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Fatal Leukemia in Interleukin-15 Transgenic Mice
Fehniger et al.
Blood Cells, Molecules, and Diseases (2001) 27(1) Jan/Feb: 223–230 doi:10.1006/bcmd.2001.0379, available online at http://www.idealibrary.com on
Fatal Leukemia in Interleukin-15 Transgenic Mice Submitted 12/18/00 (Communicated by M. Lichtman, M.D., 12/22/00)
Todd A. Fehniger,1,2 Kazuhiro Suzuki,1,3 Jeffrey B. VanDeusen,1,2 Megan A. Cooper,1,2 Aharon G. Freud,1 and Michael A. Caligiuri1,2 ABSTRACT: The role of inflammation in the early genesis of certain malignancies has recently been appreciated. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms, suggesting that inappropriate expression of IL-15 may be detrimental to the host. We recently engineered a transgenic mouse in which the normal posttranscriptional control of IL-15 is eliminated, thereby overexpressing the murine IL-15 protein. IL-15 transgenic mice have early expansions in NK and CD8⫹ T lymphocytes and later develop fatal lymphocytic leukemia with a T-NK phenotype. This article recapitulates the phenotype of these IL-15 transgenic mice and discusses the utility of this model as a tool to further our understanding of leukemogenesis. © 2001 Academic Press
of chronic inflammation, exposure to inflammatory mediators during the process of normal tissue regeneration may predispose proliferating cells to DNA damage, downregulation of tumor suppressor gene products, and ultimately malignant transformation. Additional studies are needed to examine the potential role of inflammation and proinflammatory cytokine expression in the genesis of various malignant neoplasms. Leukemia is a complex, heterogeneous disorder with multiple molecular etiologies (4). However, direct evidence that mediators of inflammation can, via alterations in proliferation or survival, contribute to leukemogenesis is lacking. We hypothesized that deregulation of IL-15 gene expression, resulting in alterations in lymphocyte homeostasis, could promote malignant transformation in lymphocytes. We tested this hypothesis in vivo by engineering transgenic mice that lack posttranscriptional control of IL-15 gene expression, thereby efficiently translating and secreting murine IL-15 protein. This article
INTRODUCTION Recently, a connection between chronic inflammatory processes and the genesis of cancer has been appreciated. Inflammation resulting from persistent infections has been linked to malignancies, including Helicobacter pylori and gastric carcinoma, schistosomiasis and bladder cancer, as well as hepatitis C virus and hepatocellular carcinoma (1). The increased production of macrophage migration inhibitory factor (MIF) during H. pylori infection, a molecule that downregulates the p53 tumor suppressor gene during inflammation, provided one direct mechanism whereby the proinflammatory state may result in susceptibility to transforming genetic mutation (2). Further, individuals with polymorphisms at the IL-1 gene, resulting in increased expression of proinflammatory IL-1 during H. pylori infection, have a higher risk of developing gastric cancers (3). Collectively, these studies suggest that in situations
Correspondence and reprint requests to: Michael A. Caligiuri, Ohio State University, 458A Starling-Loving Hall, 320 West 10th Avenue, Columbus, OH 43210. Fax: (614) 293-7522. E-mail: [email protected]. 1 Department of Internal Medicine, Division of Hematology/Oncology, Ohio State University, Columbus, Ohio 43210. 2 Department of Molecular Virology, Immunology, and Medical Genetics, Division of Human Cancer Genetics and the Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210. 3 Department of Urology, Gunma University School of Medicine, Gunma, Japan.
1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved
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FIG. 1. Structure of the IL-15 transgene. Three primary posttranscriptional checkpoints were eliminated: 5⬘ AUGs, the inefficient IL-15 signal peptide, and a C-terminus retention sequence. Near global overexpression of this modified IL-15 cDNA was driven by the MHC class I Dd promoter (36), and the 3⬘ portion of the human growth hormone gene was spliced downstream and out of frame to maximize transcription, translation, and processing of the transgene in vivo (37).
