Murine Models of Thymic Lymphomas: Premalignant Scenarios Amenable to Prophylactic Therapy

AD\’ANCES IN IMMUNOLOGY. VOI. 73

Murine Models of Thymic Lymphomas: Premalignant Scenarios Amenable to Prophylactic Therapy ElTAN YEFENOF The butenberg Center for General and Tumor Immunology, The Habrew University-Hadassah Medical

Center, knrM/em 91 120, Ismel

1. Introduction

Retroviruses that induce thymic lymphomas became important in the field of immunology owing to their instrumental role in the discovery of T lymphocytes and T cell immunological function (Miller, 1961 a,b). Concomitantly, they became a powerful tool for the study of the multistep nature of tumor development and the genomic modifications involved in lymphoma progression (Corcoran et nl., 1984; Cuypers et al., 1984; Li et nl., 1984; O’Donnel et al., 1985; Selten et al., 1984; Steffen, 1984). The landmark discovery by Ludwig Gross (1951) that spontaneous leukemias of thymic origin in susceptible AKR mice are induced by an endogenous retrovirus introduced a new era of molecular cancer research, which eventually led to the discovery of viral and cellular oncogenes (reviewed in Coffin, 1990). On another front, the work by Gross motivated the attempt to isolate retroviruses from many other types of cancers and to ascribe a viral etiology in a whole range of malignancies (Gross, 1978,1980).Although this search was futile in most instances (Weinberg, 1996; McCann, 1998),virdy induced tumors in general, and retrovirally induced thymic lymphomas in particular, provided experimental systems for the study of genetic, biochemical, cellular, and immunologcal aspects of tumorigenesis (reviewed in Tsichlis and Lazo, 1991). Oncogenic retroviruses isolated from a variety of animal species have been classified into two major categories (Varmus, 1984; Nusse, 1986): the acute viruses, which transform susceptible cells in vitro and induce tumors in vivo shortly after their inoculation, and the chronic viruses, which do not transform cells in vitro but induce tumors in vivo after a prolonged latency period. Chronic retroviruses are heterogeneous with regard to their structure, genome, susceptible host, tissue tropism, and the type of tumor induced. However, they share an essential common feature: they are nondefective, replication-competent viruses whose oncogenic activity depends on repeated cycles of infection and integration (Teich, 1982). Yet, because the genome of such viruses does not contain an oncogene, proviral integration into cellular DNA does not lead to immediate neoplastic transformation. Rather, it initiates a complex sequence of 51 1

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cellular and molecular events that gradually progress toward the development of a frank tumor (Varmus, 1988). A multitude of Factors underlying this progressive malignant transformation have been characterized (Tsichlis and L z o , 1991). Some of them (e.g., insertional mutagenesis) occur at the end stage of the latent period, whereas others (e.g., transactivation of cellular genes or receptor-mediated growth stimulation) begin to operate shortly after the initial infection. Early isolates of chronic retroviruses, such as Gross/AKR and Moloney leukemia virus, predominantly induced lymphomas and leukemias of thymic origin (reviewed in Teich et nl., 1983).Because identical tumors could be induced by X-rays, gamma irradiation, and alkalizing carcinogens (e.g., methylnitrosourea), it was proposed that these mutagens activate an endogenous retrovirus that is the common etiologic agent of thymic lymphomas (Fischinger et nl., 1981, 1982; Frei, 1980; Janowski et al., 1986). Indeed, in sporadic experiments, an oncogenic retrovirus was isolated from lymphomas arising in mice exposed to X-ray irradiation (Haran-Ghera, 1966; Kaplan, 1974). This virus induced primary thymic lymphomas when inoculated into susceptible mouse strains and accordingly has been termed radiation leukemia virus (RadLV) (Haran-Ghera, 1966; Kaplan, 1967; Haran-Ghera, 1971; Decleve et al., 1974; Lieberman et al., 1978). Subsequent experiments, however, have indicated that induction of lymphomas by radiation does not involve a viral etiology, the resemblance between X-ray- and RadLV-induced lymphomas notwithstanding (Ihle et al., 1976a,b, Yefenof, 1980; Yefenof et al., 1980a,b, Janowski et al., 1990). RadLV is, thus, yet another isolate of an oncogenic endogenous retrovirus that is reactivated in radiation lymphomas on repeated transfers in uiuo or propagation in vivo, and is not the primary cause of X-ray-induced lymphomagenesis. Even though murine leukemia retroviruses can infect a range of cell types and tissues in uitro, the outcome of their in vivo inoculation is, by and large, a lymphoma of thymic origin (Coffin, 1990; Tsichlis and Lazo, 1991). Moreover, in the case of AKR lymphomas, the genetically transmitted ecotropic virus is not leukeogenic by itself (Rowe, 1972; Chattopadhyay et al., 1980; Yanagihara et nl., 1982). Paradoxically, however, its recombination with an endogenous xenotropic virus, which increases the spectrum of susceptible target cells to viral infection on the one hand, generates an oncogenic virus that induces exclusively thymic lymphomas on the other hand (O’Donnell et al., 1981;Herr and Gilbert, 1983). Likewise, irradiation or methylnitrosourea is mutagenic in a whole range of cells in uitro, yet in vivo such treatments produce malignancies of a predominantly thymic source (Lieberman and Kaplan, 1959; Frei, 1980; Frei and Lawley, 1980; Newcornb et al., 1988). A general conclusion that can be drawn from

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these observations is that the mutagenic potential of several, independent carcinogens is realized most effectively within the thymic microenvironment. What makes the lymphocytes of the thymus a preferential target for leukemogenesis? Research on thymocyte development during the past decade has highlighted the unique function of the thymus as a primary lymphoid gland that sustains the differentiation of T lymphocytes and shapes the repertoire of immunocompetent T cells (reviewed in Fowlkes and Pardoll, 1989; Anderson et al., 1996). These features, summarized in the following section, account for the high susceptibility of thymic lymphocytes to transformation by the aforementioned experimental modalities. II. lmmunobiology of the Thymus in Relation to Lymphomagenesis

