In vitro generation of DCs

DCs were generated from peripheral blood mononuclear cells (PBMCs) from each PLC patient as described previously [23] with some modifications. Briefly, PBMCs were isolated from 60 ml peripheral blood using a Ficoll-Hypaque gradient (Amersham, Uppsala, Sweden). Two patients (Nos. 9 and 10) underwent leukapheresis using a cell separator (Kobe Spectra, Japan) in order to obtain a sufficient number of PBMCs. The cells were resuspended in culture medium comprising RPMI-1640 supplemented with 2 mM l-glutamine, 50 μg/ml streptomycin, 50 U/ml penicillin, and 1% autologous serum, and allowed to adhere to the plastic culture flask (Becton Dickinson, Franklin Lakes, N.J.). After 2 h at 37°C, non-adherent cells were removed and adherent cells were cultured in medium supplemented with 50 ng/ml GM-CSF (kindly provided by Novartis Pharmaceutical Corporation, Tokyo, Japan) and 50 ng/ml IL-4 (purchased from Pepro Tech, Rocky Hill, N.J.). Fresh medium was added and the medium was supplemented with cytokines every other day. On day 6 of culture, cells were pulsed with prepared TL (100 μg/ml) for 12 h. Then TNF-α (100 ng/ml; R&D Systems, Minneapolis, Minn.) and keyhole limpet hemocyanin (KLH) (50 μg/ml; Calbiochem, Bad Soden, Germany) were added to the culture on day 7. Cultured DCs were harvested by vigorous washing with culture medium on day 9. Aliquots were taken for cell counting and viability staining by trypan blue. Cell differentiation was monitored by light microscopy.