Gp96 immunization inhibits Con A-induced immune hyperactivation by induction of Tregs.

<p>BALB/c mice immunized with gp96 three times were challenged with Con A. At 12 h after the Con A challenge, serum levels of pro-inflammatory cytokines (A) and IL-10 (B) were measured by ELISA. Flow cytometric analysis was performed to determine the frequency of IFN-γ ⁺ CD8⁺ and IFN-γ ⁺ CD4⁺ T cells in the spleen (C) or liver (D), CD4⁺CD25⁺Foxp3⁺ Tregs in the spleen (E) or liver (F), and expression of Ki-67 by Tregs in the spleen of mice (G). (H) Different subtypes of T cells from gp96-immunized mice (the bulk of total CD4⁺ population, and the sorted CD4⁺CD25⁺ and CD4⁺CD25^- subpopulations) were adoptively transferred to naïve mice as indicated, and 24 h later, the mice were challenged with Con A. At 12 h after Con A treatment, serum ALT levels were measured. (I) Foxp3-EGFP⁺ Tregs were sorted by FACS from gp96-immunized Foxp3-EGFP transgenic mice and were adoptively transferred to naïve recipient mice. Serum ALT levels were measured 12 h after Con A injection. (J) Mice immunized with gp96 or MSA were i.p. injected with 250 µg of mice anti-IL-10 receptor antibody (aIL-10r) or control antibody one day prior to Con A injection. Serum ALT levels were detected 12 h after Con A treatment. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; and ***, <i>p</i> < 0.001. Data are representative of two independent experiments and FACS analyses were performed for at least three times in each experiment.