Figure 3.

Heat shock protein gp96 induces Foxp3 expression and promotes Treg proliferation

(A) Frequency of CD4⁺Foxp3⁺ Treg in spleen of C57BL/6 mice immunized with the indicated dose of recombinant gp96.

(B and C) Foxp3 expression in Tregs from mice immunized with 200 μg gp96 or saline as control was measured using flow cytometry analysis (B) and real-time PCR (C).

(D) FACS-sorted CD4⁺Foxp3-GFP⁺ Tregs and CD4⁺Foxp3-GFP^– Tconv cells from Foxp3-GFP KI mice were transferred to Rag2^−/− mice. Mice were then immunized with gp96 or saline three times. Foxp3-GFP levels in CD4⁺ T cells from the spleen were analyzed 1 week after the last immunization.

(E) Proliferation of Tregs was measured using in vivo BrdU labeling in the indicated organs of gp96-immunized and saline-immunized C57BL/6 mice.

(F) Chromatin immunoprecipitation (ChIP) analysis of H3K27ac, p300 and H3K4 monomethylation (H3K4me1) at Foxp3 locus and control loci (Hsp90ab, Hspa2, Rpl30, and Gm5069). The data are representative of two independent experiments with similar results. n = 5 mice/group. Mean ± SD is shown. The Student's t-test was used for statistical analysis. P < 0.05 was considered statistically significant. ns = not significant.