Mril2

Splenic B cells were enriched by depletion of CD43⁺ cells with anti-CD43 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) that reach 95–98% of B cell purity. MACS purified B cells or FACS purified T cells were cultured in DMEM supplemented with 10% FCS, 2 mM L-Glutamine, 1 mM Sodium pyruvate, 10 mM HEPES, 52 μM β-Mercaptoethanol, Non-essential amino acids, and Penicillin-streptomycin (10% FCS-DMEM). To establish LMP1/2A⁺ B lymphoma cell lines, MACS purified B cells from GCB-LMP1/2 mice spleen were stained for CD19 and CD3 surface markers, and CD19⁺CD3^- B cells were FACS sorted to eliminate T cells completely. Those B cells were intravenously transferred into Rag2^−/− or Rag2^−/− γC^−/− recipient mice to obtain lymphoma cell lines. For in vitro T cell culture, FACS purified CD4⁺ T or CD8⁺ T cells from spleen were co-cultured with γ-irradiated (3–4 Gy) LMP1/2A⁺ B lymphoma cells [19 (link)] at a 1:1–0.1 T cell to lymphoma cell ratio in 10% FCS-DMEM supplemented with 20 ng/mL mrIL-2 (Peprotech, Cranbury, NJ, USA).