Soluble anti cd3

For the proliferation assay, one day after OVA challenge, cell suspensions were generated from the pooled LN and spleens from individual mice as described above. Cells were incubated in RPMI-1640 containing 10 % FCS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1.25 mg/mL amphotericin B (all Gibco BRL, CA, USA) (complete medium) in the presence of 1, 10, and 100 mg/mL OVA at 37 °C in 5 % CO₂. Cell proliferation was evaluated by [³H] thymidine (³H-TdR) incorporation. Cytokine content was analysed in culture supernatants by ELISA from Bender Med Systems, Vienna, Austria.
For suppression assays, 1 × 10⁵ CD4⁺CD25⁻ T-cells/well, 5 × 10⁴ CD4⁺CD25⁺ T-cells/well, or both populations were cultured in 96-well U-bottom plates with 1 × 10⁵ APCs/well for 72 h at 37 °C in complete RPMI 1640 medium (0.2 mL/well) in triplicate. Cultures were stimulated with 1 μg/mL soluble anti-CD3 (BD PharMingen, San Diego, CA, USA) with or without 0.1 μg/mL SJMHE1. Certain wells were added with 3 μg/mL rat IgG1 anti-mouse IL-10 (Biolegend Inc., San Diego, CA, USA), 0.5 μg/mL rat IgG1 anti-mouse TGF-β1 (US Biological, Swampscott, MA, USA), or 3 μg/mL rat IgG1 (Biolegend). Proliferation was assessed by incubation with 0.5 μCi/well ³H-thymidine and measuring the incorporation during the final 16 h of culture.

For T_FH stimulation assays, 2 × 10⁴ CD4⁺CXCR5⁺CD25⁻ T_FH cells and CD4⁺CXCR5⁻CD25⁻ T cells from mice of the responder group were plated with 5 × 10⁴ CD19⁺ B cells (all purified from spleen of responder group) and 2 μg/mL soluble anti-CD3 (BD Biosciences) plus 5 μg/mL anti-IgM (Jackson Immunoresearch, West Grove, PA). Cells were harvested and analyzed 6 days later.