Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4⁺ T Cells

During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4⁺ T cell subsets in central memory (T_CM), transitional memory (T_TM), and effector memory (T_EM) CD4⁺ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4⁺ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the T_EM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the T_EM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. T_CM cells were triggered to differentiate into T_EM cells when they were exposed to LRAs, and this increase of T_EM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24⁺ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4⁺ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a T_EM phenotype in facilitating latency reversal.IMPORTANCE By performing phenotypic analysis of latency reversal in CD4⁺ T cells from virally suppressed individuals, we identify the T_EM subset 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 vitro modeling 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 our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4⁺ T cells.