IL-33 mediates p38 MAPK-dependent signaling to promote the expansion of proliferating Tregs expressing ST2. (A) Sorted ST2⁺ and ST2⁻ CD4⁺ Foxp3(RFP)⁺Thy1.1⁺ cells from B6 OT-II FIR were labeled with CellTrace Violet (CTV), and infused into WT or il33^−/− B6N mice that had been exposed to 1100 cGy 1 day prior. Recipient mice also received BM cells matched to recipient IL-33 status to create IL-33⁺ or IL-33 KO conditions. On day 5 posttransplant, splenocytes were isolated and CD90.1⁺ T cells assessed by flow cytometry for Foxp3 and ST2 expression, as well as proliferation (CTV dilution). Right flow plots, Proliferation and Foxp3 expression of the transferred CD90.1⁺ Tregs. Left panel, The ST2 expression on proliferating (P1) vs nonproliferating (P2) CD90.1⁺ cells. (B-C) Graphs present the average and SEM for (B) ST2 expression and (C) percentage proliferation (4 mice per group). Significant differences were calculated using unpaired Student t tests (*P < .05, **P < .01, ***P < .001, ****P < .0001). (D) CD4⁺Foxp3(RFP)⁺ Tregs were flow-sorted based on ST2 expression from B6 FIR mice and treated with IL-33 (100 ng/mL; bolded line) or (phorbol myristate acetate [PMA]/ionomycin; thin line) for 4 minutes before assessment for phospho-p38 or phospho-NF-κB p65 by flow cytometry. Representative histograms are presented. Unstimulated cells (filled histogram) served as a negative control. Graphs depict average calculated fold change in MFI of phospho-p38 or phospho-NF-κB p65 between IL-33–treated and untreated samples from 3 independent experiments. Statistical significance between groups calculated using the Student t test (*P < .05, **P < .01). (C) Bulk CD4⁺ T cells cultured with BALB/c CD11c⁺ BMDCs in media, stimulated with IL-33 alone, or IL-33 in combination with (E) the NF-κB inhibitors TPCA-1 or MG 132 or (F) the p38 MAPK inhibitor SB 203580. Flow plots depict ST2 expression vs CTV on CD3⁺CD4⁺Foxp3⁺-gated cells. (G) Average of results from 3 independent experiments represented in panel F. Statistical significance between groups calculated using the Student t test (*P < .05, **P < .01). AU, arbitrary unit.