Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen

Abstract

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

Figures

Figure 1.

Generation of TACI:Fc and BCMA:Fc transgenic mice. (A) Constructs used for the generation of transgenic mouse. AAT, human α1-antitrypsin promoter; Fc, human IgG1 linker, CH2, and CH3 domains; SP, Ig heavy chain signal peptide. Intron and poly A addition sequences were from rabbit β-globin gene. (B) Genomic screen of transgenic mice. The 252-bp band amplified by PCR is specific for human α1-anti-trypsin promoter. (C) Western blot analysis of transgene expression. Serum (0.5 μl) of two independent lines of each TACI:Fc and BCMA:Fc transgenic mice and of nontransgenic controls were analyzed under nonreducing conditions and revealed with horseradish peroxidase–coupled goat anti–human IgG antibody. Recombinant proteins (6 ng of BCMA:Fc and 12 ng of TACI:Fc) in normal serum were loaded as standard. For transgenic mice, the serum concentration of the transgenic protein is indicated. Molecular weight standard are in kDa. (D) Receptor-ligand interaction ELISA. Binding of recombinant murine BAFF (black circles), murine APRIL (white circles), or control murine TNF-α (white diamonds) to immobilized recombinant or transgenic BCMA:Fc and TACI:Fc was monitored by ELISA.

Figure 1.

Generation of TACI:Fc and BCMA:Fc transgenic mice. (A) Constructs used for the generation of transgenic mouse. AAT, human α1-antitrypsin promoter; Fc, human IgG1 linker, CH2, and CH3 domains; SP, Ig heavy chain signal peptide. Intron and poly A addition sequences were from rabbit β-globin gene. (B) Genomic screen of transgenic mice. The 252-bp band amplified by PCR is specific for human α1-anti-trypsin promoter. (C) Western blot analysis of transgene expression. Serum (0.5 μl) of two independent lines of each TACI:Fc and BCMA:Fc transgenic mice and of nontransgenic controls were analyzed under nonreducing conditions and revealed with horseradish peroxidase–coupled goat anti–human IgG antibody. Recombinant proteins (6 ng of BCMA:Fc and 12 ng of TACI:Fc) in normal serum were loaded as standard. For transgenic mice, the serum concentration of the transgenic protein is indicated. Molecular weight standard are in kDa. (D) Receptor-ligand interaction ELISA. Binding of recombinant murine BAFF (black circles), murine APRIL (white circles), or control murine TNF-α (white diamonds) to immobilized recombinant or transgenic BCMA:Fc and TACI:Fc was monitored by ELISA.

Figure 1.

Generation of TACI:Fc and BCMA:Fc transgenic mice. (A) Constructs used for the generation of transgenic mouse. AAT, human α1-antitrypsin promoter; Fc, human IgG1 linker, CH2, and CH3 domains; SP, Ig heavy chain signal peptide. Intron and poly A addition sequences were from rabbit β-globin gene. (B) Genomic screen of transgenic mice. The 252-bp band amplified by PCR is specific for human α1-anti-trypsin promoter. (C) Western blot analysis of transgene expression. Serum (0.5 μl) of two independent lines of each TACI:Fc and BCMA:Fc transgenic mice and of nontransgenic controls were analyzed under nonreducing conditions and revealed with horseradish peroxidase–coupled goat anti–human IgG antibody. Recombinant proteins (6 ng of BCMA:Fc and 12 ng of TACI:Fc) in normal serum were loaded as standard. For transgenic mice, the serum concentration of the transgenic protein is indicated. Molecular weight standard are in kDa. (D) Receptor-ligand interaction ELISA. Binding of recombinant murine BAFF (black circles), murine APRIL (white circles), or control murine TNF-α (white diamonds) to immobilized recombinant or transgenic BCMA:Fc and TACI:Fc was monitored by ELISA.

Figure 1.

Generation of TACI:Fc and BCMA:Fc transgenic mice. (A) Constructs used for the generation of transgenic mouse. AAT, human α1-antitrypsin promoter; Fc, human IgG1 linker, CH2, and CH3 domains; SP, Ig heavy chain signal peptide. Intron and poly A addition sequences were from rabbit β-globin gene. (B) Genomic screen of transgenic mice. The 252-bp band amplified by PCR is specific for human α1-anti-trypsin promoter. (C) Western blot analysis of transgene expression. Serum (0.5 μl) of two independent lines of each TACI:Fc and BCMA:Fc transgenic mice and of nontransgenic controls were analyzed under nonreducing conditions and revealed with horseradish peroxidase–coupled goat anti–human IgG antibody. Recombinant proteins (6 ng of BCMA:Fc and 12 ng of TACI:Fc) in normal serum were loaded as standard. For transgenic mice, the serum concentration of the transgenic protein is indicated. Molecular weight standard are in kDa. (D) Receptor-ligand interaction ELISA. Binding of recombinant murine BAFF (black circles), murine APRIL (white circles), or control murine TNF-α (white diamonds) to immobilized recombinant or transgenic BCMA:Fc and TACI:Fc was monitored by ELISA.

Figure 2.

FACS® analysis of lymphoid organs of TACI:Fc and BCMA:Fc transgenic mice. The indicated percentages refer to gated lymphocytes. (A) Bone marrow. Lymphocytes expressing the B cell lineage marker B220 were separated according to CD43 expression and further analyzed with CD24 and BP-1 (for CD43+ cells) and IgM and IgD (for CD43− cells). Populations A (pre-pro-B), B (pro-B), C (pro-B/PreB), D (pre-B), E (transitional B), and F (recirculating mature B) are labeled according to Hardy's nomenclature (reference 29). (B) Spleen. Analysis of splenic B (B220+) and T (CD3+) cell populations, and four color FACS® analysis of splenic B cell populations based on B220, CD23, CD21, and surface IgM expression (references 2 and 18). FO, follicular B cells; MZ, marginal zone B cells; T1, transitional T1 B cells; T2, transitional T2 B cells. (C) Inguinal lymph nodes. Analysis of B cell populations based on B220 and IgD. (D) Peripheral blood lymphocytes. Analysis of B cell populations based on the expression of B220, CD62L (L-selectin), and CD21. (E) Peritoneal exudate lymphocytes. Peritoneal B lymphocytes are classified into B1 and B2 cells based on the expression level of CD23, and further analyzed for CD5 and IgM expression. B1a, CD5+ B1 B cells; B1b, CD5− B1 B cells. (F) Thymus. Thymocytes are analyzed based on the expression of CD4, CD8, TCR-αβ, and TCR-γδ. ISP, immature CD8 single positive precursor T cells; NKT, NK T cells.

Figure 3.

Immunohistochemistry of frozen spleen, lymph node, and Peyer's patches sections. Spleen. Serial sections were double stained with B cells (anti-B220, brown), T cells (anti-CD3ε, purple), follicular dendritic cells (anti-CD35/Cr1, purple), macrophages (CD11b, brown) and dendritic cells (CD11c, purple) markers, as indicated. Inguinal lymph nodes and Peyer's patches were doubly stained for B and T cells. Peyer's patches were present in normal number in TACI:Fc mice, but had a smaller size. Bars = 100 μm.

Figure 4.

Reduced Ig levels in TACI:Fc mice. Ig levels were determined in normal serum of TACI:Fc transgenic mice and matched control littermates. P values: *0.2 > P ≥ 0.05; **0.05 > P ≥ 0.01; ***P < 0.01.