The in vivo function of a noncanonical TRAF2-binding domain in the C-terminus of CD40 in driving B-cell growth and differentiation

Abstract

The recruitment of tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 to the CD40 cytoplasmic tail upon CD40 trimerization results in downstream signaling events that ultimately lead to CD40-dependent, thymus-dependent (TD) humoral immune responses. Previously, we have shown signaling through the C-terminal tail of CD40 in the absence of canonical TRAF-binding sites is capable of signaling through an alternative TRAF2-binding site. Here, we demonstrate that B cells from mice harboring CD40 with only the C-terminal tail can activate both canonical and noncanonical NFkappaB signaling pathways. Moreover, while lacking germinal center formation, several hallmarks of humoral immune responses including clonal B-cell activation/expansion, antibody isotype switching, and affinity maturation remain normal. This study demonstrates a new functional domain in CD40 that controls critical aspects of B-cell immunity in an in vivo setting.

Figure 1

Generation of chimeric CD40 transgenic mice. (A) Schematic representation of the constructs used to generate transgenic mice expressing chimeric CD40 molecules consisted of the extracellular domain of the human CD40 and the transmembrane and varied cytoplasmic domains of murine CD40 under the MHC class II promoter control. Point substitutions are labeled by asterisks. Restriction enzyme site SalI has been inserted to connect 2 CD40 cytoplasmic tail fragments into the pDOI-5 vector. (B) Chimeric CD40 transgenic receptor expression on B220⁺ B cells were confirmed by FACS analysis. Filled histograms represented antihuman CD40 staining of the transgenic receptor; open histograms, the isotype control.

Figure 2

CD40 induced NFκB activation. (A) CD19⁺ splenic B cells purified from different CD40 transgenic mice were cultured in vitro with hCD40L (1 μg/mL) stimulation for indicated time points. Canonical NFκB activation was measured by phosphorylated IκBα blotting. Anti-α/β actin antibody was used for loading control. (B) For noncanonical NFκB activation, B cells were cultured in 24-well plates and stimulated with 1 μg/mL hCD40L for 16 hours. Cells were then harvested and cytoplasmic and nuclear fractions were isolated. The level of noncanonical NFκB activation was detected by p52 blotting in the nuclear fraction. Anti-SAM68 antibody was used for loading control. Data are representative of more than 3 independent experiments. (C) The ratio of intensity values of p52 and SAM68 signals from the nucleus fraction was presented as quantitative analysis for measuring noncanonical NFκB activation.

Figure 3

CD40-mediated B-cell proliferation and up-regulation of surface markers. (A) Proliferation was measured in splenic B cells treated with IL-4 either with or without hCD40L for 3 days. Cultures were pulsed with [³H]TdR for the last 8 hours of culture (*P = .005; **P = .008). (B) For the induction of surface markers, splenic B cells were cultured in vitro for 48 hours with or without hCD40L (400 ng/mL). Cells were harvested and stained for CD23 and CD80 followed by FACS analysis. Data are representative of more than 3 independent experiments as the average ± standard deviation (SD).

Figure 4

Plasmacytic differentiation and Ig secretion.(A-B) Real-time analysis of Bcl-6 and Blimp expression from splenic B cells cultured in vitro with IL-4 either with or without hCD40L stimulation for 24 hours and 5 days, respectively. Relative expression of various gene targets normalized to β-actin was calculated as (2^{−(experimental CT − β-actin CT)}) × 1000, where CT is the cycle threshold of signal detection. (C-D) IgM and IgG secretion from B cells was accessed by ELISA analysis using supernatant collected from 5 days of in vitro culture of splenic B cells with or without 400 ng/mL hCD40L and 10 ng/mL IL-4 (*P = .004; **P = .001). Data are representative of 3 independent experiments (mean ± SD).

Figure 5

CD40-mediated GC formation. (A) FACS analysis of GC formation was performed from mice 10 days after sheep erythrocyte immunization. Splenocytes were isolated and stained with B220, PNA, and GL7. Fluorescence was quantified by flow cytometry and profiles were gated on B220⁺ cells. (B) Immunohistologic analysis of GC formation was performed from the spleen treated similarly as mentioned in “The role of the C-terminus of the CD40 cytoplasmic tail in GC formation” section. In brief, spleens from sheep erythrocyte–immunized mice were cryocut and stained with B220 (green), PNA (red), and CD4 (blue). GC is indicated by white arrow. Data are representative of 8 to 12 mice from 2 independent experiments.

Figure 6

Humoral immune responses in chimeric CD40 transgenic mice. Mice were immunized with 100 μg NP₃₀-KLH emulsified in CFA. Circulating antibodies were measured by an NP-specific, isotype-specific ELISA. (A) Both total NP-specific IgG1 (NP30) and high-affinity NP-specific IgG1 (NP4) responses from different chimeric CD40 transgenic mice were followed over time (days 14, 28, 42, and 56) (*P < .05 by analysis of variance versus values for CD40^−/− at indicated time points). (B) The alterations of IgG1 responses (total versus high affinity) to NP antigen in Δ260 mice over time were shown. Data represent 2 independent experiments (mean ± SD).

Figure 7

Schematic representation of the potential interaction between CD40 and its adaptor molecules. (A) CD40-mediated signaling results from a combination of both positive (right) and negative (left) signals. (B) WT CD40 (left) recruits TRAF6 in its membrane-proximal domain; TRAF1, TRAF2, TRAF3, and TRAF5 through oligomerization in the canonical TRAF2-binding site; and another TRAF2 in the C-terminus of the CD40 cytoplasmic tail. Site-directed mutagenesis resulted in the disruption of TRAF6 recruitment, much reduced TRAF2, TRAF3 binding in the canonical TRAF2 site but left the second TRAF2 site intact in ΔT2,3,6 (middle). Δ260 (right) recruits TRAF2 to the alternative site and while less likely it may also recruit other TRAF and non-TRAF molecules.