APRIL-deficient mice have normal immune system development

Abstract

APRIL (a proliferation-inducing ligand) is a member of the tumor necrosis factor (TNF) superfamily. APRIL mRNA shows high levels of expression in tumors of different origin and a low level of expression in normal cells. APRIL shares two TNF receptor family members, TACI and BCMA, with another TNF homolog, BLyS/BAFF. BLyS is involved in regulation of B-cell activation and survival and also binds to a third receptor, BR3/BAFF-R, which is not shared with APRIL. Recombinant APRIL and BLyS induce accumulation of B cells in mice, while BLyS deficiency results in severe B-cell dysfunction. To investigate the physiological role of APRIL, we generated mice that are deficient in its encoding gene. APRIL(-/-) mice were viable and fertile and lacked any gross abnormality. Detailed histological analysis did not reveal any defects in major tissues and organs, including the primary and secondary immune organs. T- and B-cell development and in vitro function were normal as well, as were T-cell-dependent and -independent in vivo humoral responses to antigenic challenge. These data indicate that APRIL is dispensable in the mouse for proper development. Thus, BLyS may be capable of fulfilling APRIL's main functions.

FIG. 1.

Targeting of the mouse APRIL gene. (A) Structure of the mouse APRIL genomic locus. Boxes correspond to the genomic regions containing the TWEAK (white bars), APRIL (black bars), and SMT3IP1 (grey bars) genes. The orientations of the three genes are marked by arrows. (B) Schematic representation of the targeting construct designed to replace part of the first exon and all downstream exons of the APRIL gene with a neo cassette. (C) Structure of the mutated region in the APRIL gene. The positions of the 5′ and 3′ external probes used for Southern blot analysis of ES cell are indicated by bars. The positions of the primer sets used for genotype analysis of mouse tail DNA are indicated by black (external) and grey (internal) arrowheads. (D) Southern blot analysis of recombination of the APRIL gene. Analysis of BsmI (DI) and SpeI (DII) digested DNA derived from several ES cell clones. DNA was digested and fractionated on a 0.7% agarose gel, blotted onto a nylon membrane, and hybridized with 5′ (DI) and 3′ (DII) probes. (E) Genotyping of APRIL^−/− mice by PCR. Tail-derived genomic DNA was subjected to PCR amplification with nested external and internal sets of primers to visualize wild-type and deletion mutant APRIL genes as 3.2-kb and 3.0-kb fragments, respectively. (F) Expression of APRIL in total splenocytes derived from APRIL^+/+ and APRIL^−/− mice was determined by fluorescence-activated cell sorting with anti-mouse APRIL monoclonal antibody (black) or an isotype-matched irrelevant antibody control (grey line and filled area). (G) Quantitative real-time PCR analysis of APRIL (black bars), TWEAK (white bars), and SMT3IP (grey bars) mRNA expression in spleens of APRIL^+/+ (WT), APRIL^+/− (HET), and APRIL^−/− (KO) mice. All values were normalized to an RPL19 RNA internal control. Standard deviations were calculated from triplicate reactions.

FIG. 2.

APRIL^−/− mice have normal lymphoid organ structure. Histologic analysis of APRIL^−/− mice. Sections from spleens (A) and mesenteric lymph nodes (B) from APRIL^+/+ mice (left panel) and APRIL^−/− mice (right panel) were stained with hematoxylin and eosin. Stained sections were photographed under a light microscope at 40×.

FIG. 3.

APRIL^−/− mice have normal T-cell development. Thymic T-cell populations in APRIL^+/+ and APRIL^−/− mice were determined by fluorescence-activated cell sorting. Numbers indicate the percentages of cells within the lymphocyte forward and side scatter gates. The data are representative of at least four analyses.

FIG. 4.

T and B cells from APRIL^−/− mice have normal responses to different types of stimulation in vitro. (A) Total splenic T cells from APRIL^+/+ (♦) or APRIL^−/− mice (▪) were cultured in the presence of monoclonal antibody to CD3 (a) or concanavalin A (ConA) (b). Proliferation was measured by [³H]thymidine uptake on day 3. (B) Positively (a and b) and negatively (c) selected B cells from APRIL^+/+ and from APRIL^−/− mice were cultured in the presence of lipopolysaccharide (LPS) (a) or monoclonal antibody to CD40 plus interleukin-4 (IL4) (b and c). Proliferation was measured by [³H]thymidine uptake on day 5 (a and b) or day 3 (c). (C) B cells from APRIL^−/− mice produce normal amounts of immunoglobulins upon in vitro stimulation. Purified B cells from APRIL^+/+ and APRIL^−/− mice were cultured in the presence of lipopolysaccharide (a) or monoclonal antibody to CD40 plus interleukin-4 (a and b). Five days later, culture supernatants were examined for the presence of IgG1 (a and b). Data are means ± standard deviation of four mice in each group.

FIG. 5.

APRIL^−/− mice have normal T-cell responses to KLH. (A) APRIL^+/+ mice (⧫) and APRIL^−/− mice (▪) were immunized with KLH in complete Freund's adjuvant. After 5 days, dendritic lymph nodes were collected and restimulated in vitro with KLH at the indicated concentrations. Proliferation was measured by [³H]thymidine uptake on day 5. (B to D) Cytokine secretion was measured in KLH-activated T cells by ELISA.

FIG. 6.

B cells from APRIL^−/− mice have normal humoral responses to different types of stimulation in vivo. (A) Basal serum titers for various immunoglobulin isotypes were measured from age-matched APRIL^+/+ (▪) and APRIL^−/− mice (□) determined by ELISA. (B-D) Mice were immunized with NP₂₃-chicken gamma globulin (a thymus-dependent antigen) or trinitrophenyl (TNP)-KLH (a type 2 T-cell-independent antigen). Antinitrophenyl serum titers in NP₂₃-chicken gamma globulin-immunized and antitrinitrophenyl serum titers in TNP-KLH-immunized mice were measured 10 days after challenge. ELISA was used to measure the indicated immunoglobulin isotypes with high affinity to antigen (B) and total immunoglobulin (C) in anti-NP₂₃-chicken gamma globulin-immunized mice and in anti-trinitrophenyl-KLH-immunized mice (D). White bars, APRIL^+/+ mice; black bars, APRIL^−/− mice. Data are means ± standard deviations of four mice in each group.

FIG. 7.

Thymus-independent antigen-specific plasmablast differentiation in APRIL^−/− mice is normal. APRIL^+/+ mice (▪) and APRIL^−/− mice (□) were injected intravenously with 10⁸ (high dose) or 2 × 10⁷ (low dose) heat-inactivated Streptococcus pneumoniae bacteria or phosphate-buffered saline. After 3 days, splenocytes were collected and analyzed by fluorescence-activated cell sorting for phosphorylcholine-positive (PC⁺) CD138⁺ plasmablasts.