IL-15 TRANSGENIC MICE LACK THE NORMAL POSTTRANSCRIPTIONAL REGULATION OF IL-15 AND THEREBY OVEREXPRESS MURINE IL-15 PROTEIN
summarizes the phenotype and utility of IL-15 transgenic mice as a model of leukemogenesis. IL-15 BIOLOGY
Despite an abundance of transcript in multiple tissues and cell types, IL-15 is poorly translated and secreted. Three primary posttranscriptional checkpoints are responsible for this observation: multiple AUGs in the 5⬘ UTR (6, 22), inefficient long (LSP) and short (SSP) signal peptides (23, 24), and a negative regulator near the C-terminus of the precursor proteins (24). Through the systematic elimination of these three checkpoints the synthesis of bioactive human IL-15 protein increased 250-fold in vitro (25). Such tight posttranscriptional control of the IL-15 gene product is unusual for most cytokines thus far characterized, suggesting that constitutively abundant IL-15 protein may somehow be deleterious to the host. Previous reports have identified major posttranscriptional regulatory mechanisms that control human IL-15 translation and secretion (9, 24, 25). We constructed a modified murine IL-15 cDNA that lacked the three primary posttranscriptional checkpoints controlling wild type IL-15, thereby optimizing for IL-15 overexpression (Fig. 1) (26). The transgene transcript, now efficiently translated and secreted, was quantified and found to be abundantly expressed in multiple tissues
Interleukin (IL)-15 is a pleiotropic cytokine that is important for both innate and adaptive immune cell homeostasis, as well as peripheral immune function (5). IL-15 shares the common ␥ chain (␥c) and IL-2/15R with IL-2 for signaling, but also utilizes a private IL-15R␣ subunit for high affinity binding (6 – 8). Numerous in vitro and in vivo studies have documented a critical role for IL-15 in the development, survival, and function of the natural killer (NK) cell lineage (9 –16). Further, IL-15 is required for the normal expansion and/or survival of nonclassical T cells and memory-phenotype TCR␣ CD8⫹ T cells, while not being essential for their development (15–17). These studies are consistent with the broad expression of IL-15 and IL-15R␣ by multiple cell types and tissues, suggesting that this ligand/receptor may mediate a wide range of functions in vivo (6, 8). Indeed, the phenotypes of mice deficient in IL-15/IL-15R␣ (15, 16), compared to mice deficient in IL-2/IL-2R␣ (18 –20), definitively demonstrate a large variety of unique in vivo functions mediated by IL-15 (21). 224
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from IL-15tg mice, in a similar pattern as the endogenous IL-15 transcript (6). The transgenic protein was detected by immunoblot analysis of the FLAG epitope and in serum by ELISA. The phenotype of IL-15tg mice, reviewed below, has recently been described in detail (26).
signs progress for about 3–7 days when the mice become acutely moribund and are sacrificed (⬃20 weeks of age). In evaluable IL-15tg mice, a massive elevation in peripheral blood leukocyte number, consisting mainly of lymphocytes, was observed (average 150,000 –200,000/l blood). Flow cytometric analysis revealed that while DX5⫹CD3⫺ NK cells remained elevated, the major lymphocyte population was consistently CD3⫹TCR⫹DX5hi/lo/neg T cells. Further, the expanded CD3⫹ T cells and NK cells were observed in multiple lymphoid tissues including blood, spleen, and bone marrow (Fig. 2A). More detailed analysis of the expanded lymphocyte populations demonstrated a consistent phenotype of CD3⫹TCR⫹CD2⫹CD5⫹CD44⫹CD8⫹/⫺DX5hi/lo/neg. In two additional cases, CD3⫺TCR⫺CD8⫺CD4⫺DX5⫹ lymphocyte populations were expanded, likely representing a murine NK cell leukemia, while in no case was CD4 expression observed. Analysis of peripheral blood smears confirmed the dramatic lymphocyte expansion, some of which had striking blast morphology features and prominent nucleoli (Fig. 2B). Molecular studies and flow cytometry were used to evaluate clonality, and through such experiments expanded lymphocytes from 9 of 19 (48%) IL-15tg mice were identified as clonal, consistent with leukemia.
