Latently infected CD4 T cells provide a highly durable reservoir for HIV-1, but the ability to support viral persistence appears to differ considerably among different CD4 T cell populations. Here, we used single-template near full-length viral deep sequencing to map proviral sequences across nine different CD4 T cell subsets from blood and lymph nodes (LN).
PBMC from three HIV-infected patients treated with suppressive ART were sorted into the following CD3+ CD4+ T cell subsets: CD45RA- cells (total memory), CXCR3+CXCR5- (Th1-enriched), CCR4+CCR6- (Th2-enriched), CCR4+CCR6+ (Th17-enriched), CXCR3-CXCR5+ (circulating Tfh-like cells) and CXCR3+CXCR5+ (enriched for Th1/Tfh-like cells). Simultaneously, the following CD3+ CD4+ subsets were isolated from autologous LN: CD45RA- (total memory), PD-1- CXCR5-, PD-1- CXCR5+, PD-1+ CXCR5+/- (enriched for Tfh), and CD45RA+ naïve T cells. Total DNA was extracted, diluted for single-template full-HIV-1 genome PCR, and Illumina deep-sequenced. HIV sequences were classified into ‘genome-intact’ or ‘defective’ using established criteria.
A total of 312 HIV proviral sequences were detected from 3 patients. Frequencies of genome-intact sequences relative to defective copies did not notably differ between blood (17/135, 12.6%) and lymph nodes (20/177, 11.3%) (p=0.73, Fisher’s). Except for lymph node naïve cells, genome-intact HIV DNA was detected in all analyzed CD4 T cell subsets from both blood and lymph nodes. In LN, proportions of intact proviruses were highest in CXCR5+ or PD-1+ CD4 T cells, while in blood, distribution of intact proviral sequences was more variable. Proviral sequences from blood and LN phylogenetically intermingled without obvious compartmentalization (Slatkin-Maddison test p=1, 0.2, 0.1). Notably, we observed a cluster of clonally-expanded intact provirus derived from cells with discrete functional polarization from blood and LN (Figure1). Clonally-expanded defective HIV-1 sequences with mixed contributions from blood and lymph nodes were also observed.
This study suggests dynamic interchanges between ‘genome-intact’ viral reservoir cell populations from blood and lymphoid tissues. Clusters of clonally-expanded intact proviruses encompassing sequences from cells with distinct anatomic localization and functional polarization suggest infection of common precursor cells as a driving force for viral reservoir stabilization.