Suppressor and reactive lymphocytes in radiation leukemia virus (RadLV)-induced leukemogenesis

Suppressor cells capable of enhancing tumor growth in vivo and of abrogating a potential anti-tumor immunity in vitro are generated in C57BL/6 mice inoculated with the high-leukemogenic A-RadLV. Mice inoculated with low-leukemogenic D-RadLV do not develop suppressor cells but contain anti-tumor reactive lymphocytes that can inhibit in vivo tumor growth. Cyclophosphamide (CyF) treatment of mice inoculated with A-RadLV hampered suppressor cell function and rendered the animals' lymphocytes responsive to A-RadLV induced tumor cells in vitro. Administration of CyF also reduced leukemia incidence in mice inoculated with A-RadLV, but had no effect on leukemia induction by D-RadLV in irradiated mice. It is suggested that the high leukemogenic activity of A-RadLV depends on the virus' ability to recruit CyF-sensitive suppressor cells early in latency and that tumor progression in mice inoculated with D-RadLV is arrested due to the host immune response.     

Characteristics of preleukemia cells induced in mice.

A high proportion of irradiated C57BL/6 mice inoculated with the radiation leukemia virus D-RadLV develop overt T-cell leukemias originating in the thymus. In unirradiated hosts the incidence is much lower. As early as 10 days after injection of D-RadLV the bone marrow contains “preleukemia” cells which, although not frankly leukemic, will develop into leukemia cells if transferred into a specially pretreated recipient mouse. In the present report, certain properties of D-RadLV-induced leukemia and preleukemia cells are compared. In this model system, leukemia cells express the T-cell surface component Thy-1 (Thy-1⁺) whereas preleukemia cells do not (Thy-1^?). But preleukemia cells could be induced in vitro by thymopoietin or ubiquitin to become Thy-1⁺, suggesting that they are prothymocytes. Unlike leukemia cells, preleukemia cells injected into normal recipients immunized them against transplants of leukemias induced by the same D-RadLV virus. Evidently D-RadLV virus induces a critical change in prothymocytes which in a later (Thy-1⁺) phase of differentiation is manifest in overt leukemia transformation.     

Suppressor and reactive lymphocytes in radiation leukemia virus (RadLV)-induced leukemogenesis

Summary Suppressor cells capable of enhancing tumor growth in vivo and of abrogating a potential anti-tumor immunity in vitro are generated in C57BL/6 mice inoculated with the high-leukemogenic A-RadLV. Mice inoculated with low-leukemogenic D-RadLV do not develop suppressor cells but contain anti-tumor reactive lymphocytes that can inhibit in vivo tumor growth. Cyclophosphamide (CyF) treatment of mice inoculated with A-RadLV hampered suppressor cell function and rendered the animals' lymphocytes responsive to A-RadLV induced tumor cells in vitro. Administration of CyF also reduced leukemia incidence in mice inoculated with A-RadLV, but had no effect on leukemia induction by D-RadLV in irradiated mice. It is suggested that the high leukemogenic activity of A-RadLV depends on the virus' ability to recruit CyF-sensitive suppressor cells early in latency and that tumor progression in mice inoculated with D-RadLV is arrested due to the host immune response.     

Delayed type hypersensitivity responses to radiation leukemia virus variants

DTH responses were evaluated in different strains of mice shown to be resistant or sensitive to leukemogenesis by the radiation leukemia virus variants A-RadLV and D-RadLV. A significant response was observed only in the H-2 complex-linked resistant haplotypes to RadLV leukemogenesis. The DTH response could be transferred by immune cells of mice resistant to the appropriate RadLV variant. Thus, an inverse relationship between the leukemogenic activity of the virus and its immunization ability expressed by DTH response was demonstrated in different mouse strains.     

H-21-linked control of immunological resistance to viral leukemogenesis as a response to preleukemic cells

