Exhausted T cells were prepared as described.(17 (link)) CD8+ T cells were purified from C57BL/6-Tg(TcraTcrb)1100Mjb/J (commonly known as OT-I) mouse splenocytes with magnetic beads (Miltenyi Biotec, Auburn, CA). Cells were cultured at a final density of 5 × 105/mL in complete medium (RPMI 1640, 10% FBS, 1% 2 mM l -glutamine, 1% 1 M HEPES, 1% 100 mM sodium pyruvate, 1% nonessential amino acids, 1% penicillin–streptomycin, 0.05 mM beta-mercaptoethanol) with interleukin (IL)-15 (5 ng/mL; Peprotech, East Windsor, NJ) and IL-7 (5 ng/mL; Peprotech) with 10 ng/mL OVA(257–264) peptide (Anaspec, Fremont, CA).
Cells were repetitively stimulated with 10 ng/mL OVA(257–264) peptide daily for 5 to 7 days. Cells were checked daily, and when confluent, split, and cultured with fresh complete medium containing cytokines. For flow cytometry analysis, cells were first incubated with anti-Fc receptor antibody clone 2.4G2 (1 μg per well) for 30 minutes at room temperature. The indicated concentration of PE-conjugated PD-1 mAb 1A12, RMP1-14, or RMP1-30 or isotype control mAb was added and incubated for 30 minutes at 4°C. Cells were washed twice and binding was analyzed by flow cytometry.
Cells were repetitively stimulated with 10 ng/mL OVA(257–264) peptide daily for 5 to 7 days. Cells were checked daily, and when confluent, split, and cultured with fresh complete medium containing cytokines. For flow cytometry analysis, cells were first incubated with anti-Fc receptor antibody clone 2.4G2 (1 μg per well) for 30 minutes at room temperature. The indicated concentration of PE-conjugated PD-1 mAb 1A12, RMP1-14, or RMP1-30 or isotype control mAb was added and incubated for 30 minutes at 4°C. Cells were washed twice and binding was analyzed by flow cytometry.
