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[Cancer Research 62, 5828-5834, October 15, 2002]
© 2002 American Association for Cancer Research

Immunology

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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transformed lymphocytes exhibit aberrant growth potential resulting from enhanced proliferation and resistance to apoptotic stimuli. These mechanisms also influence the development of autoimmune disease, where dysregulated lymphocyte homeostasis has been implicated in expansion of autoreactive T cells. In the nonobese diabetic (NOD) mouse, a murine model of autoimmune type 1 diabetes and Sjögren’s syndrome, T cells are apoptosis resistant compared with other mouse strains, a feature thought to potentiate their autoimmune function. NOD mice congenic for the severe combined immunodeficiency scid mutation (NOD.scid) have an incidence of pro-T-cell lymphoma far in excess of scid mutants on other genetic backgrounds. This mutation arrests lymphocyte development secondary to a generalized defect in double-strand DNA break repair that compromises V(D)J recombination. To distinguish between the contributions of immunodeficiency and defective double-strand DNA break repair to lymphoma susceptibility on the NOD background, we examined the incidence, phenotype, and molecular mechanisms of lymphoma development in two immunodeficient NOD strains with normal DNA repair function. We report that NOD mice deficient in mature B cells (NOD.µMT) or mature T and B cells (NOD.RAG-2-/-) display a high incidence of lymphoma of both T- and B-cell origin compared with these mutations on other genetic backgrounds. Strikingly, the lymphoma incidence in both strains was greater in females, mirroring the greater incidence of autoimmune type 1 diabetes in NOD females than in males. The high incidence of autoimmune diabetes and lymphoma in immunodeficiency NOD mice suggests the presence of genetic modifiers that affect lymphocyte homeostasis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The immune system is thought to play a vital role in the surveillance of and protection against the growth of tumor cells (reviewed in Ref. 1 ). Acquired immunodeficiencies caused by cancer chemotherapy (2) , posttransplant immunosuppressive therapy (3) , and HIV infection (reviewed in Ref. 4 ) are associated with an increased incidence of malignancies, particularly lymphoma and leukemia. Lymphomas of both T- and B-cell origin are also common among patients with primary immunodeficiencies arising from genetic defects in DNA repair (reviewed in Ref. 5 ). Faithful execution of the site-specific recombination process that assembles functional T- and B-cell antigen receptor genes from dispersed variable (V), diversity (D), and joining (J) coding segments [V(D)J3 recombination] is required for T- and B-cell maturation and depends upon ubiquitous DSB repair pathways (6) . Thus, genetic defects in DSB repair components arrest lymphocyte development, causing severe immunodeficiency (reviewed in Ref. 7 ). However, it is unclear whether the elevated cancer risk in these patients results from DNA repair defects or defective immunosurveillance in the absence of mature lymphocytes.

To distinguish the contributions of these factors to lymphoma susceptibility, we examined mouse mutants in which DNA repair and genome stability were separated from defective lymphocyte development and immune surveillance. The inbred NOD mouse is a well-established model for type 1 (also called insulin-dependent) diabetes (T1D; Ref. 8 ) and is also susceptible to other autoimmune diseases, including Sjögren’s syndrome and thyroiditis (9) . In both NOD mice and humans, T1D is a complex, multigenic disease with many loci controlling susceptibility (reviewed in Ref. 10 ). With the exception of the clearly defined role of the MHC haplotype to T1D, most of the remaining genes have not yet been identified. In addition to T1D, elderly NOD mice are prone to the development of lymphoid tumors (11 , 12) . Interestingly, the introduction of mutations in the DSB repair pathway onto the NOD background greatly enhanced the incidence of lymphoid tumors. The Prkdcscid (scid) mutation creates a premature stop codon in the gene encoding the DNA-dependent protein kinase, causing inefficient V(D)J recombination and arrested maturation of T-cell and B-cell precursors (6) . As a result, these animals have severe combined immune deficiency and elevated sensitivity to ionizing 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 of thymic tumors (15% by 15 months of age) in this strain (14) . However, the scid mutation on the NOD background results in a thymic lymphoma incidence of 76% by 6 months of age, with more females affected than males (15, 16, 17) . These findings suggest that modifier gene(s) sensitize immunodeficient NOD mice to the development of lymphoid tumors in the context of defective DSB repair.

