Cell-associated Unspliced RNA Research Articles

CCL5-Secreting Virtual Memory CD8+ T Cells Inversely Associate With Viral Reservoir Size in HIV-1-Infected Individuals on Antiretroviral Therapy.

Recent studies highlighted that CD8+ T cells are necessary for restraining reservoir in HIV-1-infected individuals who undergo antiretroviral therapy (ART), whereas the underlying cellular and molecular mechanisms remain largely unknown. Here, we enrolled 60 virologically suppressed HIV-1-infected individuals, to assess the correlations of the effector molecules and phenotypic subsets of CD8+ T cells with HIV-1 DNA and cell-associated unspliced RNA (CA usRNA). We found that the levels of HIV-1 DNA and usRNA correlated positively with the percentage of CCL4+CCL5- CD8+ central memory cells (TCM) while negatively with CCL4-CCL5+ CD8+ terminally differentiated effector memory cells (TEMRA). Moreover, a virtual memory CD8+ T cell (TVM) subset was enriched in CCL4-CCL5+ TEMRA cells and phenotypically distinctive from CCL4+ TCM subset, supported by single-cell RNA-Seq data. Specifically, TVM cells showed superior cytotoxicity potentially driven by T-bet and RUNX3, while CCL4+ TCM subset displayed a suppressive phenotype dominated by JUNB and CREM. In viral inhibition assays, TVM cells inhibited HIV-1 reactivation more effectively than non-TVM CD8+ T cells, which was dependent on CCL5 secretion. Our study highlights CCL5-secreting TVM cells subset as a potential determinant of HIV-1 reservoir size. This might be helpful to design CD8+ T cell-based therapeutic strategies for cure of the disease.

O17.1 Histone deacetylase inhibitors alter the accumulation of cell-associated spliced hiv mrna – implications for reactivating the reservoir

Background Clinical trials in HIV-infected patients on antiretroviral therapy with histone deacetylase inhibitors (HDACi) have demonstrated an increase in cell-associated unspliced (CA-US) HIV RNA, variable changes in plasma HIV RNA and no change in the number of latently infected cells. We aimed to define the effects of latency reversing agents (LRAs) on HIV mRNA splicing. Methods Resting CD4 + T cells isolated from the blood of HIV-negative individuals were treated with the chemokine CCL19 and infected with wild type HIV NL4.3 to establish latency (n = 9). Latently infected CCL19-stimulated cells were then cultured with vorinostat, romidepsin, JQ1, romidepsin+JQ1 or PMA/PHA, all in the presence of an integrase inhibitor (L8). Cells and supernatant were harvested at 6, 24, 48, and 72 h. Reverse transcriptase (RT) was quantified in supernatant and CA-US and multiply spliced (MS) HIV RNA were quantified by real time qPCR. Results In latently infected CCL19-treated CD4 + T-cells, stimulation with PMA/PHA led to a significant exponential increase in both US-RNA and MS-RNA by 72 h and reached a mean fold increase above baseline of 34-fold for US-RNA and 54-fold for MS-RNA (p = 0.0003, p = 0.0005 respectively, relative to DMSO). In contrast, following stimulation with each LRA, there was only a modest increase in CA-US RNA that was not statistically significantly different from DMSO (p = 0.89). MS-RNA increased transiently (mean 1.65-fold change at 6hr with romidepsin) and then significantly declined over time with a reduction to 0.18-fold by 72 h relative to DMSO (p = 0.008 romidepsin compared to baseline) in the absence of any cellular cytotoxicity. Conclusions In this in vitro model of latency, PMA/PHA and the potent HDACi romidepsin had strikingly different effects on the accumulation of US-RNA, MS-RNA and virus production. Changes in HIV RNA splicing may limit the efficacy of HDACi in activating latent HIV.