IL-6 secretion stimulated by hCD40-P227A versus WT hCD40. (A) CH12.hCD40WT (hCD40WT) or CH12.P227A (P227A.1, P227A.2, P227A.3) cells were stimulated as indicated with Hi5 cells (1:5 ratio with B cells) plus or minus 10 μg/mL α-μ chain F(ab)′2 Ig for 48 hours and IL-6 ELISA was performed. Two separate subclones of CH12.LX expressing hCD40WT were tested with similar results (not shown). Data represent means plus or minus SD of triplicate cultures. A representative experiment of 5 independent experiments with similar results is shown. Hi5-WTBV; Hi5 infected with Wt baculovirus. (B) Cells were stimulated as in panel A for the indicated times and IL-6 ELISA was performed. IL-6 was undetectable in supernatants from cells stimulated with media (BCM) or with uninfected Hi5 cells. A representative of 3 independent experiments with similar results is shown. (C) Cells were stimulated with 10 μg/mL agonistic α-mCD40, α-hCD40, or isotype control mAbs plus or minus 10 μg/mL α-μ chain F(ab)′2 Ab (α-BCR) or media (BCM) for 48 hours. IL-6 ELISA was performed. Data represent means plus or minus SD of 3 independent experiments. Two separate subclones of CH12.LX expressing hCD40WT were tested with similar results (not shown). (D) Cells were stimulated for 20 hours as indicated with Hi5 cells (1:5 ratio with B cells) plus or minus 25 nM CpG 2084 or control CpG 2087 oligonucleotides. Culture supernatants were analyzed by ELISA. Data are representative of 3 independent experiments with similar results, and a second hCD40WT subclone was tested with similar results (not shown). Asterisks denotes statistically significant difference between hCD40WT and hCD40-P227A: *P < .05, **P < .001 by 2-tailed Student t test.
