Figure 2. OX40 induces RORγt expression but impairs its transcriptional activities in CD4⁺ T cells.

(A) Flow cytometry plot showing expression of RORγt by WT CD4⁺ T cells activated under neutral control- or Th17-polarizing conditions for 3 days.

(B) Immunoblot analysis of RORγt in the nucleus of naive CD4⁺ T cells activated with anti-CD3 plus WT APCs (OX40 −) or OX40L-TG APCs (OX40 ⁺) under Th17-polarizing conditions for 24–96 h.

(C) Real-time PCR quantification of Rora, Batf, Rel, and Rela mRNA in naive WT CD4⁺ T cells activated under Th17-polarizing conditions for 48 h with anti-CD3 plus WT APC or OX40L-TG APCs. Results are relative expression normalized to GAPDH mRNA (glyceraldehyde phosphate dehydrogenase).

(D) Immunoblot analysis of the induction of RORa, Batf, c-Rel, and RelA in the nucleus of naive CD4⁺ T cells activated for 48 h with or without OX40 stimulation.

(E and F) Flow cytometry analysis of the induction of Th17 cells from WT naive CD4⁺ T cells transduced with empty retroviral vector (Ctrl) or retrovirus expressing RORγt, cultured Th17-polarizing conditions with WT APCs or OX40L-TG APCs for 3 days (E). Graphs in (F) depict Mean ± SEM of 3 experiments with triplicate cultures. Data are presented as mean ± SD (C) and are representative of at least three individual experiments in (A–E). (G) ChIP analysis of the enrichment of RORγt at Il17a promoter and CNS5 regions in WT CD4⁺ T cells activated for 48 h as in Figure 2D. Values are presented as relative binding based on normalization to control IgG. Data are presented as mean ± SD (n=4). (H–M) ChIP analysis of H3K9me2 (H), H3K9Me3 (I), H3K4Me3 (J), H3K27Me2 (K), H3K36Me3 (L) and H3K79Me3 (M) at the Il17a promoter (RORE1, RORE2/3) and CNS5 (RORE4) regions in WT CD4⁺ T cells activated under Th17-polarizing conditions for 2 days. Data are presented as mean ± SD (n=3). ** p <0.01.