The mechanism of resistance to leukemogenesis by two radiation leukemia virus variants, A-RadLV and D-RadLV, was investigated. Resistance to these viruses is linked to H-21 in both B10.S and C57BL/10 mice. The resistance of virus-infected mice to transplantation of syngeneic. A- or D-RadLV-induced lymphoma cells was similar to their resistance to leukemogenesis by the same viruses. This resistance could be transferred by lymphoid cells from immune donors to normal recipients, and it was specific for RadLV lymphomas. Virus-primed (responder x sensitive)F1 hybrids rejected only resistant-type parental lymphoma cells. Hence, it appears that H-21-linked resistance to RadLV leukemogenesis is regulated by Ir genes. Resistant mice immunized by A- or D-RadLV rejected syngeneic lymphoma cells, irrespective of whether they were sensitive or resistant to the RadLV variant used for the induction of the lymphoma cells. It follows that resistant and sensitive type lymphomas are antigenically similar for the effector mechanism, and that the Ir genes may be expressed in the sensitization phase of the reaction. In virus-infected mice which are resistant to A- or D-RadLV we were able to demonstrate the presence of preleukemic lymphocytes. Normal mice could be immunized by these preleukemic cells against lymphoma challenge. These data are interpreted to suggest that mice having H-21-linked resistance to RadLV infection may be sensitized by their preleukemic cells, and that these preleukemic cells are then arrested in their development as a result of the immune response.     

H-2I-linked control of immunological resistance to viral leukemogenesis as a response to preleukemic cells

The mechanism of resistance to leukemogenesis by two radiation leukemia virus variants, A-RadLV and D-RadLV, was investigated. Resistance to these viruses is linked toH-2I in both B10.S and C57BL/10 mice. The resistance of virus-infected mice to transplantation of syngeneic, A- or D-RadLV-induced lymphoma cells was similar to their resistance to leukemogenesis by the same viruses. This resistance could be transferred by lymphoid cells from immune donors to normal recipients, and it was specific for RadLV lymphomas. Virus-primed (responder x sensitive)F₁ hybrids rejected only resistant-type parental lymphoma cells. Hence, it appears thatH-2I-linked resistance to RadLV leukemogenesis is regulated byIr genes. Resistant mice immunized by A- or D-RadLV rejected syngeneic lymphoma cells, irrespective of whether they were sensitive or resistant to the RadLV variant used for the induction of the lymphoma cells. It follows that resistant and sensitive type lymphomas are antigenically similar for the effector mechanism, and that theIr genes may be expressed in the sensitization phase of the reaction. In virus-infected mice which are resistant to A- or D-RadLV we were able to demonstrate the presence of preleukemic lymphocytes. Normal mice could be immunized by these preleukemic cells against lymphoma challenge. These data are interpreted to suggest that mice havingH-2I-linked resistance to RadLV infection may be sensitized by their preleukemic cells, and that these preleukemic cells are then arrested in their development as a result of the immune response.     

Virus-induced immunosuppression: immune system-mediated destruction of virus-infected dendritic cells results in generalized immune suppression.

Despite the clinical importance of virus-induced immunosuppression, how virus infection may lead to a generalized suppression of the host immune response is poorly understood. To elucidate the principles involved, we analyzed the mechanism by which a lymphocytic choriomeningitis virus (LCMV) variant produces a generalized immune suppression in its natural host, the mouse. Whereas adult mice inoculated intravenously with LCMV Armstrong rapidly clear the infection and remain immunocompetent, inoculation with the Armstrong-derived LCMV variant clone 13, which differs from its parent virus at only two amino acid positions, by contrast results in persistent infection and a generalized deficit in responsiveness to subsequent immune challenge. Here we show that the immune suppression induced by LCMV clone 13 is associated with a CD8-dependent loss of interdigitating dendritic cells from periarteriolar lymphoid sheaths in the spleen and, functionally, with a deficit in the ability of splenocytes from infected mice to stimulate the proliferation of naive T cells in a primary mixed lymphocyte reaction. Dendritic cells are not depleted in immunocompetent Armstrong-infected mice. LCMV Armstrong and clone 13 exhibit differences in their tropism within the spleen, with clone 13 causing a higher level of infection of antigen-presenting cells in the white pulp, including periarterial interdigitating dendritic cells, than Armstrong, thereby rendering these cells targets for destruction by the antiviral CD8+ cytotoxic T-lymphocyte response which is induced at early times following infection with either virus. Our findings illustrate the key role that virus tropism may play in determining pathogenicity and, further, document a mechanism for virus-induced immunosuppression which may contribute to the clinically important immune suppression associated with many virus infections, including human immunodeficiency virus type 1.     

I-region linked complementing loci in resistance to viral leukemogenesis in the mouse

A-RadLV, a variant of the radiation leukemia virus, inoculated intrathymically into adult mice, causes a high frequency of leukemia in haplotypes b, f, k, d, p and j on the B10 background, whereas H-2s mice are resistant. Resistance is dominant and segregates with H-2s in the offspring of (b x s)b and (b x t2)b backcrosses. Analysis of recombinant strains revealed that resistance is associated with I-A and I-B. B10.A(5R), a recombinant of two sensitive haplotypes, was found to be resistant, suggesting intra-H-2-gene complementation. The resistance of such complementing loci was demonstrated also in the trans position by testing F1 mice bred from sensitive parents. These data are taken to suggest that I-region linked complementing loci, similar to classical Ir genes, may be involved in resistance to murine leukemia.     

