Isolation and Stimulation of Anergic T Cells

For anergic cell induction, spleens were harvested from NOD.Ocab^fl/flCD4-cre or control NOD.Ocab^fl/fl animals. Single-cell suspensions were generated by grinding and passage through 70-µm strainers. Cells were isolated using a mouse CD4⁺ T cell isolation kit (Miltenyi Biotec) and stimulated with 5 µg/ml plate-bound anti-CD3ε (BD Bioscience) ± 2 µg/ml anti-CD28 antibody (eBioscience) as described previously (Kim et al., 2019 (link)). For OCA-B inhibitor peptide treatments, cells were isolated from WT C57BL/6 spleens using a mouse naive CD4⁺ T cell isolation kit (Miltenyi Biotec) and cultured as described previously (Shakya et al., 2011 (link)). Indicated concentrations of peptides were incubated with cells, with media changes every 2 d after primary stimulation. Activation of C57BL/6 CD4⁺ cells and subsequent profiling of anergic responses were performed identically to the procedure by (Kim et al.,2019 (link)) For peptide restimulation, total WBCs from PLNs were incubated with 10 µg/ml specific peptide for 4 h in the presence of brefeldin A. The peptides used were as follows: GAD65_206-214, TYEIAPVFV; GAD65_546-554, SYQPLGDKV; IGRP_21-29, TYYGFLNFM; IGRP_206-214, VYLKTNVFL; IGRP_225-233, LRLFGIDLL; IGRP_241-249, KWCANPDWI; and IGRP_324-332, SFCKSASIP. Cells were then fixed and stained for flow cytometry.