Fig 1: Gp96 activates CD4+Foxp3+ Tregs through MyD88 signal(A–C) Foxp3CreMyd88fl/fl and Foxp3CreMyd88+/+ mice were treated as in Figure 6B. OVA-specific IgG levels were determined in serum on day 20 post OVA challenge using ELISA (A). FACS analysis of CXCR5+PD-1+ cells (Tfr) (B) in spleen and CD4+Foxp3+ Tregs (C) in the indicated organs.(D) FACS analysis of Foxp3, Helios, CD62L, CD69, ICOS and CD137 expression on CD4+Foxp3+ Tregs in spleens of Foxp3CreMyd88fl/fl and Foxp3CreMyd88+/+ mice immunized with 200 µg gp96 or saline (control).(E) Flow cytometry analysis of ICOS, KLRG1, CD69 and CD137 expression in CD4+Foxp3+ Tregs from CD45.1+ cells and CD45.2+ cells.(F) Foxp3CreMyd88fl/fl and Foxp3CreMyd88+/+ mice were immunized with gp96 or saline (control) before induction of EAE. Disease score was measured daily after EAE induction. Dots represent data from individual mice. The data are representative of two independent experiments with similar results. Mean ± SD is shown. The Student's t test or two-way ANOVA was used for statistical analysis. P < 0.05 were considered statistically significant. ns = not significant.
Fig 2: TLR binding domain is essential for gp96-induced Treg activation(A) Low- (20 µg) or high- (200 µg) dose Cy5-labelled gp96 was subcutaneously injected into the backs of Foxp3-GFP KI mice. Flow cytometry analysis of Cy5-positive Tregs (CD4+Foxp3-GFP+), DCs (CD11c+), B cells (CD3–CD19+), conventional CD4+ T cells (CD4+Foxp3-GFP–) and CD8+ T cells 6 h post treatment.(B–D) Ovalbumin (OVA)-specific IgG in serum on day 10 and day 21 post OVA treatment was measured using ELISA (B). FACS analysis of Treg cells (C) and Tfr cells (D) in spleen.(E) Flow cytometry analysis of Foxp3, Helios, CD62L, CD69, ICOS, and CD137 expression on CD4+Foxp3+ Tregs in the spleen of saline, mutant or WT gp96-immunized mice. n = 5 mice/group.(F) Serum anti-dsDNA antibodies were determined by ELISA in Lyn–/– mice at the indicated weeks of age one week after mutant gp96 treatment.(G) FACS analysis of surface TLR1/2 expression in CD4+Foxp3+ Tregs from C57BL/6 mice.(H) Tregs were cultured with 100 µg/mL His-gp96 for 4 h before immunoprecipitation with anti-His antibody and western blotting.(I) Luciferase assay using reporter plasmids containing the Foxp3 promoter in 293T cells. Expression plasmids of TLR1 and TLR2 were transfected as indicated. Cells were either unstimulated or stimulated with 100 µg/mL gp96 for 12 h before analysis.(J) Frequency of Tregs in the spleen from Tlr2-/- mice immunized with 200 µg gp96 or saline (control, n = 5/group).(K) Mean Fluorescence intensity (MFI) of indicated markers in the spleen from Tlr2-/- mice immunized with 200 µg gp96 or saline (control) (n = 5/group).(L and M) Chimeric mouse model was produced and immunized with gp96 (L). Flow cytometry analysis of ICOS, KLRG1, CD69, and CD137 expression on CD4+Foxp3+ Tregs from CD45.1+ cells and CD45.2+ cells (M). Dots represent data from individual mice. 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.
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