EARLY LYMPHOCYTOSIS IN IL-15 TRANSGENIC MICE Previous studies have documented a role for IL-15 in the homeostasis of several lymphocyte subsets (5). Consistent with this, IL-15tg mice exhibited a significant increase in lymphocytes at 6 – 8 weeks of age, compared to sex- and agematched nontransgenic littermate controls. Examination of peripheral blood smears from IL-15tg mice confirmed the expansion of small and large granular lymphocytes. IL-15, acting through the IL-15R complex, has a requisite role in NK cell lineage development (15, 16), and provision of exogenous IL-15 expands murine NK cells in vivo (16, 27). Immunophenotyping the peripheral blood of IL-15tg mice at 6 – 8 weeks of age revealed that the major population of expanded lymphocytes was DX5⫹CD3⫺ NK cells. IL-15 has also been shown to be critical for memoryphenotype CD8⫹ T cell expansion and/or survival in mice (15–17, 28). While there was no difference in absolute CD4⫹ T cell counts between IL-15tg and wild type mice, CD8⫹ T cells were significantly increased in the transgenics. Further phenotypic analysis of these expanded CD8⫹ T cells showed that they are CD44hiCD69⫺Ly6Chi, consistent with a memory-phenotype. Thus, transgenic overexpression of murine IL-15 results in early expansion in NK and CD8⫹CD44hi T cells in mice, in vivo.
PROGRESSIVE ALOPECIA AND MULTIORGAN LYMPHOCYTIC INFILTRATION OF IL-15 TRANSGENIC MICE IL-15tg mice that developed striking lymphocyte expansions were remarkable for whole-body alopecia, hepatosplenomegaly, and lymphadenopathy. Microscopic examination of tissues harvested from these animals showed diffuse lymphocytic infiltrates, most prominent in the peritoneum and surrounding intraabdominal organs. In addition, all showed marked skin involvement with effacement of normal architecture, expansion of splenic white pulp, and prominent lung and liver infiltrates (Fig. 3). Interestingly, the pattern of cutaneous involvement found in these animals is very similar to that seen in patients with NK/T cell lymphomas (29).
IL-15 TRANSGENIC MICE DEVELOP FATAL LYMPHOCYTIC LEUKEMIA WITH AGE, FOLLOWING CHRONIC LYMPHOCYTOSIS Between 12 and 30 weeks of age, most IL-15tg mice develop a cluster of signs including decreased activity, weight loss, and difficulty breathing. These 225
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FIG. 2. IL-15tg mice develop fatal lymphocytic leukemia with age. (A) Flow cytometric analyses of peripheral blood lymphocytes, spleen, and bone marrow isolated from a representative IL-15tg mice immediately prior to death. While the DX5⫹CD3⫺ NK cell expansion persisted, the major lymphocyte population is CD3⫹TCR⫹DX5⫹ T cells. (B) Photomicrographs (100⫻) illustrating the morphology of the leukemic lymphocytes from peripheral blood smears of four representative IL-15tg mice.
THE EVOLUTION OF LEUKEMIA IN IL-15 TRANSGENIC MICE: A USEFUL TOOL TO BETTER UNDERSTAND LEUKEMOGENESIS?
IL-15 has been shown to drive NK cell differentiation from human (11, 30) and murine (31) IL-2/15R⫹ NK cell precursors in the bone marrow, as well as support mature NK cell survival in the absence of serum or other factors (12). This suggests that multiple mechanisms, including increased differentiation and prolonged survival in the periphery, are likely responsible for the accumulation of NK cells observed in IL-15tg mice. Considering the greatest expansion is in the NK cell compartment, why then do primarily T cellphenotype leukemias develop? Intriguingly, this pattern mimics the phenotypic distribution found within LGL leukemia patients (32). We hypothesize that the early, limited exposure to IL-15 benignly expands NK and CD8⫹ T cells. However, during this expansion there occurs abnormally prolonged survival and/or abnormally high transit through the cell cycle of IL-15responsive lymphocytes. Such chronic proliferation and/or extended survival of lymphocytes in IL-15tg mice likely contribute to the accumula-
IL-15tg mice exhibit peripheral blood lymphocytosis, with significant expansions in both the NK cell and memory-phenotype CD8⫹ TCR␣ T cell compartments, early in life. These results demonstrate that IL-15 is a growth factor for these cell types in vivo and are consistent with gene targeting studies demonstrating that IL15R␣⫺/⫺ and IL-15⫺/⫺ mice lack NK cells and are severely deficient in memory/activated-phenotype CD8⫹ T cells (15, 16). However, continual translation and secretion of the proinflammatory cytokine in the IL-15tg mice over several months eventually led to the manifestation of clonal lymphocytic leukemia in a significant fraction of mice. What is the mechanism whereby the benign lymphocyte expansion transforms into malignancy? 226
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FIG. 3. Multiorgan lymphocytic infiltration in IL-15tg mice. Histology sections of skin and lung stained with hematoxylin and eosin. Low-power (10⫻) micrographs contrast wild-type and IL-15tg tissues. High power (40⫻) micrographs demonstrate the lymphocytic morphology of the infiltrating cells in the IL-15tg mice.
