Expansion and activation of cTfh in patients with pES. (A) Representative FItSNE plots showing frequency and activation status of cTfh cells in patient with pES and HC where CD4⁺ T cells were first manually gated for total memory cells (CD45RA⁻) and then down-sampled to equal number of cells. CXCR5⁺ cells were gated on CD45RA⁻CD4⁺ T cells and subsequently gated for HLA-DR⁺ or PD-1⁺ cells. Expression of cTfh and activation markers on cTfh (ie, PD-1⁺ and HLA-DR⁺) are shown in different colors. Overlaid expression of PD-1⁺, HLA-DR⁺, and CXCR5⁺ markers are shown at the extreme right for both HC and pES. (B-C) Plots showing percentage of cTfh as CXCR5⁺CD45RA⁻CD4⁺ T and CXCR5⁺PD-1⁺CD45RA⁻CD4⁺ T subsets in HCs (n = 24), patients with pES (n = 24), and patients with cITP (n = 22). (D) Plot showing ratio of PD-1⁺ cTfh vs PD-1⁻ cTfh in HCs, patients with pES, and patients with cITP. (E-G) Percentage of CXCR5⁺HLA-DR⁺, CXCR5⁺Tim3⁺, and CXCR5⁺CD57⁺ expression on total memory CD4⁺ T cells in different patient groups. Data represent mean ± standard error of the mean (SEM) values for each group. Kruskal-Wallis 1-way analysis of variance (ANOVA) followed by Dunn’s multiple comparison test for nonnormally distributed samples and ordinary 1-way ANOVA followed by Tukey’s multiple comparison test for normally distributed samples were used for statistical comparison. ***P < .001; ****P < .0001; ns, not significant.