The mechanism of lymphomagenesis in NOD.scid mice remains unclear. Molecular analysis revealed multiple integrations of the endogenous ecotropic provirus, Emv30, in some NOD.scid thymic lymphomas, suggesting that insertional mutagenesis by mobilized retrovirus occurs in this model (15) . However, congenic replacement of the Emv30-containing segment of chromosome 11 with this segment from the NOR strain (Emv30-/-NOD.scid) slowed the growth but did not significantly lower the incidence of thymic lymphoma compared with the NOD.scid strain (17) . Another consequence of NOD genetic modifiers is a functional deficit in natural killer cells in NOD.scid compared with C.B.-17.scid or C57BL/6.scid mice (18 , 19) , a feature thought to render them superior hosts for human hematopoietic precursors (20) . Evidence from wild-type mice suggests that natural killer cells function in tumor immunosurveillance (21) ; therefore, their deficiency may further enhance lymphoma susceptibility in the absence of mature T and B cells.

We have examined the incidence and cell surface and molecular phenotype of lymphoid tumors in two NOD congenic strains with variable degrees of immunodeficiency and normal DNA repair. The RAG proteins, RAG-1 and RAG-2, introduce site-specific DSB at the recombination signal sequence flanking the V, D, and J gene segments of the T-cell and B-cell receptor gene loci. Disabling mutations in either RAG gene results in arrested lymphoid development at the CD4-CD8- pro-T and B220+CD43+ pro-B-cell stages (reviewed in Ref. 22 ). Importantly, neither RAG-1 nor RAG-2 deficiency has been reported to enhance tumor development on the 129/Sv x C57BL/6 (129/B6) backgrounds (23 , 24) . The targeted mutation in RAG-2 was bred to NOD background to generate a congenic strain (NOD.RAG-2-/-; Ref. 25 ). A second targeted germ-line mutation (IgµMT) that disables production of membrane-bound Igµ causing a profound block in B-cell development but allows normal maturation of the T-cell compartment was transferred onto the NOD background by repeated backcrossing (NOD.µMT; Ref. 26 ). Although neither of these mutations has been shown to enhance cancer risk on other genetic backgrounds, we found that both NOD.RAG-2-/- and NOD.µMT mice display a high incidence of lymphomas and leukemias in some cases involving both B-cell and T-cell precursors. Therefore, under conditions of normal DNA repair pathways and variable degrees of immunodeficiency, modifier genes in the NOD background confer high risk for lymphoid malignancy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Mice used in these studies were maintained in a pathogen-free facility at The Hospital for Sick Children Research Institute. NOD.RAG-2-/- 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. B-cell-deficient NOD.µMT mice were generated as described (26) and used at the N10 backcross to NOD. NOD.scid mice (The Jackson Laboratory) were at N10 backcross to NOD. All of the mice were monitored until 40 weeks of age and were sacrificed at the first clinical signs of tumor development. The mice were euthanized if there were any clinical signs of cachexia, anorexia, weakness, or airway obstruction. Tumor diagnosis was made at the time of necropsy.

Antibodies and Reagents.
Antibodies used in flow cytometric analyses of murine lymphocytes and myeloid cells were affinity purified from tissue culture supernatants: anti-CD3{epsilon} (YCD3–1), anti-TCR Cß (H57.597), anti-CD4 (YTS 191.1), anti-CD8 (YTS 169.4), anti-I-Ag7 (10–2.16), anti-B220 (RA3–6B2), anti-Mac-1 (M1/70), and anti-CD69 (H1.2F3). Biotinylated anti-CD25 (7D4), biotinylated anti-CD19 (1D3), FITC-conjugated and biotinylated isotype control antibodies were purchased from PharMingen (San Diego, CA). AV-PE was purchased from Caltag (South San Francisco, CA). PI for dead cell exclusion was purchased from Sigma Chemical Co. (St. Louis, MO).

Flow Cytometry.
Preparation and staining of LN, spleen, bone marrow, and thymus cell suspensions were performed as described (27) using antibodies listed above. Two-color FACS was performed on the FACScalibur (Becton Dickinson, Mountain View, CA). Analyses of FACS data were performed using CellQuest software (Becton Dickinson).

