Background
HIV persists in distinct anatomical reservoirs despite effective antiretroviral therapy (ART). Clonal expansion of HIV-infected cells has been reported in peripheral blood mononuclear cells (PBMCs) and lymph node (LN)-derived follicular helper (Tfh) cells during viremia, but remains undefined during ART. We analyzed HIV integration sites (IS) and clonal dynamics in blood and LN to define tissue-specific reservoir features, aiming at targeted HIV cure strategies.
Methods
Tfh cells were sorted by flow cytometry from LNs (CXCR5+PD1hi), and total CD4+ T cells were enriched from PBMCs. IS were determined using integration site loop amplification (ISLA), excluding low-quality reads (<3% HQ threshold). Genomic annotation was performed using the Fred Hutch Integration Site Analysis pipeline. Hi-C compartment information was obtained from GM12878 (human B-lymphoblastoid) sub compartment annotations (NCBI GEO: GSE63525). Histone marks were derived from ROADMAP ChIP-seq datasets. Our preliminary analyses included samples from individuals with acute untreated infection (n = 2), chronic untreated infection (n = 2), and on ART (> 2 years, n = 2), with additional donors ongoing.
Results
Across all samples, 101 and 125 unique HIV IS were identified in peripheral CD4⁺ T cells and LN Tfh cells, respectively. Tfh cells exhibited a significantly higher proportion of IS within transcriptionally active genomic regions compared with peripheral CD4⁺ T cells (72.0% vs. 51.5%, p=0.0132). Clonal expansion was observed during untreated infection in both compartments. Notably, we provide the first evidence that LN Tfh cells also undergo clonal expansion during ART. All expanded clones were integrated within active chromatin regions, a genomic context permissive to viral transcription. In ART-treated individuals, HIV integrations in Tfh cells were significantly enriched near histone marks associated with both active and repressive chromatin compared with PBMC-derived CD4⁺ T cells (H3K27ac: p=0.0015; H3K9me3: p<0.0001; H3K27me3: p=0.0007).
Conclusions
This study provides the first evidence that LN-derived Tfh cells harbor clonally expanded HIV-infected cells during ART which display a distinct integration landscape enriched in epigenetically active regions. These findings confirm the critical importance of tissue-targeted cure strategies.