The thymic microenviroriment is unique in its ability to uphold the complex process required for generation of immunocompetent T lymphocytes. Similar to all other types of blood cells, T cells are descended from hematopoietic stem cells (HSCs) residing in the bone marrow (Ikuta et al., 1992). An intermediate step in the differentiation of HSCs along the T lymphoid lineage is the formation of prothymocyte precursor cells in the bone marrow, which are recruited to populate the thymus by chemoattractants produced in thymic stromal cells (Champion et al., 1986; Imhof et al., 1988; Bauvois et al., 1989; Deugnier et al., 1989; Carr et al., 1994). On entering the thymus, the prothymocytes engage in a complex sequence of interactions with nonlymphoid stromal cells, leading to their further differentiation and maturation (Boyd et al., 1993; Scollay and Godfrey, 1995). During their intrathymic residence, which lasts 4-5 days, the prothymocytes travel from the corticomedullary junction through the thymic parenchyma and toward the subcapsular cortex (Boyd and Hugo, 1991; Van-Ewijk, 1991; Van-Ewijk et al., 1994). From there the differentiating thymocytes migrate down the cortex and enter the thymic medulla as mature T lymphocytes. While migrating through the intrathymic compartments, the thymocytes proliferate, generating a sufficiently large number of TCR-aP+ and TCR-yF cells for repertoire selection (Von-Boehmer, 1990; Jameson et al., 1995). Concomitantly, they acquire receptors for growth factors and adhesion molecules, and costimulatory receptors such as CD4, CD8, and CD28 (Scollay, 1991; Anderson et al., 1996; Anderson and Jenkinson, 1997). The temporal order of these events ensures the shaping of the T cell repertoire such that T cells acquiring receptors with a high affinity for self-antigenshelf-MHC are eliminated via apoptosis, whereas those expressing receptors with an affinity to foreign peptided self-MHC mature and survive (Huesmann et al., 1991; Ignatowicz et al.,

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1996, Janeway et al., 1998). Eventually, the positively selected T cells leave the thymus and populate secondary lymph nodes, where they provide immune protection against potentially hazardous foreign antigens. Hence, the thymus is a dynamic gland in which lymphocytes undergo extensive proliferation, differentiation, and maturation processes predicated on genetic activation, repression, and recombinatorial events operating in individual cells within a short window of time. These modifications are mandatory for the maintenance of thymic physiology and function, but at the same time they create a state of genetic instability that renders the population of thymic lymphocytes susceptible to viral, chemical, and radiation carcinogenesis. These events also contribute to the activation of endogeneous retroviral sequences and their recombinations, leading to the generation of a carcinogenic retrovirus, as in the highly leukemic AKR mouse strain. 111. Thymic lymphomas of AKR Mice

The AKR inbred mouse strain has been instrumental in stud>ringthe steps underlying retroviral transformation of thymic lymphomas (Rowe, 1978). At least two proviral sequences are carried by the AKR germ line at two distant chromosomal loci designated AKV-1 and AKV-2 (Rowe, 1972; Chattopadhyay et al., 1980).These endogenous viruses are expressed early in the course of embryogenesis, causing virernia from birth on (O’Donnelet nl., 1981; Famulari, 1983),but none of them is leukemogenic per se (Yanagihara et al., 1982; Fredrickson et al., 1984). However in mice 5-6 months old, the viruses undergo recombinational events with endogenous, xenotropic viral sequences, resulting in the cle navo production of a dual tropic virus (DTV),also known as mink cell focus-forming (MCF) virus, due to its ability to infect mink cells in v i t m (Fischinger et nl., 1975; Hartley et al., 1977). The DTV is expressed predominantly in the aged thymus and is the etiologic agent of thymic lymphomas that begin to arise 2-3 months later (Pedersen et al., 1981; Hays et al., 1982; Herr and Gilbert, 1983). Injection of DTV into thymuses of young AKH mice accelerates significantly the onset of disease and reduces the latent period to 60-90 days following viral administration (Cloyd, 1983; O’Donnell et al., 1984; S t a t and Hartley, 1988; Hays et al., 1989a). This is the length of time required for selection of a clonal lymphoma with a proviral integration upstream from tlie c - q c locus or with a duplication of chromosoine 15 that carries an activated c-rnyc (Corcoran et al., 1984; Li et d., 1984; Steffen, 1984; Wirschubsky et nl., 1984a,b;O’Donnell et al., 1985). Hence, the thymic compartment in which premature T lymphocytes proliferate and differentiate provides a microenvironment that favors the sequential occurrence of two stochastic molecular events mandatory for lymphoma

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progression: (1)recombination between ecotropic and xenotropic endogenous viral sequences, resulting in the formation of the DTV, and (2)specific proviral insertion or chromosomal duplication. Whereas the former step involves multiple cycles of virus infection and propagation in an oligoclonal population of thymic lymphocytes, the latter stage entails the selection of a single cell that becomes the sole progenitor of a clonal lymphoma (O’Donnell et al., 1981; Takeuchi et al., 1984; O’Donnell et al., 1985; Cuypers et al., 1986; Hays et al., 1989b, 1990).An inquiry into the nature of lymphocyte populations that provide a cellular source for the evolving lymphoma has led to the characterization of potential lymphoma cells in AKR and other mouse strains susceptible to the induction of primary thymic lymphomas. IV. Prelymphoma Cells in AKR Mice

The term “prelymphoma cells” (PLCs) was introduced by Haran-Ghera (1980a) to depict the presence of potential lymphoma cells in young AKR mice long before the development of overt thymic lymphoma. The concept of PLCs evolved through studies demonstrating that thymectomy of AKR mice at the age of 1-3 months prevented development of spontaneous lymphomas (McEndy et al., 1944; Peled and Haran-Ghera, 1985). However, grafting of a thymus to thymectomized mice enabled progression of the disease, culminating in the emergence of thymic lymphomas arising from the host rather than from the thymic donor cells (Hays, 1982;Takeuchi et nl., 1984; Haran-Ghera et al., 1987; Haran-Ghera, 1994). Reciprocal experiments demonstrated that transfer of AKR bone marrow cells to irradiated F1 recipients results in thymic lymphomas of donor origin. These results were interpreted as indicating that cells with a malignant potential are present in the bone marrow of young AKR mice, and later seed in the thymus, where they acquire a fully malignant phenotype. Another line of evidence advocating the existence of PLCs in the bone marrow was the 20-30% incidence of B cell lymphomas developing in thymectomized, aged AKR mice (Peled and Haran-Ghera, 1985).These data, collected in the 198Os, indicated that the DTV replicating in the thymus was not the sole factor triggering lymphoma progression. Another prerequisite is the generation in the bone marrow of PLCs, which, following migration to the thymus, become susceptible targets for the DTV. This concept, however, should be revisited in light of population dynamic studies demonstrating that thymic lymphocytes do not persist in the thymus for more than 4-5 days, the time required to accomplish a full course of positive and negative selection of maturing T cells (Huesmann et al., 1991; Fowlkes and Pardoll, 1989). At the end of this selection interval T lymphocytes must emigrate from the thymus and populate a secondary lymph node or die via apoptosis

