The majority of HIV replication occurs in follicular helper T cells (TFH) in secondary lymphoid follicles during asymptomatic disease. Follicular regulatory T cells (TFR) are a subset of TFH that control germinal center (GC) responses. Their role in HIV-1 replication is unknown. We hypothesized that TFR are highly susceptible to HIV and evaluated potential causative mechanisms.
Cells were disaggregated from healthy human tonsils or HIV-uninfected human lymph nodes (LN) and spinoculated with R5-tropic GFP reporter virus or mock spinoculated, cultured for 2 days, and analyzed for GFP expression by flow cytometry. Due to downregulation of CD4 by HIV, cells were gated on CD3+CD8- cells and subsets within were defined as TFR (CXCR5+CD25+CD127-), GC TFR (CXCR5+PD1+CD25+CD127-), TFH (CXCR5+CD25-), and GC TFH (CXCR5+PD1+CD25-). In select experiments, tonsil TFR and TFH were sorted prior to spinoculation. CCR5 and Ki67 expression were determined at baseline prior to spinoculation in a subset of subjects. Statistical analyses were performed by Wilcoxon matched-pairs signed rank test using GraphPad Prism 6.
%GFP+ is higher in TFR compared to TFH (median, 4.8-fold higher; p=0.008) and in GC TFR compared to GC TFH (median, 4.0-fold higher; p=0.008; n=8). GFP MFI is not significantly different in TFR compared to TFH (medians, TFR=4599, TFH=6952; p=0.09, n=6), but is lower in GC TFR compared to GC TFH (medians, GC TFR=4114, GC TFH=6427; p=0.03, n=6). Spinoculated cultures of sorted tonsil TFH and TFR (n=4) confirm differences observed in unsorted cultures (median fold difference %GFP+ TFR:TFH = 3.5). In HIV-uninfected LN (n=2), %GFP+ is also higher in TFR compared to TFH (average, 4.7-fold higher) and GC TFR compared to GC TFH (average, 3.4-fold higher). In tonsil cells prior to spinoculation, %CCR5+ is higher in TFR (37%) compared to TFH (11%; p=0.03, n=6) and GC TFR (38%) compared to GC TFH (11%). %Ki67+ (n=3) is higher in TFR compared to TFH (medians, TFR=51%, TFH=14%) and in GC TFR compared to GC TFH (medians, GC TFR=63%, GC TFH=14%).
TFR are highly permissive to R5-tropic HIV ex vivo, which may promote HIV infection and replication within secondary lymphoid follicles in vivo. Potential mechanisms underlying heightened permissiveness of TFR include elevated proliferation and CCR5 expression.