Figure 5. Acquisition of CXCR5, enhanced proliferation and reduced apoptosis promote T_FR expansion.

(a) T_FH and CXCR5⁻ T-cell populations were sorted and cultured without stimulation, or T_FH were cultured in the presence of exogenous TGF-β (100 ng ml⁻¹). CXCR5 expression was analysed on sorted CXCR5⁻ cells after 2 days and cells expressing CXCR5 are labelled as ‘new T_FR'. The Foxp3 expression levels of T_FH, T_FH cultured with TGF-β and ‘new' T_FR were determined (n=3). (b) T_FH and T_FR were mock-, X4- or R5-spinoculated and cell proliferation measured by BrdU incorporation after 2 days of culture (n=7). (c,d) A 5-day time course was performed to monitor T-cell population counts and rates of cell death with mock- or X4-spinoculated tonsil cells (n=3). (c) Average counts of total (CD3⁺CD8⁻, circles), T_FH (CXCR5⁺CD25^lo/−, squares) and T_FR (CXCR5⁺CD25^hiCD127⁻, triangles) are shown for the duration of culture (n=3). (d) Stages of cell death are shown at day 5 and defined as early apoptosis (AnnexinV⁺), late or advanced apoptosis (AnnexinV⁺ PI⁺) and necrotic death (PI⁺; n=3). (e) Cell subsets from disaggregated lymphoid tissues of chronically SIV-infected (n=6) and uninfected rhesus macaques (n=8) were analysed for apoptosis by percent Annexin-V binding. Cell phenotypes were defined as total (CD3⁺CD8⁻), Treg (CD3⁺CD8-CD25^hiCD127⁻), T_FH (CD3⁺CD8-CXCR5⁺CD25^lo/−), T_FR (CD3⁺CD8-CXCR5⁺CD25^hiCD127⁻), GC T_FH (CD3⁺CD8-CXCR5⁺PD1^hiCD25^lo/−) and GC T_FR (CD3⁺CD8-CXCR5⁺PD1^hiCD25^hiCD127⁻). Statistical analyses were performed by Wilcoxon matched-pairs tests (b) or Mann–Whitney tests (e) and significance is denoted by asterisks where **P<0.01 and ***P<0.001.