Susceptibility to Lymphoid Neoplasia in Immunodeficient Strains of Nonobese Diabetic Mice1

[CANCER RESEARCH 62, 5828–5834, October 15, 2002] Susceptibility to Lymphoid Neoplasia in Immunodeficient Strains of Nonobese Diabetic Mice1

Priscilla P. L. Chiu, Evgueni Ivakine, Steven Mortin-Toth, and Jayne S. Danska2 Program in Developmental Biology, The Hospital for Sick Children Research Institute [P. P. L. C., E. I., S. M-T., J. S. D.], Departments of Surgery [P. P. L. C.] and Immunology [E. I., J. S. D.] and the Institute of Medical Science [P. P. L. C., J. S. D.], Faculty of Medicine, University of Toronto, Toronto, Ontario, M5G 1X8 Canada

ABSTRACT pathways (6). Thus, genetic defects in DSB repair components arrest lymphocyte development, causing severe immunodeficiency (re- Transformed lymphocytes exhibit aberrant growth potential resulting viewed in Ref. 7). However, it is unclear whether the elevated cancer from enhanced proliferation and resistance to apoptotic stimuli. These risk in these patients results from DNA repair defects or defective mechanisms also influence the development of autoimmune disease, where dysregulated lymphocyte homeostasis has been implicated in expansion of immunosurveillance in the absence of mature lymphocytes. autoreactive T cells. In the nonobese diabetic (NOD) mouse, a murine To distinguish the contributions of these factors to lymphoma model of autoimmune type 1 diabetes and Sjo¨gren’s syndrome, T cells are susceptibility, we examined mouse mutants in which DNA repair and apoptosis resistant compared with other mouse strains, a feature thought genome stability were separated from defective lymphocyte develop- to potentiate their autoimmune function. NOD mice congenic for the ment and immune surveillance. The inbred NOD mouse is a well- severe combined immunodeficiency scid mutation (NOD.scid) have an established model for type 1 (also called insulin-dependent) diabetes incidence of pro-T-cell lymphoma far in excess of scid mutants on other (T1D; Ref. 8) and is also susceptible to other autoimmune diseases, genetic backgrounds. This mutation arrests lymphocyte development sec- including Sjo¨gren’s syndrome and thyroiditis (9). In both NOD mice ondary to a generalized defect in double-strand DNA break repair that compromises V(D)J recombination. To distinguish between the contribu- and humans, T1D is a complex, multigenic disease with many loci tions of immunodeficiency and defective double-strand DNA break repair controlling susceptibility (reviewed in Ref. 10). With the exception of to lymphoma susceptibility on the NOD background, we examined the the clearly defined role of the MHC haplotype to T1D, most of the incidence, phenotype, and molecular mechanisms of lymphoma develop- remaining genes have not yet been identified. In addition to T1D, ment in two immunodeficient NOD strains with normal DNA repair elderly NOD mice are prone to the development of lymphoid tumors function. We report that NOD mice deficient in mature B cells (11, 12). Interestingly, the introduction of mutations in the DSB repair ␮ Ϫ/Ϫ (NOD. MT) or mature T and B cells (NOD.RAG-2 ) display a high pathway onto the NOD background greatly enhanced the incidence of incidence of lymphoma of both T- and B-cell origin compared with these lymphoid tumors. The Prkdcscid (scid) mutation creates a premature mutations on other genetic backgrounds. Strikingly, the lymphoma incidence in both strains was greater in females, mirroring the greater stop codon in the gene encoding the DNA-dependent protein kinase, incidence of autoimmune type 1 diabetes in NOD females than in males. causing inefficient V(D)J recombination and arrested maturation of The high incidence of autoimmune diabetes and lymphoma in immunod- T-cell and B-cell precursors (6). As a result, these animals have severe eficiency NOD mice suggests the presence of genetic modifiers that affect combined immune