TRAF2 Deficiency in B Cells Impairs CD40-Induced Isotype Switching That Can Be Rescued by Restoring NF-κB1 Activation

Abstract

Effective humoral immunity requires class switch recombination (CSR) catalyzed by activation-induced cytidine deaminase (AID). In response to T cell-dependent (TD) Ags, CSR can be induced by CD40 signaling in B cells. TNFR-associated factors 2 and 3 (TRAF2/TRAF3) function as adaptors of the CD40 signaling pathway. B cell-intrinsic TRAF2 or TRAF3 (B-TRAF2 or B-TRAF3) knockout mice were previously reported to have indistinguishable phenotypes in gene expression, B cell survival and development, and enlarged peripheral lymphoid organs. However, it remains unknown whether deficiency of B-TRAF2 or B-TRAF3 differentially affects TD humoral immune responses and CD40-induced CSR. In this article, we show that B-TRAF2 is essential for optimal isotype switching induced by in vivo TD Ag immunization or by engaging CD40 in vitro. Our data clarify the controversial role of B-TRAF3 and confirm its dispensability in CD40-induced CSR. Mechanistically, CD40-induced AID expression was markedly impaired by B-TRAF2, but not B-TRAF3, deficiency. Moreover, B-TRAF2 deficiency causes defective activation of the NF-κB1 complex in a CD40-autonomous manner, and restoring CD40-induced NF-κB1 activation in TRAF2-deficient B cells rescues AID expression and CSR. We conclude that TRAF2 is essential but TRAF3 is dispensable for TD humoral immunity and CD40-induced CSR. Our studies provide significant biological bases for optimizing treatment of B cell-associated immune disorders by targeting CD40 signaling.

Figure 1.. Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on TD Ag-induced Ab isotype-switching.

(A) Representative FACS data showing the gating strategy and the percentage of IgG1⁺ B cells. 28 days post first immunization, splenocytes were isolated from mice immunized with TNP-BGG Ag and analyzed by flow cytometry. Numbers indicate the percentage of IgG1⁺ B cells in the B220⁺CD19⁺ population. (B) Quantification of FACS data. The percentage of IgG1⁺ isotype-switched B cells were significantly reduced in B-TRAF2-KO but not in B-TRAF3-KO mice (n=6 per group). (C) Representative ELISA data of serum IgG1, IgG2a and IgE Abs against TNP-BGG. Data are presented as mean±s.e.m (n=5 or 6 per group). All experiments were repeated three times independently and representative data from one experiment are shown. P values were calculated by a Student’s t-test, *p≤0.05, **p≤0.01, which was applied to graphic data in all figures. LMC: littermate control (TRAF2^fl/fl or TRAF3^fl/fl, respectively). KO: knockout. TNP-BGG: 2,4,6-trinitrophenol-conjugated bovine γ-globulin.

Figure 2.. B cell-intrinsic TRAF2 but not TRAF3 deficiency impairs CD40-induced CSR ex vivo.

(A) Representative FACS data of IgG1 and IgE CSR. Purified B cells from various genotypes were stimulated with anti-CD40/IL-4 or LPS/IL-4 for 4 days and analyzed by flow cytometry. (B) Quantification of FACS data. The percentage of IgG1⁺ or IgE⁺ isotype-switched B cells was calculated from triplicates of each indicated genotype in one experiment. LMC: littermate control (TRAF2^fl/fl or TRAF3^fl/fl); 2KO: B-TRAF2-KO; 3KO: B-TRAF3-KO. Statistical analysis was performed as described in Figure 1. For panel A and B, all experiments were repeated five times independently and representative data from one experiment were shown. (C) Representative FACS data of purified MZ and FO B cells. FO B cells are CD23^highCD21^low, whereas MZ B cells are CD21^highCD23^low. (D) Representative FACS data of IgG1 CSR of FO and MZ B cells. Purified FO vs. MZ B cells from B-TRAF2-KO or B-TRAF3-KO mice were stimulated with anti-CD40/IL-4 or LPS/IL-4 for 4 days. For panel C and D, all experiments were repeated three times independently and representative data from one experiment are shown.

Figure 3.. Effects of B cell-intrinsic TRAF2 or TRAF3 deficiency on AID expression and GLT of Cγ1 or Cε induced by CD40.

