Fig. 3.

Anti-LFA-1 treatment alters the phenotype but not the stability or function of iTreg cells. A: Expression of CD62L, Neuropilin-1 (NRP-1), CD103 and Helios on Foxp3⁺ iTreg cells after 7-day differentiation culture with 1 μg/ml MBP Ac1-9 in the presence or absence of 10 μg/ml anti-LFA-1. Pooled data, n = 6–7 per condition. **P < 0.01, *** P < 0.001,unpaired, 2-tailed t-test. B: Analysis of the relative level of methylation of 9 CpG in the foxp3 CNS2 region of naive CD4⁺ T cells, CD4⁺CD25⁺ splenic Treg cells or iTreg cells generated under various conditions, in the presence or absence of 10 μg/ml soluble anti-LFA-1 (all Tg4). Blocks in each column represent the mean values of three replicate samples. C: GFP-Foxp3 retention in Tg4 Foxp3^gfp iTreg cells in vivo. FACS-sorted iTreg cells labeled with CPD-ef450 were transferred to Tg4 recipients. Mice were challenged with a high affinity variant of MBP Ac1-9 48 h post transfer. 72 h post challenge iTreg cells retrieved from the spleen were analyzed for proliferation and Foxp3 retention by FACS. One experiment representative of 3 is shown. D: Tg4 mice received 5 × 10⁶ Tg4 iTreg cells generated using either stimulation with 1 μl/ml MBP Ac1-9 and soluble anti-LFA-1 (Ag iTreg) or anti-CD3 and anti-CD28 (Ab iTreg) in PBS, or PBS only, i.p. 3 days prior to EAE induction with MBP Ac1-9 in CFA and were monitored daily for disease. n = 3 per group, representative of 2 similar experiments.