Figure 2.

Inhibiting NFATc1 SUMOylation triggers CD4⁺ T cells to express more IL-2 but less IFN-γ and IL-17A. (A) Th1 and Th17 in vitro differentiation of naive CD4⁺ T cells from WT and Nfatc1^deltaSUMO mice. On day 3, CD4⁺ T cells were restimulated with T/I followed by intracellular cytokine staining of IL-2 and IL-4 or IL-17A and IFN-γ. Representative flow cytometry plots and comparison of WT versus Nfatc1^deltaSUMO CD4⁺ T cells (Student’s t test ± SEM; n = 10). (B) Chromatin of Th1 and Th17, prepared as for A, was precipitated by anti–H3-Ac. Isolated genomic DNA was subjected to PCR with primers specific for the Il2 and Actb promoters. Student’s two-tailed t test (*, P < 0.05; **, P < 0.005; ***, P < 0.001); mean + SD; n > 3. (C) RT-PCR with primers specific for exon 9 + 11 (Δexon10) showed deletion of the C terminus of Nfatc1 mRNA in CD3⁺ T cells of Nfatc1^deltaBC [Nfatc1^deltaSUMO × Cd4cre] but not in CD3⁻ cells nor in WT cells. Primers binding to exon 9 + 10 detected WT Nfatc1 mRNA; n > 3. (D) Immunoblot analysis of unstimulated (w/o) and 6 h stimulated (T/I) CD4⁺ T cells from WT and Nfatc1^deltaBC mice illustrated the differential isoform expression of NFATc1/ΔBC; n > 3. (E) Th1 and Th17 in vitro differentiation of naive CD4⁺ T cells from WT and Nfatc1^deltaBC mice. Analyses as in A; n > 10. (F) ELISA of IL-2 in the supernatant of CD4⁺ T cells, derived from WT, Nfatc1^deltaSUMO, and Nfatc1^deltaBC mice and stimulated by anti-CD3 or anti-CD3/CD28 for 48 h. Student’s t test (*, P < 0.05; **, P < 0.005); n = 3.