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(Fowlkes and Schweighhoffer, 1995; Jameson et al., 1995). It is, thus, obvious that thymic lymphomas arise from lymphocytes that transiently reside in the thymus for a few days. The source of such lymphocytes is bone marrow stein cells that differentiate along the lymphoid lineage into prothymocytes, which then seed in the thymus, where they mature into a functional repertoire of mature T cell clones in a relatively short period of time (Ikuta et al., 1992). Once a DTV has appeared in the thymus following recombination between ecotropic and xenotropic endogenous viral sequences, it will continue to propagate by repeated cycles of infections, affecting lymphocytes trafficking through the thymus and thymic stroinal cells (Hays et al., 1984). Such cells are not transformed by the DTV infection and continue to differentiate to maturity or die. However, when a stochastic integration of the provinis upstream from c-myc, duplication of chromosome 15, or another as yet unidentified genetic event has occurred in a single infected T cell, it will become the progenitor of a clonal lymphoma. The introduction of the PLC concept through the study of AKR lymphomagenesis constituted an important contribution to the field of experimental o n c o l o ~in , that it motivated the search for and the identification of PLCs in other murine lymphoma models. These then provided the tools for the study of prophylactic intervention in multistage oncogenesis by targeting and attacking potentially malignant cells, as will be discussed in Section VIII. V. Carcinogen-Induced lymphomas

AKR mice are susceptible to the induction of thymic lymphomas by the chemical carcinogen N-methyl-N-nitrosourea ( N M U). A single injection of N M U into AKR mice at 6 weeks of age results in the accelerated development of thymic lymphomas 2-4 months later, prior to the onset of the spontaneous disease (Frei, 1980; Richie et al., 1985). It was, therefore, assumed that NMU induces genetic recombination between endogenous retroviruses, thereby expediting the generation of a lymphomagenic DTV ( Frei, 1980). However, molecular analysis revealed that carcinogeninduced AKR lymphomas contain proviral sequences of ecotropic origin only, which are distinct from the lymphomagenic DTV prevalent in the genome of spontaneous AKR lymphomas (Richie et al., 1985, 1988). Somatic integration of ecotropic proviral sequences in N M U-induced AKR lymphomas does not contribute to lymphomagenesis, based on the finding that AKRIFV-I’ congenic mice were equally sensitive to N M U carcinogenesis (Richie et al., 1991).The B allele of the F V - I locus restricts the integration and replication of endogenous N-tropic viruses, thereby

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protecting AKR ( F V - l N )mice against the development of spontaneous lymphomas (Lilly and Pincus, 1973; Jolicoeur and Rassart, 1980). On the other hand, such mice are equally sensitive to NMU-induced lymphomagenesis, and the resulting lymphomas do not contain somatically acquired proviral integrations (Richie et al., 1991). In addition, treatment of AKR mice with antiviral envelope antibodies, which drastically reduces the incidence of spontaneous lymphomas, had no effect on NMU-induced lymphomagenesis (Haran-Ghera, 1994).Finally, NMU induces thymic lymphomas in mouse strains (e.g., C57L/6J) that do not carry an endogenous ecotropic viral genome, albeit with a longer latency and lower incidence as compared with the AKR strain (Newcomb et aZ., 1988, 1990; Brathwaite et al., 1992; Kubota et aZ., 1995). It may, therefore, be surmised that induction of thymic lymphomas by NMU does not involve somatic integration of the proviral genome, which is crucial for the development of spontaneous AKR lymphomas. This conclusion is corroborated by the finding that a high proportion of the NMU-induced lymphomas in AKR (and other mouse strains) had an activated K-ras, displaying point mutations at codons 12, 61, or 146 of the gene (Geurrero and Pellicer, 1987; Corominas et al., 1991). By contrast, activation of K-ras has not been detected in spontaneous or virally induced lymphomas of these mouse strains (Warren et al., 1987). Nevertheless, the higher incidence and shorter latency of NMU-induced lymphomas in AKR mice, as compared to other inbred strains, suggest cooperation between the chemical carcinogen and endogenous murine leukemia viruses (MuLVs) in lymphoma development. Indeed, NMU treatment stimulated high-level expression of infectious ecotropic MuLV, suggesting that viral products enhance lymphoma progression (Richie et al., 1988). Likewise, the presence of the AKV-1 locus in congenic NFS-N mice, which are otherwise relatively resistant to NMU carcinogenesis, strongly enhanced the formation of thymic lymphomas following NMU treatment (Becker, 1990). This experiment indicates that the AKV-1 locus confers susceptibility to lymphoma induction by increasing the number of target cells responding to NMU. This may occur through multiple infection with the AKV-1 -encoded ecotropic virus or by the action of another gene linked to the AKV-1 locus. It seems clear, accordingly, that the etiologies of viral- and NMU-induced lymphomas are distinct. Yet, it is striking that these independent modalities converge to develop a common, tissue-specific tumor. This outcome emphasizes the uniqueness of the thymic microenvironment as an optimal target for lymphomagenesis. The continuous progression of prothymocytes migrating from the bone marrow to the thymus, followed by extensive proliferation and differentiation before emigration to secondary lymph nodes or death, sustains the high frequency of recombinational events

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between endogenous viruses on the one hand, and the acquisition of specific point mutations that are induced by the chemical carcinogen on the other hand. VI. Thymic lymphomas Induced by Fractionated Irradiation

