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Blood, Vol. 113, Issue 19, 4595-4603, May 7, 2009
Lymphocyte-specific TRAF3 transgenic mice have enhanced humoral responses and develop plasmacytosis, autoimmunity, inflammation, and cancer
Blood Zapata et al.
113: 4595
Supplemental materials for: Zapata et al
Files in this Data Supplement:
- Figure S1 (JPG, 29.5 KB)
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(A) Analysis of TRAF3 transgene expression in transgenic mice. Immunoblot analysis of the hTRAF3 transgene expression in spleen from three TRAF3 transgenic lines (TRAF3 α, ε, and µ) (+) and wild-type littermates (−). hTRAF3 expression was detected using an antibody that specifically recognizes human TRAF3, but not mouse TRAF3.16 (B) Histological analysis of tissues from TRAF3 mice. Tissues from wild-type mice (n=20) and the three TRAF3 lines (TRAF3α (n=13), TRAF3ε (n=48) and TRAF3 µ (n=3)) were fixed in Z-fix solution and embedded in paraffin. Tissue sections were stained with H&E and evaluated for evidence of plasmacytosis or plasmadyscrasias and inflammation. The percentage of animals displaying such pathology is indicated.
- Figure S2. Macroscopic assessment of phenotype of TRAF3 mice (JPG, 97.5 KB)
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(A) Photograph showing a comparison between a cachectic TRAF3 female mouse (top) and an age-matched wild-type female littermate (ε-line). (B) Gross analysis of abdominal cavity of the mice shown in panel (A) Note the absence of abdominal fat in the TRAF3 mouse. (C) Analysis of the weights of wild-type and TRAF3 female (wt, n=9; TRAF3, n=11) and male (wt, n=10; TRAF3, n=11) mice between 12 and 15 month old. Statistical significance was determined by unpaired t-test. (D) Graphic representation of the weight variation over a period of 10 months of wild-type and TRAF3 littermates. Only data from female mice were included in the figure, although TRAF3 male mice also suffer from weight loss (E) Comparative analysis of spleens from 2 months and 14 month old TRAF3 and wild-type mice. The 14 month old TRAF3 mouse was cachexic. (F) Analysis of submaxillary gland lymph nodes and spleens from a cachexic 13 month old TRAF3 mouse and a wild-type littermate. Note the enlarged LN and reduced spleen of the TRAF3 mouse.
- Figure S3. Analysis of B-cell and T-cell populations from lymphoid organs isolated from TRAF3 transgenic mice and wild-type littermates (JPG, 102 KB)
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(A) Single cell suspensions were prepared from spleens and lymph nodes (LN) from a 14 month old TRAF3 (ε-line) mouse suffering from weight loss (bottom) or from a wild-type littermate (top). Mononuclear cells were blocked with human γ globulin, and stained anti-B220 mAb and anti-CD3 mAb. FACS profiles of gated lymphocytes are shown in the figure. The percentage of lymphocytes in each quadrant is indicated. (B) Lymphocytes from the spleens of the mice used in A were stained with anti-B220 mAb, anti-IgD and anti-IgM (left) or with anti-CD3 mAb, anti-CD4 and anti-CD8 (right). The IgM/IgD profile of the B220 positive population (left) and the CD4/CD8 profile of the CD3 positive population (right) are shown. (C) Total number of splenocytes from TRAF3 transgenic (n=7) and wild-type mice (n=7) were enumerated. The percentage of B cells (B220+) and T cells (CD3+) in each sample were determined by FACS analysis and the total number of B cells and T cells was determined. Data represent mean ± SEM. Statistical significance was determined by unpaired t-test.
