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. 2021 May 5;8(14):2004973. doi: 10.1002/advs.202004973

Figure 1.

Figure 1

The cytoplasmic domain of the CTLA‐4 peptide induces functional Foxp3+ Tregs in vitro. a) Schematic of the synthetic CTLA‐4 signaling peptide. b–e) Mouse naïve CD4 T cells were stimulated with anti‐CD3/CD28 mAb in the presence or absence of 2 × 10−6 m dNP2‐ctCTLA‐4 for 4 days. Representative flow cytometry dot plots (left) and bar graphs (right) of Foxp3 induction under b) Th0, c) Th1, d) Th17, or e) iTreg differentiation conditions (n = 6–10). f) Treg markers including CD25, CD73, and CTLA‐4 under Th17 differentiation conditions were analyzed by flow cytometry (n = 5–7). g–i) In vitro suppression assay based on coculture of Foxp3+ T cells (CD45.1‐ GFP+) and responder T cells (CD45.1+ CD4+ CD25− CD62L+ CD44−) for 3 days (n = 4–5). Foxp3+ T cells induced by 2 × 10−6 m of CTLA‐4 signaling peptide were sorted and then cocultured with responder T cells for 3 days. Naïve CD4 responder T cells were isolated from splenocytes, labeled with carboxyfluorescein succinimidyl ester (CFSE), and stimulated with CD3/28 dynabeads. g) Representative CFSE histogram of responder T cells cultured under each differentiation condition. h) Suppression of proliferation (%) was analyzed by flow cytometry. i) Treg stability was determined by assessing the remaining Foxp3+ cells among CD45.1− CD4+ T cells after 3 days. All data are presented as mean ± S.D. Statistical significance was determined by Mann–Whitney test in b–e) and h–i) Wilcoxon test in f). n.s. = nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001.