Another modality by which murine thymic lymphomas are induced is whole-body exposure to irradiation (Kaplan, 1967). Most of the studies on radiation-induced lymphomagenesis have been performed in the C57BL mouse strain, which has an extremely low incidence of spontaneous lymphoma. Kaplan and Brown (1952) demonstrated that exposure of C57BL/ Ka mice to four weekly doses of 1.7Gy induced the development of thymic lymphomas in the majority of the mice, after 4-7 months. Because thymic lymphoma is the only type of tumor emerging in split-irradiated mice, it was initially assumed that the thyinus is a direct target organ for cell transformation by fractionated irradiation (Kaplan, 1967, 1974). However, later experiments indicated that X-ray lymphomagenesis proceeds through an indirect mechanism of induction. Thus, irradiation of only the thymus was insufficient to produce an overt disease (Kaplan, 1967). Furthermore, full or partial shielding of the bone marrow during irradiation, as well as infusion of bone marrow cells from unirradiated syngeneic mice, protected the irradiated mouse against lymphoma development (Wallis et nl., 1966; Ilbery, 1967; Peled and Haran-Ghera, 1969).Finally, whereas thymectomy rescued irradiated inice from subsequent development of lymphomas, progression of the disease could be restored by subscutaneous grafting of a nonirradiated thymus (Kaplan and Brown, 1954). These observations pointed to involvement of an endogeneous infectious virus in the etiology of the disease, one that is activated in the irradiated bone tnarrow but infects, and subsequently transforms, lymphocytes of the thymus. A search for a radiation-induced leukemogenic virus led to the isolation of the radiation leukemia virus (HadLV) from the cells of i i n X-ray-induced thymic lymphoma (Lieberrnan and Kaplan, 1959; ManteuilBrutlag et al., 1980). This virus induced thymic lymphomas when injected intrathymically (i.t.) into adult C57BL inice (Kaplan, 1961; Decleve’et d , 1978; Haran-Ghera et nl., 1966). It was thus inferred that RadLV is an endogenous v i m activated by fractionated irradiation and that lymphocytes proliferating in the postirradiated thymus are targets for infection arid transformation by the reactivated virus. Later experiments, however, revealed that the vast majority of X-ray-induced lymphomas do not prodiice a leukemogenic virus and that exposure of mice to fractionated irr‘i(1iation does not activate an endogeneous virus (Lieberman et d . , 1976; Oecleve 1977). This led to two alternative hypotheses: I-Iaran-Chcw (1976) ct d.,

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suggested that fractionated irradiation eliminates the bulk of the thymic lymphocytes and at the same time causes a genetic lesion in premature bone inarrow cells, rendering them potentially lymphomagenic (PLCs). Migration of the PLCs to the thymus and their proliferation in order to regenerate the intrathymic lymphoid pool result in further progression to full malignancy. Lieberman ct al. (1987), on the other hand, postulated that irradiation activates in the bone marrow a leukeinogenic factor that is transmitted to target cells in the thymus. In both cases, the thymus provides the appropriate microenvironment in which initial premalignant events that take place in bone marrow cells can eventually be altered into a mature lymphoma. To assess the validity of these hypotheses, Boniver et al. (1981) transplanted bone inarrow or thymic cells froin mice previously exposed to fractionated irradiation into niice receiving a single, nonleukeinogenic irradiation treatment. Because the donors were C57BL/Thy1.2 and the recipients C57BL/Thyl.l congenic mice, the origin of the ensuing thymic lymphomas could be traced by Thyl phenotyping. The findings indicated the presence of PLCs in the thymus 30-60 days after irradiation, which progressed to full lymphoma on transfer to syngeneic recipients. No PLCs were detected among bone marrow cells in this study. Haran-Ghera (1980b) performed similar experiments in which thymic or bone inarrow cells from irradiated C57BL/6 mice were transferred to (BALB/CXC57BL/G)Fl mice, thus allowing for a distinction between donor- and recipient-type cells by H-2 phenotyping. I n this study the recipient mice developed thymic lymphomas of donor origin when transfer was performed with bone marrow, but not with thymic lymphocytes, indicating the presence of PLCs in the bone marrow ofthe mice followingtheir exposure to fractionated irradiation. In another study (Liebernian et al., 1987), thymectomized, irradiated C57BL/Ka/Thy1.2 mice were grafted with a thymus from a Thyl.1 congeiiic, neonatal m o u e before or after exposure to four fractions of irradiation. The genetic origin of thymic lymphomas developing in the treated mice was analyzed by Thyl phenotyping. The results indicated that when the mice were grafted before irradiation, most of the lymphomas were of host origin. In contrast, lymphomas in mice receiving a thymic graft after irradiation were predominantly of donor origin. These results were interpreted by the investigators as suggesting that split-dose irradiation induces production and secretion froin cells in the bone marrow of a transmittable leukemogenic factor that acts on lymphocytes proliferating in the thymus, bringing about their transformation into lymphoma cells. The leukernogenic factor was not an endogenous KadLV, because neither the bone inarrow nor the lymphoma cells produced a detectable amount of virus.

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Lieberman et al. (1987) also maintained that the variance between their study and the study of Haran-Ghera (1980b), regarding involvement of the bone marrow in lymphoma progression, was attributable to the experitnental systems used, namely, transfer of parental cells into F1 hybrid recipients versus Thyl. 1 cells into Thyl.2 congenic mice. Kotler et al. (1994) transferred graded numbers of thymic lymphocytes from an irradiated mouse into syngeneic recipients at various intervals following termination of the leukemogenic treattnent (four doses of 1.7 Gy). By recording the frequency of the thymic lymphomas developing in the recipient inice they estimated that PLCs first appear in the thymus 6 weeks after irradiation and 9-15 weeks prior to the development of overt lymphoma. The initial proportion of PLCs in the thymus is their frequency continouslyincreases with time, reaching L at 10weeks after termination of irradiation. These results indicate that the transition of the leukemogenic process from the bone marrow to the thymus is lengthy and gradual, whether manifested by the migration of PLCs or by the transfer of a transmittable leukemogen. A common conclusion that can be drawn from these studies is that the initial effect of fractionated irradiation is the emergence of PLCs, which appear in the bone marrow several months before the thymus is populated with lymphoma cells. The existence of PLCs for an extended period of time during the latent phase of the disease provided an experimental system in which it was possible to examine the effect of prophylactic intervention on lymphoma progression, by targeting the cells producing a leukemogenic Factor or by interference in the transfer of PLCs from the bone marrow to the thymus. These studies are dscussed in Section VIII. VII. RadlV-Induced Lymphomagenesis

The existence of thymic PLCs in the early stages of lytnpho~nagenesis has been most persuasively demonstrated in mice inoculated with RadLV. RadLV was isolated frotn an X-ray-induced thymic lymphoma of a CS7BL/ Ka mouse (Lieberman and Kaplan, 1959). The first isolate was weakly leukemogenic, but serial passage of the virus in newborn mice resulted in the selection of a highly leukemogenic variant that induced thymic lymphomas when inoculated i t . into adult mice (Kaplan, 1967). Another RadLV variant was isolated from bone marrow cells of a C57BL/6 mouse that had been exposed to fractionated irradiation ( I Iaran-Ghera, 1966). This virus induced a high incidence of thymic lymphomas in inice receiving a single, nonleukemogenic dose of 4 Gy irradiation. Accordingly, it was designated radiation-dependent RadLV (D-RadLV). Repeated passage of D-RadLV in C57BL/6 mice yielded a highly leukemogenic, autonomous

