Fig. 2.

LFA-1 modulates antigen-specific Foxp3 induction. A: Foxp3 induced in CD4⁺CD62L⁺ Tg4 T cells on plates coated with anti-CD3 and anti-CD28, with or without anti-CTLA-4, − PD-1, − LFA-1 or -LAG3, either plate-bound or soluble (all at 10 μg/ml). Mean of triplicates + SEM. Each bar is representative of 3 or more similar experiments. ***P < 0.001, One-way ANOVA with Dunnett's post-test. B: Foxp3 induction in and proliferation of CPD-ef450-labeled CD4⁺ Tg4 T cells stimulated with 1 μg/ml MBP Ac1-9 and irradiated APC, in the presence or absence of soluble anti-LFA-1, − CTLA-4 or -IL-10R at 10 μg/ml. Representative of three similar experiments. C: Foxp3 induction in CD4⁺CD62L⁺ Tg4 T cells after 7-day culture with titrated amounts of MBP Ac1-9 and irradiated APC, with or without 10 μg/ml soluble anti-LFA-1. Top; percentage of Foxp3⁺ cells among live CD4⁺ cells. Bottom; median fluorescence of Foxp3 staining, gated on CD4⁺Foxp3⁺ cells. n = 6–8 individual experiments per condition. **P < 0.01, ***P < 0.001,2-tailed paired t-test. D: Proliferation and division indexes of T cells labeled with CPD-ef450 after 7-day iTreg cell differentiation culture, stimulated with 1 μg/ml MBP Ac1-9 with or without 10 μg/ml anti-LFA-1 in the culture. Gated on live CD4⁺ Foxp3⁺ cells. Differences not significant, unpaired, 2-tailed t-test. E: LFA-1^hi, CD4^hi, CD62L, Ki67 and Foxp3 expression on live CD4⁺ T cells during 7-day iTreg cell differentiation culture, stimulated with 1 μg/ml MBP Ac1-9 and irradiated APC.