(A) Sorted CXCR5⁺CXCR3⁺PD-1^lo Tfh-like cells were cultured with autologous B cells and SEB for 6 days (left plot). PD-1^lo and PD-1^hi events from these cultures were selectively resorted and cocultured for 6 additional days with B cells and SEB. Right plots show PD-1 expression on resorted PD-1^lo and PD-1^hi cells after the second 6-day culture assay. Dot plots from a representative experiment are shown. Numbers on the gates represent the proportion of cells. (B) Proportions of remaining PD-1^lo and PD-1^hi cells detected on day 6 of coculture of freshly sorted (F) or resorted (R) CXCR5⁺CXCR3⁺PD-1^lo (n = 5), CXCR5⁺CXCR3⁺PD-1^hi (n = 5), and CXCR5⁺CXCR3^–PD-1^hi (n = 5) pTfh cells with total B cells and SEB. Statistical significance was calculated using a Friedman test followed by Dunn’s test (*P < 0.05; **P < 0.01). (C) Mean fluorescence intensity of Notch 1, 2, and 4 and intracellular Notch (ICN) in pTfh subsets and total CD4⁺ T cells (n = 8). Statistical significance was calculated using a Friedman test followed by a Dunn’s test (*P < 0.05; **P < 0.01; *** P < 0.01). (D) Proportions of PD-1^lo or PD-1^hi Tfh-like cells generated upon coculture of naive CD4⁺ T cells with autologous B cells and allogeneic mDCs in the presence of gamma secretase inhibitors (GSI) in comparison to 100 nM DMSO (n = 6). Statistical significance was calculated using a 2-tailed Wilcoxon matched-pairs signed-rank test (*P < 0.05). (E) Proportions of PD-1^hi T cells derived from CXCR5⁺CXCR3⁺PD-1^lo CD4⁺ T cells after 6 days of coculture with autologous B cells and SEB in the presence of GSI compared with DMSO (n = 6). Statistical significance was calculated using a two-tailed Wilcoxon matched-pairs signed-rank test (*P < 0.05).