Unique CD8⁺ T Cell Populations Expand during ART and Predict Delayed HIV-1 Rebound

Antiretroviral therapy (ART) suppresses HIV-1 replication but does not eliminate the latent reservoir, resulting in viral rebound with variable kinetics after treatment interruption. How the immune cell states established during ART influences timing of rebound is not fully understood. In this study, we analyzed 111 participants across multiple cohorts, with 188 single-cell multiomic samples generated and integrated for joint analysis. Longitudinal profiling of peripheral blood mononuclear cells from individuals with acute HIV-1 infection on ART, spanning early infection through sustained therapy and pre-analytical treatment interruption, revealed that time to viral rebound was driven not by global changes in immune composition but by dynamic transcriptional programs within CD8⁺ T cells. During ART, there was a dramatic expansion of a unique cluster of poised naïve CD8⁺ T cells, with a distinct immune state positioned upstream of stem-like memory CD8⁺ T cells along a cell differentiation continuum. The differential abundance of this poised naïve CD8⁺ T cell population was enriched in participants with delayed rebound and showed strong predictive power for discriminating time to rebound. Mechanistically, the poised naïve CD8⁺ T cells exhibited features of a precursor phenotype of stem-like memory CD8⁺ T cells, and showed activation of the TNFα- NF-κB signaling pathway and increased chromatin accessibility at AP-1 motifs. Notably, both poised naïve CD8⁺ T cells and stem-like memory CD8⁺ T cells were consistently enhanced during ART in both acute and chronic infection. In participants who received investigational therapeutic vaccination, the dominant predictive signal shifted downstream along the differentiation trajectory, with stem-like memory CD8⁺ T cells emerging as the primary determinant of delayed rebound. Together, these findings identify a dynamic CD8⁺ T cell state continuum as a central determinant of HIV-1 rebound, even in the absence of antigen-specificity, where ART establishes a predictive poised naïve state that can be further leveraged by vaccination to enhance protective stem-like memory responses.