Figure 5. NR2F6 Suppresses Il21 Expression in Tfh Cells In Vitro and In Vivo.

(A) Naive Nr2f6^+/+ CD4⁺ T cells were stimulated for 24 h with indicated amounts of anti-CD3 and a fixed amount (2 μg/mL) of anti-CD28 (left panel). Alternatively, anti-CD3 was kept constant, and anti-CD28 was added at the indicated dose (right panel). qRT-PCR performed for Nr2f6 expression is displayed; values are shown relative to gapdh expression.

(B) Wild-type CD4 T cells were cultured under Tfh cell-polarizing conditions, and qRT-PCR was used to determine Nr2f6 expression at the indicated time points. Expression of the closely related nuclear receptor Nr2f2 was also determined under these conditions (right panel).

(C–E) Cytokine expression of (C) Il2, (D) Il4, and (E) Il21 was determined in in vitro Tfh cell culture by qRT-PCR at the indicated time points.

(F) IL-21 secretion into culture media was measured using Bioplex technology.

(G) Scheme for Nr2f6^+/+ OT-II and Nr2f6^−/− OT-II mouse immunization and Tfh cell sorting.

(H) Day 3 and day 7 Nr2f6 expression from sorted Nr2f6^+/+ OT-II Tfh cells.

(I) Nr2f2 expression from Nr2f6^+/+ OT-II and Nr2f6^−/− OT-II Tfh cells sorted as in (H).

(J) Il21 expression from day 7 sorted Nr2f6^+/+ OT-II or Nr2f6^−/− OT-II Tfh cells.

Data shown are from two independent experiments with n ≥ 4. The middle bar represents the dataset average. The data are presented as the percentage of input samples before immunoprecipitation. Error bars represent SD, and an asterisk indicates statistically significant differences between genotypes calculated using Student’s t test. A p value of <0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001.