Tumor cell–specific BCR-mediated Btk, Syk, Erk1/2, and p38 signaling. Flow cytometry contour plots of FL patient biopsy cells (FL-P09) stimulated by BCR cross-linking plus H₂O₂ (α-μ/γ + H₂O₂) for 4 or 30 minutes or left unstimulated (0 minute). BCR-mediated phosphorylation of Btk, Syk, Erk1/2, and p38 was compared in FL B cells (CD20⁺ Bcl-2^hi λ^–, dark arrow) and nonmalignant B cells (CD20^hi Bcl-2^lo, light arrow).

Rapid activation of BCR-mediated signaling in the presence of H₂O₂ is a common feature of FL sample B cells and differs from normal PBMC B cells. Differences in initiation of BCR-mediated signaling were compared in 5 tumor biopsy samples from individuals with FL and 5 PBMC samples from healthy donors. Samples were stimulated for 4 minutes by a combination of BCR cross-linking and H₂O₂ (α-μ/γ + H₂O₂) or left unstimulated. The average MFI of each phosphoprotein in CD20⁺ B cells has been graphed. Error bars indicate standard deviation.

Altered BCR signaling kinetics distinguish individual FL samples. BCR-mediated phosphorylation of Btk, Syk, Erk1/2, and p38 was observed at numerous times over a 2-hour time course in 3 representative FL samples (FL-P07, FL-P10, FL-P11). Cells were stimulated by BCR cross-linking and H₂O₂ (α-μ/γ + H₂O₂) or left unstimulated (0 minute), and the MFI of CD20⁺ B cells was graphed for each phosphoprotein. The area under the curve has been shaded to indicate the sum of signaling induction over time in B cells from each FL sample. As a reference, the same plot of the average MFI and standard deviation observed in 3 samples of normal PBMCs (PBMC Average) is shown on each graph.

Tumor cell–specific BCR-mediated Syk signaling. (A) Flow cytometry contour plots of FL patient biopsy cells (FL-P10) stimulated by BCR cross-linking alone (α-μ/γ) or by a combination of BCR cross-linking and H₂O₂ (α-μ/γ+ H₂O₂) for various times (4, 16, 30, 60, 90 minutes) or left unstimulated (0 minute). BCR-mediated phosphorylation of Syk was compared in CD20⁺ Bcl-2⁺ FL B cells and CD20⁺ Bcl-2^– nonmalignant B cells (refer to Figure 1). Dark arrows indicate greater Syk signaling in Bcl-2⁺ FL tumor B cells than in nonmalignant host B cells. Light arrows indicate λ isotype B cells that failed to activate Syk in response to BCR cross-linking. (B) Flow cytometry contour plots of FL patient biopsy cells (FL-P10) stimulated by a combination of BCR cross-linking and H₂O₂ (α-μ/γ + H₂O₂) for various times (4, 16, 30, 60, or 90 minutes) or left unstimulated (0 minute). BCR-mediated phosphorylation of Syk was measured in CD20⁺ κ isotype B cells and compared with that in λ isotype nonmalignant B cells (all λ^– cells are κ⁺). Dark arrows indicate sustained Syk signaling in Bcl-2⁺ FL tumor B cells. Light arrows indicate where Syk signaling in λ isotype B cells differed from κ isotype B cells.

Tumor cell–specific BCR-mediated signaling is common in FL. (A) BCR-mediated signaling at 4 and 30 minutes following BCR cross-linking in the presence of H₂O₂ (α-μ/γ+ H₂O₂) was measured in subsets of the CD20⁺ B cells from 4 FL patient samples (FL-P07, FL-P08, FL-P09, FL-P11). FL B cells (CD20⁺ Bcl-2^hi nontumor light chain^–, dark arrow) and tumor-infiltrating nonmalignant B cells (CD20⁺ Bcl-2^lo nontumor light chain⁺, light arrow) were distinguished and their signaling was compared by coloring heat map squares relative to the fold induction of phosphorylation relative to the unstimulated sample (0 minute). (B) Flow cytometry analysis of FL patient biopsy cells (FL-P12) stimulated by a combination of BCR cross-linking and H₂O₂ (α-μ/γ+ H₂O₂) for various times (4, 30, 60, or 90 minutes) or left unstimulated (0 minute). BCR-mediated phosphorylation of Erk1/2 and p38 was measured in nonmalignant B cells (CD20⁺ λ isotype, light arrow) and compared with that in FL B cells (CD20⁺ Bcl-2⁺ λ^–, dark arrow).

Activation of BCR signaling varies among primary FL samples and contrasts with that of normal B cells and a lymphoma cell line. (A) Flow cytometry analysis of signaling in PBMCs from a healthy blood donor, Ramos cells, and 5 FL patient samples (FL-P07–11) stimulated by BCR cross-linking (α-μ/γ) for various times in a short time course (1, 2, 4, 8, or 16 minutes) or left unstimulated (0 minute). BCR-mediated signaling in the CD20⁺ B-cell subset was compared by coloring heat map squares relative to the unstimulated PBMC sample. (B) As in panel A, with the addition of H₂O₂ just prior to BCR cross-linking. (C) Histogram data underlying the heat maps in panels A and B are shown for 2 samples, the lymphoma cell line Ramos and FL-P08.

Distinguishing BCR signaling events and FL biopsy cell subsets by flow cytometry. (A) Phosphoproteins detected by flow cytometry (dark blue rings) are highlighted on a model of BCR signaling that includes regulation by protein tyrosine phosphatases. The BCR can instruct B cells to proliferate, alter innate immune signaling thresholds, induce B-cell anergy, or initiate cell death in a manner thought to depend on the strength, duration, and path of signaling. We detected BCR-mediated phosphorylation of Syk, Btk, Erk1/2, and p38 in subsets of primary human B cells. (B) Flow cytometry analysis of light chain isotype of CD20⁺ cells from a peripheral blood mononuclear cell (PBMC) sample (healthy donor), an FL tumor biopsy specimen from a different patient (FL-P12), and the Ramos lymphoma cell line. Ramos cells are clonal in origin and were of λ isotype. FL tumor cells in FL-P12 were κ isotype and vastly outnumbered the TIL B cells, as is commonly observed in FL tumor biopsy specimens. (C) Flow cytometry analysis of an FL biopsy specimen with an unusually large number of infiltrating nonmalignant B cells (FL-P10). Analysis of light chain isotypes present in the CD20⁺ subset of cells suggested that the tumor B cells were κ isotype and indicated that all B cells in the sample were exclusively κ or λ isotype. Expression of λ isotype was compared with Bcl-2 expression to identify FL tumor and nonmalignant B-cell populations. FL tumor B cells were κ isotype and overexpressed Bcl-2. Nonmalignant tumor-infiltrating host B cells did not overexpress Bcl-2 and were either κ or λ isotype. CD20 expression was also compared with Bcl-2 and with λ light chain expression in the total population of cells in the sample.