HIV replication is concentrated within follicular T cells in B cell follicles (F) during asymptomatic disease. Whether T follicular regulatory cells (TFR) are more permissive than other subsets and what factors drive permissivity are not understood.
Disaggregated HIV- human tonsil cells were spinoculated with R5-tropic GFP reporter virus (NLYUV3-GFP), 3 R5-tropic transmitted/founder viruses (T/F) (CH58, CH470, CH40) or mock spinoculated at day 0 and cultured for 2 days. Some cells were cultured for 2 days alone or with imatinib, a T cell proliferation inhibitor, prior to spinoculation. GFP or p24 expression were determined by flow cytometry. Due to CD4 downregulation by HIV, we gated on CD3+CD8- cells, and defined subsets as: extrafollicular (EF; CXCR5-CD25-CD127+/-), EF Treg (CXCR5-CD25+CD127-), TFH (CXC5+CD25-CD127+/-) and TFR (CXCR5+CD25+CD127-). CCR5, HLA-DR/CD38, CD69 and Ki67 expression were determined prior to spinoculation. Lymph node cells (LN) from HIV+ untreated subjects were sorted into EF, EF Treg, TFH and TFR, and RNA quantified by RT PCR. Data were log transformed. Mixed-effects models and repeated-measures ANOVA were used with pairwise tests if overall p<0.05. Geometric means and 95% CIs are reported.
GFP expression differed by cell type (p≤0.001) with %GFP+ cells in TFR (2.0%; 1.3, 2.8) > EF Treg (0.6%; 0.4, 0.9), TFH (0.3%; 0.2, 0.5) > EF (0.09%; 0.04, 0.18), and TFR > TFH (n=6; p<0.01). The same pattern was observed for p24 Ag expression in T/F infections (n=6; overall p<0.01). %Ki67 expression differences between cell subsets at day 0 (n=6; p<0.001) paralleled differences in %GFP and %p24 expression, but CCR5, CD95, and CD69 expression did not. At day 0 for each 1 log10 increase in %Ki67 expression, there was a 0.8 (0.6, 1.1) log10 increase in %GFP expression (p<0.001). Compared to untreated cells, imatinib reduced Ki67 expression by 34% (6, 53; p=0.02) and reduced GFP expression by 77% (61, 87; p<0. 001), without affecting viability. In sorted HIV+ LN, TFR harbored more (1167; 207, 6577) HIV RNA copies/ng total RNA than TFH (585; 113, 3020), EF Treg (516; 163, 1641), and EF (71; 10, 507) (n=6; overall p=0.04; pairwise p<0.05). LN Ki67 expression was higher in TFR (20%; 13, 30) than TFH (10%; 7, 15) and higher in EF Treg (19%; 15, 24) than EF (4.7%; 2, 10) (n=6; overall p=0.01; pairwise p<0.05).
TFR are highly permissive to HIV, likely due to heightened proliferation, and harbor the highest concentrations of HIV RNA in vivo.