tion of additional transforming mutations (Fig. 4), as has been postulated for other cancers (1). In support of this, IL-15 has been shown to costimulate stem cell proliferation (11), and extend the survival of normal NK cells (12) and T cells (33) by preventing apoptosis. Current experiments are focused on determining whether extended survival and/or increased cell cycle transit are responsible for the benign lymphocyte expansion and subsequent leukemic phase. Further, studies to sequentially analyze these cells for such secondary genetic alterations are also underway, with the hope of providing new insights into the pathogenesis of lymphocytic leukemia.
toward potential similarities to cutaneous lymphoid malignancies (e.g., Sezary syndrome or mycosis fungoides), and possibly rare forms of T-chronic lymphocytic leukemia (CLL). Further, the chronic lymphocytosis and subsequent leukemia observed in IL-15tg mice share many clinical features and manifestations with the human disease large granular lymphocytic (LGL) leukemia (32). These include extra-lymphoid involvement, a chronic course evolving to an acute expansion, and the prevalence of T cell subtypes. Likewise, the histopathology of the lymphocytic infiltrates in IL-15tg mice resembles those observed in NK-T lymphomas (29, 34). Lymphocytes isolated from patients with LGL leukemia express all three components of the IL-15R complex (␣␥), and increased IL-15 expression was noted in macrophages from these patients (35). In addition, early in vitro propagation of LGL leukemia cell lines can be maintained in the presence of IL-15. We
IS THE LYMPHOCYTIC LEUKEMIA WITHIN IL-15 TRANSGENIC MICE RELATED TO HUMAN MALIGNANCY? The striking skin involvement, as well as the pattern and phenotype of leukemic cells points 227
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proinflammatory cytokine stimulation. In a subset of mice, these expansions are clonal and portray a clinical picture consistent with T-NK leukemia. Thus, the IL-15 transgenic mouse may provide a useful model to study the molecular changes during the evolution of lymphocytic leukemia, thereby identifying genes important in leukemogenesis. Equally important, the phenotype of IL15tg mice adds to the growing body of evidence linking chronic inflammation and proinflammatory cytokine expression to the subsequent development of malignancy.
ACKNOWLEDGMENTS This work was supported by Grants CA-68458, CA65670, and P30CA-16058 from the National Institutes of Health. T. A. Fehniger is the recipient of Medical Scientist Program (MSP) and Bennett Fellowships from the Ohio State University College of Medicine. J. B. VanDeusen is supported by T-32 CA-09498 and M. A. Cooper by the HHMI Medical Student Research Fellowship. This paper was presented at a Focused Workshop on Animal Models of Leukemia and Lymphoma jointly sponsored by the Leukemia & Lymphoma Society and the Mouse Models of Human Cancer Consortium of the National Cancer Institute on September 19 –20, 2000.
FIG. 4. The evolution of leukemia in IL-15tg mice: A model to study the molecular events during lymphocyte transformation. This schematic summarizes the hypothesis that early, limited exposure to IL-15 results in a benign lymphocytosis in IL-15tg mice. Later, after months of chronic IL-15 stimulation, some lymphocytes within IL15tg accumulate genetic mutations that ultimately lead to malignant transformation and leukemia. Comparison of gene expression during benign lymphocytosis to the subsequent leukemic cells may allow identification of genes important in leukemogenesis.
