Figure 3.

Effects of TBS mutation on TNF-α, IL-6, and IgM production. A, M12.4.1 B cells stably transfected with hCD40LMP1, hCD40, hCD40ADD, or hCD40LMP1AEDL were stimulated for 4 hours with agonistic mouse or human CD40-specific mAbs or isotype control mAbs. TNF-α secreted in response to stimulation was assayed from cell culture supernatants by ELISA. Data are presented as hCD40-induced TNF-α normalized to mCD40-induced TNF-α. TNF-α secreted in response to isotype control mAbs was subtracted from CD40 stimulated groups prior to normalization. B, CH12.LX B cells stably transfected with hCD40LMP1, hCD40, hCD40ADD, or hCD40LMP1AEDL were stimulated for 2 days with Hi5 insect cells expressing hCD154, agonistic anti-mouse or human anti-CD40 mAbs, or isotype control mAbs. IL-6 was assayed from cell culture supernatants by ELISA. Black bars represent cells stimulated by Hi5 insect cells expressing hCD154, open bars represent cells stimulated by agonistic anti-mCD40 mAb, gray bars represent cells stimulated by agonistic anti-hCD40 mAb. Open bars are generally below the level of detection because anti-CD40 mAb does not induce IL-6 production via hCD40 (49). IL-6 secreted in cultures containing isotype control mAbs or untransformed Hi5 insect cells was subtracted from values obtained from cultures stimulated via hCD40 extracellular domains. C, CH12.LX B cells stably transfected with hCD40LMP1, hCD40, hCD40ADD, or hCD40LMP1AEDL were stimulated for 3 days with agonistic mouse or human anti-CD40 mAbs or isotype control mAbs. Data are presented as the mean number of Ab secreting cells ± S.E. of replicate cultures induced in response to hCD40 stimulation normalized to the number of Ab secreting cells induced in response to mCD40 stimulation. Ab secreted by cells cultured with isotype control mAbs was subtracted from CD40 stimulated groups prior to normalization. Data shown are representative of three or more experiments performed with two or more clones of each transfectant.