Splenic B2 and Peritoneal B1a Cell Assay

Splenic CD4⁺ T cells and CD43⁻ B cells (sB2) were isolated by negative selection with magnetic beads (Miltenyi Biotec, Auburn, CA, USA) yielding sB2 and CD4⁺ T cell population with a purity >95%. Peritoneal B1-a cells (pB1a) were isolated as previously described (33 (link)). Briefly, non-adherent peritoneal cells were first negatively selected with biotinylated anti-Thy1.2 (53-2.1) and anti-CD3ε (145-2C11) Abs, and then positively selected with biotinylated anti-CD5 (53-7.3) Ab. This protocol led to a B220^int CD5⁺ cell population with an average of 80% purity. CD4⁺ T cells (2 × 10⁵) from bm12 mice were co-cultured with pB1a or sB2 from the congenic NZM strains or B6 mice (1 × 10⁵) for 5 d in T cell polarizing media without anti-CD3 or anti-CD28 Abs. Th17 polarizing media contained TGFβ (3 ng/ml), IL-6 (50 ng/ml), 6-Formylindolo (3,2-b) carbazole (FICZ, 300 nM; Enzo Life Sciences, Farmingdale, NY, USA), anti-IL-4 Ab and anti-IFNγ Ab (10 ug/ml each). Th1 polarizing media contained IL-12 (10 ng/ml) and anti-IL-4 Ab (10 ug/ml), and Treg polarizing media contained TGFβ (20 ng/ml) and IL-2 (100 U). All cytokines were purchased from Peprotech (Rocky Hill, NJ, USA). In some experiments, blocking Abs to CD86 (GL1, 10 ug/ml), CD44 (either IM7 or KM114 clones, 20 ug/ml) or IL-6 (1 ug/ml, Peprotech) were added to the co-cultures.