αcd28

Purified T cells were stimulated with plate-bound 5 µg/ml αCD3 (145-2C11; BioLegend) and 5 µg/ml αCD28 (37.51; Tonbo), polarized to the indicated lineages, and harvested at the designated time points. Cells were washed twice with PBS and lysed with RIPA buffer. Protein concentrations were quantified using the Bradford Assay, and equal amounts of protein were resolved on 4–12% Bis-Tris Plus gels (Thermo Fisher Scientific), transferred to PVDF membrane (EMD Millipore), and blotted for BCAP (1 µg/ml; 501813; R&D Systems) or H3 histone (1:3,000; Cell Signaling). Blots were then probed with appropriate secondary antibodies conjugated to fluorophores (IRDye 800CW goat anti–mouse, IRDye 680RD goat anti–rabbit), and membranes were imaged using LI-COR Odyssey CLx.

For polarizations using plate-bound αCD3 + αCD28, tissue culture plates were coated with 5 µg/ml αCD3 (145-2C11; BioLegend) and 5 µg/ml αCD28 (37.51; Tonbo), incubated at 37°C for 4 h, and washed three times immediately before use. For differentiation to Th17 cells, naive CD4⁺CD62L^highCD44^low T cells were incubated with IL-6 (20 ng/ml; Peprotech), hTGF-β (5 ng/ml; Peprotech), αIL-4 (10 µg/ml; 11B11; BioLegend), and αIFN-γ (10 µg/ml; XMG1.2; BioLegend), with or without IL-1β (2–10 ng/ml; Peprotech). For differentiation to Th1 cells, naive CD4⁺CD62L^highCD44^low T cells were incubated with IL-12 (10 ng/ml; Peprotech), IL-2 (50 U/ml; eBioscience), and αIL-4 (10 µg/ml; 11B11; BioLegend), with or without IL-18 (0.3–3 ng/ml; Gibco). Th2 cell differentiation was achieved by incubating naive CD4⁺CD62L^highCD44^low T cells with IL-4 (4 ng/ml; Peprotech), IL-2 (50 U/ml; eBioscience), and αIFN-γ (10 µg/ml; XMG1.2; BioLegend).