Figure 5

Efficient induction of Tregs by GMPs.

Non-specific T cell proliferation assays were performed as described in Fig. 1A and Tregs were analyzed by FACS. In (C,D), the cultured cells were recovered and sorted for CD4⁺CD25⁺ cells. For analyzing Foxp3 expression (C), sorted CD4⁺CD25⁺ cells were used for total RNA extraction and Foxp3 expression levels determined by RT-qPCR. For assessing the functional activities of induced Tregs, sorted CD4⁺CD25⁺ cells were re-cultured with fresch CFSE-labeled splenocytes at 1:4 ratio as described above. (A) Example FACS dot plots gated on CD4⁺ cells. (B) Percentages of Foxp3⁺CD25⁺ cells induced in the CD4 T cells. (C) Relative Foxp3 expression in CD4⁺CD25⁺ cells recovered from indicated cultures. Foxp3 expression level in BM cells-added groups was calculated as fold of that of medium-only samples. (D) Inhibition of T cell proliferation by GMP-induced Tregs. 1: splenocytes only; 2, 3, 4: splenocytes + CD4⁺CD25⁺ cells recovered from original cultures with presence of MEP (2), T-GMP (3), or N-GMP (4). Data shown are mean ± SD of triplicate samples from three (A,B) or two (C,D) independent experiments^***p < 0.001; ^**p < 0.01; ^*p < 0.05.