Figure 6. Maf is a critical downstream target of the CIC–ETV5 axis in the process of T_FH cell differentiation.

(a) qRT-PCR analysis for levels of T_FH-related genes in sorted non-T_FH and T_FH cells from Cic^f/f and Cic^f/fCd4-Cre mice. Splenocytes from 5 to 6 Cic^f/f mice or 3 to 4 Cic^f/fCd4-Cre mice were pooled and subjected to cell sorting. Three independent experiments were performed. (b) FACS-sorted splenicT_FH cells from Cic^f/f and Cic^f/fCd4-Cre mice were subjected to western blot analysis for MAF expression. The images are representative of two independent experiments. (c) qRT-PCR analysis of T_FH-related genes in CD4⁺ T cells infected with control or ETV5-expressing retrovirus. The cells were incubated in the presence of IL-6 and IL-21, and re-stimulated with anti-CD3 for 2 h prior to RNA extraction. Four independent experiments were performed. (d) qRT-PCR analysis for Maf levels in CD4⁺ T cells infected with control or ETV5-expressing retrovirus in the absence of IL-6 and IL-21. Before RNA extraction, the cells were activated with anti-CD3 for 2 h. Four independent experiments were performed. (a,c,d) Error bars indicate s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (two-tailed two-sample unequal variance student t-test). (e) Western blot analysis for MAF levels in CD4⁺ T cells infected with control or ETV5-expressing retrovirus. The cells were cultured in the presence or absence of IL-6 and IL-21, and then re-stimulated with anti-CD3 for 12 h. The images are representative of two independent experiments. (f) WT Thy1.1⁺ OT-II cells infected with control retrovirus and Cic null Thy1.1⁺ OT-II cells infected with control or Maf shRNA (shMAF) expressing retrovirus were transferred into Thy1.2⁺ B6 recipient mice. Seven days after immunization with NP-OVA in alum, the Thy1.1⁺ OT-II cells were analysed for T_FH cell differentiation using flow cytometry. WT:shNC, n=11; cKO:shNC, n=9; cKO:shMAF, n=8. **P<0.01 (two-tailed two-sample unequal variance Student t-test). (g) Schematic illustration on how CIC deficiency induces T-cell activation, T_FH cell differentiation and autoimmunity. CIC deficiency in T cells makes the TCR response hypersensitive, thereby inducing T-cell activation. During T_FH cell differentiation, de-repression of Etv5 induces Maf expression in Cic null T_FH cells, subsequently promoting T_FH cell differentiation. These T-cell abnormalities could contribute to autoimmunity in the Cic-deficient mice.