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Fatal Leukemia in Interleukin-15 Transgenic Mice Submitted 12/18/00 (Communicated by M. Lichtman, M.D., 12/22/00)
Todd A. Fehniger,1,2 Kazuhiro Suzuki,1,3 Jeffrey B. VanDeusen,1,2 Megan A. Cooper,1,2 Aharon G. Freud,1 and Michael A. Caligiuri1,2 ABSTRACT: The role of inflammation in the early genesis of certain malignancies has recently been appreciated. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms, suggesting that inappropriate expression of IL-15 may be detrimental to the host. We recently engineered a transgenic mouse in which the normal posttranscriptional control of IL-15 is eliminated, thereby overexpressing the murine IL-15 protein. IL-15 transgenic mice have early expansions in NK and CD8⫹ T lymphocytes and later develop fatal lymphocytic leukemia with a T-NK phenotype. This article recapitulates the phenotype of these IL-15 transgenic mice and discusses the utility of this model as a tool to further our understanding of leukemogenesis. © 2001 Academic Press
of chronic inflammation, exposure to inflammatory mediators during the process of normal tissue regeneration may predispose proliferating cells to DNA damage, downregulation of tumor suppressor gene products, and ultimately malignant transformation. Additional studies are needed to examine the potential role of inflammation and proinflammatory cytokine expression in the genesis of various malignant neoplasms. Leukemia is a complex, heterogeneous disorder with multiple molecular etiologies (4). However, direct evidence that mediators of inflammation can, via alterations in proliferation or survival, contribute to leukemogenesis is lacking. We hypothesized that deregulation of IL-15 gene expression, resulting in alterations in lymphocyte homeostasis, could promote malignant transformation in lymphocytes. We tested this hypothesis in vivo by engineering transgenic mice that lack posttranscriptional control of IL-15 gene expression, thereby efficiently translating and secreting murine IL-15 protein. This article
INTRODUCTION Recently, a connection between chronic inflammatory processes and the genesis of cancer has been appreciated. Inflammation resulting from persistent infections has been linked to malignancies, including Helicobacter pylori and gastric carcinoma, schistosomiasis and bladder cancer, as well as hepatitis C virus and hepatocellular carcinoma (1). The increased production of macrophage migration inhibitory factor (MIF) during H. pylori infection, a molecule that downregulates the p53 tumor suppressor gene during inflammation, provided one direct mechanism whereby the proinflammatory state may result in susceptibility to transforming genetic mutation (2). Further, individuals with polymorphisms at the IL-1 gene, resulting in increased expression of proinflammatory IL-1 during H. pylori infection, have a higher risk of developing gastric cancers (3). Collectively, these studies suggest that in situations
Correspondence and reprint requests to: Michael A. Caligiuri, Ohio State University, 458A Starling-Loving Hall, 320 West 10th Avenue, Columbus, OH 43210. Fax: (614) 293-7522. E-mail: [email protected]. 1 Department of Internal Medicine, Division of Hematology/Oncology, Ohio State University, Columbus, Ohio 43210. 2 Department of Molecular Virology, Immunology, and Medical Genetics, Division of Human Cancer Genetics and the Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210. 3 Department of Urology, Gunma University School of Medicine, Gunma, Japan.
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FIG. 1. Structure of the IL-15 transgene. Three primary posttranscriptional checkpoints were eliminated: 5⬘ AUGs, the inefficient IL-15 signal peptide, and a C-terminus retention sequence. Near global overexpression of this modified IL-15 cDNA was driven by the MHC class I Dd promoter (36), and the 3⬘ portion of the human growth hormone gene was spliced downstream and out of frame to maximize transcription, translation, and processing of the transgene in vivo (37).
