High-dose gp96 promotes Treg proliferation and function by up-regulation of Foxp3 expression and IL-10 secretion.

<p>(A) Mice splenocytes cultured in the presence of 1 µg/ml anti-CD3, anti-CD28 Abs and 50 IU/ml IL-2 were stimulated with increasing amounts of gp96 for 24 h, and the TNF-α and IL-10 levels in the supernatants were analyzed by ELISA. Mice splenocytes (B–E) or Treg cells (F, G) were incubated with 100 µg/ml gp96 <i>in vitro</i> in the presence of anti-CD3 and anti-CD28 Abs and IL-2. The Treg frequency was measured by FACS after 48 h (B). Proliferation of Tregs was determined by CSFE staining 3 d later (C). A total of 3×10⁵ CSFE-labeled CD4⁺CD25^- Teff cells were cultured with Tregs at a ratio of 3:1 for 3 d. The Teff cell division cycle was measured by FACS (D). The suppression rate for proliferation was calculated (E). The IL-10 levels in the supernatants of Tregs were measured by ELISA (F). The Foxp3 expression of Tregs was detected by Western blotting (G). *, <i>p</i> < 0.05; **, <i>p</i> < 0.01. Results are representative of three independent experiments and FACS analyses were performed for at least five times in each experiment.