Fig. 4
Effect of Anti-CD40 Abs KPL-404 and G28-5 on T cell-dependent B cell activation in healthy donors. PBMCs were cultured in the presence of 10 μg/ml IgG4 isotype control or anti-CD40 Abs KPL-404, or G28-5 (16–18 h of cell culture). Cells were left unstimulated (media control) or stimulated with CD3/CD28 cross-linker IC or F(ab′)2 goat anti-human IgM (anti-IgM). a, b Flow cytometry data from one representative experiment, showing CD69 (a) and CD86 (b) expression on gated live CD19+ B cells. c, d Cumulative data, depicting the expression of CD69 (c) and CD86 (d) in six individual HD donors. Data are expressed as fold change MFI over IgG4-teated, media controls. Data were analyzed using one-way ANOVA on log-transformed data with matched mixed-effects modeling for multiple comparisons tests of significance between different conditions; *p < 0.05. Only comparisons between IgG4 vs KPL-404 of IC-stimulated samples are shown. Complete statistical analysis of the data is presented as Supplemental Fig. S3. e, f PBMCs were stimulated with IC in the presence of varying concentrations (20 to 0.01 μg/ml) of IgG4 isotype antibody control, KPL-404, or G28-5 anti-CD40 antibodies. Graphs show the expression of CD69 (e) and CD86 (f) on gated CD19+ B cells, relative to isotype control (100% max. stimulation) for each Ab. Cumulative data from three independent experiments using different donors