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variant (A-RadLV) that induced high-incidence thymic lymphomas when injected i.t. into adult, nonirradiated mice (Haran-Ghera, 1971). Establishment of cell lines from RadLV-induced lymphomas enabled molecular analysis of the virus. Ben-David et nl. (1987a) cloned a highly leukemogenic thymotropic virus from the A-RadLV-induced lymphoma cell line 136.5 (Haas, 1974). The virus displayed a unique genomic RNA containing ecotropic and xenotropic endogenous sequences. Rassart et al. (1986) isolated a highly leukemogenic RadLV produced by the BL/VL3 cell line (Lieberman et al., 1979), whose RNA contained xenotropic sequences in the long-terminal repeat (LTR) and envelope(env) regions. It also had 43-base-pair tandem repeats in the LTR-US region, as well as additional point mutations (Merregaert et nl., 1985; Rassart et al., 1986; Gorska-Flipot et nl., 1992).It is conceivable that the unique LTR sequence of RadLV restricts the tissue tropism of the virus and that the tandem repeats function as transcriptional enhancers. Indeed, Gorska-Flipot and Joulicoeur (1990) identified a factor, termed Rad-1, that was present in T cells but not in fibroblasts. The Rad-1 protein binds to a unique RNA sequence located immediately downstreain of the core consensus region, which has a sequence inotif in its minus-DNA strand. Rad-1 may also interact with other factors bound to the LTR core sequence. The LTR-US of RadLV produced by the BL/VL3 cell line induces in fibroblasts synthesis of a suppressive factor that blocks the replication of RadLV and other MuLVs (Rassart et nl., 1988).This finding indicates that the U3 restricts the tropism of RadLV by inducing in nonlymphoid cells a state of resistance, which interferes with virus replication following infection (Gorska-Flipot et nl., 1992). The env protein plays a significant role in the pathogenesis of certain MuLV-induced tumors. To assess the function of the RadLV env, Poliquin (1992) constructed a series of recombinant viruses, using RadLV and a nonleukeinogenic endogenous virus derived from a BALB/c mouse. Infectivity assays with these variant viruses indicated that the thymotropisin of RadLV is conferred by the en0 region of its genome. However, the BALB/c endogenous virus could be rendered thyinotropic by replacing its env or LTR with those of RadLV. It was concluded, accordingly, that the thymotropism of RadLV is determined by a complementarity between the env and the LTR of the virus. Thus, the restricted infection and transformation of thymic lymphocytes by RadLV might be dependent on the formation of a particular cnv-LTR-target cell combination within the t hyinic microenvironinent . Activation of cellular protooncogenes via proviral integration has been implicated in the induction of malignancies by a number of chronic retroviruses (Peters, 1990). Thus, c-~nyc,lck, pim-1, gin-1, and nzlvi have been

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identified as common integration sites for MuLVS,and genomic rearrangement of these regions was found to correlate with development of T cell lymphomas and leukemias (Tsichlis et nl., 1983, 1984; Corcoran et al., 1984; Cuypers et al., 1984; Lemay and Jolicoeur, 1984; Li et al., 1984; Selten et d , 1984; Steffen, 1984; Graham et d, 1985; O’Donnell et nl., 1985;Villeneuve et al., 1986). However, integration of HadLV in the vicinity of these genetic loci in thymic lymphomas has not been demonstrated. On the other hand, Tremblayet al. (1992)detected a novel gene, designated vin-1, which underwent rearrangement by insertion of proviral RadLV in some RadLV-induced lymphomas. The viti-1 gene was later identified by Hanna et (11. (1993) a s coding for the cycline-D2 protein, which regulates cell cycle transition from GI to the S phase (Hunter and Pines, 1991). These investigators suggested that constitutive activation of cycline-D2 following insertional inutagenesis may contribute to oncogenesis by driving the cells to continuous proliferation. It is unlikely, however, that cyclineD2 expression per se could account for the malignant transformation of T cells by RadLV. Proviral integration at the vin-llcycline-D2 locus occurred in only 5% of the RadLV-induced lymphomas tested, indicating that cycline-D2 activation is neither a mandatory nor a common genetic alteration occurring during RadLV-induced leukemogenesis. The process apparently requires additional genetic events (Tremblay et nl., 1992). The involvement of c-myc and rrilvi in RadLV leukemogenesis was indicated by the high frequency (>60%) of RadLV-induced lymphomas displaying chromosome 15 trisomy (Wiener et al., 1978a).The essential segment involved in chromosome 15 duplication was localized to the distal region in which c-myc and mlvi are located (Wiener et al., 1978b).Acquisition of chromosome 15 trisomy is a late event in the genesis of T cell lymphomas and occurs independently of insertional mutagenesis or c-niyc rearrangement (Wirschubsky et nl., 19844. The genetic overdose of c-myc due to chromosome 15 duplication apparently has a decisive influence on the expression of the malignant phenotype (Wirschubsky et al., 1986). The multiplicity of mechanisms presumably underlying RadLV-induced leukemogenesis suggests the existence of alternative pathways related to cellular and molecular events that are activated by primary infection with RadLV, eventually leading to the development of a common malignant phenotype. This conclusion is reinforced by data demonstrating that regardless of the eventual transforming event, RadLV-induced lymphomas stem from a population of potentially malignant cells that appear shortly after virus inoculation and persist in the thymus during the entire premalignant latency. Using a transplantation assay that distinguishes between donorand recipient-type lymphomas, Haran-Ghera (1980b) demonstrated that

