Figure 5. Mechanism of Blimp1-Dependent Control of T_FR Differentiation: Contribution of the IL-23R-STAT3 Axis and CXCR5-CCR7 Expression.

(A) WT and KO mice (6 weeks old) were immunized with NP-OVA in CFA. Seven days later, T_FR cells (CD4⁺CD3⁺PD-1⁺CXCR5⁺YFP⁺) were sorted for microarray analysis. Differential gene expression in T_FR from WT and KO mice (>1.5-fold) is shown.

(B) Pathway analysis revealed control of genes associated with T_H cell differentiation by Blimp1.

(C) Genes related to cytokine-cytokine receptor interaction by DAVID.

(D) IL-23R expression by donor T_FR cells in Tcra^−/− hosts after tamoxifen-induced deletion of Blimp1, as in Figure 4.

(E) IL-23R expression in the CD25-expressing T_FR subsets from WT and KO mice (6–8 weeks old) 10 days post-immunization.

(F) FoxP3-GFP reporter mice were immunized with NP-OVA in CFA. Seven days later, Treg (CD4⁺CD3⁺GFP⁺CD25⁺) and CD4⁺ naive T cells (CD4⁺CD3⁺GFP⁻CD44⁻) were sorted, chromatin prepared, and ChIP-PCR analyses performed for Blimp1, acetylated H3 (AcH3), and H3K27me3 at Blimp1-binding sites at the 3^rd intron of Il23r, the 1^st intron of Cxcr5, the 5′ distal element of CTLA-4, the 3^rd intron of Ccr7, the FoxP3 CNS2, the 1^st intron of Il2ra, and a non-specific region (C, control) of Il2ra. Data are shown as the percentage of input. Naive cells were used as controls for the anti-Blimp1 assay. Rabbit IgG isotype (R-iso) and mouse IgG isotype (M-iso) served as controls for the anti-AcH3 assay and anti-H3K27 assay, respectively.

(G) Prdm1^fl/fliCre⁻ (WT) and Prdm1^fl/fliCre⁺ (Del) mice (8 weeks old) were treated with tamoxifen at day 0, followed by immunization with NP-OVA in CFA at day 1 and injection of tamoxifen daily for 4 days. Expression of pSTAT3 and pSTAT5 in T_FR cells at day 7 post-immunization. At right, pSTAT3:pSTAT5 ratios in T_FR cells are shown (n = 4/group).

In (D), (E), and (G), the data represent one of two experiments. In (F), the data represent one of three independent experiments. **p < 0.01 (unpaired two-tailed Student’s t test). Error bars indicate means ± SEMs.

See also Figure S4.