Spontaneous HIV expression during suppressive ART is associated with the magnitude and function of HIV CD4⁺ and CD8⁺ T cells

Spontaneous transcription and translation of HIV can persist during suppressive antiretroviral therapy (ART). The quantity, phenotype, and biological relevance of this spontaneously "active"reservoir remain unclear. Using multiplexed single-cell RNAflow-fluorescence in situ hybridization (FISH), we detect active HIV transcription in 14/18 people with HIV on suppressive ART, with a median of 28/million CD4(+) T cells. While these cells predominantly exhibit abortive transcription, p24(-)expressing cells are evident in 39% of participants. Phenotypically diverse, active reservoirs are enriched in central memory T cells and CCR6(-)and activation marker-expressing cells. The magnitude of the active reservoir positively correlates with total HIV-specific CD4(+) and CD8(+) T cell responses and with multiple HIV-specific T cell clusters identified by unsupervised analysis. These associations are particularly strong with p24-expressing active reservoir cells. Single-cell vDNA sequencing shows that active reservoirs are largely dominated by defective proviruses. Our data suggest that these reservoirs maintain HIV-specific CD4(+) and CD8(+)T responses during suppressive ART.