Quantitative RT-PCR (qPCR) data of relative transcripts of Cγ1 GLT (A), Cμ GLT (B) and AID (C). Purified B cells of indicated genotypes were stimulated with anti-CD40/IL-4 or LPS/IL-4 for 2 days. qPCR was conducted as described in Materials and Methods. Statistical significance analysis was performed by student t test, **p<0.01, *p<0.05. (D) Western blot of AID protein expression in the indicated B cells that were either unstimulated (day 0) or stimulated with anti-CD40/IL-4 or LPS/IL-4 for 3 days. β-actin is the loading control. LMC: littermate control (TRAF2^fl/fl or TRAF3^fl/fl). 2KO: B-TRAF2-KO. 3KO: B-TRAF3-KO. All experiments were repeated three times independently and representative data from one experiment are shown.

Figure 4.. TRAF2 is required for CD40 signaling to activate the NF-κB1 complex and induce isotype-switching.

(A) Western blot of IκBα degradation and RelA phosphorylation in CD40-stimulated B cells. Purified B cells of indicated genotypes were stimulated with anti-CD40 for 0, 5 or 30 mins. CD40 stimulation did not induce IκBα degradation or RelA phosphorylation in B-TRAF2-KO B cells. (B) Western blot of IκBα degradation and RelA phosphorylation in LPS-stimulated B cells. Purified B cells of indicated genotypes were stimulated with LPS for 0, 30 or 240 mins that exhibited comparable levels of IκBα degradation and RelA phosphorylation. (C) Representative FACS data of IgG1 CSR (top panel) and CFSE dilution (bottom panel). LMC B cells were labeled with CFSE, then either treated with 5Z-7 (0.5μM) or untreated (Med), and stimulated with anti-CD40/IL-4 for 3 days. Top panel: representative of FACS data for IgG1 CSR and averaged percentage of IgG1⁺ B cells from triplicates. Bottom panel: representative FACS data for CFSE dilution (Med: red; 5Z-7 treated: blue) and the averaged proliferation index from triplicates. Proliferation index was calculated using Flowjo proliferation analysis program. P values were calculated by a Student’s t-test, **p<0.01, NS: not significant. (D) Western blot data show that 5Z-7 also significantly reduced AID expression. (E) Representative FACS data of IgG1 and IgE CSR. B cells of different genotypes were stimulated by anti-CD40/IL-4 for 4 days and analyzed by flow cytometry. TRAF3 deficiency in B-TRAF2-KO B cells did not rescue CD40-induced CSR. (F) Western blot of IκBα degradation in CD40- or LPS-stimulated LMC B cells (top panel) and B-DKO B cells (bottom panel). B cells were stimulated by anti-CD40 or LPS for 0, 30 or 240 mins and subjected to western blotting. TRAF3 deficiency in B-TRAF2-KO B cells did not rescue the defective CD40-induced IκBα degradation. B-DKO: double knockout of B-TRAF2 and B-TRAF3; LMC: littermate control (CD19Cre⁻/TRAF2^fl/fl/TRAF3^fl/fl). Med: culture medium with anti-CD40 plus IL-4. All experiments were repeated independently three or five times and representative data from one experiment are shown.

Figure 5.. PMA rescues the defective CD40-induced NF-κB1 complex activation and CSR in B-TRAF2-KO B cells.

(A) Western blot of IκBα degradation induced by PMA stimulation alone. LMC and B-TRAF2-KO B cells were stimulated with PMA (1μM) for 0, 5 or 30 min and comparable IκBα degradation was observed in both groups. (B) Western blot of IκBα degradation and RelA phosphorylation in anti-CD40- vs. anti-CD40/PMA-stimulated B cells. LMC and B-TRAF2-KO B cells were stimulated as indicated in (A). (C) Representative FACS data of IgG1 CSR. Med: culture medium with anti-CD40/IL-4. B-TRAF2-KO (group A-C) or LMC (group D) B cells were stimulated in medium with anti-CD40/IL-4 and stimulator or inhibitor as indicated for 4 days. PMA (2nM) rescued CD40-induced CSR in B-TRAF2-KO B cells (A vs. B). However, the rescue effect of PMA was completely abrogated by 5Z-7 treatment (B vs. C). (D) Western blot of AID expression in different groups (A-D). B cells were stimulated as indicated in panel (C) and subjected to western blot analysis. P-RelA: phospho-RelA. All experiments were repeated independently three or five times and representative data from one experiment are shown.