IL-15 TRANSGENIC MICE LACK THE NORMAL POSTTRANSCRIPTIONAL REGULATION OF IL-15 AND THEREBY OVEREXPRESS MURINE IL-15 PROTEIN
summarizes the phenotype and utility of IL-15 transgenic mice as a model of leukemogenesis. IL-15 BIOLOGY
Despite an abundance of transcript in multiple tissues and cell types, IL-15 is poorly translated and secreted. Three primary posttranscriptional checkpoints are responsible for this observation: multiple AUGs in the 5⬘ UTR (6, 22), inefficient long (LSP) and short (SSP) signal peptides (23, 24), and a negative regulator near the C-terminus of the precursor proteins (24). Through the systematic elimination of these three checkpoints the synthesis of bioactive human IL-15 protein increased 250-fold in vitro (25). Such tight posttranscriptional control of the IL-15 gene product is unusual for most cytokines thus far characterized, suggesting that constitutively abundant IL-15 protein may somehow be deleterious to the host. Previous reports have identified major posttranscriptional regulatory mechanisms that control human IL-15 translation and secretion (9, 24, 25). We constructed a modified murine IL-15 cDNA that lacked the three primary posttranscriptional checkpoints controlling wild type IL-15, thereby optimizing for IL-15 overexpression (Fig. 1) (26). The transgene transcript, now efficiently translated and secreted, was quantified and found to be abundantly expressed in multiple tissues
Interleukin (IL)-15 is a pleiotropic cytokine that is important for both innate and adaptive immune cell homeostasis, as well as peripheral immune function (5). IL-15 shares the common ␥ chain (␥c) and IL-2/15R with IL-2 for signaling, but also utilizes a private IL-15R␣ subunit for high affinity binding (6 – 8). Numerous in vitro and in vivo studies have documented a critical role for IL-15 in the development, survival, and function of the natural killer (NK) cell lineage (9 –16). Further, IL-15 is required for the normal expansion and/or survival of nonclassical T cells and memory-phenotype TCR␣ CD8⫹ T cells, while not being essential for their development (15–17). These studies are consistent with the broad expression of IL-15 and IL-15R␣ by multiple cell types and tissues, suggesting that this ligand/receptor may mediate a wide range of functions in vivo (6, 8). Indeed, the phenotypes of mice deficient in IL-15/IL-15R␣ (15, 16), compared to mice deficient in IL-2/IL-2R␣ (18 –20), definitively demonstrate a large variety of unique in vivo functions mediated by IL-15 (21). 224
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from IL-15tg mice, in a similar pattern as the endogenous IL-15 transcript (6). The transgenic protein was detected by immunoblot analysis of the FLAG epitope and in serum by ELISA. The phenotype of IL-15tg mice, reviewed below, has recently been described in detail (26).
signs progress for about 3–7 days when the mice become acutely moribund and are sacrificed (⬃20 weeks of age). In evaluable IL-15tg mice, a massive elevation in peripheral blood leukocyte number, consisting mainly of lymphocytes, was observed (average 150,000 –200,000/l blood). Flow cytometric analysis revealed that while DX5⫹CD3⫺ NK cells remained elevated, the major lymphocyte population was consistently CD3⫹TCR⫹DX5hi/lo/neg T cells. Further, the expanded CD3⫹ T cells and NK cells were observed in multiple lymphoid tissues including blood, spleen, and bone marrow (Fig. 2A). More detailed analysis of the expanded lymphocyte populations demonstrated a consistent phenotype of CD3⫹TCR⫹CD2⫹CD5⫹CD44⫹CD8⫹/⫺DX5hi/lo/neg. In two additional cases, CD3⫺TCR⫺CD8⫺CD4⫺DX5⫹ lymphocyte populations were expanded, likely representing a murine NK cell leukemia, while in no case was CD4 expression observed. Analysis of peripheral blood smears confirmed the dramatic lymphocyte expansion, some of which had striking blast morphology features and prominent nucleoli (Fig. 2B). Molecular studies and flow cytometry were used to evaluate clonality, and through such experiments expanded lymphocytes from 9 of 19 (48%) IL-15tg mice were identified as clonal, consistent with leukemia.
