Spleen cells were prepared by gently flushing the spleen with IMDM supplemented with antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), 10% FCS, 50 mM b-ME, and 2 mM Lglutamine. Erythrocytes were lysed for 2 min in ACK buffer (0.15 M NH 4 Cl, 10 mM KHCO 3 , and 0.1 mM EDTA) and cells were washed two times with PBS. Cell clumps were removed by passaging through a 50 mm nylon filter. Preparation was conducted strictly at 0uC.