Adoptive Transfer of NOD.µMT-derived and NOD.RAG-2-/--derived Tumor Cells.
Splenic or thymic tumors isolated from NOD.RAG-2-/- or NOD.µMT mice were prepared as cell suspensions. After red cell lysis, 1–5 x 106 cells were injected i.v. into NOD.scid recipients, 4 weeks of age. Age-matched NOD.scid adoptive transfer recipients injected with PBS served as negative controls. All NOD.scid recipients were monitored for tumor development for a period of 28 days and sacrificed at 28 days after adoptive transfer or at the first signs of morbidity.

DNA Preparation.
High molecular weight DNA was extracted from LN, spleen, and thymus cell suspension as described (28) . Briefly, 1 x 107 cells were resuspended in 400 µl of solution A [10 mM Tris-HCl (pH 7.5), 10 mM 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 final concentration of 100 µg/ml. The samples were incubated at 55°C overnight. The DNA was isolated by sequential extractions through phenol pH 8.0, phenol:chloroform:isoamyl alcohol (ratio, 25:24:1) and chloroform, and then precipitated in ethanol. DNA was spooled in a heat-sealed glass pipette, air-dried, and resuspended in 300 µl of TE [10 mM Tris (pH 7.5), 5 mM EDTA].

Molecular Probes.
The pEco-env probe (15 , 17) and oligonucleotides for the PCR amplification of genomic DNA were provided by Dr. D. V. Serreze (The Jackson Laboratory, Bar Harbor, Maine). The probes were purified using QIAEX II gel extraction (Qiagen, Chatworth, CA). Fragments were labeled to high specific activity with [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham Canada, Oakville, Ontario, Canada) by random hexamer labeling using standard techniques.

Southern Blot Analysis.
For each sample, 15 µg of genomic DNA were digested at 37°C with restriction enzymes PvuII (Life Technologies, Inc., Burlington, Ontario, Canada) for 12 h. Restriction products were separated by agarose gel electrophoresis, transferred to nylon membrane (ZetaProbe; Bio-Rad Laboratories, Hercules, CA), and immobilized with UV light (Stratagene, La Jolla, CA). [{alpha}-32P]-Labeled DNA probes were prepared and hybridized using standard techniques, followed by exposure to a phosphorscreen. Images were collected on a phosphorscreen (Molecular Dynamics, Sunnyvale, CA) and analyzed by ImageQuant software (Molecular Dynamics).

Statistical Analysis.
To calculate for the difference in survival in NOD, NOD.RAG-2-/-, and NOD.µMT mice, Kaplan-Meier log-rank statistical analysis was performed using SPSS 10.1 for Windows software (SPSS, Inc., Chicago, IL). Statistical significance was achieved for P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Development of Lymphoid Tumors in NOD.RAG-2-/- Mice.
As described above, the elevated lymphoma incidence in NOD.scid mice may reflect either immunodeficiency or defective DSB repair. To distinguish between these two alternatives, we examined NOD congenic mice lacking mature T and B lymphocytes caused by a targeted mutation in the RAG-2 gene (25 , 29) , a mutation that has no effect on generalized DSB repair. The incidence of lymphoid tumors in NOD.RAG-2-/- mice was 44% with disease onset starting at 21 weeks of age (Fig. 1ACitation ; Table 1Citation ). Interestingly, the gender bias (female:male) in lymphoma incidence was 10:1in NOD.RAG-2-/- lymphoma incidence (Fig. 1BCitation ; Table 1Citation ), similar to the gender disparity reported in NOD.scid mice (15) and T1D in standard NOD mice (12) . As was reported previously in NOD.scid mice (15) , NOD.RAG-2-/- mice develop thymic tumors with dissemination to peripheral lymphoid organs. In contrast to previous observations in NOD.scid animals, NOD.RAG-2-/- mice also developed non-thymic lymphoid tumors primarily involving the spleen (Table 2)Citation and bone marrow (see below). Some affected NOD.RAG-2-/- mice displayed massive enlargement of the spleen but minimal or no thymic enlargement (Table 2Citation , and data not shown). Adoptive transfer of either thymic or splenic tumor cells from NOD.RAG-2-/- mice into young NOD.scid hosts resulted in rapid onset of disease in all recipients (data not shown). Thus, NOD.RAG-2-/- mice, particularly females, are susceptible to the development of thymic and disseminated lymphoid malignancies. Furthermore, the high incidence of these malignancies on the NOD background does not depend upon defective DNA repair mechanisms or the initiation of V(D)J recombination of immune receptor genes. Rather, the absence of mature lymphocytes is sufficient to allow the emergence of lymphoid malignancies in mice of NOD background.



