Lymphocyte-specific TRAF3 transgenic mice have enhanced humoral responses and develop plasmacytosis, autoimmunity, inflammation, and cancer

Abstract

Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) regulates both innate and adaptive immunity by modulating signaling by Toll-like receptors (TLR) and TNF receptors. TRAF3 was recently identified as a tumor suppressor in human multiple myeloma, suggesting a prominent role in plasma cell homeostasis. We have generated transgenic mice expressing human TRAF3 in lymphocytes. These mice are normal at birth, but they develop over time plasmacytosis and hypergammaglobulinemia, as well as systemic inflammation and tertiary lymphoid organ formation. The analysis of the humoral responses of the TRAF3 mice demonstrated increased responses to T-dependent and T-independent antigens with increased production of antigen-specific immunoglobulin Gs (IgGs) compared with wild-type mice. Furthermore, TLR-mediated IgG production is also increased in TRAF3 B cells. In addition, TRAF3 mice develop autoimmunity and are predisposed to cancer, particularly squamous cell carcinomas of the tongue ( approximately 50% incidence) and salivary gland tumors. In summary, TRAF3 renders B cells hyperreactive to antigens and TLR agonists, promoting autoimmunity, inflammation, and cancer, hereby providing a new model for studying de novo carcinogenesis promoted by B cell-initiated chronic inflammation.

Figure 1

Generation of TRAF3 transgenic mice. (A) Representation of the TRAF3 transgenic constructs. The various components of transgenic cassette are indicated. (B) Immunoblot analysis of the hTRAF3 transgene expression in various tissues from the TRAF3 transgenic mice (ϵ-line) and wild-type littermates. hTRAF3 expression was detected using an antibody that specifically recognizes human TRAF3, but not mouse TRAF3. (C) Analysis of TRAF3 and IκBα expression in total splenocytes and in affinity-purified B lymphocytes and T lymphocytes isolated from TRAF3 transgenic mice and wild-type littermates. The TRAF3 antibody used recognizes only human TRAF3. The purity of B cells and T cells was greater than 95%. (D) Purified B cells from spleens of the various transgenic mice indicated and wild-type littermates were lysed in Laemmli buffer and sonicated. Lysates were normalized for protein content, and 25 μg were immunoblotted. Total TRAF3 and IκBα levels are shown. The TRAF3 antibody used (C-20) recognizes both human and mouse TRAF3. (E) B cells from wild-type mice were left resting or stimulated with 2 μg/mL LPS for 16 or 72 hours. TRAF3 and Bcl-2 expression were analyzed by immunoblotting. (F) Kaplan-Meier analysis of the lifespan of wild-type and TRAF3 littermates (17 mice per group). Statistical significance was calculated by log-rank analysis. (G) The weight of spleens from mice up to 8 months (TRAF3, n = 7; wild-type, n = 5) or more than 16 months old (TRAF3, n = 27; wild-type, n = 19) was determined. Statistical significance was determined by unpaired Student t test.

Figure 2

TRAF3 mice develop plasmacytosis. (A) Phenotypic analysis of plasma cells. Submaxillary lymph nodes (LN) and spleen lymphoid populations were analyzed by 3-color flow cytometry in a representative 14-month-old TRAF3 transgenic mouse (TRAF3) and wild-type littermate (WT). Total nucleated cells were stained for B220, Syndecan-1 and IgM surface expression and analyzed with no gate (top) or selecting the IgM null cells (bottom). Numbers indicate the percentage of cells in that gate. (B) Analysis of plasma cells in lymph nodes. Immunohistochemical analysis of a representative lymph node from a diseased TRAF3 mouse showing severe plasmacytosis. Plasma cells were identified by cytosolic Ig staining. Bars, 100 μm. (C) Plasma cell infiltrates in tissues from TRAF3 mice. Immunohistochemical analysis of the tongue (left) and pancreas (right) of a representative diseased TRAF3 mouse (top and middle panels) and a wild-type littermate (bottom panels). Left, H&E (top and bottom) or anti-IgG (middle) staining of sections of the tongue. Right, H&E staining of infiltrating plasma cell in the pancreas. Bars, 50 μm. (D) Analysis of the expression XBP-1 and IgG in purified B cells. B-cell extracts (20 μg) were analyzed by SDS-PAGE and immunoblotting. (E) TRAF3 mice develop hypergammaglobulinemia. Serum IgG_2a, IgG_2b, and IgM concentrations from TRAF3 mice (ϵ-line) and wild-type littermates (10-15 months old) were determined using isotype-specific ELISA. P values are indicated in the figure. Statistical significance was determined by unpaired Student t test.

