Figure 3. HIV entry and replication promote T_FR expansion.

Disaggregated tonsil cells were spinoculated with X4 or R5 HIV and T_FR populations were analysed by flow cytometry (n=15). (a) A representative example of tonsil cell flow gating. From viable CD3⁺CD8⁻ cells, T_FR are defined as CXCR5⁺ and CD25^hiCD127^–. T_FR cells contain Foxp3⁺ cells, whereas remaining T_FH (CXCR5⁺CD25^lo/−) cells are Foxp3⁻. (b) Percentages of T_FR determined by gating strategies in a are shown. Experimental conditions include mock-spinoculated cells cultured with PMA (50 ng ml⁻¹) and ionomycin (1 μg ml⁻¹) or exogenous TGF-β (100 ng ml⁻¹) for 24 h and cells pretreated to block CXCR4 (AMD, 200 μM) and CCR5 (MVC, 2 μM). (c) Using flow cytometry counting beads, the number of cells per μl were determined for total (CD3⁺CD8⁻), T_FH (CXCR5⁺CD25^lo/−) and T_FR (CXCR5⁺CD25^hiCD127⁻) subsets in mock- and X4-spinoculated samples (n=3). (d) Bcl-6 expression is shown in CXCR5− (grey), T_FH (blue) and T_FR (red) populations after mock-, X4- or R5-spinoculation (n=5). (e) Blimp-1 expression was also determined as in d. The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed by Friedman nonparametric tests (b,d,e) and significance is denoted by asterisks where *P<0.05, **P<0.01 and ***P<0.001.