Methods: Sigmoid colon biopsies were obtained from aviremic HIV+ subjects on antiretroviral therapy. Mucosal mononuclear cells (MMCs) were isolated then stained for CD45, CD3 and CD4. After gating on CD45+ cells, this population was sorted into double positive, single positive, and double negative populations using CD3 and CD4. Genomic DNA was isolated from each subset and used for proviral qPCR as well as sequencing of nef, env, gag, and pol.
Results: Cell sorting of isolated MMCs yielded distinct cellular subsets for all subjects with the following ranges: CD3+ CD4+ (167,178 – 460,494 cells), CD3- CD4+ (262,925 – 300,115 cells), CD3+ CD4- (113,858 – 377,367 cells), CD3- CD4- (42,835 – 466,436 cells). In all subjects, the majority of quantifiable provirus was detected in the CD3+ CD4+ cell population (2677 ± 3649 copies/million cells), though there was also detectable provirus in the CD4 single positive (224 ± 551 copies/million cells) and double negative (52 ± 87 copies/million cells) populations. No provirus was detected in the CD3 single positive population. Phylogenetic analysis using a minimum of 10 clones per population showed little sequence diversity for all genes examined (nef, env, gag, pol), with nearly identical clones observed in sequences obtain from the CD3+ CD4+ population and total MMCs. Finally, no PRO or RT drug resistance mutations were detected in sequences from any cell compartment.
Conclusion: CD4+ T cells appear to be the dominant source of persistent HIV in the gut of aviremic individuals. This represents an important target for new therapy and eradication strategies. Further studies to examine the longitudinal dynamics of this compartment may provide additional insight into the mechanism of persistence.
P. Anton, None
M. Lewis, None