Figure 2.

Activation-induced signaling reveals contrasting differences between NHL subtypes, and potentiated α-BCR–induced signaling in MCL. Patient samples and control samples were thawed and rested for 20 minutes before activation with CD40L, IL-4, IL-7, IL-10, IL-21, or IFN-γ for 15 minutes, CpG for 45 minutes, BCR engagement with F(ab′)₂ anti-IgM and anti-IgG (α-BCR) for 4 minutes or 45 minutes, or left untreated (unstim) (see supplemental Figure 1). Tumor cells were identified as described in Figure 1. (A) Histogram overlays of α-BCR– or CD40L-induced p-SYK and p-p65 NF-κB in CLL and MCL cells, shown relative to unstimulated cells. (B) Typical activation-induced signaling profiles shown as heatmaps of lymphoma cells from CLL and MCL patient samples as compared with healthy donor PBMC B cells. (C) Gating on lymphoma cells (CD20⁺BCL2⁺CD5⁺CD3⁻) and nonmalignant B cells (CD20⁺BCL2⁻CD5⁻CD3⁻) within the same patient sample revealed potentiated signaling in malignant B cells. (D) Heatmap of major activation-induced phosphorylation levels in lymphoma cells, across different types of NHLs. Each column represents a patient sample, and the rows were clustered for visual clarity (dendrogram not shown). Samples were annotated for tissue (gray, LN; black, peripheral blood), DLBCL subtype (gray, GCB; black, non-GCB; white, n.d.), IGHV status (gray, unmutated; black, mutated; white, n.d.), and Ig heavy chain (gray, IgM; black, IgG). (E) Scatter plots of α-BCR–induced phosphorylation levels in lymphoma cells, relative to unstimulated cells. Each dot represents a single patient sample. Phosphorylation levels are relative to unstimulated lymphoma cells from the same patient. DLBCL (n = 12), FL (n = 27), CLL (n = 14), MCL (n = 42). Healthy donor controls: tonsillar B cells (n = 4) and PBMC B cells (n = 8). Statistical difference was calculated using the Mann-Whitney nonparametric test (***P < .0001, **P < .005, *P < .05). iono, ionomycin.