Tracking the CD40 signature in normal B cells. The CD40 gene expression signature (left panel) was used to track the activity of CD40 signaling in normal B cells with the use of the gene expression profiles of pre-GC naive B cells (N), GC B-cell populations (CB and CC), and post-GC memory B cells (M). The expression of the CD40 signature genes in the normal B-cell subpopulations was analyzed by hierarchical clustering with the use of the average linkage method (right panel). The color scale identifies relative gene expression changes normalized by the standard deviation, with 0 representing the mean expression level of a given gene across the panel. The first branching in the dendrogram separates the GC (CB and CC) from the non-GC (N and M) B cells. The majority of the 106 CD40 up-regulated genes showed higher expression in naive and memory B cells compared with CBs and CCs. Reciprocal gene expression patterns were observed for the CD40 down-regulated genes that are mostly expressed at lower levels in naive and memory B cells compared with GC B cells. The expression of the CD40 signature genes in CBs and CCs mimics the expression of those genes in unstimulated cells, suggesting that CD40 signaling is not active in GC B cells. To further quantify the presence of the CD40 signature, we used 2 binary scoring approaches (see “Materials and methods”), showing that the CD40 signature of stimulated cells was absent from GC B cells and detectable in naive and memory B cells at P ≤ 10^-7.