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the thymus of a C57BL mouse inoculated with A-RadLV contains PLCs, which are detectable as early as 10 days after virus inoculation. The virally induced PLCs were characterized as premature, continuously dividing CD4+8+thymic lymphocytes (Gokhman et al., 1990). These cells constitute the major thymic population of lymphocytes subject to positive and negative selection. We estimated the number of PLCs in the thymus of RadLV-inoculated mice by transplanting graded numbers of thymocytes from mice inoculated with RadLV into a number of recipients (Yefenof et al., 1991). The minimum number of thymocytes required to convey lymphomagenesis was lo3/ mouse. Because the average number of lymphocytes in the thymus is 8 X lo’, we assumed that 3 weeks after virus inoculation, the thymus contains some -8 X lo4“leukemogenic units,” each capable of initiating independent lymphoma progression when transferred to a susceptible thymus. Ben-David et al. (1987b) identified virus-producing cells in the premalignant thymus, by staining them with a monoclonal antibody directed against the RadLV envelope glycoprotein (gp70). Virus-producing cells were first detected 10-15 hr following inoculation of the virus, their frequency steadily increasing and reaching one-third of the total thymic cell population 1-4 days later. Thereafter, the percentage of virus-positive cells declined, plummeting to 2-3% in the third week following virus inoculation. This low level of virus-positive cells remained constant during the remainder of the premalignant latency and until the outbreak of overt lymphoma, when the thymus was repopulated by lymphoma cells, all of which were infected by the virus. The fluctuation in the relative number of viruspositive cells in the premalignant thymus indicates that shortly after its inoculation, RadLV infects a large population of thymic lymphocytes, the majority of which are subsequently eliminated due to intrathymic lymphocyte turnover. However, several millions of virus-infected cells are retained in the thymus over an extended period of time, constituting a pool of PLCs from which a mature thymic lymphoma eventually arises. The relatively large number of PLCs in the thymus of RadLV-inoculated mice led to the assumption that the cells constitute a pleioclonal population of T lymphocytes.The monoclonal origin of mature RadLV-induced lymphomas has been proved by detection of unique rearrangements in the TP gene of the T cell receptor (Ben-David et al., 198%; Yefenof et al., 1991). To determine the clonal nature of PLCs we developed a split-transfer assay in which thymocytes from a single RadLV-inoculated mouse were injected i.t. into several syngeneic recipients (Avni et al., 1995). Donortype lymphomas were then analyzed by Southern hybridization using a TP-specific probe. The clonal makeup of the PLCs could be inferred, as

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different T p rearrangements within the array of generated lymphomas reflected the clonal heterogeneity of the PLC progenitors at the time of transfer. These experiments revealed that when recipient inice are injected with limited numbers ( lo3-lo4)of thymic cells, explanted 3-6 weeks after virus inoculation, each of the ensuing donor-type lymphomas displays a unique and distinct T p rearrangement (Yefenof et nl., 1991). This pattern indicates that the PLCs constitute a pleioclonal population of thymic lymphocytes, with a single cell becoming the sole progenitor of a malignant lymphoma. When the PLCs remain within the thymus of the virusinoculated mouse, a clonal lymphoma eventually develops due to the selection of a particular PLC that has progressively acquired a fully inalignant phenotype. However, split injection of PLCs from a single thymus into several mice results in the independent progression of leukeinogenesis in each recipient, yielding clonal lymphomas derived from different PL precursors. The long-term persistence of PLCs in the thymus during the premalignant latency of the disease required particular attention in view of the short-term residency of maturing T cells in the thymus, which does not exceed 4-5 days (Fowlkes and Pardoll, 1989; Huesmann et nl., 1991). Indeed, most of the RadLV-infected cells are eliminated from the thymus within the first week following virus infection (Ben-David et nl., 1987b). However, 1-3 million virus-infected PLCs remain in the thymus, where they survive for several months. It was, therefore, postulated that infection by RadLV confers on such cells the ability to survive in the thymus for at least 12-14 weeks, the period required for the development of a fiilly mature lymphoma. Because RadLV-induced PLCs have been characterized as activated, continuously dividing T lymphocytes (Gokhman et d., l99O), it seemed likely that their maintenance in the thymus is facilitated by the self-renewal of a PLC pool. Haas et al. (1984) reported that early developing T cell lymphomas induced by RadLV are dependent on a continuous response to an undefined growth Factor. In a subsequent study we found that some, but not all, RadLV-induced lymphomas secrete interleukin-4 (IL-4),even though their growth is not dependent on the factor ( Yefenof et nl., 1992b). RadLV does not infect or transform T lymphocytes in vitro. Howrwr, incubation of thymic cells with RadLV resulted in 1L-4 secretion that was inhibited in the presence of anti-RadLV antibodies (Yefenof p t nl., lSS2b). Whereas untreated normal thymic cells did not secrete 1L-4, thymocytes from RadLV-inoculated prelymphoina mice constitutively produced the factor, and production was enhanced by RadLV. As opposed to mature lymphomas, in uitro growth of PLCs was dependent on IL-4 ( Yefenof et nl., 1991). It is, therefore, conceivable that IL-4 secretion in the former

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is a vestige of the PLC progenitors, whose long-term survival is dependent on an IL-4-driven autocrine stimulus, because progression to the malignant state involves the selected outgrowth of a PLC that is emancipated from IL-4 dependency. Such a cell will become the progenitor of a clonal lymphoma that is no longer dependent on the factor for continuous growth. How does RadLV induce the secretion of IL-4 in a manner that is inhibited by anti-RadLV antibodies? O’Neil et al. (1987)found that RadLV cross-ligates CD3/TCR and CD4 expressed on the membrane of THcells. These molecules are directly involved in relaying activation signals to T helper (Th) cells, recognizing an antigen presented by MHC class I1 molecules. On presentation to Th2 cells, an IL-4-dependent autocrine growth stimulation loop begins to operate, bringing about the clonal expansion of cells bearing receptors specific for the stimulating antigens ( Fernandez-Botran et al., 1986). This finding suggests that through its binding to CD3 and CD4, RadLV mimics the action of antigen on Th2 cells and induces autocrine growth mediated by IL-4. Two basic features, however, distinguish antigen from RadLV-driven stimulation. ( 1)Whereas antigen activates an autocrine growth stimulation loop for clonal expansion of antigen-specific T cells, RadLV acts on a pleioclonal population of T lymphocytes. ( 2 ) In the course of an immune response to an antigen, IL-4 secretion is limited and subsides as soon as the antigen is eliminated. Autocrine activation by RadLV, on the other hand, is continuous, because the virus integrates and replicates within the infected cells. A persistent autocrine response to IL-4 enables the cells to proliferate in the thymus and maintain a population of pleioclonal PLCs, one of which is eventually transformed. This model may be construed as a modification of the receptor-mediated lymphomagenesis model proposed by Weissman and McGrath (1982), which implies that RadLV transforms T cell clones expressing a TCR specific for the virus envelope glycoprotein. Binding of the virus or its enu products to a clonotypic TCR could induce antigenic stimulation resulting in continuous proliferation. This hypothesis, however, cannot be reconciled with the need for an antigen to be processed and presented by MHC molecules of antigen-presenting cells before it can be recognized by a specific TCH (Germain, 1993). Our model proposes the interaction of the transforming retrovirus with the antigen receptor as a growth-promoting event during the premalignant phase of the disease, but excludes the elements of specific antigenic recognition of viral antigens by clones expressing a unique TCR for the viral envelope glycoprotein. VIII. Preventive Therapy of Prelymphoma Mice