deficiency and elevated sensitivity to ionizing lymphocyte homeostasis. radiation and radiomimetic drugs (13). The Prkdcscid mutation, identified in 1980, arose spontaneously on the C.B-17 background, an isogenic strain to BALB/c. Only one group has reported observation INTRODUCTION of thymic tumors (15% by 15 months of age) in this strain (14). The immune system is thought to play a vital role in the surveil- However, the scid mutation on the NOD background results in a lance of and protection against the growth of tumor cells (reviewed in thymic lymphoma incidence of 76% by 6 months of age, with more Ref. 1). Acquired immunodeficiencies caused by cancer chemother- females affected than males (15–17). These findings suggest that apy (2), posttransplant immunosuppressive therapy (3), and HIV modifier gene(s) sensitize immunodeficient NOD mice to the devel- infection (reviewed in Ref. 4) are associated with an increased inci- opment of lymphoid tumors in the context of defective DSB repair. dence of malignancies, particularly lymphoma and leukemia. Lym- The mechanism of lymphomagenesis in NOD.scid mice remains phomas of both T- and B-cell origin are also common among patients unclear. Molecular analysis revealed multiple integrations of the en- with primary immunodeficiencies arising from genetic defects in dogenous ecotropic provirus, Emv30, in some NOD.scid thymic lym- DNA repair (reviewed in Ref. 5). Faithful execution of the site- phomas, suggesting that insertional mutagenesis by mobilized retro- specific recombination process that assembles functional T- and B- virus occurs in this model (15). However, congenic replacement of the cell antigen receptor genes from dispersed variable (V), diversity (D), Emv30-containing segment of chromosome 11 with this segment and joining (J) coding segments [V(D)J3 recombination] is required from the NOR strain (Emv30Ϫ/ϪNOD.scid) slowed the growth but did for T- and B-cell maturation and depends upon ubiquitous DSB repair not significantly lower the incidence of thymic lymphoma compared with the NOD.scid strain (17). Another consequence of NOD genetic Received 5/1/02; accepted 8/16/02. modifiers is a functional deficit in natural killer cells in NOD.scid The costs of publication of this article were defrayed in part by the payment of page compared with C.B.-17.scid or C57BL/6.scid mice (18, 19), a feature charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. thought to render them superior hosts for human hematopoietic pre- 1 This work was supported by grants from the Juvenile Diabetes Research Foundation cursors (20). Evidence from wild-type mice suggests that natural killer and the National Cancer Institute of Canada. J. S. D. is a Research Scientist of the National Cancer Institute of Canada and Principal Investigator of the Canadian Genetic cells function in tumor immunosurveillance (21); therefore, their Disease Network. P. P. L. C. is the recipient of a Juvenile Diabetes Research Foundation deficiency may further enhance lymphoma susceptibility in the ab- Postdoctoral Fellowship. sence of mature T and B cells. 2 To whom requests for reprints should be addressed, at Program in Developmental Biology, The Hospital for Sick Children Research Institute, 555 University Avenue, We have examined the incidence and cell surface and molecular Toronto, Ontario, M5G 1X8 Canada. Phone: (416) 813-6450; Fax: (416) 813-8823; E- phenotype of lymphoid tumors in two NOD congenic strains with mail: [email protected]. 3 The abbreviations used are: V(D)J, variable(diversity)joining; DSB, double-strand DNA break; NOD, nonobese diabetic; T1D, autoimmune type diabetes; scid, severe phycoerythrin; PI, propidium iodide; LN, lymph node; FACS, fluorescence-activated cell combined immune deficiency; RAG, recombinase-activating gene; AV-PE, streptavidin- sorting; TCR T-cell receptor. 5828