- Figure S4. TRAF3 overexpression does not alter B-cell proliferation (JPG, 97.6 KB)
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For these experiments, mouse spleens were carefully crushed to release the lymphocytes. Cell suspensions were depleted of red cells and neutrophils by density centrifugation (Lympholite-M, Cedarlane laboratories, Hornby, Ontario). B cells were purified using the murine B-cell enrichment cocktail from Stem Cells technologies (Vancouver, Canada). (A) Effect of TLR-agonists on B-cell proliferation. Purified B-cells (5 × 104) from TRAF3 transgenic mice (n=4) and wild-type littermates (n=4) were incubated with Pam3CSK4 (1 µg/ml; TLR1/2 agonist), HKLM (108 cells/ml; TLR2 agonist), Poly(I:C) (25 µg/ml; TLR3 agonist), LPS (1 µg/ml; TLR4 agonist), Flagelin (2.5 µg/ml; TLR5 agonist), FLS1 (1 µg/ml; TLR2/6 agonist), ssRNA40 (2.5 µg/ml; mouse TLR7 agonist), ODN1826 (CpG, 2 µM; TLR9 agonist) (all from Invivogen, San Diego, CA) or 10 or 25 µg/ml of anti-µ mAb (F(ab)′2 fragment; Jackson Immunoresearch Lab. Inc. West Grove, PA) for 62h at 37°C Then, 3H-Thymidine (0.5 µCi/well) was added and 10 hours later cells were harvested and 3H-thymidine incorporation into DNA was measured. No statistically significant differences were detected for TRAF3 versus WT mice (mean ± SEM; n=3). (B) Effects of IL-4 and CD40L on B-cell proliferation. Purified B cells (5 × 104) from TRAF3 transgenic mice and wild-type littermates (control) were incubated with IL-4 alone (4 ng/ml), CD40L alone (1 µg/ml) or a mixture of IL-4 (4 ng/ml) plus 100 ng/ml or 1 µg/ml of CD40L for 62 h. 3H-thymidine incorporation into DNA was subsequently measured as described above. Trimeric CD40L was kindly provided by Immunex Corporation (Thousand Oaks, CA). Data are mean ± SEM (n=3). (C) Effect of BAFF on B-cell proliferation. Purified B cells (n=3 for each genotype) were incubated with 100 ng/ml of purified BAFF (R&D systems, Minneapolis, MN) for 68 h. Then 3H-thymidine incorporation into DNA was measured as described above. Data represent fold induction of DNA synthesis relative to unstimulated cells. (D) Effect of BCR and BAFF on proliferation of B cells. B cells were cultured with or without anti–B-cell Receptor (BCR) reagent (1 µg/ml anti-µ mAb, F(ab)′2 fragment; Jackson Immunoresearch Lab. Inc. West Grove, PA) alone or in combination with 100 ng/ml of mouse BAFF (R&D systems). DNA incorporation was measured and data presented as fold-increase relative to unstimulated cells.
- Figure S5. CD40-mediated signaling events in TRAF3 mice (JPG, 64.7 KB)
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(A) TRAF3 transgenic B cells have reduced Nik but most CD40-induced signal transduction events are intact. Purified B cells (5 × 106) from a pair of age-matched, sex matched wild-type and TRAF3 transgenic littermates were left untreated or treated with 1 µg/ml CD40L (R&D systems) for the indicated times (t). Cell lysates were prepared (10 µg total protein) and analyzed by SDS-PAGE/immunoblotting using various antibodies specific for TRAF3, TRAF2, IκBα and RelA (Santa Cruz Biotech. Santa Cruz, CA), JNK, phospho-JNK, ERK, phospho-ERK, p38, phospho-p38, p100/p52 NFκB2, Nik (Cell Signaling Technology, Danvers, MA), Tubulin (Sigma, San Louis, MO), and c-Rel (R&D Systems). Antibodies were detected by an ECL method. (B) Quantification of Nik protein levels in B cells before and after CD40 activation. B cells were isolated from the spleens of control and TRAF3 transgenic mice (two mice each) and were left untreated or activated with CD40L as described above. Cell lysates were prepared and 10 µg total protein were analyzed by SDS-PAGE/immunoblotting. The Kodak Digital Science 1D (v 3.0) was used for densitometry.
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