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  		Fig. 1. Tumor incidence and distribution in NOD, NOD.RAG-2-/-, and NOD.µMT mice. All mice were monitored for tumor development until 40 weeks of age. A, Kaplan-Meier analysis. NOD mice (n = 10) do not develop lymphoid tumors but are prone to the development of diabetes. The onset of lymphoma started at 21 weeks of age in NOD.RAG-2-/- mice (n = 25), with 44% affected by 40 weeks of age. The onset of disease in NOD.µMT mice (n = 47) started at 32 weeks of age and reached 23% by 40 weeks of age. Log-rank statistical analysis: NOD versus NOD.RAG-2-/-, P < 0.0001; NOD versus NOD.µMT, P = 0.0003; NOD.RAG-2-/- versus NOD.µMT, P = 0.029. B, gender bias in lymphoma development in NOD.RAG-2-/- and NOD.µMT mice. *, among NOD and immunodeficient NOD females: NOD versus NOD.RAG-2-/-, P < 0.0001; NOD versus NOD.µMT, P = 0.0006; NOD.RAG-2-/- versus NOD.µMT, P = 0.048. **, for male NOD and immunodeficient NOD mice: NOD versus NOD.RAG-2-/-, P = 0.0077; NOD versus NOD.µMT, P = 0.277; NOD.RAG-2-/- versus NOD.µMT, P = 0.07.

 

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  		Table 1 Tumor development in NOD.scid, NOD.RAG-2-/-, and NOD.µMT mice

NOD.RAG-2-/- and NOD.µMT mice were observed for the onset of lethargy, cachexia, anorexia, or breathing difficulties or until 40 weeks of age. The mice were sacrificed and examined at the first signs of illness for evidence of thymic, splenic, or LN enlargement. Thymuses, spleens, and LNs from mice with lymphoid organomegaly were evaluated as described in "Materials and Methods."

 

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  		Table 2 Cell counts and immunological phenotypes of thymic and splenic tumors of NOD.scid, NOD.RAG-2-/-, and NOD.µMT mice

NOD.scid, NOD.RAG-2-/-, and NOD.µMT mice were sacrificed at 40 weeks of age or at the onset of disease. Cell suspensions were prepared, and cell counts were recorded using trypan blue to exclude dead cells. Immune-deficient NOD mice with thymic enlargement displayed marked cellularity in thymic cell counts, whereas mice with splenic enlargement consistently demonstrated elevated splenic cell counts. Tumor cells from thymus and spleen were assessed using FACS analysis for the surface expression of immunological markers as described in "Materials and Methods."

 
Lymphoma Development in B-cell-deficient NOD.µMT Mice.
Although the cellular basis of tumor immunosurveillance remains poorly understood, T cells and natural killer cells are thought to be critical to recognition of tumor antigens (21) . To determine whether the development of a normal T-cell compartment alone would protect against lymphoma development, we studied NOD.µMT mice that lack mature B cells (26) . The original report of the µMT mutation on 129/Sv x C57BL/6 (129/B6) background did not comment on tumor development (30) , and subsequent studies on µMT on the NOD background have focused on the impact of the B-cell compartment on T1D (26 , 31) . To investigate the impact of a B-cell deficiency on NOD lymphoma development, NOD.µMT mice were monitored to 40 weeks of age. Twenty-three % of these mice displayed lymphoid tumors beginning at 30 weeks of age (Fig. 1ACitation ; Table 1Citation ), with more females affected than males (Fig. 1BCitation ; Table 1Citation ). These lymphomas were associated with massive enlargement of the thymus and spleen (Table 2)Citation . In comparison with NOD.RAG-2-/- mice, NOD.µMT animals showed a lower lymphoma incidence and increased disease latency (Fig. 1A)Citation . Thus, these data suggest that the presence of a mature T-cell compartment provides incomplete protection against the onset of lymphoid malignancies, and B-cell immunodeficiency constitutes a significant risk factor in NOD mice.