variable degrees of immunodeficiency and normal DNA repair. The precipitated in ethanol. DNA was spooled in a heat-sealed glass pipette, RAG proteins, RAG-1 and RAG-2, introduce site-specific DSB at the air-dried, and resuspended in 300 ␮lofTE[10mM Tris (pH 7.5), 5 mM recombination signal sequence flanking the V, D, and J gene segments EDTA]. of the T-cell and B-cell receptor gene loci. Disabling mutations in Molecular Probes. The pEco-env probe (15, 17) and oligonucleotides for either RAG gene results in arrested lymphoid development at the the PCR amplification of genomic DNA were provided by Dr. D. V. Serreze Ϫ Ϫ ϩ ϩ (The Jackson Laboratory, Bar Harbor, Maine). The probes were purified using CD4 CD8 pro-T and B220 CD43 pro-B-cell stages (reviewed in QIAEX II gel extraction (Qiagen, Chatworth, CA). Fragments were labeled to Ref. 22). Importantly, neither RAG-1 nor RAG-2 deficiency has been high specific activity with [␣-32P]dCTP (3000 Ci/mmol; Amersham Canada, ϫ reported to enhance tumor development on the 129/Sv C57BL/6 Oakville, Ontario, Canada) by random hexamer labeling using standard tech- (129/B6) backgrounds (23, 24). The targeted mutation in RAG-2 was niques. bred to NOD background to generate a congenic strain (NOD.RAG- Southern Blot Analysis. For each sample, 15 ␮g of genomic DNA were Ϫ Ϫ 2 / ; Ref. 25). A second targeted germ-line mutation (Ig␮MT) that digested at 37°C with restriction enzymes PvuII (Life Technologies, Inc., disables production of membrane-bound Ig␮ causing a profound Burlington, Ontario, Canada) for 12 h. Restriction products were separated by block in B-cell development but allows normal maturation of the agarose gel electrophoresis, transferred to nylon membrane (ZetaProbe; Bio- T-cell compartment was transferred onto the NOD background by Rad Laboratories, Hercules, CA), and immobilized with UV light (Stratagene, ␣ 32 repeated backcrossing (NOD.␮MT; Ref. 26). Although neither of La Jolla, CA). [ - P]-Labeled DNA probes were prepared and hybridized using standard techniques, followed by exposure to a phosphorscreen. Images these mutations has been shown to enhance cancer risk on other Ϫ/Ϫ were collected on a phosphorscreen (Molecular Dynamics, Sunnyvale, CA) genetic backgrounds, we found that both NOD.RAG-2 and and analyzed by ImageQuant software (Molecular Dynamics). ␮ NOD. MT mice display a high incidence of lymphomas and leuke- Statistical Analysis. To calculate for the difference in survival in NOD, mias in some cases involving both B-cell and T-cell precursors. NOD.RAG-2Ϫ/Ϫ, and NOD.␮MT mice, Kaplan-Meier log-rank statistical anal- Therefore, under conditions of normal DNA repair pathways and ysis was performed using SPSS 10.1 for Windows software (SPSS, Inc., variable degrees of immunodeficiency, modifier genes in the NOD Chicago, IL). Statistical significance was achieved for P Ͻ 0.05. background confer high risk for lymphoid malignancy. RESULTS

MATERIALS AND METHODS Development of Lymphoid Tumors in NOD.RAG-2Ϫ/Ϫ Mice. As described above, the elevated lymphoma incidence in NOD.scid Mice. Mice used in these studies were maintained in a pathogen-free mice may reflect either immunodeficiency or defective DSB repair. facility at The Hospital for Sick Children Research Institute. NOD.RAG-2Ϫ/Ϫ To distinguish between these two alternatives, we examined NOD mice were a gift of D. Holmberg (University of Umea, Sweden). These mice were generated as described (25) and used at the N9 backcross to NOD. congenic mice lacking mature T and B lymphocytes caused by a B-cell-deficient NOD.