Total IgM levels in the EBV-transformed B-cell cultures enhanced by feeder cells. (A) Total IgM levels in the EBV-transformed B-cell cultures derived from CLL246 PBMCs under different culture conditions. After EBV infection, PBMCs were plated as follows: lane a indicates 10 000 PBMCs per well without feeder cells; lane b, 5000 PBMCs per well without feeder cells; lane c, 5000 PBMCs per well with 50 000 cells per well of J774A.1; lane d, 2500 PBMCs per well with 50 000 cells per well of J774A.1; lane e, 2500 PBMCs per well with 25 000 cells per well of J774A.1; and lane f, 2500 PBMCs per well with 12 500 cells per well of J774A.1. The J774A.1 feeder cells were γ-irradiated before use. Levels of IgM in the culture supernatants were measured by ELISA. Each data point represents the level of IgM in each well (54-60 wells per culture condition). U indicates unmutated type; and M, > 2% mutated compared with germline according to ImMunoGeneTics.27 Total IgM levels in the EBV-transformed B-cell cultures derived from additional B-CLL samples were measured in 240 wells per sample (B) or in 20 wells per sample (C). After EBV infection, 5000 PBMCs were incubated in the presence of 50 000 cells per well of irradiated J774A.1 cells. Each data point represents the level of IgM in each well. To determine the minimal number of PBMCs necessary for B-CLL cell activation as defined by IgM production, total of 100 000 PBMCs were plated in total of 20 wells (5000 PBMCs per well) in panel C.