EARLY LYMPHOCYTOSIS IN IL-15 TRANSGENIC MICE Previous studies have documented a role for IL-15 in the homeostasis of several lymphocyte subsets (5). Consistent with this, IL-15tg mice exhibited a significant increase in lymphocytes at 6 – 8 weeks of age, compared to sex- and agematched nontransgenic littermate controls. Examination of peripheral blood smears from IL-15tg mice confirmed the expansion of small and large granular lymphocytes. IL-15, acting through the IL-15R complex, has a requisite role in NK cell lineage development (15, 16), and provision of exogenous IL-15 expands murine NK cells in vivo (16, 27). Immunophenotyping the peripheral blood of IL-15tg mice at 6 – 8 weeks of age revealed that the major population of expanded lymphocytes was DX5⫹CD3⫺ NK cells. IL-15 has also been shown to be critical for memoryphenotype CD8⫹ T cell expansion and/or survival in mice (15–17, 28). While there was no difference in absolute CD4⫹ T cell counts between IL-15tg and wild type mice, CD8⫹ T cells were significantly increased in the transgenics. Further phenotypic analysis of these expanded CD8⫹ T cells showed that they are CD44hiCD69⫺Ly6Chi, consistent with a memory-phenotype. Thus, transgenic overexpression of murine IL-15 results in early expansion in NK and CD8⫹CD44hi T cells in mice, in vivo.
PROGRESSIVE ALOPECIA AND MULTIORGAN LYMPHOCYTIC INFILTRATION OF IL-15 TRANSGENIC MICE IL-15tg mice that developed striking lymphocyte expansions were remarkable for whole-body alopecia, hepatosplenomegaly, and lymphadenopathy. Microscopic examination of tissues harvested from these animals showed diffuse lymphocytic infiltrates, most prominent in the peritoneum and surrounding intraabdominal organs. In addition, all showed marked skin involvement with effacement of normal architecture, expansion of splenic white pulp, and prominent lung and liver infiltrates (Fig. 3). Interestingly, the pattern of cutaneous involvement found in these animals is very similar to that seen in patients with NK/T cell lymphomas (29).
IL-15 TRANSGENIC MICE DEVELOP FATAL LYMPHOCYTIC LEUKEMIA WITH AGE, FOLLOWING CHRONIC LYMPHOCYTOSIS Between 12 and 30 weeks of age, most IL-15tg mice develop a cluster of signs including decreased activity, weight loss, and difficulty breathing. These 225
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FIG. 2. IL-15tg mice develop fatal lymphocytic leukemia with age. (A) Flow cytometric analyses of peripheral blood lymphocytes, spleen, and bone marrow isolated from a representative IL-15tg mice immediately prior to death. While the DX5⫹CD3⫺ NK cell expansion persisted, the major lymphocyte population is CD3⫹TCR⫹DX5⫹ T cells. (B) Photomicrographs (100⫻) illustrating the morphology of the leukemic lymphocytes from peripheral blood smears of four representative IL-15tg mice.
THE EVOLUTION OF LEUKEMIA IN IL-15 TRANSGENIC MICE: A USEFUL TOOL TO BETTER UNDERSTAND LEUKEMOGENESIS?
IL-15 has been shown to drive NK cell differentiation from human (11, 30) and murine (31) IL-2/15R⫹ NK cell precursors in the bone marrow, as well as support mature NK cell survival in the absence of serum or other factors (12). This suggests that multiple mechanisms, including increased differentiation and prolonged survival in the periphery, are likely responsible for the accumulation of NK cells observed in IL-15tg mice. Considering the greatest expansion is in the NK cell compartment, why then do primarily T cellphenotype leukemias develop? Intriguingly, this pattern mimics the phenotypic distribution found within LGL leukemia patients (32). We hypothesize that the early, limited exposure to IL-15 benignly expands NK and CD8⫹ T cells. However, during this expansion there occurs abnormally prolonged survival and/or abnormally high transit through the cell cycle of IL-15responsive lymphocytes. Such chronic proliferation and/or extended survival of lymphocytes in IL-15tg mice likely contribute to the accumula-
IL-15tg mice exhibit peripheral blood lymphocytosis, with significant expansions in both the NK cell and memory-phenotype CD8⫹ TCR␣ T cell compartments, early in life. These results demonstrate that IL-15 is a growth factor for these cell types in vivo and are consistent with gene targeting studies demonstrating that IL15R␣⫺/⫺ and IL-15⫺/⫺ mice lack NK cells and are severely deficient in memory/activated-phenotype CD8⫹ T cells (15, 16). However, continual translation and secretion of the proinflammatory cytokine in the IL-15tg mice over several months eventually led to the manifestation of clonal lymphocytic leukemia in a significant fraction of mice. What is the mechanism whereby the benign lymphocyte expansion transforms into malignancy? 226
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FIG. 3. Multiorgan lymphocytic infiltration in IL-15tg mice. Histology sections of skin and lung stained with hematoxylin and eosin. Low-power (10⫻) micrographs contrast wild-type and IL-15tg tissues. High power (40⫻) micrographs demonstrate the lymphocytic morphology of the infiltrating cells in the IL-15tg mice.
