Dose-response assay and kinetics of huCD40LT stimulation of primary MCL cell cultures.
(A) Dose-response assay: Isolated mononuclear cells from 7 MCL patients were plated at 2 × 106 cells/mL and cultured in the absence or presence of increasing doses of huCD40LT for 4 days. Cultured cells were pulsed with 3HTdR 16 hours before harvest and measurement of thymidine incorporation. The maximal response in the presence of huCD40LT (mean, 8162 cpm; range, 61-48 000 cpm) was obtained at doses between 100 and 1000 ng/mL, and huCD40LT-induced 3HTdR incorporation was significantly higher than in the controls (mean, 1813 cpm; range, 4-12 500 cpm) from doses of 1 ng/mL and above (P = .03, Wilcoxon). Data are shown as mean and range of the triplicate cultures. (B) Kinetics: Mononuclear cells from 6 MCL patients were plated at 2 × 106 cells/mL and cultured in the presence of 400 ng/mL of huCD40LT, and 3HTdR incorporation was measured after 1, 2, 4, or 8 days of culture. The difference between days 1 and day 4 was significant (P < .04, Wilcoxon). Data are shown as mean and range of the triplicate cultures. (C) Spontaneous DNA synthesis: Control cultures were plated at 2 × 106cells/mL in medium alone, and 3HTdR incorporation was measured after 1, 2, 4, or 8 days of culture. No significant spontaneous 3HTdR incorporation could be demonstrated as compared with the stimulated cultures. Data are shown as mean and range of triplicate cultures. UPN indicates unique patient number.
