Effects of complement upon rATG-induced cell death. (A) Addition of complement augments the ability of rATG to kill CD138+ myeloma cells. Once isolated by Ficoll centrifugation and washed, 105 cells/well were incubated with rIgG control, rATG alone, or rATG plus rabbit complement. Compared with rATG alone, rATG with added complement significantly increased death of CD40L-stimulated B cells, H-Sultan, NCI-H929, RPMI-8226, MC-CAR, and U266 cell lines. However, it had no effect on CHO or ARH-77 cell lines. Error bars indicate the mean ± standard deviation. (B) rATG F(ab′)2 fragment activity was assessed by reconstituting with anti-FcR-specific monoclonal antibodies. CD40L-stimulated human B-cell blasts or myeloma cell lines (105 cells/well) were incubated with 250 μg/mL rATG F(ab′)2 fragments alone or coincubated with either human Fc fragments, anti-CD32, or anti-CD64 (♦). The response is measured against the intact rATG molecule (▪). Controls without rATG F(ab′)2 but with unimmunized rIgG, Fc fragments, anti-CD32, or anti-CD64 are also shown (⋄). rATG F(ab′)2 fragment with or without human Fc fragment showed a weaker apoptotic induction potential than intact rATG (P < .05, all samples). For CD40L sBc, RPMI-8226, and NCI-H929 cells, rATG F(ab′)2 fragments coincubated with anti-CD32 or anti-CD64 increased apoptosis of myeloma cells, although still somewhat less than intact rATG. No effect was observed on U266 cells.