The AKR inbred strain became the first murine model for investigating the long-term progression of thymic lymphomas (Hays et al., 1989a,b,

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1990).Apart froin allowing identification of the DTV as the etiologic agent of the disease, it provided an experimental tool for studying the prospects of prophylactic therapy in protecting the inice against later development of overt lymphoma. The drastic reduction in lymphoina incidence aniong AKR mice that have been thymectomized at 1-2 months of age (McEiidy et al., 1944), encouraged the search for additional nonsurgical means that could be applied during the latent period to rescue the mice from early death. Because DTV is the cause of thymic lymphomas in AKK mice, treatments targeting viral production or infectivity are useful in preventing lymphoma progression. Indeed, injection of a nonleukeinogenic virus (24666) into young AKR inice (1-2 months old) drastically reduced the incidence of spontaneous thymic lymphomas in the adult aniinals (l'eled and Haran-Ghera, 1991). The antilymphomagenic activity of the 24-666 virus depended on its ability to abolish the DTV expression in the thymus, which retained its intact structure and cellular composition over time. Another effective inodality that protects AKR mice against lymphoma development is passive immunotherapy with antiviral antibodies (HaranGhera et al., 1995). Administration of monoclonal antibodies (18-5 on Hy-72) directed against the envelope glycoprotein of DTV, to young AKR mice (Portiset al., 1982),reduced the incidence of spontaneous lymphomas from 87 to 7% and abolished the changes in thymic cell populations characteristic of the adult aniinals (Maran-Ghera and Peled, 1991). Active, nonspecific immune stimulation also effectively reduced the incidence of thymic lymphomas. Thus, injection of heat-killed Lactobacillus casei, which acts as a nonspecific iinmunopotentiator, into 2- to 4-inonth-old AKH mice protected the aniinals against later development of lynphonias (Watanbe, 1996). The reduction in lymphoma incidence correlated with the development of antiviral iininunity and a reduction in the level of DTV produced in the thymus. Another modality that provides prophylactic interference is low doses of total-body irradiation. Exposure of 2-month-old AKK mice to fractions of 15 cGy twice a week for 40 weeks reduced the incidencc of lymphomas by 40% (Ishii et al., 1996). Ishii and co-workers suggested that the protective effect of total-body irradiation against lymphoma tlevelopinent results from potentiated immunity, previously described in clironically irradiated mice (Anderson et aE., 1988). Another possibility is the direct suppressive influence of irradiation on intrathyinic production of DTV, which is mandatory for the development of spontaneous lymphoma. All of the modalities that effectively reduce the incidence of spontaneous lymphomas directly or indirectly affect the level of DTV produced in the thymus or the infectivity of thymic lymphocytes by that virus. The n l d i t y to induce a prelyinphoma state by viral or nonviral carcinogenesis has facilitated the study of prophylactic intervention using treatments that

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interfere with the persistence of PLCs or their progression to full lymphoma. As discussed in Section VI, the induction of thymic lymphomas by fractionated X-ray irradiation is preceded by the emergence of PLCs in the bone marrow. The protective effect of partial bone shielding during irradiation (Wallis et al., 1966) indicated that nonirradiated marrow cells interfere in the progression of PLCs to overt thymic lymphoma. Indeed, administration of 10' bone marrow cells from a nonirradiated syngeneic donor 2 hr after termination of X-ray irradiation was sufficient to protect the mice against the development of thymic lymphomas (Boniver et al., 1981; Van Bekkum et al., 1984). When the bone marrow cells are grafted 1 month later, this maneuver fails to inhibit the emergence of the tumors (Humblet et al., 1997). Gorelick et al. (1984) suggested that NK cells residing in the bone marrow recognize and eliminate X-ray-induced PLCs. Such effector lymphocytes are radiation sensitive and are therefore destroyed by fractionated irradiation, but infusion of nonirradiated bone marrow restores this inhibitory activity. However, later studies by Lieberman et al. (1992) demonstrated that bone marrow of severe combined immune deficiency (SCID) mice is not protective during radiation leukemogenesis, even though the activity of natural killer (NK) cells in the SCID marrow is intact. The inability of SCID bone marrow to prevent X-ray lymphomagenesis may thus be attributed to the SCID mutation, which does not allow stem cells in the thymus to mature into immunocompetent T lymphocytes (Bosma and Carroll, 1991). Because radiation leukemogenesis requires gradual intrathymic accumulation of PLCs that originate in radiation-damaged stem cells in the bone marrow, reconstitution with nonirradiated bone marrow may interfere with this process by competing with irradiated stem cells for thymic repopulation. However, such interference can occur only with bone marrow cells capable of maturing into functional T lymphocytes, whereas stem cells defective in differentiation competence along the T lineage lack this ability. Boniver et al. (1989, 1992) found that repeated injection of TNF-a or interferon-? following fractionated irradiation prevented the onset of thymic lymphomas. Administration of 2.5 X lo4 units of T N F - a or 4 X 104 units of interferon-y, three times a week during the first 6 weeks after irradiation, reduced the incidence of lymphomas by more than SO%. Both treatments eliminated the presence of PLCs and restored the size and cellular composition of the irradiated thymus. In addition to the direct cytotoxic effect of TNF-a against PLCs, both TNF-a and interferon-y may have acted indirectly by restoring thymic lymphopoiesis, thereby interfering in the seeding of PLCs in the thymus (Humblet et al., 1994). The protective activity of nonirradiated bone marrow infused