␮MT mice were generated as described (26) and used at targeted mutation in the RAG-2 gene (25, 29), a mutation that has no the N10 backcross to NOD. NOD.scid mice (The Jackson Laboratory) were at effect on generalized DSB repair. The incidence of lymphoid tumors Ϫ/Ϫ N10 backcross to NOD. All of the mice were monitored until 40 weeks of age in NOD.RAG-2 mice was 44% with disease onset starting at 21 and were sacrificed at the first clinical signs of tumor development. The mice weeks of age (Fig. 1A; Table 1). Interestingly, the gender bias (female: were euthanized if there were any clinical signs of cachexia, anorexia, weak- male) in lymphoma incidence was 10:1in NOD.RAG-2Ϫ/Ϫ lymphoma ness, or airway obstruction. Tumor diagnosis was made at the time of ne- incidence (Fig. 1B; Table 1), similar to the gender disparity reported cropsy. in NOD.scid mice (15) and T1D in standard NOD mice (12). As was Antibodies and Reagents. Antibodies used in flow cytometric analyses of reported previously in NOD.scid mice (15), NOD.RAG-2Ϫ/Ϫ mice murine lymphocytes and myeloid cells were affinity purified from tissue develop thymic tumors with dissemination to peripheral lymphoid culture supernatants: anti-CD3⑀ (YCD3–1), anti-TCR C␤ (H57.597), anti-CD4 g7 organs. In contrast to previous observations in NOD.scid animals, (YTS 191.1), anti-CD8 (YTS 169.4), anti-I-A (10–2.16), anti-B220 (RA3– Ϫ/Ϫ 6B2), anti-Mac-1 (M1/70), and anti-CD69 (H1.2F3). Biotinylated anti-CD25 NOD.RAG-2 mice also developed non-thymic lymphoid tumors (7D4), biotinylated anti-CD19 (1D3), FITC-conjugated and biotinylated iso- primarily involving the spleen (Table 2) and bone marrow (see be- Ϫ/Ϫ type control antibodies were purchased from PharMingen (San Diego, CA). low). Some affected NOD.RAG-2 mice displayed massive en- AV-PE was purchased from Caltag (South San Francisco, CA). PI for dead cell largement of the spleen but minimal or no thymic enlargement (Table exclusion was purchased from Sigma Chemical Co. (St. Louis, MO). 2, and data not shown). Adoptive transfer of either thymic or splenic Flow Cytometry. Preparation and staining of LN, spleen, bone marrow, tumor cells from NOD.RAG-2Ϫ/Ϫ mice into young NOD.scid hosts and thymus cell suspensions were performed as described (27) using antibod- resulted in rapid onset of disease in all recipients (data not shown). ies listed above. Two-color FACS was performed on the FACScalibur (Becton Thus, NOD.RAG-2Ϫ/Ϫ mice, particularly females, are susceptible to Dickinson, Mountain View, CA). Analyses of FACS data were performed the development of thymic and disseminated lymphoid malignancies. using CellQuest software (Becton Dickinson). Adoptive Transfer of NOD.␮MT-derived and NOD.RAG-2Ϫ/Ϫ-derived Furthermore, the high incidence of these malignancies on the NOD Tumor Cells. Splenic or thymic tumors isolated from NOD.RAG-2Ϫ/Ϫ or background does not depend upon defective DNA repair mechanisms NOD.␮MT mice were prepared as cell suspensions. After red cell lysis, or the initiation of V(D)J recombination of immune receptor genes. 1–5 ϫ 106 cells were injected i.v. into NOD.scid recipients, 4 weeks of age. Rather, the absence of mature lymphocytes is sufficient to allow the Age-matched NOD.scid adoptive transfer recipients injected with PBS served emergence of lymphoid malignancies in mice of NOD background. as negative controls. All NOD.scid recipients were monitored for tumor Lymphoma Development in B-cell-deficient NOD.␮MT Mice. development for a period of 28 days and sacrificed at 28 days after adoptive Although the cellular basis of tumor immunosurveillance remains transfer or at the first signs of morbidity. poorly understood, T cells and natural killer cells are thought to be DNA Preparation. High molecular weight DNA was extracted from LN, critical to recognition of tumor antigens (21). To determine whether spleen, and thymus cell suspension as described (28). Briefly, 1 ϫ 107 cells the development of a normal T-cell compartment alone would protect were resuspended in 400 ␮l of solution A [10 mM Tris-HCl (pH 7.5), 10 mM against lymphoma development, we studied NOD.␮MT mice that lack EDTA, and 10 mM NaCl] and an equivalent volume of solution B (solution A ␮ with 2% SDS). Proteinase K (Promega Corp., Madison, WI) was added to a mature B cells (26). The original report of the MT mutation on final concentration of 100 ␮g/ml. The samples were incubated at 55°C over- 129/Sv ϫ C57BL/6 (129/B6) background did not comment on tumor night. The DNA was isolated by sequential extractions through phenol pH 8.0, development (30), and subsequent studies on ␮MT on the NOD phenol:chloroform:isoamyl alcohol (ratio, 25:24:1) and chloroform, and then background have focused on the impact of the B-cell compartment on 5829