Figure 3

TRAF3 transgenic mice develop systemic inflammation. (A) Histologic analysis. H&E staining shows lymphoid neogenesis (tertiary lymphoid organ [TLO] formation) in omentum and pancreatic parenchyma partly replaced by inflamed adipose tissue in TRAF3 mouse. (B) B cells in TLO were stained with B220 mAb. (C) T cells in TLO were stained with anti-CD3 monoclonal antibody (mAb). (D) H&E staining of an inflammatory cuff surrounding renal artery with thickened tunica media. (E) H&E staining of a joint from a representative TRAF3 mouse with arthritis. Note the distended joint spaces, pannus formation, proliferation of synoviocytes, the irregular proliferation of cartilage at the junction of cartilage bone, and synovium or cartilage erosion. Bars in panels A through E represent 100 μm. (F) Analysis of proinflammatory proteins in serum. Sera from TRAF3 transgenic mice and wild-type littermates (10-15 months old) were analyzed to determine the concentrations of inflammatory cytokines and CRP. Statistical significance was determined by unpaired Student t test. P values are indicated in figure. Hash marks indicate means.

Figure 4

TRAF3 mice develop autoimmunity. (A) Analysis of circulating autoantibodies in TRAF3 mice and wild-type littermates. Blood from TRAF3 mice (12-17 months old) and from wild-type littermates was extracted for serum collection. The presence of autoantibodies in serum was determined by ELISA (anti-dsDNA, 1:150 dilution; anti-ANA, anti-ssDNA, and anti-SSA/Ro, 1:50 dilution). Statistical significance was determined by unpaired Student t test. (B) Pathologic alterations in kidneys from TRAF3 mice. Morphologic lesions encompass both glomerular (i-xi) and tubulointerstitial (i,iii) alterations, demonstrated by H&E (i-iii), Jones silver methanamine (iv,v), and Masson trichrome (vi,vii) stains. Depositions of IgG (viii,ix) and C3 complement (x,xi) were detected by immunostaining. Bars, 50 μm.

Figure 5

TRAF3 mice exhibit enhanced humoral responses to TD and TI antigens. (A) Mice were challenged with TNP-BSA (T-dependent antigen) in complete Freund adjuvant or with TNP-LPS (T-independent antigen). Blood was collected before and after injection of TRAF3 mice (◇) and wild-type littermates (■) with antigens at the days indicated. Titers of anti-TNP IgM and anti-TNP IgG₂ were determined by ELISA using TNP-KLH–coated plates. Data represent mean (± standard error of the mean [SEM]; n = 4). (B) B-cell antibody responses to TLR agonists. B cells were cultured in 12-well plates (2 × 10⁵ cells/mL) and either untreated or treated with 2 μg/mL LPS or 1 μM CpG (ODN1826). Culture supernatants were collected at various days, and secreted IgM, IgG₁, and IgG_2a were quantified. Data represent mean (± SEM) for TRAF3 mice (n = 5, ) and wild-type littermates (n = 4, ■).

Figure 6

Cancer development in TRAF3 transgenic mice. (A) Percentage of tumors found in wild-type mice (n = 24; 17 ± 1.5 months) and in TRAF3 mice (n = 70; 18 ± 0.8 months). (B) Graphic representation of percentage of mice bearing tumors in the tongue (squamous cell carcinoma, n = 19; papilloma, n = 2), other oral tumors (pleomorphic adenomas, cystadenomas, and adenocarcinomas), lung carcinomas (papillary bronchioloalveolar carcinoma, n = 15; adenoma, n = 2), and hepatocarcinoma (n = 7). (C) Comparative analysis of tongues from 2-month-old (bottom) and 14-month-old (top) TRAF3 mice and wild-type littermates. The arrow indicates the squamous cell carcinoma in the tongue. (D,E) Squamous cell carcinoma of the tongue (H&E). (F,G) Example of lymphoid infiltration in submaxillary gland and parotid gland, respectively, in TRAF3 mice. (H,I) H&E staining of parotid glands with pleomorphic adenoma and sarcoma, respectively. (J) Papillary bronchioloalveolar carcinoma of lung in a TRAF3 mouse. (K) H&E staining of hepatocellular carcinoma in TRAF3 mouse. Note the presence of subcapsular fat necrosis with chronic inflammatory cells. Bars (D-K), 100 μm.