A BAFF-R mutation associated with non-Hodgkin lymphoma alters TRAF recruitment and reveals new insights into BAFF-R signaling

Abstract

The cytokine B cell activating factor (BAFF) and its receptor, BAFF receptor (BAFF-R), modulate signaling cascades critical for B cell development and survival. We identified a novel mutation in TNFRSF13C, the gene encoding human BAFF-R, that is present in both tumor and germline tissue from a subset of patients with non-Hodgkin lymphoma. This mutation encodes a His159Tyr substitution in the cytoplasmic tail of BAFF-R adjacent to the TRAF3 binding motif. Signaling through this mutant BAFF-R results in increased NF-κB1 and NF-κB2 activity and increased immunoglobulin production compared with the wild-type (WT) BAFF-R. This correlates with increased TRAF2, TRAF3, and TRAF6 recruitment to His159Tyr BAFF-R. In addition, we document a requirement for TRAF6 in WT BAFF-R signaling. Together, these data identify a novel lymphoma-associated mutation in human BAFF-R that results in NF-κB activation and reveals TRAF6 as a necessary component of normal BAFF-R signaling.

Figure 1.

Identification of the BAFF-R His159Tyr SNP. (A) TNFRSR13C was sequenced on both sense (bottom chromatogram) and antisense (top chromatogram) strands, and a heterozygous cytosine-to-thymidine transition at position 519 (C519T) was identified. (B) A multiple sequence alignment of a sample of mammalian BAFF-R amino acid sequences, showing evolutionary conservation of the cytoplasmic portion and the location of the missense substitution of tyrosine (Y) for histidine (H) in codon 159. Symbols below the sequence alignment indicate when residues across species encode for identical (*), conserved (:), or semiconserved (.) amino acids.

Figure 2.

BAFF-R His159Tyr exhibits enhanced signaling ability. (A) HEK-293 cells expressing BAFF-R_WT, BAFF-R_H159Y, BAFF-R_AVAAA, or a vector control were stimulated with 200 or 50 ng/ml BAFF for 0, 3, or 6 h. NF-κB2 p100 processing to p52 was analyzed by immunoblotting. Levels of p52 and actin were quantified by densitometry, and combined analysis of three independent experiments is shown in the bar graph. (B) NF-κB luciferase reporter assay in HEK-293 cells expressing BAFF-R_WT, BAFF-R_H159Y, or BAFF-R_AVAAA. (C) A20.2J B cells expressing hCD40–BAFF-R_WT or hCD40–BAFF-R_H159Y chimeras were incubated with media alone (M), uninfected Hi5 cells (0), or Hi5-hCD154 cells for the indicated times. Levels of phospho-IκBα, total IκBα, and actin were examined by Western blotting and quantified by densitometry. Combined analysis of four independent experiments is shown in the bar graph. (D) CH12.LX B cells expressing hCD40–BAFF-R_WT, hCD40–BAFF-R_H159Y, or hCD40–BAFF-R_AVAAA were incubated with anti–human CD40 (G28.5) or isotype control (0). Levels of p52, RelB, and YY1 in the nuclear cell fraction were examined by Western blotting and quantified by densitometry. Combined analysis of four independent experiments is shown in the bar graph. In all analyses, error bars represent the ± SEM and statistical comparisons were made between BAFF-R_WT and BAFF-R_H159Y. *, P < 0.05.

Figure 3.

BAFF-R His159Tyr stimulates enhanced immunoglobulin production. CH12.LX cells stably expressing hCD40–BAFF-R_WT or hCD40–BAFF-R_H159Y were stimulated for 72 h with 2 µg/ml of agonistic mCD40-specific antibody, hCD40-specific antibody, or isotype control. Numbers of IgM-secreting B cells (±SEM) from three independent experiments are shown. *, P < 0.05. Pfc, plaque forming cell.

Figure 4.

The His159Tyr BAFF-R mutant recruits increased amounts of TRAF2, 3, and 6. (A) A20.2J B cells expressing hCD40–BAFF-R_WT or hCD40–BAFF-R_His159Tyr chimeras were subjected to combined stimulation and immunoprecipitation with the hCD40-specific antibody G28.5. (iso 15, 15 min with isotype control). The immunoprecipitate was probed for the presence of TRAF2, TRAF3, TRAF6, and hCD40–BAFF-R. (B) Levels of coimmunoprecipitated TRAF2, 3, and 6 were normalized to the amount of hCD40–BAFF-R immunoprecipitated. Histogram represents the mean ± SEM of four independent experiments. *, P < 0.05 (BAFF-R_H159Y vs. BAFF-R_WT). (C) HEK-293 cells expressing HA-tagged BAFF-R_WT, BAFF-R_H159Y, BAFF-R_AVAAA, or a vector control were stimulated with BAFF and subjected to immunoprecipitation using an HA-specific antibody. Associated protein or total lysates were probed for the presence of TRAF2 or TRAF6 by Western blotting.

Figure 5.

TRAF6 is recruited to BAFF-R in B cells and is required for BAFF- and BAFF-R–induced prosurvival signals. (A–C) Mouse A20.2J and A20.2J TRAF6 KO cells (A), primary mouse T cell–depleted splenocytes (B), or human Karpas B cells (C) were stimulated with BAFF, and the Brij58 insoluble (lipid raft enriched) fraction (lysate) was subjected to immunoprecipitation with a mouse or human BAFF-R–specific antibody or isotype control (iso). Immunoprecipitated sample was probed for the presence of TRAF6, 2, or 3. A and C are representative of three independent experiments and B is representative of two independent experiments. (D) A20.2J WT, A20.2J TRAF6 KO, and A20.2J TRAF6 KO cells expressing full-length TRAF6 were incubated with BAFF for the indicated times. (E) A20.2J WT and A20.2J TRAF6 KO cells expressing the hCD40-BAFFR chimera were incubated with hCD154 for the indicated times. Western blots were performed to detect phosphorylated IκBα, total IκBα, and actin. D and E are representative of three independent experiments with similar results. (F) A20.2J WT and A20.2J TRAF6 KO cells expressing the hCD40-BAFFR chimera were incubated with agonistic mCD95/Fas-specific antibody to induce apoptosis. Agonistic hCD40-specific antibody was used to activate the hCD40–BAFF-R chimera. Agonistic mouse CD40-specific antibody rescues in a TRAF6-dependent manner and was used as a control (Benson et al., 2006). Isotype controls for each of these antibodies are named isotype 1, 2, and 3, respectively. Subdiploid (apoptotic) cells were measured by flow cytometric analysis after propidium iodide staining. Data are plotted as the mean ± SEM of five independent experiments, with the mean percentage of rescue calculated as described in Materials and methods. *, P < 0.05 (percentage of rescue in WT vs. TRAF6 KO cells).