The immunophenotype and proviral landscape of HIV-infected CD4 T cells during antiretroviral therapy

In individuals on effective antiretroviral therapy (ART), integrated HIV proviruses persist within CD4 T cells, forming a viral reservoir that rebounds if treatment is stopped. Identifying and targeting these rare, infected cells is critical for advancing therapies, but methods to study reservoir cells are limited, and their unique properties remain largely unknown. We applied DAb-seq, a high-throughput method that combines single-cell DNA and surface protein sequencing, to profile ~527,000 CD4 T cells from the blood of six individuals on ART. Infected cells were distributed across all CD4 T cell subsets but were enriched in central memory subsets and in a CD4 subset with Th17-like signatures expressing high levels of integrin β7. Attempts to identify surface markers distinguishing infected from uninfected cells revealed epitopes that largely reflected the subsets most enriched for infection. However, while central memory T cells harbored the majority of HIV, proviruses with a greater number of genomic regions were enriched relative to their more defective counterparts in the CD4 Naïve, transitional memory (Ttm), and regulatory (Treg) subsets, suggesting that these subsets differentially maintain more complete proviral genomes. In summary, we developed DAb-seq as an open-source platform to link proviral sequences to cellular phenotypes, revealing heterogeneity in surface protein expression and proviral fate across infected subsets.