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immediately after split-dose irradiation may also be mediated, in part, by the TNF-a and interferon-? produced by the grafted cells, because

antibodies directed against these cytokines partially restored lymphoma induction (Humblet et nl , 1996). Potworowski et al. (1996) showed that mice exposed to fractionated irradiation have a reduced incidence of thymic lymphomas if injected i.t. with lo5dendritic cells every fifth week after termination of the radiation treatment. Transfer of thymic cells from treated to untreated mice indicated a drastic reduction in the number of PLCs following the administration of dendritic cells. The antilymphoma activity has been attributed to the ability of dendritic cells to augment specific immunity against PLCs by restoring the capacity of antigen presentation in the irradiated thymus, and to interact directly with PLCs, thereby leading to their death via apoptosis. These studies demonstrate that the development of a full-fledged lymphoma may be prevented by prophylactic intervention that aborts the progression of PLCs to overt disease. The most compelling evidence that this approach is indeed appropriate comes from studies of mice inoculated with RadLV. Both the initiation and the promotion of a RadLV-induced lymphoma occurred in the thymus, the site where PLCs are generated, propagated, and progress to primary lymphoma. Hence, treatments designed to target biological properties enabling the persistence of PLCs in the thymus are presumed to be prophylactically effective. Because survival of PLCs in the thymus is dependent on IL-4-driven autocrine growth stimulation, treatments antagonizing IL-4 activity should presumably prevent lymphoma development. A suitable drug for this purpose is cyclosporin A (CSA), an immunosuppressive cyclic peptide that inhibits cytokine production by activated T cells (Schreiber and Crabtree, 1992). Because CSA suppresses T cell immunity, it is of no benefit for cancer therapy, which seeks to improve, rather than to suppress, the iminunocompetence of the host. However, as the intrathymic persistence of PLCs induced by HadLV is enabled by IL-4, suppressed production of the cytokine could be effective in retarding lymphoma progression. In vitro studies (Yefenof et nl., 1992a) demonstrated that CSA markedly reduces the secretion of IL-4 by RadLV-induced lymphomas and PLCs. These results provided the basis for applying CSA in prophylactic intervention during the period of premalignant latency. Administration of CSA 3-6 weeks after virus inoculation significantly delayed the onset of lymphoma, the most effective regimen being intraperitoneal (i.p.) injections of 50 mg/kg CSA twice a week for 2 weeks (Yefenof et n l , 1992a): Although optimal CSA treatment prolonged the latency of the disease by 5-7 weeks, it did not prevent the onset of lymphoma.

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A similar effect was obtained by specific targeting of PLCs with an immunotoxin (IT) consisting of a deglycosylated ricin A chain and a monoclonal antibody directed against the RadLV envelope glycoprotein (gp70), which is expressed on the surface of virus-infected cells (Yefenof et al., 1991). Biweekly intravenous (i.v.) administration of 1 mgkg IT during the fourth and fifth week after virus inoculation extended the survival of the mice by 40-45 days (Yefenof et al., 1992b) but did not prevent lymphoma development. When, however, the two drugs were given together, they synergistically protected more than 80% of the mice against lymphoma development for up to 1 year after virus inoculation. It is, therefore, assumed that the virus-specific IT effectively eradicates a great majority, but not all, of the virus-infected PLCs, thereby delaying, but not preventing the development of lymphoma. When CSA is concomitantly administered, it arrests the growth of the few residual PLCs escaping IT treatment, whose survival is dependent on IL-4 secretion. These complementary activities prevent the progression of the disease and provide full protection to the mice. Another antagonist of IL-4 is a monoclonal antibody (11B11) that specifically neutralizes the activity of IL-4 (O’Hara and Paul, 1985). By inhibiting IL-4 binding to its receptors, l l B l l effectively and specifically curbed the growth of IL-4-dependent cells in vitro (Yefenof et al., 1991). It is also an effective immunomodulatory drug in vivo owing to its specific IL-4 antagonistic activity (Vicari and Papiernik, 1993; Cheever et al., 1994). Based on these data, we postulated that administration of l l B l l to RadLVinoculated mice could interfere with lymphoma progression. Indeed, when the premalignant mice were twice injected with 2 m g k g l l B l l in the early latent period, a significant delay in the onset of the disease was recorded (Yefenof et al., 1992a). Moreover, if the dose of l l B l l was increased to 20 m&/kg, two injections during the third week of latency were sufficient to afford full protection, the animals remaining diseasefree for 1 year after inoculation of the virus (Epszteyn et d., 1997). The multiple procedures and relative ease with which the development of primary thymic lymphomas may be delayed and prevented allow the prediction that prophylactic therapy may be adequate and effective in various premalignant conditions, whether of a lymphoid or a nonlymphoid nature. Once the existence and unique biological properties of PLCs are identified, it should be possible to intervene in their progression to fullfledged disease by designing modalities that decrease the size, growth rate, and survival of the PLC population. The laboratory experience with murine thymic lymphomas suggests that this approach would be efficient, reliable, and curative.

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IX. Concluding Remarks

The significance of the thymus for the ontogeny of the immune system became apparent through investigations into its involvement in retrovirally induced oncogenesis. This discovery led to a sequence of studies that unraveled the role of the thymus as a primary lymphoid organ where bone marrow-derived progenitors proliferate, differentiate, and undergo complex selection procedures, yielding a functional population of T lymphocytes. Alongside with these developments in the itnmunological arena, the thymus continued to attract the attention of experimental oncologists as a preferential site for malignant transformation induced by viral, chemical, or physical carcinogens. The present article highlights several distinct pathways of murine lymphomagenesis, all leading to the development of thymic lymphomas, and outlines the unique features of the thymus, which provides an optitnal tnicroenvironment sustaining the stepwise progression of the oncogenic process, initiated by various independent agents. The long interval of latency that precedes the development of a fullblown lymphoma, and its confinement to the thymus, offered an experimental tool for investigating premalignant steps in the development of the disease. Analysis of these steps led to the detection of PLCs, whose survival and progression to overt lymphoma depend on their interaction with cells and factors in the thymic microenvironment. Their characterization enabled the design and testing of prophylactic modalities targeted at various biological characteristics of PLC populations and aiming to restrict their proliferation and progression toward full malignancy. These experiments demonstrated that the ultimate outcome of lymphomagenesis may be delayed, and sometimes prevented, by applying regimens that perturb unique aspects of PLC physiology. As such, the study of experimental thymic lymphomas has generated knowledge extending beyond its relevance to a specific experimental tumor. It argues in favor of the concept that prenialignant cells emerging in the early stages of tumor progression could become potential targets for prophylactic therapy at a time when it could be categorically effective. Identification and characterization of potential malignant cells in other experimental and clinical settings may expand the scope of opportunities for chemoimmuno-prevention of malignant diseases, creating a better alternative to conventional therapy of an already existing malignancy.

ACKNOWLEDGMENTS I would like t o thank Drs. E. Klein, D. Weiss, and A. Mahler for their critical review of the manuscript, Ms.G . Rubinstein for library services, and Ms. 1).Ben-Dov and Ms. S . Saiinders for secretarial assistance. Studies perfomled in the author’s laboratory were sup-

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ported by the U.S.-Israel Binational Science Foundation, the Israel Science Foundation, and Concern Foundation, Los Angeles.

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