TNF receptor-associated factor (TRAF) domain and Bcl-2 cooperate to induce small B cell lymphoma/chronic lymphocytic leukemia in transgenic mice

Abstract

Transgenic mice overexpressing in B lymphocytes either Bcl-2 or a TNF receptor-associated factor (TRAF)2 mutant lacking the N-terminal RING and zinc finger domains located at the N terminus of the molecule (TRAF2DN), which mimics TRAF1, developed lymphadenopathy and splenomegaly due to polyclonal B cell expansion. Remarkably, TRAF2DN/Bcl-2 double-transgenic mice contained B cell populations similar to those observed in TRAF2DN mice. However, over time, they developed severe splenomegaly and lymphadenopathy, and most animals also developed leukemia, pleural effusion, and, in some cases, ascites associated with monoclonal and oligoclonal B cell neoplasms. The life span of TRAF2DN/Bcl-2 mice was markedly reduced compared with Bcl-2 and TRAF2DN single-transgenics or wild-type littermates. The expanded B cell population of TRAF2DN/Bcl-2 double-transgenic mice was primarily comprised of small/medium-size noncycling B220(M)/IgM(H)/IgD(L)/CD21(L)/CD23(NULL)/CD11b(+)/CD5+ cells that were Bcl-6-negative, consistent with a B-1 phenotype. The cells also expressed high levels of CD54 and other adhesion molecules. In vitro, these B cells showed comparable proliferation rates to those of wild-type counterparts but exhibited markedly increased survival and were resistant to apoptosis induced by chemotherapeutic agents and glucocorticoids. Histopathologic features were consistent with mouse small B cell lymphoma progressing to leukemia with many similarities to human chronic lymphocytic leukemia. Given that many human chronic lymphocytic leukemias overexpress TRAF1 and Bcl-2, our findings suggest that cooperation between Bcl-2 and TRAF pathways contributes to the development of this type of leukemia.

Fig. 1.

Characterization of transgenic mice. (A) Schematic representation of the Bcl-2 (Upper) and TRAF2DN (Lower) transgenic constructs. The Bcl-2 transgene is of human origin and mimics the t(14, 18)(q32-q21) chromosomal translocation found in most non-Hodgkin's B cell lymphomas by fusing a Bcl-2 minigene to a 14;18 breakpoint and adjacent IgH enhancer (12). TRAF2DN transgenic mice express a murine TRAF2 mutant lacking the RING finger domain and the four N-terminal zinc finger domains under the control of an H-2k promoter and an IgH enhancer that drives the transgene expression only in lymphocytes (14). (B) Immunoblot analysis was performed by using lysates from splenocytes isolated from age-matching mice having different genotypes, as indicated. Samples were normalized for protein content and blotted with Abs recognizing mouse TRAF2, human Bcl-2, mouse Bcl-2, lung Krüppel-like factor, and T cell lymphoma-1. +/+, TRAF2DN/Bcl-2 double-transgenic mice. (C) Spleen weights are compared from euthanized mice (gray columns) or from mice that died from disease or old age (black columns). The average weight ± SE of spleens from the different transgenic mice was calculated. WT, 138 ± 13.9 mg, n = 10; Bcl-2, 205 ± 6.3 mg, n = 9; TRAF2DN, 496 ± 37.7 mg, n = 10; TRAF2DN/Bcl-2, 647 ± 54 mg, n = 7; TRAF2DN/Bcl-2 dead from disease or old age, 1,247 ± 180 mg, n = 16. *, P < 0.05 when TRAF2DN/Bcl-2 (dead by natural causes) are compared with euthanized TRAF2DN/Bcl-2 littermates, and P < 0.0001 compared with age-matched wild-type mice by unpaired t test. (D) Representative examples of spleens and submaxilary lymph nodes from age-matched (12–13 months) transgenic mice. WT, wild-type. The spleen of a TRAF2DN/Bcl-2 double-transgenic mouse dead from disease is shown. (E) Kaplan–Meier analysis of survival of wild-type, Bcl-2 single-, TRAF2DN single-, and TRAF2DN/Bcl-2 double-transgenic mice (n = 14 per genotype). The proportion of animals remaining alive over time is plotted. Survival analysis was performed by using the nonparametric model of Kaplan–Meier, and statistical significance was determined by using the log-rank test.

Fig. 2.

Histochemical analysis of tissues from the TRAF2DN/Bcl-2 double-transgenic mice. Immunohistochemical analysis of organs and tissues from the TRAF2DN/Bcl-2 double-transgenic mice was performed. Spleen (A), prostate (B), lung (C and D), epididymis (F and G), submaxillary gland lymph node (H), ascites (I), and bone marrow (J). Hematoxylin/eosin staining is shown in A–C, E, and G. Staining with anti-B220 mAb is shown in D, F, and I to illustrate that the infiltrating cells are predominately B lymphocytes. Wright–Giemsa staining of ascites cells is shown in H.

Fig. 3.

