B-cell maturation protein, which binds the tumor necrosis factor family members BAFF and APRIL, is dispensable for humoral immune responses

Abstract

B-cell maturation protein (BCMA) is a member of the tumor necrosis factor (TNF) receptor family and is expressed in B lymphocytes. BCMA binds two TNF family members, BAFF and APRIL, that stimulate cellular proliferation. BAFF in particular has been shown to influence B-cell survival and activation, and transgenic mice overexpressing BAFF have a lupus-like autoimmune disorder. We have inactivated BCMA in the mouse germ line. BCMA(-/-) mice have normal B-cell development, and the life span of mutant B lymphocytes is comparable to that of wild-type B cells. The humoral immune responses of BCMA(-/-) mice to T-cell-independent antigens as well as high and low doses of T-cell-dependent antigens are also intact. In addition, mutant mice have normal splenic architecture, and germinal centers are formed during an ongoing immune response. These data suggest a functional redundancy of BCMA in B-cell physiology that is probably due to the presence of TACI, another TNF receptor family member that is expressed on B cells and that can also bind BAFF and APRIL.

Figures

FIG. 1

Gene inactivation of BCMA. (A) Partial restriction endonuclease map of the wild-type allele, the targeting vector, and the inactivated allele of BCMA (B, BamHI; E, EcoRI; H, HindIII; X, XhoI; pBKS, pBluescript KS). The black boxes represent exons. HindIII digestion of the genomic DNA will yield fragments of 8 and 6 kb for the wild-type and targeted alleles, respectively, as revealed by the external probe A. (B) Southern blot analysis of HindIII-digested tail DNA obtained from wild-type, BCMA+/− and BCMA−/− mice. (C) RT-PCR of splenic RNA samples obtained from wild-type and BCMA−/− mice. The 5′ RT-PCR identified the region corresponding to exon I of the gene. The housekeeping gene GADPH is included as control.

FIG. 2

B-cell populations in BCMA−/− mice, determined by flow cytometry analyses of B-cell populations found in bone marrow (A), spleens (B and C), and peritoneal cavities (D) of wild-type and BCMA−/− mice. Only IgM+ B cells are shown in panel C. Numbers indicate the percentages of cells within the lymphocyte forward and side scatter gates (A, B, and D) and percentages of IgM+ B cells for (D). The data shown are representative of more than five analyses.

FIG. 3

Distribution of various B-cell populations in spleens of BCMA−/− mice. The distribution of marginal zone (MZ), immature (Im), and follicular B cells as defined in Fig. 2C was examined in four wild-type (white boxes) and five mutant (black boxes) mice and expressed as percentage of total splenic B cells.

FIG. 4

Turnover of wild-type and BCMA-deficient B cells. Mice were continuously fed with BrdU in drinking water for a period of 1 or 3 weeks. B220+ IgM+ splenic and peritoneal cavity B cells were stained for intracellular BrdU content. Groups of four wild-type (white box) and five mutant (black box) mice were analyzed for each time point.

FIG. 5

Basal serum immunoglobulin levels in BCMA−/− mice. The concentrations of various serum immunoglobulin isotypes were measured by ELISA, and the value for each wild-type (open circles) and mutant (filled circles) mouse was plotted.

FIG. 6

BCMA−/− mice have normal T-cell-independent immune responses. Wild-type (open circles) and mutant (filled circles) mice were immunized with 10 μg of the T-cell-independent antigen NP-Ficoll. The amount of antigen-specific antibodies of the IgM and IgG3 class was measured in an ELISA 8 days after the challenge. The value for each mouse was plotted. Preimmune sera were negative for the antigen-specific antibodies and are not shown.

FIG. 7

Primary and secondary immune responses of BCMA-deficient mice to a high dose of T-cell-dependent antigen. Wild-type (open circles) and mutant (filled circles) mice were immunized with 200 μg of the alum-precipitated T-cell-dependent antigen NP-CG for the primary (10) response and reboosted at day 45 with 5 μg of the antigen for the secondary (20) immune response. Sera were collected from the mice at various time points after primary and secondary immunizations and quantified for the presence of NP-specific antibodies of the IgM and IgG1 classes. The immune sera were diluted as indicated. The value for each mouse was plotted. Preimmune sera were negative for the antigen-specific antibodies and are not shown.

FIG. 8

Primary and secondary immune responses of BCMA-deficient mice to a low dose of T-cell-dependent antigen. Wild-type (open circles) and mutant (filled circles) mice were immunized with 5 μg of alum-precipitated NP-CG and reboosted at day 45 with the same amount of the antigen. The ELISA was performed as for Fig. 7.

FIG. 9

Distribution of Syndecan-1-expressing B cells in BCMA−/− mice, determined by flow cytometry analyses of Syndecan-1-expressing B cells found in the spleens of naive mice (A) and wild-type and BCMA−/− mice 10 days post-NP-CG challenge (B). Numbers indicate percentages of total B cells present.

FIG. 10

Structures of primary follicles and germinal centers in spleens of BCMA-deficient mice. Cryosections of spleens from wild-type and mutant mice were stained with the indicated antibodies conjugated to FITC (green) or Texas red (red). Anti-IgM and anti-IgD stain B cells, anti-CD3 stains T cells, and peanut agglutinin (PNA) stains germinal center B cells.

FIG. 11

Semiquantitative RT-PCR of TACI expression in BCMA−/− mice. Total RNA was extracted from splenocytes of wild-type and BCMA−/− mice, and the reverse-transcribed cDNA was subjected to PCR for the numbers of cycles indicated. GADPH was amplified as a control for the amount of template present in the reaction.