Lymphoma Phenotypes in NOD.µMT and NOD.RAG-2-/- Mice.
Stages of intrathymic T-cell maturation are defined by the sequential expression of cell surface markers, and these transitions are tightly coupled to V(D)J recombination at TCR loci (reviewed in Ref. 32 ). Early pro-T-cell precursors lack surface expression of CD4 and CD8 ("double negative") coreceptors and are actively engaged in recombination at the TCRß locus. Productive TCRß rearrangement is accompanied by extensive proliferation and maturation to the CD4+CD8+ ("double positive") pre-T-cell stage, when recombination at the TCR{alpha} locus is initiated. Subsequently, thymocytes up-regulate expression of the TCR{alpha}ß heterodimer and extinguish expression of either CD4 or CD8 coreceptors to become "single positive" thymocytes (27) . Because the RAG-2-/- 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 extremely 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 CD4+CD8+TCRß- cells (Fig. 2A)Citation , reflecting the pre-T-cell stage of thymocyte development in the absence of productive TCRß rearrangement. Similarly, we found that thymic tumors in NOD.RAG-2-/- mice express markers consistent with this stage of T-cell development (Fig. 2A)Citation . Thymic lymphoma in NOD.µMT mice were also CD4+CD8+ but expressed cell surface TCR (Fig. 2A)Citation , 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-2-/- and NOD.µMT animals. In these mice, the tumor phenotypes were distinct from the double-positive thymic lymphomas (Fig. 3A)Citation , and thymic cellularity was normal (Table 2)Citation . Splenic and bone marrow lymphoma cells in NOD.RAG-2-/- mice expressed cell surface markers B220 and CD19 (Fig. 3)Citation , suggestive of precursor B-cell lymphoblastic lymphoma (33) . In contrast, splenic tumor cells from NOD.µMT mice were either B220+CD19- pro-B cells (Fig. 3A)Citation or mature single-positive CD4+ TCRß+ T cells (Fig. 2B)Citation , the latter phenotype being consistent with small T-cell lymphoma (33) and reminiscent of human small T-cell lymphoma (34) . Therefore, both NOD.µMT and NOD.RAG-2-/- mice are prone to the development of both T- and B-lymphoid malignancies and suggest that the malignant NOD lymphocytes represent different stages of lymphocyte development.



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  		Fig. 2. Cell surface markers expressed on NOD, NOD.scid, NOD.RAG-2-/-, and NOD.µMT tumor cells. A, thymuses from NOD, NOD.scid, NOD.RAG-2-/-, and NOD.µMT mice were prepared in cell suspensions. Two x 106 thymocytes were stained with FITC-conjugated anti-CD8 (YTS 169.4) and biotinylated anti-CD4 (YTS 191.1) or anti-TCRß (H57.597) antibodies, followed by AV-PE. Dead cells were excluded by PI staining. Results are from one experiment using one animal from each genotype. B, spleens from NOD, NOD.RAG-2-/-, and NOD.µMT mice were prepared into cell suspensions. Two x 106 splenocytes were stained with FITC-conjugated YTS 169.4 and biotinylated YTS 191.1or H57.597 antibodies, followed by AV-PE. Dead cells were excluded by PI staining. Results are from one experiment using one animal from each genotype.

 


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  		Fig. 3. B-cell marker expression on NOD, NOD.scid, NOD.RAG-2-/-, and NOD.µMT splenic and bone marrow tumor cells. A, spleens from NOD, NOD.RAG-2-/-, and NOD.µMT mice were prepared in cell suspensions. Two x 106 splenocytes were stained with FITC-conjugated YTS 169.4 and biotinylated YTS 191.1or FITC-conjugated anti-B220 (RA3-6B2) and biotinylated anti-CD19 (1D3) antibodies, followed by AV-PE. Dead cells were excluded by PI staining. Results are from one experiment using one animal from each genotype. B, bone marrow cells were isolated from one femur from NOD (left panels), NOD.RAG-2-/- mouse with splenomegaly (center panels), and NOD.RAG-2-/- mouse with thymic enlargement (right panels). One x 106 bone marrow cells were stained with FITC-conjugated anti-B220 (RA3-6B2) and biotinylated anti-CD19 (1D3) or FITC-conjugated H57.597 and biotinylated anti-CD3{epsilon} (YCD3-1) or FITC-conjugated YTS 169.4 and biotinylated YTS 191.1 antibodies, followed by AV-PE. Dead cells were excluded by PI staining. Results are from one experiment with one animal from each genotype as indicated.