tion of additional transforming mutations (Fig. 4), as has been postulated for other cancers (1). In support of this, IL-15 has been shown to costimulate stem cell proliferation (11), and extend the survival of normal NK cells (12) and T cells (33) by preventing apoptosis. Current experiments are focused on determining whether extended survival and/or increased cell cycle transit are responsible for the benign lymphocyte expansion and subsequent leukemic phase. Further, studies to sequentially analyze these cells for such secondary genetic alterations are also underway, with the hope of providing new insights into the pathogenesis of lymphocytic leukemia.
toward potential similarities to cutaneous lymphoid malignancies (e.g., Sezary syndrome or mycosis fungoides), and possibly rare forms of T-chronic lymphocytic leukemia (CLL). Further, the chronic lymphocytosis and subsequent leukemia observed in IL-15tg mice share many clinical features and manifestations with the human disease large granular lymphocytic (LGL) leukemia (32). These include extra-lymphoid involvement, a chronic course evolving to an acute expansion, and the prevalence of T cell subtypes. Likewise, the histopathology of the lymphocytic infiltrates in IL-15tg mice resembles those observed in NK-T lymphomas (29, 34). Lymphocytes isolated from patients with LGL leukemia express all three components of the IL-15R complex (␣␥), and increased IL-15 expression was noted in macrophages from these patients (35). In addition, early in vitro propagation of LGL leukemia cell lines can be maintained in the presence of IL-15. We
IS THE LYMPHOCYTIC LEUKEMIA WITHIN IL-15 TRANSGENIC MICE RELATED TO HUMAN MALIGNANCY? The striking skin involvement, as well as the pattern and phenotype of leukemic cells points 227
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proinflammatory cytokine stimulation. In a subset of mice, these expansions are clonal and portray a clinical picture consistent with T-NK leukemia. Thus, the IL-15 transgenic mouse may provide a useful model to study the molecular changes during the evolution of lymphocytic leukemia, thereby identifying genes important in leukemogenesis. Equally important, the phenotype of IL15tg mice adds to the growing body of evidence linking chronic inflammation and proinflammatory cytokine expression to the subsequent development of malignancy.
ACKNOWLEDGMENTS This work was supported by Grants CA-68458, CA65670, and P30CA-16058 from the National Institutes of Health. T. A. Fehniger is the recipient of Medical Scientist Program (MSP) and Bennett Fellowships from the Ohio State University College of Medicine. J. B. VanDeusen is supported by T-32 CA-09498 and M. A. Cooper by the HHMI Medical Student Research Fellowship. This paper was presented at a Focused Workshop on Animal Models of Leukemia and Lymphoma jointly sponsored by the Leukemia & Lymphoma Society and the Mouse Models of Human Cancer Consortium of the National Cancer Institute on September 19 –20, 2000.
FIG. 4. The evolution of leukemia in IL-15tg mice: A model to study the molecular events during lymphocyte transformation. This schematic summarizes the hypothesis that early, limited exposure to IL-15 results in a benign lymphocytosis in IL-15tg mice. Later, after months of chronic IL-15 stimulation, some lymphocytes within IL15tg accumulate genetic mutations that ultimately lead to malignant transformation and leukemia. Comparison of gene expression during benign lymphocytosis to the subsequent leukemic cells may allow identification of genes important in leukemogenesis.
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