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.

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.

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.

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.