CD4⁺ iNKT cells promote an expansion of donor Tregs. (A) FoxP3 expression of live Thy1.1⁺CD4⁺ T cells. Gates are set on isotype controls. Shown are representative dot plots from 1 of at least 5 independent experiments. (B) Relative (percentage of live Thy1.1⁺CD4⁺) and (C) absolute cell numbers of FoxP3-expressing donor CD4⁺ T cells. Shown are 3 animals per group from 1 of at least 5 independent experiments. (D) Expression of CD25, cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte-activation gene 3 (Lag3), and transforming growth factor β (latency-associated peptide) (TGF-β [LAP]) on live Thy1.1⁺CD4⁺FoxP3⁺ T cells. Shaded curves depict isotype controls. Shown are pooled data of 3 mice from 1 of at least 2 independent experiments. (E) Mixed lymphocyte reaction (MLR) of donor Thy1.1⁺ Tregs pooled and sorted from BALB/c mice, donor Tcon effectors, and irradiated allogeneic BALB/c stimulators. The MLR was performed in triplicate. Shown is 1 of 3 independent experiments. Error bars indicate standard error of the mean. E, effector (Tcon); I, inhibitor (Thy1.1⁺ Treg); ITN, intestine. *P ≤ .05; **P ≤ .01; ***P ≤ .001.