HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals.

A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART).Here, we developed ultrasensitive assays to precisely measure the frequency of cells harboring total HIV DNA, integrated HIV DNA, and two long terminal repeat (2-LTR) circles. These assays are performed on cell lysates, which circumvents the labor-intensive step of DNA extraction, and rely on the coquantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_A/E, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA, and 2-LTR circles in CD4 ؉ T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naive subjects were positive for the three HIV molecular forms (n ‫؍‬ 15). Total HIV DNA, integrated HIV DNA, and 2-LTR circles were detected in, respectively, 100%, 94%, and 77% of the samples from individuals in which HIV was suppressed by ART. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects than individuals on ART (P ‫؍‬ 0.0003 and P ‫؍‬ 0.0004, respectively), while the frequency of CD4 ؉ T cells harboring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA of multiple HIV subtypes without the need for nucleic acid extraction, making them well suited for the monitoring of viral persistence in large populations of HIV-infected individuals. IMPORTANCESince the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction, and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV-infected individuals and, given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.

Key Points IL-7 does not disrupt viral latency in highly pure resting latently infected CD4+ T cells from HIV-infected subjects receiving ART. IL-7 therapy leads to a 70% increase in the absolute number of circulating CD4+ T cells harboring integrated HIV DNA 4 weeks posttherapy.

NCT00510497.

In the majority of HIV-1 infected individuals, the adaptive immune response drives virus escape resulting in persistent viremia and a lack of immune-mediated control. The expression of negative regulatory molecules such as PD-1 during chronic HIV infection provides a useful marker to differentiate functional memory T cell subsets and the frequency of T cells with an exhausted phenotype. In addition, cell-based measurements of virus persistence equate with activation markers and the frequency of CD4 T cells expressing PD-1. High-level expression of PD-1 and its ligands PD-L1 and - L2 are found on hematopoietic and non-hematopoietic cells, which are regulated by chronic antigen stimulation, Type 1 and Type II interferons (IFNs), and homeostatic cytokines. In HIV infected subjects, PD-1 levels on CD4 and CD8 T cells continue to remain high following combination anti-retroviral therapy (cART). System biology approaches have begun to elucidate signal transduction pathways regulated by PD-1 expression in CD4 and CD8 T cell subsets that become dysfunctional through chronic TCR activation and PD-1 signaling. In this review, we summarize our current understanding of transcriptional signatures and signal transduction pathways associated with immune exhaustion with a focus on recent work in our laboratory characterizing the role of PD-1 in T cell dysfunction and HIV pathogenesis. We also highlight the therapeutic potential of blocking PD-1-PD-L1 and other immune checkpoints for activating potent cellular immune responses against chronic viral infections and cancer.