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. 2004 Mar 16;101(11):3903-8.
doi: 10.1073/pnas.0307348101. Epub 2004 Feb 26.

Impaired IgA class switching in APRIL-deficient mice (VSports手机版)

Emanuela Castigli  1 , Sumi Scott, Fatma Dedeoglu, Paul Bryce, Haifa Jabara, Atul K Bhan, Emiko Mizoguchi, Raif S Geha
Affiliations

V体育ios版 - Impaired IgA class switching in APRIL-deficient mice

"VSports在线直播" Emanuela Castigli et al. Proc Natl Acad Sci U S A. .

Abstract

The tumor necrosis factor (TNF) family member APRIL binds to the receptors BCMA on B cells and TACI on B and T cells VSports手机版. To investigate the role of APRIL in immunity, we generated APRIL-deficient mice. APRIL(-/-) mice have normal T and B lymphocyte development, normal T and B cell proliferation in vitro, but increased numbers of CD44(hi)CD62L(lo) CD4(+) effector/memory T cells and increased IgG responses to T-dependent antigens. Serum IgA levels were significantly decreased, and serum IgA antibody responses to mucosal immunization with TD antigens and to type 1 T-independent antigens were impaired in APRIL(-/-) mice. APRIL by itself induced IgA as well as IgG1 isotype switching in CD40-deficient IgM(+)IgD(+) sorted B cells. These results suggest that APRIL down-regulates T cell-dependent antibody responses and promotes IgA class switching. .

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VSports app下载 - Figures

Fig. 1.
Fig. 1.
Targeting the APRIL locus. (A) Partial restriction map of the APRIL gene (Top), APRIL targeting construct (Middle), and mutated APRIL allele after homologous recombination (Bottom). Exons are represented as black boxes. NEO, neomycin resistance gene; TK, thymidine kinase gene. A 0.65-kb fragment at the 3′ end of the gene was used as external probe. (B) Southern blot analysis of genomic DNA from untransfected (+/+) ES cells and heterozygous (+/-) clone (Left) and from tails of WT (+/+), heterozygous (+/-), and homozygous (-/-) APRIL mutant mice (Right). Ten micrograms of genomic DNA were digested with BamHI and hybridized with the 3′ external probe. The upper band (13.5 kb) indicates the WT allele, and the lower band (11.5 kb) corresponds to the targeted allele. (C) RT-PCR analysis of APRIL mRNA expression in spleen and bone marrow of WT (+/+), heterozygous, (+/-), and homozygous (-/-) APRIL mutant mice. RT-PCR of β2-microglobulin (β2m) was used as control. (D) RT-PCR of TWEAK and Smt3ip-1 mRNA expression in spleens of WT (+/+) and APRIL mutant (-/-) mice.
Fig. 2.
Fig. 2.
Antibody response, affinity maturation, and germinal center formation in response to TD antigen in APRIL-/- mice. (A) IgM, IgG subclasses, and IgA antibody responses to NP after immunization with the TD antigen NP28-CGG in APRIL-/- mice (KO, n = 5, blue curve; n = 4 for IgA) and controls (WT, n = 5, red curve; n = 4 for IgA). Statistical analysis was performed by using two-way ANOVA; ns, not significant. (B) Affinity of NP-specific IgG1 antibodies measured as ratio of the OD at 405 nm of the same serum dilution tested on NP4-BSA- and NP25-BSA-coated plates. The dilutions used were in the linear part of the titration curve. (C) Germinal centers in representative spleen sections (×40) from mice immunized with NP28-CGG examined for PNA binding by immunohistochemistry.
Fig. 3.
Fig. 3.
Serum Ig in APRIL-/- mice. Serum Ig levels from 10- to 12-week-old nonimmunized APRIL-/- mice (n = 15 for IgA and IgE; n = 13 for the other isotypes) and WT littermates (n = 8 for IgA and IgE; n = 7 for the other isotypes). Bars represent the mean. Mann-Whitney test was used for statistical analysis (ns, not significant).
Fig. 4.
Fig. 4.
IgA antibody responses to mucosal immunization and to TI antigen in APRIL-/- mice. (A) IgM, IgG, and IgA antibody responses to CGG of APRIL-/- mice, KO (n = 5) and WT littermates (n = 5) after mucosal (i.g. plus i.n.) immunization. Statistical analysis was performed by using two-way ANOVA. (B) Frozen sections of the small intestine (×20) from APRIL-/- (KO, Right) and WT mice (Left) stained for IgA by the immunoperoxidase method. The frozen sections shown are representative of four APRIL-/- or four WT control mice analyzed. (C and D) IgM, IgG, and IgA antibody responses to NP after i.p. immunization with the TI-1 (NP-LPS, seven WT mice and seven APRIL-/- mice) (C) or TI-2 (NP-Ficoll, five WT mice and six APRIL-/- mice) antigens (D). Statistical analysis was performed by using two-way ANOVA.
Fig. 5.
Fig. 5.
APRIL and IgA class switching in murine B cells. (A) In vitro secretion of IgA, IgG1, and IgE by IgM+IgD+ B cells derived from CD40-deficient mice. Cells were stimulated for 6 days with APRIL, BAFF, LPS+TGFβ as control for IgA synthesis, and LPS+IL-4 as control for IgG1 and IgE synthesis. Similar results were obtained in two other experiments. (B) Expression of α, γ1 and εGLT, Iμ-Cα Iμ-Cγ1 and Iμ-Cε mature pST and AID transcripts measured by RT-PCR in B cells stimulated for 4 days with IL-4, APRIL, BAFF, and LPS plus TGFβ or LPS plus IL-4. RT-PCR for β2-microglobulin (β2-m) was used as control. (C) Sμ → Sα,Sμ → Sγ1, and Sμ → Sε deletional switch recombination measured by DC-PCR at day 6. DC-PCR for the acetylcholine receptor (nAchR) was used as control.
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