Figure 4.

Grp78-treated cells could suppress T-cell proliferation and induce formation of Tregs in vitro. (A) CD11c⁺ cells and CD4⁺CD25⁻, CD8⁺ T cells from splenocytes were isolated using Miltenyi MACS. The purity of CD11c⁺ cells (left), CD4⁺CD25⁻ Teff (median), and CD8⁺ T (right) cells were tested. (B) CFSE-stained T cells (2 × 10⁵) were cocultured with αCD3 (0.3 μg/ml) and DCs at titration indicated, for 72 h. CFSE dilution of CD4⁺CD25⁻ Teff (a) and CD8⁺ T cells (b) were analyzed. Proliferation index (left) and representative flow cytometric histograms (right) were depicted. (C) 7-AAD-stained CD4⁺CD25⁻ Teff (left) and CD8⁺ T (right) cells were cocultured with CD11c⁺ cells, as described above. 7-AAD⁺ cells were analyzed. Percentage of apoptosis (upper) and representative images (lower) were depicted. (D) CD4⁺CD25⁻ cells were cocultured with CD11c⁺ cells as described. Cells were gated for CD4⁺. CD25⁺FoxP3⁺ expressing cells were analyzed. Percentage of CD25⁺FoxP3⁺ cells (upper) and representative flow cytometric dot plots (lower) were depicted. The results were representative of three independent experiments. Statistical comparison was conducted as indicated. Error bars were presented in SEM. *P < 0.05, **P < 0.01.