Analysis of B lymphocyte populations in wild-type vs. transgenic mice. Four-color flow-cytometry analysis was performed to determine the phenotype of B lymphocytes. Gating was performed on the B220⁺ population (R1 and R2, Left) in the case of CD23/CD21 and IgM/IgD analyses or on the lymphocyte population for B220/CD5 and isotype control analyses. Splenocytes were analyzed from representative 11-month-old wild-type, Bcl-2, TRAF2DN, and TRAF2DN/Bcl-2 double-transgenic mice at a premalignant stage and splenocytes or blood lymphocytes of a representative TRAF2DN/Bcl-2 mouse (12 months old) with acute disease.

Fig. 4.

Increased levels of adhesion molecules on the surface of B lymphocytes from TRAF2DN and TRAF2DN/Bcl-2 transgenic mice. (A) Surface expression of intercellular adhesion molecule-1 (CD54) and β1-integrin (CD29) was measured on the B220-positive B lymphocytes by flow cytometry. FITC anti-CD29 and PE-anti-CD54 mAbs were used to stain splenic B cells from age-matched wild-type, Bcl-2, TRAF2DN, premalignant TRAF2DN/Bcl-2 double-positive, and TRAF2DN/Bcl-2 double-positive in the acute phase of the disease and blood lymphocytes from this TRAF2DN/Bcl-2 double-positive mouse in the acute phase. (B) A similar analysis was performed by using blood from another representative wild-type mouse or lymphocytes from spleen, lungs, blood, or pleural effusion of a TRAF2DN/Bcl-2 mouse that had developed leukemia. (C) The percentage of B220-positive lymphocytes with high levels of surface CD54/CD29 expression (upper right quadrant) was compared. B cells from spleen (black dots) or lung (white dots) were analyzed. Each dot represents an individual mouse. Statistical significance was determined by unpaired t test.

Fig. 5.

Southern blot analysis of IgH rearrangement in B cells from TRAF2DN/Bcl-2 mice. DNA was isolated from spleen, liver (1), or pleural effusion (2), and digested with EcoR1 and analyzed by Southern blotting by using a IgH probe. The band of 6 kb corresponds to the germ line (GL) IgH locus. IgH rearrangements corresponding to clonal expansions are indicated with an arrowhead. Wild-type, TRAF2DN, and TRAF2DN/Bcl-2 mice either asymptomatic (premalignant) or with overt lymphoma (disease) were analyzed. The ages in months of the mice (identified by number) analyzed were 17 (102), 15 (137), 14 (158, 157, and 148), 13 (83 and 316), and 12 (302, 327, 87, 89, and 95).

Fig. 6.

(A) Analysis of lymphocyte proliferation. Lymphocytes were isolated from spleens of wild-type (▪), Bcl-2 (♦), TRAF2DN (○), and TRAF2DN/Bcl-2 (Δ) transgenic mice. Cells (5 × 10⁴) were seeded in triplicate in 96-well plates and pulsed with 1 μCi (1 Ci = 37 GBq) of [³H]-methylthymidine for 12 h at days 0 (immediately after purification), 3, and 6. Proliferation was measured as the mean of [³H]-methylthymidine incorporation (cpm) ± SEM (n = 3) from three mice of each genotype. (B) DNA content analysis; 10⁶ lymphocytes from spleen (a–d), ascitic fluid (e), and lung (f) from wild-type (a; n = 4), Bcl-2 (b; n = 3), TRAF2DN (c; n = 4), and symptomatic TRAF2DN/Bcl-2 (d–f; n = 4) mice were lysed in hypotonic buffer and stained with PI. Cells with DNA content ≥ of 2n were gated, and the percentage of G₁ cells (DNA content = 2n) and cells in S, G₂, or M phases (>2n) was quantified. Shown are representative DNA content profiles of the various genotypes. The statistical analysis in the text corresponds to mean ± SEM. (C) Bcl-2 protects cells from spontaneous apoptosis. Splenocytes isolated from wild-type, Bcl-2, TRAF2DN, and TRAF2DN/Bcl-2 transgenic mice were cultured for 24 h and then incubated with APC-anti-B220 mAb for 1 h, washed, and incubated with annexinV-FITC/PI. B lymphocytes (B220⁺ cells) were gated, and the average percentage of annexin V-FITC-positive (apoptotic) B lymphocytes and the SD (n = 3) is shown (P = 0.01) comparing apoptosis of wild-type vs. Bcl-2 (*) or TRAF2DN/Bcl-2 (**). B cells are compared by unpaired t test. (D) Effects of chemotherapeutic drugs on apoptosis. Splenocytes isolated from wild-type (▪), Bcl-2 (♦), TRAF2DN (○), and TRAF2DN/Bcl-2 (Δ) transgenic mice were incubated with or without dexamethasone (0.01, 0.1, and 0.5 μM) or F-ara-A (1.25, 2.5, and 5 μM). Cells were harvested after 24 (Dex) or 48 (F-ara-A) h and incubated with APC-anti-B220 mAb for 1 h, then washed and incubated with annexin-V-FITC and PI. B lymphocytes (B220⁺ cells) were gated, and the percentage of apoptotic cells (annexin V⁺) was determined by flow cytometry. Data were corrected for differences in spontaneous apoptosis (mean ± SEM) (n = 6).