 
Novel Integrations of Emv30 in NOD.µMT and NOD.RAG-2-/- Tumors.
Previous studies have demonstrated somatic acquisition of proviral insertions of the murine endogenous retrovirus Emv30 in double-positive thymic lymphomas from NOD.scid mice (15) . Southern analysis was performed to determine whether mobilization of Emv30 was associated with lymphoma development in NOD.RAG-2-/- and NOD.µMT mice (Fig. 4)Citation . Two of three NOD.RAG-2-/- and none of three NOD.µMT tumors analyzed showed evidence of novel Emv30 integration. Thus, somatic acquisition of proviral insertions is not a consistent feature of lymphomas in NOD.RAG-2-/- and NOD.µMT mice, similar to a published observation of NOD.scid animals (17) . Importantly, our data show that mobilization of Emv30 in NOD lymphocyte precursors occurs in immunodeficient mice with wild-type DNA repair pathways, suggesting that NOD background genes are responsible for this phenotype.



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  		Fig. 4. Emv30 somatic integration in NOD.scid, NOD.µMT, and NOD.RAG-2-/- tumors. Southern blot analysis of endogenous proviral integrations was performed using high molecular weight DNA isolated from NOD thymus (control) and thymic tumor cells from each genotype and digested with PvuII as described in "Materials and Methods." The blot was probed with 32P-labeled pEco-env probe, which only hybridizes to a single 3.7-kb germ-line fragment in NOD control (arrow). Each lane represents the DNA isolated from the thymus of one animal from each genotype. The results are from one experiment representing analysis of DNA from two NOD, one NOD.scid, three NOD.µMT, and three NOD.RAG-2-/- mice.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NOD was first described as an inbred strain with spontaneous autoimmune T1D (8) . It has since become a model for defects in T-cell tolerance, apoptosis, antigen-presenting and natural killer cells, leukocyte infiltration into pancreatic islets, thyroid, salivary and lacrimal glands, and when congenic for the scid mutation, an optimal host to support human hematopoiesis and leukemia cell growth (16 , 35) . However, the high incidence of thymic lymphoma in NOD.scid has imposed limitations on its use as a xenograft recipient. Here we show that either B-cell or T + B-cell immunodeficiency can reveal the susceptibility of NOD mice to lymphoma that can affect both the T- and B-cell lineages. Importantly, this lymphoma risk is greatest in NOD females, just as has been observed for T1D incidence. One implication of these data is that susceptibility to T1D and lymphoma in NOD mice reflects defects in lymphocyte homeostasis, resulting in dysregulated proliferation and apoptosis resistance. The contribution of apoptosis resistance to both autoimmune disease and lymphoma is well described in autoimmune lymphoproliferative syndrome of childhood. In autoimmune lymphoproliferative syndrome patients, inactivating mutation of the TNFRSF6 gene that encodes the Fas protein results in autoimmune disease and a high incidence of lymphoma (36) . In Sjögren’s syndrome, an autoimmune disease of the salivary glands, the severity of lymphoproliferation ranges from benign to dysplastic lymphoma (37) . In both Sjögren’s and autoimmune thyroiditis patients, lymphoid follicle-like structures involving germinal centers that resemble frank lymph nodes are observed in the involved secretory tissues. In NOD mice, genetic linkage analysis of T1D disease susceptibility has identified loci that include genes controlling lymphocyte survival, proliferation, and apoptosis (38, 39, 40, 41) . Furthermore, functional studies of NOD T and B lymphocytes demonstrate abnormally vigorous proliferation in response to self-antigen (42 , 43) and aberrant CTLA-4 regulation (44 , 45) , as well as resistance to apoptotic stimuli (39 , 46 , 47) . These results support the contribution of enhanced lymphocyte survival and proliferation to the pathogenesis of both T1D and lymphoid malignancy. Because the genetic loci controlling these cellular phenotypes are identified and isolated in congenic strains, it will be possible to directly test their contribution to both disease processes. Future experiments to further delineate the role of these genetic loci in NOD lymphoma susceptibility include backcrossing the RAG-2-/- and IgµMT mutations to NOD strains congenic for T1D susceptibility genes linked to lymphocyte homeostasis functions. Such studies will provide valuable insights into the genes that regulate lymphocyte response to immune deficiency and contribute to NOD lymphoma development.

