For in vitro activation, CD4+ cells were purified by flow cytometry sorting or with magnetic beads and stimulated with plate-bound antibodies against CD3 (10 μg/ml; 2C11) and CD28 (1 μg/ml; 37.51) (both from BD Biosciences), Con A (2 μg/ml; Sigma), or an analog of PCC peptide (5 μM; PCC50V54A) in the presence of antigen-presenting cells in αMEM media (HyClone) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), 2 mM l-glutamine, dextrose, essential and nonessential amino acids, sodium pyruvate, sodium bicarbonate, antibiotics, and 2-β-mercaptoethanol.

To produce polarized effector cells, CD4+ T cells were purified by flow cytometry sorting or with magnetic beads and stimulated with plate-bound antibodies against CD3 and CD28. For TH1 differentiation, cells were stimulated in the presence of IL-12 (10 ng/ml; PeproTech) and antibody against IL-4 (10 μg/ml; BD Biosciences). For TH2 differentiation, cells were stimulated in the presence of IL-4 (10 ng/ml; PeproTech) and antibody against IFN-γ (10 μg/ml; BD Biosciences) and IL-12 (10 μg/ml; BD Biosciences). For TH17 differentiation, cells were stimulated in the presence of IL-6 (20 ng/ml; PeproTech) and TGF-β (3 ng/ml; PeproTech). Cells were cultured and analyzed after 4 days. To examine BMP signaling, T cells were cultured with BMP2, BMP4, and BMP7 (3 ng/ml each; PeproTech). To inhibit TGF-β signaling, SB525334 (10 nM; Selleckchem) was added to cell culture. To inhibit BMP signaling, T cells were incubated in the presence of dorsomorphin (1 μM; Tocris Bioscience) or DMH1 (10 μM; Tocris Bioscience).

For coculture, total lymph nodes and spleen from PCC transgenic mice were combined. The cells from wild-type and BMPR1αT− mice were then mixed 1:1 and stimulated with PCC peptide and either IL-6 (20 ng/ml; PeproTech) and TGF-β (3 ng/ml; PeproTech) or IL-21 (100 ng/ml; PeproTech) and TGF-β (3 ng/ml; PeproTech). Cell proportions in the coculture were determined to be 60% BMPR1α-sufficient and 40% BMPR1α-deficient using CD45.1 and CD45.2 staining. Cells were cultured and analyzed after 6 days.

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