Figure 6. NR2F6 Directly Binds to the Il21 Promoter and CNS –36 in Resting CD4 T Cells, and Inhibition of IL-21 Signaling Reduces Tfh Cell Accumulation.

(A) Putative NR2F6 binding sites of the mouse Il21 promoter and CNS regions are shown.

(B) NR2F6 binding to the Il21 promoter at −1.5 kb, −2.2 kb, or CNS −36 by EMSA nuclear extracts from Jurkat cells (J+ NR2F6) transfected with pEFneo Nr2f6 plasmid. To validate binding specificity, consensus (c) or mutated (mc) oligonucleotides were added in excess as unlabeled competition oligonucleotides. Where indicated, an anti-NR2F6 antibody was added. One representative experiment out of three is shown.

(C) NR2F6 binding to the Il21 promoter at −1 to −2.2 kb or CNS −36 was investigated by ChIP. Nr2f6^+/+ or Nr2f6^–/– CD4 cells either resting or activated under Tfh-differentiating conditions were used with anti-NR2F6 or IgG control precipitation, Il21 promoter or CNS sequence was quantified by qPCR. Data are presented as the percentage of input. Differences between genotypes were calculated using log-transformed data following a linear mixed-effects model fit by restricted maximum likelihood (REML) and was analyzed via ANOVA; interaction plots are shown in Figures S6F–S6H.

(D) Scheme for anti-IL-21R block within OVA-alum-immunized Nr2f6^+/+ or Nr2f6^−/− mice. Wild-type or Nr2f6^−/− mice were treated with anti-IL-21R antibody or isotype control at the same time as OVA immunization (50 μg) and 3 days later (30 μg).

(E and F) Representative Tfh cell flow cytometry data (E) and total Tfh cell numbers (F) are shown.

Data shown are from two independent experiments with n ≥ 4. Error bars represent SD, and an asterisk indicates statistically significant differences between genotypes calculated using Student’s t test, Mann-Whitney U test, or ANOVA. A p value of <0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001.