A surprising aspect of lymphoma incidence in NOD.scid, NOD.RAG-2-/-, and NOD.µMT mice was the bias toward females. Consistent with this observation, a gender bias in lymphoma incidence has also been documented in human common variable immunodeficiency (48) . Together, these observations suggest that immunodeficiency alone may be a significant risk factor for lymphoma development in females. This gender bias is also reminiscent of the higher T1D incidence in females compared with NOD males (8 , 12) . Numerous studies in NOD mice show that suppression of female hormones by oophorectomy or the castration of males depresses or elevates T1D incidence, respectively (reviewed in Ref. 49 ). Presumably, the higher incidences of T1D in female NOD and lymphoma in female immunodeficient NOD mice reflect hormonal contribution in lymphocyte homeostasis. The effects of male hormones on lymphocyte growth regulation have been described (50 , 51) . However, the molecular basis of these observations has not been fully elucidated. Additional experiments are warranted to determine whether hormonal manipulation in immunodeficient NOD mice can reverse the incidence of tumor development in the respective gender groups.

The RAG-1 and RAG-2 mutations bred onto the 129/B6 backgrounds result in scid but has not been shown to be associated with an elevated risk of lymphoma development (23 , 24) . Recently, it was reported that NOD mice mutant in RAG-1 (NOD.RAG-1-/-) are lymphoma prone (29) , consistent with our observations of NOD.RAG-2-/- mice. The concordant results from both RAG-deficient NOD strains demonstrate that defective DSB repair is not required for the high incidence of NOD lymphoid tumors. In addition, we showed that lymphomas in NOD.RAG-2-/- mice can be of pro-T- or pro-B-cell origin, demonstrating that both lineages are susceptible to malignant transformation. Furthermore, we show here that NOD.µMT lymphomas can display a mature CD4+ T-cell phenotype, reminiscent of human acute T-cell lymphoma, suggesting that NOD lymphocytes at various stages of maturation are vulnerable to malignant transformation. Interestingly, the onset of lymphoma occurs at an age coincident with the onset of T1D in some NOD.µMT mice (52) . Previous studies have reported that NOD.µMT mice are diabetes resistant (26 , 31) or develop diabetes at a lower incidence (52) compared with age-matched NOD mice. Perhaps progression to lymphoma protects NOD.µMT from T1D because (pre-)neoplastic clones compete favorably against normal T cells for expansion, and the frequency of neoplastic CD4+ T-cell clones bearing an islet ß-cell-reactive antigen receptor would be very low.

