IgM secretion stimulated by hCD40-P227A versus WT hCD40. (A) CH12.LX cells stably expressing hCD40WT (WT) or hCD40-P227A (P227A.1, P227A.2) were stimulated for 72 hours with insect cells infected with WT baculovirus (WT-Hi5), mCD154 expressing baculovirus (mCD154), or hCD154 expressing baculovirus (hCD154). IgM-secreting cells in replicate cultures are enumerated (± SE) as described.25 Results are representative of 2 similar experiments. (B) Cells were stimulated with 2 μg/mL agonistic α-mCD40 mAb, α-hCD40 mAb, or isotype control mAbs for 72 hours. Data represent fold increase in Pfc: mean Pfc of replicate cultures stimulated through CD40 divided by mean Pfc of replicate cultures of cells stimulated with isotype control mAbs, and are the mean values of 3 separate experiments. (C) CH12.LX cells stably expressing hCD40WT, hCD40-P227A (P227A.1) or a chimeric molecule consisting of the extracellular and transmembrane domains of hCD40 and the intracellular domain of LMP-1 (hCD40-LMP1) were stimulated as in panel B. Data represent mean fold increase in Pfc over cells stimulated with isotype control antibodies, as in panel B and are representative of 3 similar experiments. (D) Cells were stimulated as indicated with 2 μg/mL agonistic α-mCD40 or α-hCD40 mAb plus or minus 0.1% SRBC (Ag). IgM production was measured as in panel A and is representative of 2 similar experiments. Similar results were observed with a second subclone expressing hCD40-P227A. Asterisk denotes statistical difference (*P < .05) between hCD40WT and hCD40-P227A by 2-tailed Student t test.