32). Early pro-T-cell precursors lack surface expression of CD4 and CD8 (“double negative”) coreceptors and are actively engaged in recombination at the TCR␤ locus. Productive TCR␤ rearrangement is accompanied by extensive proliferation and maturation to the CD4ϩCD8ϩ (“double positive”) pre-T-cell stage, when recombination at the TCR␣ locus is initiated. Subsequently, thymocytes up- regulate expression of the TCR␣␤ heterodimer and extinguish expression of either CD4 or CD8 coreceptors to become “single positive” thymocytes (27). Because the RAG-2Ϫ/Ϫ and scid mutations disable V(D)J recombination, T-cell development in these animals is blocked at the double-negative pro-T-cell stage, and their thymuses are ex- tremely small. To evaluate the developmental stage(s) represented by the lymphomas observed on the NOD background, multiparameter FACS analyses were performed. Consistent with previous reports (15, 16), NOD.scid thymic tumors were lymphoblastic CD4ϩCD8ϩTCR␤Ϫ cells (Fig. 2A), reflecting the pre-T-cell stage of thymocyte development in the absence of productive TCR␤ rearrangement. Similarly, we found that thymic tumors in NOD.RAG-2Ϫ/Ϫ mice express markers consistent with this stage of T-cell development (Fig. 2A). Thymic lymphoma in NOD.␮MT mice were also CD4ϩCD8ϩ but expressed cell surface TCR (Fig. 2A), consistent with the normal V(D)J recombinase activity in these mice. Massive splenomegaly as a primary feature of disease was observed in a subset of both NOD.RAG-2Ϫ/Ϫ and NOD.␮MT animals. In these mice, the tumor phenotypes were distinct from the double-positive thymic lymphomas (Fig. 3A), and thymic cellularity was normal (Table 2). Splenic and bone marrow lymphoma cells in NOD.RAG- 2Ϫ/Ϫ mice expressed cell surface markers B220 and CD19 (Fig. 3), suggestive of precursor B-cell lymphoblastic lymphoma (33). In contrast, splenic tumor cells from NOD.␮MT mice were either B220ϩCD19Ϫ pro-B cells (Fig. 3A) or mature single-positive CD4ϩ TCR␤ϩ T cells (Fig. 2B), the latter phenotype being consistent with small T-cell lymphoma (33) and reminiscent of human small T-cell lymphoma (34). Therefore, both NOD.␮MT and NOD.RAG-2Ϫ/Ϫ mice are prone to the development of both T- and B-lymphoid Fig. 1. Tumor incidence and distribution in NOD, NOD.RAG-2Ϫ/Ϫ, and NOD.␮MT mice. All mice were monitored for tumor development until 40 weeks of age. A, malignancies and suggest that the malignant NOD lymphocytes rep- Kaplan-Meier analysis. NOD mice (n ϭ 10) do not develop lymphoid tumors but are resent different stages of lymphocyte development. prone to the development of diabetes. The onset of lymphoma started at 21 weeks of age ␮ in NOD.RAG-2Ϫ/Ϫ mice (n ϭ 25), with 44% affected by 40 weeks of age. The onset of Novel Integrations of Emv30 in NOD. MT and NOD.RAG- Ϫ/Ϫ disease in NOD.␮MT mice (n ϭ 47) started at 32 weeks of age and reached 23% by 40 2 Tumors. Previous studies have demonstrated somatic acquisi- Ϫ Ϫ weeks of age. Log-rank statistical analysis: NOD versus NOD.RAG-2 / , P Ͻ 0.0001; tion of proviral insertions of the murine endogenous retrovirus Emv30 NOD versus NOD.␮MT, P ϭ 0.0003; NOD.RAG-2Ϫ/Ϫ versus NOD.␮MT, P ϭ 0.029. B, .(in double-positive thymic lymphomas from NOD.scid mice (15 ,ء .gender bias in lymphoma development in NOD.RAG-2Ϫ/Ϫ and NOD.␮MT mice among NOD and immunodeficient NOD females: NOD versus NOD.RAG-2Ϫ/Ϫ, Southern analysis was performed to determine whether mobilization P Ͻ 0.0001; NOD versus NOD.␮MT, P ϭ 0.0006; NOD.RAG-2Ϫ/Ϫ v