A central rationale of this study was to uncouple the mechanism of defective DNA repair from a deficiency of mature T and B lymphocytes in the incidence, latency, and phenotype of NOD lymphoma. The high incidence of thymic lymphoma in NOD.scid mice compared with C.B.17.scid animals clearly implicates modifier genes, including those regulating mobilization of endogenous proviruses (17) , but does not reveal the contribution of defective DSB repair. We found that lymphoid malignancies develop at high incidence in the presence of wild-type DSB repair and, in some cases, in the absence of somatic integration of Emv30. Furthermore, we found that NOD.RAG-2-/- and NOD.µMT mice were susceptible to the development of multiple classes of lymphoma, including precursor T-cell lymphoblastic lymphoma and small T-cell and small B-cell lymphomas (33) . The incidence of lymphoid malignancies was higher and the latency of onset shorter in combined T- and B-immunodeficient NOD.scid and NOD.RAG-2-/- mice compared with the B-cell-deficient NOD.µMT mice, suggesting a dose-dependent effect of immunodeficiency on lymphoma development. There are two potential explanations for this observation: (a) the absence of both T- and B-lymphocyte subsets results in a greater deficit in immunosurveillance than loss of B cells alone, allowing more frequent and rapid development of neoplastic disease; and (b) alternatively, the absence of both mature T and B cells facilitates more rapid expansion of lymphoid precursors than lack of B cells alone, where a normal T-cell compartment competes for space. The lymphopenic environment induces lymphocyte proliferation to repopulate the lymphoid compartment (reviewed in Ref. 53 ). This replicative stress, in the absence of normal lymphocyte homeostatic control, may result in the accumulation of transformed lymphocytes. The variety of lymphoma phenotypes observed in NOD.RAG-2-/- and NOD.µMT mice may reflect the dysregulated responses of both mature and immature lymphocytes to expand and fill the lymphoid compartment. Thus, the critical issue is that depletion of either or both lymphoid compartments is permissive for lymphocyte expansion, providing greater opportunity for acquisition of mutations.

There is evidence that proliferative pressure to restore lymphocyte homeostasis may affect lymphocyte susceptibility to malignant transformation. Data from human immunodeficiency disorders provide correlation between lymphocyte proliferation triggered by lymphopenia and the development of lymphomas. The incidence of lymphomas but not other tumors correlates with the severity of T-cell depletion in transplant recipients (54) and in HIV-infected patients (reviewed in Ref. 55 ). Additional lymphocyte proliferation, particularly that induced by viruses such as EBV (56 , 57) , significantly increases susceptibility to lymphoma development in immune-deficient patients (reviewed in Ref. 58 ). Correspondingly, infection of EBV-like murine herpesviruses in BALB/c mice results in lymphoma development (59) that is enhanced by T-cell-specific immunosuppressive treatments (59) . Thus, homeostatic mechanisms to restore immune competence can predispose to malignant changes within the lymphocyte compartment. Perhaps the best evidence favoring this interpretation for our data is the incidence of lymphoma compared with other tumors in NOD.RAG-2-/- and NOD.µMT mice. If the absence of immune surveillance was the critical factor, a high incidence of epithelial and mesenchymal neoplasias might be expected in NOD mice as in immunosuppressed organ transplant patients (60) . Confinement of the high tumor incidence only to multiple forms of lymphoma is consistent with the idea that NOD genetic modifiers affect lymphocyte homeostasis, and in the absence of normal lymphocyte maturation, these precursors have opportunities for massive expansion. Thus, immunodeficiency is sufficient to be a significant risk factor for lymphomagenesis in genetically susceptible individuals. The contributions of immune deficiency and dysregulated lymphocyte homeostasis to NOD lymphoma development may be further assessed through reconstitution of the lymphocyte compartment in immune-deficient NOD mice with variable doses of T and/or B cells. By modulating the size of the lymphocyte compartment, the proliferative activity of both donor and recipient lymphocytes can be monitored, and their roles in NOD lymphomagenesis can be determined. These studies may help to quantify the threshold at which the severity of immune deficiency will trigger lymphocyte proliferation in immune-deficient NOD mice and perhaps the onset of lymphoma. Insights into the role of immune deficiency in lymphoma susceptibility will provide valuable tools in the prevention of lymphomas in immunosuppressed patients.


   ACKNOWLEDGMENTS
 
We thank Ildiko Grandal and the staff of The Hospital for Sick Children Research Institute vivarium for technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Juvenile Diabetes Research Foundation 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 Disease Network. P. P. L. C. is the recipient of a Juvenile Diabetes Research Foundation Postdoctoral Fellowship. Back

2 To whom requests for reprints should be addressed, at Program in Developmental Biology, The Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada. Phone: (416) 813-6450; Fax: (416) 813-8823; E-mail: jayne.danska{at}sickkids.ca Back

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 combined immune deficiency; RAG, recombinase-activating gene; AV-PE, streptavidin-phycoerythrin; PI, propidium iodide; LN, lymph node; FACS, fluorescence-activated cell sorting; TCR T-cell receptor. Back

Received 5/ 1/02. Accepted 8/16/02.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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