Up-regulation of IL-10 and PD-1 by Treg cells after heart, and bone marrow transplantation is enhanced by NKT cells in an IL-4–dependent manner. (A) Mean percentages of donor type cells at day 19 after marrow transplantation in WT BALB/c mice given heart and bone marrow transplants (TX WT, n = 27 mice), and in CD1d^−/− BALB/c mice given transplants (TX CD1d^−/−, n = 20 mice). (B) Experimental scheme: representative FACS pattern of gated recipient type H-2K^b−CD4⁺CD25⁺ and H-2K^b− CD4⁺CD25⁻ T cells among CD4⁺ splenocytes before sorting (left and middle panels) or after sorting (right panel), obtained 19 days after marrow transplantation. Sorted cells were cultured with PMA and ionomycin, and cytokine concentrations in culture supernatants were assessed by luminex beads. (C) Mean (± SEM) IL-10 (left panel), IL-4 (right panel), and IFNγ (middle panel) concentrations in supernatants from sorted CD4⁺CD25⁺ T cells, after 48 hours of culture with PMA/ Ionomycin from untreated WT BALB/c mice (UNT WT, n = 15 mice), WT BALB/c mice given heart and bone marrow transplants (TX WT, n = 27 mice) and CD1d^−/− mice given heart and bone marrow transplants (TX CD1d^−/−, n = 20 mice). Data are combined from 3 or 4 independent experiments with 2 to 3 wells in each experiment. NS, not significant, P > .05; *P < .05, **P < .01, ***P < .001 (Student 2-tailed t test of independent means). Culture supernatants from Treg cells without PMA/ionomycin had less than 20 pg/mL of IFNγ, IL-4, or IL-10. (D) Percentages (horizontal lines show means) of PD-1^hi cells among gated CD4⁺CD25⁺ T cells from spleen in untreated WT BALB/c mice (UNT WT, n = 8 mice); WT BALB/c mice given heart and bone marrow transplants (WT TX, n = 13 mice), Ja18^−/− BALB/c mice given transplants (Ja18^−/− TX, n = 6 mice); Ja18^−/− BALB/c mice given transplants with add-back of sorted 0.4 × 10⁶ NKT cells from WT BALB/c mice (Ja18^−/− TX + WT NKT, n = 7 mice), and Ja18^−/− BALB/c mice given transplants with add-back of sorted 0.4 × 10⁶ NKT cells from IL-4^−/− BALB/c mice (Ja18^−/− TX + IL-4^−/− NKT, n = 5 mice). Comparison of means (Student 2-tailed t test) gave P values shown on the panels; NS > 0.05, *P < .05, **P < .01, ***P < .001. (E) Mean IL-10 concentrations of supernatants of sorted host PD-1^lo and PD-1^hi Treg cells obtained from the spleen of WT heart and bone marrow transplants recipients on day 19 after marrow transplantation (WT TX, n = 8). There were 8 replicate cultures from 2 experiments used for stimulation of cells with PMA and ionomycin. Unstimulated cultures had less than 20 pg/mL IL-10.