TNF receptor-associated factor 3 restrains B-cell receptor signaling in normal and malignant B cells

Abstract

TRAF3 has diverse signaling functions, which vary by cell type. Uniquely in B lymphocytes, TRAF3 inhibits homeostatic survival. Highlighting the role of TRAF3 as a tumor suppressor, loss-of-function TRAF3 mutations are associated with human B-cell malignancies, while B-cell-specific deletion of TRAF3 in mice leads to autoimmunity and lymphoma development. The role of TRAF3 in inhibiting noncanonical NF-κB activation, CD40 and BAFF-R signaling to B cells is well documented. In contrast, TRAF3 enhances many T-cell effector functions, through associating with and enhancing signaling by the T-cell receptor (TCR)-CD28 complex. The present study was designed to determine the role of TRAF3 in signaling via the B-cell antigen receptor (BCR). The BCR is crucial for antigen recognition, survival, proliferation, and antibody production, and defects in BCR signaling can promote abnormal survival of malignant B cells. Here, we show that TRAF3 is associated with both CD79B and the BCR-activated kinases Syk and Btk following BCR stimulation. BCR-induced phosphorylation of Syk and additional downstream kinases was increased in TRAF3^-/- B cells, with regulation observed in both follicular and marginal zone B-cell subsets. BCR stimulation of TRAF3^-/- B cells resulted in increased surface expression of MHC-II, CD80, and CD86 molecules. Interestingly, increased survival of TRAF3^-/- primary B cells was resistant to inhibition of Btk, while TRAF3-deficient malignant B-cell lines showed enhanced sensitivity. TRAF3 serves to restrain normal and malignant BCR signaling, with important implications for its role in normal B-cell biology and abnormal survival of malignant B cells.

Keywords: B-cell receptor (BCR); Btk; MAPK; TNF receptor-associated factor (TRAF); cell signaling; immunology; inhibition mechanism; lymphocyte; lymphoma; spleen tyrosine kinase (Syk).

Figure 1

TRAF3 is a negative regulator of BCR signaling.A, representative WB and (B) graphs (mean ± SEM) of densitometry of protein lysates from WT and TRAF3^−/− mouse splenic B cells stimulated with 10 μg/ml anti-IgM Ab for indicated times (N = 3–4 mice). A two-way ANOVA (# p < 0.05, NS = not significant) followed up with an unpaired t-test with Sidak correction for multiple comparisons (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, NS = not significant) was used to determine statistical significance. C, representative tracing of the ratio of Calcium-Green-1/Fura fluorescent in WT and TRAF3^−/− mouse splenic B cells after stimulation with 5 μg/ml anti-IgM Ab. The graph represents the mean area under the curve (AUC) ± SEM (N = 3 mice). An unpaired t-test was used to evaluate differences for statistical significance (∗ p < 0.05).

Figure 2

TRAF3-mediated regulation of BCR signaling in FO and MZ B cells. Mouse splenocytes were stimulated with 10 μg/ml anti-IgM Ab for indicated times. Representative plots and graphs of p-Erk (T202/Y204) and p-Syk (Y352) for (A) total B220⁺, (B) FO (CD21^lo CD23⁺), and (C) MZ (CD21^hi CD23^-) B cells. Data were analyzed using FlowJo software, and population frequencies are expressed as percent of the parent population. Graphs represent mean ± SEM (N = 4 mice). A two-way ANOVA (## p < 0.01) followed up with an unpaired t-test with Holm–Sidak correction for multiple comparisons (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001) was used to determine statistical significance.

Figure 3

Impact of inhibition of Syk and Btk on BCR-mediated Erk activation in WT and TRAF3^−/−mouse B cells. Splenocytes from WT or B-Traf3^−/− mice were pretreated with (A) 4 μM of R406 or (B) 20 μM of ibrutinib for 1 h prior to stimulation with 10 μg/ml anti-IgM Ab. Graphs represent % p-Erk (T202/Y204) positive (mean ± SEM). A two-way ANOVA (#### p < 0.0001) was used to determine statistical significance (N = 3 mice).

Figure 4

BCR-induced association of TRAF3 with Syk and Btk.A) TRAF3 or B) Btk was immunoprecipitated from cell lysates of BJAB cells following 20 μg/ml anti-human IgM Ab stimulation. C) TRAF3, D) Btk, or E) Syk was immunoprecipitated from cell lysates of mouse splenic B cells following 20 μg/ml anti-mouse IgM Ab stimulation. Co-immunoprecipitation was assayed with WB. Images are representative of three independent experiments. Graphs represent densitometry of protein of interest normalized to the IP protein (mean ± SEM). An unpaired t-test was used to determine statistical significance (∗ p < 0.05).

Figure 5

Association of TRAF3 with CD79 B in the mouse lymphoma CH12.LX cell line.A, representative WB and graphs (mean ± SEM) of densitometry of protein lysates from TRAF3^−/−, TRAF2^−/−, and WT CH12.LX cells stimulated with 10 μg/ml anti-IgM Ab for the indicated times (N = 3 independent experiments). An unpaired t-test was used to determine statistical significance (∗ p < 0.05). B, CD79B was immunoprecipitated from cell lysates of CH12.LX cells with or without 5 min of 20 μg/ml anti-mouse IgM Ab stimulation. Images are representative of three independent experiments. Graphs represent densitometry of protein of interest normalized to the IP protein (mean ± SEM). An unpaired t-test was used to determine statistical significance (∗ p < 0.05).

Figure 6

BCR-induced activation marker expression and survival in TRAF3^−/−mouse splenic B cells. Mouse splenic B cells were stimulated with 5 μg/ml anti-mouse IgM Ab for the indicated times. (A–C, Left) Representative histograms of B cell expression of the indicated surface receptors, measured by flow cytometry. (A–C, Right), graphs of % positive (mean ± SEM) of B cells (N = 3 mice). D, graph of gMFI (mean ± SEM) of MHC-II (I-A/I-E) expression of unstimulated mouse splenic B cells (N = 3 mice). E, mouse splenic B cells were treated with indicated doses of anti-IgM Ab for 24 h (N = 3 mice). A two-way ANOVA followed up with an unpaired t-test with Sidak correction for multiple comparisons (A–C, E) or an unpaired t-test (D) was used to determine statistical significance (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001).

Figure 7

Impact of TRAF3 expression on survival of mouse splenic B cells and human B-cell lymphoma cell lines following BCR pathway inhibition.A, mouse splenic B cells were treated with indicated doses of R406 (left) or ibrutinib (right) with (black) or without (gray) 5 μg/ml anti-IgM Ab for 24 h. Cell viability was determined by exclusion of live/dead stain by flow cytometry. The percent viability was calculated as the mean viability of inhibitor-treated samples normalized to DMSO vehicle-treated sample for each genotype. Graphs indicate mean values ± SEM of percent viable cells (N = 3 mice). A two-way ANOVA followed up with unpaired t-tests with Holm–Sidak correction for multiple comparisons was used to evaluate differences for statistical significance (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). B, TRAF3 expression in the indicated human lymphoma cell lines was measured by Western blot (N = 3 independent experiments). Graphs represent the mean normalized TRAF3 expression ± SEM. C, the indicated human B lymphoma cell lines were treated with indicated doses of ibrutinib for 48 h. Cell viability was determined by exclusion of live/dead stain by flow cytometry. Line graph indicates mean values ± SEM of percent viable cells. The half maximal inhibitory concentration (IC₅₀) was calculated using Graphpad Prism (N = 3 independent experiments). D, line graph of mean relative TRAF3 protein expression versus IC₅₀. Trendline and R-squared value was calculated with Graphpad Prism.