Soluble anti cd28

For in vitro CD8⁺ T-cell culture, splenic CD8⁺ T cells specific for the OVA_257–264 (InvivoGen) antigen were sorted (>98% purity) from WT mice immunized with OVA_257–264 for 1 week (10 μg/mouse). T cells were cultured with plate bound anti-CD3 (1 μg/mL, BioXCell) and soluble anti-CD28 (2 μg/mL, BioXCell), with the addition of recombinant IL35 (rIL35, 50 ng/mL)(Chimerigen Laboratories; CHI-MF-11135) and OVA_257–264 (2 μg/mL) for 48 hours. For blocking STAT3 or STAT4 activity, cells were cultured with 50 μM fludarabine (Selleckchem), 20 μM STA-21, or 100 μM lisofylline (Santa Cruz Biotechnology) for 48 hours (Supplementary Table S2 for list of antibodies and reagents).
Mouse splenic Bregs (CD19⁺CD21^hiCD5⁺CD1d⁺) were sorted by flow cytometry from spleens of tumor bearing WT, p35^–/–, and Ebi3^–/– mice (>97% purity), as described above. 100,000 Bregs or Bcon cells and 100,000 CD8⁺ T cells (1:1 ratio) were cocultured in the 96-well Transwell plates, with B cells occupying the top chamber and CD8⁺ T cells the bottom chamber (Corning; 3381) for 48 hours. B cells were activated by anti-CD40 (1 μg/mL, eBioscience) and LPS (2 μg/mL, Sigma) for 48 hours, and CD8⁺ T cells were activated by plate bound anti-CD3 (1 μg/mL) and soluble anti-CD28 (2 μg/mL) with OVA_257–264 (2 μg/mL). Cytokine secretion of CD8⁺ T cells was evaluated by flow cytometry, as described below.