IL-21 and T follicular helper (Tfh) cells in peripheral blood of SS and NSCS patients. (A) ELISA quantification of IL-21 serum level (pg/mL) from HD (n=12), NSCS (n=37) and SS (n=37). (B) Correlations of IL-21 serum level (pg/mL) with serum level of IgG, IgM, IgA, C3, C4 and ESSDAI in NSCS (n=37) and SS (n=37) patients. Spearman and p value are shown for the significant correlation (IgG, in red). (C) IL-21 serum level (pg/mL) in SS (n=31) according to ESSDAI domains. Box and whiskers plot show median and 5–95 percentile. Statistical analysis by Kruskal-Wallis-test with Dunn’s post-test correction for multiple comparison (A). (D) Average frequencies of Tfh-cell subsets, identified on the basis of ICOS and PD-1 expression and their frequencies distribution (E) in NSCS (white dots, n=10) and SS (dark grey dots, n=42). (F) Frequency of IFN-γ+, IL-21+ and IFN-γ+-IL-21+ double producing cells as percentage of PD-1+ICOS+, gated on CXCR5+CD4+ cells, detected by flow-cytometry on PBMC stimulation with PMA and ionomycin. Frequency of IL-21+ cells segregating the SS cohort for Ro (G) (Ro- (n=17) and Ro+ (n=20)) and La presence (H) (La- (n=27) and La+ (n=10)). Spearman correlation of IL-21+ cell (I, J, O), IFN-γ+ (K, L, P) or IL-21+-IFN-γ+ cell frequency (M, N, Q) with IgG (g/L) and C4 (g/L) serum levels and SG focus score in NSCS (n=10) and SS (n=42) patients. (R) Frequency of Tfh-cells subsets identified on the basis of ICOS and PD-1 expression in SS cohort segregated for ELS presence (ELS-, light grey dots (n=17), ELS+, dark grey dots (n=25)). (S) Frequency of CXCR5-PD-1hi cells, as percentage of CD4+ cells, in NSCS (n=10) and SS (n=42), and segregating the SS cohort for the presence of ELS. Statistical analysis by Mann-Whitney U t-test in (C), (E), (F), (G), (H), (R), (S). All graphs represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ELS, ectopic lymphoid structure; HD, healthy donor; ICOS, inducible T-cell costimulator; IFN-γ, interferon-γ; IL-21, interleukin-21; NSCS, non-specific chronic sialoadenitis; PD-1, programmed cell death protein 1; PNS, peripheral nervous system; SS, Sjogren’s syndrome.