Figure 3.

Expression of transcription factors and induction of CXCR5⁺CD8⁺ T cells. (a) Heat map illustrating the relative expression of key transcription factors from RNA-Seq data in CD45RA⁺CXCR5⁻CD8⁺, CD45RA⁻CXCR5⁻CD8⁺, and CD45RA⁻CXCR5⁺CD8⁺ T cell subsets from tonsil samples. (b) mRNA expression of transcription factors determined by quantitative PCR in the three CD8⁺ T cell subsets. Experiments were repeated at least three times. (c) CD45RA⁺CXCR5⁻CD8⁺ T cells (left plot) were sorted from human tonsils and stimulated with anti-CD3 and anti-CD28 antibodies, and cultured with TGF-β and IL-23 for 2–3 days. Surface expression of CXCR5 was determined by flow cytometry (right plot). (d) CXCR5⁺CD8⁺ T cells were induced as described in (c), and stimulated with PMA/Ionomycin for 5 hours with Brefeldin A added for last 3 hours. Production of IFN-γ and TNF-α was determined by intracellular staining and flow cytometry. (e) ID2 expression was determined by qPCR in CD45RA⁻CXCR5⁻CD8⁺ (blue) and CD45RA⁻CXCR5⁺CD8⁺ T cells (red) sorted from human tonsil and CD45RA⁻CXCR5⁺CD8⁺ T cells (orange) induced in vitro as in (c). Data represents 3 independent experiments. (f) Memory B cells sorted from human tonsils were stimulated with SEB and co-cultured in the presence or absence of sorted autologous T_FH and/or CXCR5⁺CD8⁺ T cells. Differentiation of memory B cells into CD38⁺CD19^int plasmablast cells was determined by flow cytometry after 9 days of co-culture. TW (transwell) indicates cells in the transwell chamber. Ratios of T_FH:CXCR5⁺CD8⁺ T cells are shown in parentheses. Representative data from one of 6 independent experiments is shown.