By performing phenotypic analysis of latency reversal in CD4⁺T cells from virally suppressed individuals, we identify the T_EMsubset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4⁺T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry.In vitromodeling of the latent HIV reservoir in memory CD4⁺T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of T_EM-associated genes and cell surface markers shown to be associated with latency reversal in ourex vivoandin vitromodels. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4⁺T cells.

HIV infection persists during antiretroviral therapy (ART) due to a reservoir of latently infected cells that harbor replication-competent virus and evade immunity. Previous ex vivo studies suggested that CD8⁺ T cells from people with HIV may suppress HIV expression via non-cytolytic mechanisms, but the mechanisms responsible for this effect remain unclear. Here, we used a primary cell-based in vitro latency model and demonstrated that co-culture of autologous activated CD8⁺ T cells with HIV-infected memory CD4⁺ T cells promoted specific changes in metabolic and/or signaling pathways resulting in increased CD4⁺ T cell survival, quiescence, and stemness. Collectively, these pathways negatively regulated HIV expression and ultimately promoted the establishment of latency. As shown previously, we observed that macrophages, but not B cells, promoted latency in CD4⁺ T cells. The identification of CD8-specific mechanisms of pro-latency activity may favor the development of approaches to eliminate the viral reservoir in people with HIV.

Interleukin-10 (IL-10) is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper (Tfh) cell differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph nodes (LNs) were induced by infection and not normalized with antiretroviral therapy (ART). During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4⁺ memory subsets, including Tfh cells, and predicted the frequency of CD4⁺ Tfh cells and their cell-associated SIV-DNA content during ART, respectively. In ART-treated rhesus macaques, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and, by extension, LN memory CD4⁺ T cells, including Tfh cells and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4⁺ T cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV.

Studies have demonstrated that intensive ART alone is not capable of eradicating HIV-1, as the virus rebounds within a few weeks upon treatment interruption. Viral rebound may be induced from several cellular subsets; however, the majority of proviral DNA has been found in antigen experienced resting CD4+ T cells. To achieve a cure for HIV-1, eradication strategies depend upon both understanding mechanisms that drive HIV-1 persistence as well as sensitive assays to measure the frequency of infected cells after therapeutic interventions. Assays such as the quantitative viral outgrowth assay (QVOA) measure HIV-1 persistence during ART by ex vivo activation of resting CD4+ T cells to induce latency reversal; however, recent studies have shown that only a fraction of replication-competent viruses are inducible by primary mitogen stimulation. Previous studies have shown a correlation between the acquisition of effector memory phenotype and HIV-1 latency reversal in quiescent CD4+ T cell subsets that harbor the reservoir. Here, we apply our mechanistic understanding that differentiation into effector memory CD4+ T cells more effectively promotes HIV-1 latency reversal to significantly improve proviral measurements in the QVOA, termed differentiation QVOA (dQVOA), which reveals a significantly higher frequency of the inducible HIV-1 replication-competent reservoir in resting CD4+ T cells. Quantifying the number of cells harboring HIV-1 provirus is critical to evaluating HIV cure interventions, but precise quantification of the latent reservoir has proven to be technically challenging. Our data demonstrates that targeted differentiation of CD4+ T cells to an effector memory phenotype is a successful strategy for promoting latency reversal in vitro, and significantly enhances the performance of existing protocols to quantify the frequency of replication-competent HIV-1 in resting CD4+ T cells from virally-suppressed individuals. Using peripheral blood samples from well-established and characterized cohorts, we show the presence of a significantly higher frequency of replication-competent provirus than has previously been reported, raising the average estimated frequency 18-fold over the established QVOA measures. These results demonstrate that ex vivo effector memory differentiation has moved reservoir measurements closer to what may be the bona fide inducible replication-competent reservoir frequency, thus beginning to bridge the gap between viral outgrowth and molecular-based quantification. Taken together, these data support accumulating evidence that effector memory differentiation is a key pathway to HIV-1 latency reversal that may be exploited for assay development, mechanistic understanding, and therapeutic interventions.

MHC-I-restricted, virus-specific cytotoxic CD8⁺ T cells (CTLs) may control human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication via the recognition and killing of productively infected CD4⁺ T cells. Several studies in SIV-infected macaques suggest that CD8⁺ T cells may also decrease virus production by suppressing viral transcription. Here, we show that non-HIV-specific, TCR-activated non-cytolytic CD8⁺ T cells suppress HIV transcription via a virus- and MHC-independent immunoregulatory mechanism that modulates CD4⁺ T cell proliferation and activation. We also demonstrate that this CD8⁺ T cell-mediated effect promotes the survival of infected CD4⁺ T cells harboring integrated, inducible virus. Finally, we used RNA sequencing and secretome analyses to identify candidate cellular pathways that are involved in the virus-silencing mediated by these CD8⁺ T cells. This study characterizes a previously undescribed mechanism of immune-mediated HIV silencing that may be involved in the establishment and maintenance of the reservoir under antiretroviral therapy and therefore represent a major obstacle to HIV eradication. Antiretroviral therapy (ART) has revolutionized the fight against the HIV pandemic, but this approach requires life-long treatment in order to maintain suppression of virus replication, as rapid viral rebound follows ART discontinuation. Our current inability to cure HIV infection despite ART is due to a persistent reservoir of latently infected CD4⁺ T cells harboring replication competent HIV integrated in their genome. Advancing our understanding of how the virus reservoir is established and maintained in the setting of ART-treated HIV-infection may reveal novel targets for immune-based interventions that can eradicate HIV. In this study, we developed an innovative co-culture experimental system to identify and characterize an important antiviral and immunoregulatory activity of non-cytolytic CD8⁺ T lymphocytes on activated CD4⁺ T cells. This activity drives CD4⁺ T cells into a more resting state that is less conducive to HIV transcription, thereby promoting the survival of infected CD4⁺ T cells harboring integrated, inducible virus. In addition, we identified cellular pathways that may mediate the CD8⁺ T cell-mediated transcriptional silencing of HIV in CD4⁺ T cells. The possibility that this non-cytolytic activity of CD8⁺ T cells may sustain the in vivo pool of latently infected CD4⁺ T cells under ART therefore represents a key, previously unrecognized obstacle to the elimination of the virus reservoir and the eradication of HIV infection.

The pathogenesis of multi-organ dysfunction associated with severe acute SARS-CoV-2 infection remains poorly understood. Endothelial damage and microvascular thrombosis have been identified as drivers of COVID-19 severity, yet the mechanisms underlying these processes remain elusive. Here we show alterations in fluid shear stress-responsive pathways in critically ill COVID-19 adults as compared to non-COVID critically ill adults using a multiomics approach. Mechanistic in-vitro studies, using microvasculature-on-chip devices, reveal that plasma from critically ill COVID-19 adults induces fibrinogen-dependent red blood cell aggregation that mechanically damages the microvascular glycocalyx. This mechanism appears unique to COVID-19, as plasma from non-COVID sepsis patients demonstrates greater red blood cell membrane stiffness but induces less significant alterations in overall blood rheology. Multiomics analyses in pediatric patients with acute COVID-19 or the post-infectious multi-inflammatory syndrome in children (MIS-C) demonstrate little overlap in plasma cytokine and metabolite changes compared to adult COVID-19 patients. Instead, pediatric acute COVID-19 and MIS-C patients show alterations strongly associated with cytokine upregulation. These findings link high fibrinogen and red blood cell aggregation with endotheliopathy in adult COVID-19 patients and highlight differences in the key mediators of pathogenesis between adult and pediatric populations.