) in the dark. not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a encouraging step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells. IMPORTANCE One of the challenges associated with HIV-1 contamination is usually that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual computer virus by reactivating latent computer virus and eliminating the reservoir of HIV-1 (so-called shock-and-kill strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, main T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells. (21). However, a subsequent study that evaluated acitretin as an LRA in latently infected cell lines and patient-derived samples failed to lengthen these observations (22). A recent Tyrosol study demonstrated that this STING agonists 23-cGAMP and cyclic di-AMP were able to decrease the amount of simian immunodeficiency computer virus (SIV) Gag in the DNA of peripheral blood mononuclear cells (PBMCs) obtained from monkeys exhibiting natural SIV control at 40?weeks postinfection (23). However, only cyclic di-AMP reactivated latent HIV in a main CD4+ Tyrosol T cell model of HIV-1 latency established after activation through the T cell receptor and the subsequent return to quiescence. To date, it remains unclear whether a combination of LRAs and immunotherapy could be the important to clearing HIV reservoirs. In the present study, we demonstrate that this combination of the cGAS-STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells without affecting cell death. Recent achievements in malignancy immunotherapy (24,C26) have raised the possibility that the application of immunotherapeutic approaches to other fields, including HIV research, could promote the clearance of the latent HIV-1 reservoir (6, 27, 28). To evaluate the abilities of immunostimulatory biologic brokers to induce the reactivation of HIV-1 provirus and to selectively induce the death of latently HIV-1-infected cells, we first analyzed the effects of three different agonists of innate immunitythe RIG-I agonist M8, previously characterized by our group (29), and the STING agonists cGAMP and c-di-GMP (the cyclic dinucleotide of GMP)on HIV-1 reactivation in J-Lat 10.6 cells, an model of HIV-1 latency that contains a green fluorescent protein (GFP) gene substitution in Tyrosol the Nef open reading frame (ORF) (30). The HIV-1-free cell collection Jurkat E6.1 was used as GMFG an uninfected control to test the cytotoxicities of the compounds analyzed. The activities of M8, cGAMP, and c-di-GMP were compared to that of acitretin, a RIG-I agonist that was Tyrosol previously shown to reactivate latent provirus and selectively kill infected cells (21), while dimethyl sulfoxide (DMSO) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-) were used as negative and positive controls, respectively (Fig. 1). Briefly, Jurkat and J-Lat cells either were treated with cGAMP, c-di-GMP, or acitretin or were transfected with M8; cells were analyzed by fluorescence-activated cell sorting (FACS) at 24 h to evaluate GFP expression as a marker of viral reactivation; and lifeless cells were excluded from your analysis by 7-aminoactinomycin D (7-AAD) staining. Interestingly, only the STING agonist cGAMP exhibited the ability to induce a low but significant increase Tyrosol in the proportion of.