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.     

Thymocyte subsets transformed by Abelson murine leukemia virus.

The infectious complex of Abelson murine leukemia virus was altered by replacing its usual helper virus, Moloney leukemia virus, with radiation leukemia virus (RadLV). After intrathymic injection of the Abelson-RadLV complex, thymomas arose rapidly, as described previously for injection of the Abelson-Moloney complex. Cell lines were derived from thymomas induced by each Abelson virus complex and were classified according to normal thymus cell phenotypes. Each virus complex induced some cell lines which were like a 0.7% subpopulation of murine thymocytes in that they failed to express the Thy-1 cell-surface antigen. These lines are thus far indistinguishable from some Abelson-derived bone marrow transformants classified as pre-B cells. However, the Abelson-Moloney complex induced some cell lines which expressed low levels of Thy-1 and which shared most markers with immature blast cells of the thymic medulla, whereas the Abelson-RadLV complex induced some lines which were clearly like thymic cortex blast cells. Thus, Abelson virus can induce thymoma cell lines of at least two, and possibly three, distinct phenotypes corresponding to normal thymocyte blast subsets, the determination of which can be influenced by helper virus sequences.     

Restricted virus replication in the spinal cords of nude mice infected with a Theiler''s virus variant.

The Daniels strain of Theiler's murine encephalomyelitis produces a chronic disease which is an animal model for human demyelinating disorders. Previously, we selected a neutralization-resistant virus variant producing an altered and diminished central nervous system disease in immunocompetent mice which was evident during the later stage of infection (after 4 weeks) (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). The exact epitope determining neurovirulence was precisely mapped to a capsid protein, VP-1, and represents a neutralizing region (A. Zurbriggen, J. M. Hogle, and R. S. Fujinami, J. Exp. Med. 170:2037-2049, 1989). Here, we present experiments with immunoincompetent animals to determine viral replication, spread, and targeting to the central nervous system in the absence of detectable antibodies or functional T cells. Nude mice were infected orally, and the virus was monitored by plaque assay, immunohistochemistry, and in situ hybridization. Early during the infection (1 week), the variant virus induced an acute disease comparable to that induced by the wild-type virus in these nude mice. Alterations in tropism in the central nervous system were not apparent when wild-type parental Daniels strain virus was compared with the variant virus. Moreover, variant virus replicated in tissue culture (BHK-21 cells) to similarly high titers in a time course identical to that of the wild-type virus (A. Zurbriggen and R. S. Fujinami, J. Virol. 63:1505-1513, 1989). However, replication of the variant virus versus the wild-type virus within the spinal cord of athymic nude mice infected per os was substantially restricted by 6 weeks postinfection. Therefore, the reduced neurovirulence in the later stage (6 weeks) of the disease is most likely due to a diminished growth rate or spread of the variant virus in the central nervous system rather than to marked differences in viral tropism.     

Identification and quantitation of human T-cell antigen by antisera purified from antibodies crossreacting with hemopoietic progenitors and other blood cells

Anti-T cell globulin (ATCG) prepared from antihuman thymocyte serum by absorption with kidney, cells from patients with chronic lymphatic leukemias, and several lymphoblastoid cell lines was shown to react specifically with human thymus-derived lymphocytes. While high activity against thymocytes and a T-lymphoblastoid cell line could be demonstrated, ATCG remained negative against several chronic lymphatic leukemias and B-lymphoblastoid cell lines. The ATCG was used in the cytotoxic test, electronmicroscopy, and immunoautoradiography for identification of T cells in thymus, tonsils, spleen, blood, bone marrow, lymphatic leukemias, and lymphoblastoid cell lines. A comparison of these results with the ability to form spontaneous SRBC-rosettes revealed remarkable deviations between both markers in leukemias. Absorption with human brain failed to remove specific activity of ATCG. Labeling experiments by immunoautoradiography and investigations by complement fixation permitted quantitation of relative T-cell antigen concentration on different cell populations. As further evidence for specificity it could be shown that ATCG was no longer toxic for hemopoietic progenitors, whereas unabsorbed globulin reduced the number of colonyforming cells considerably.Abbreviations ALL acute lymphatic leukemia - ATCG anti-human T cell globulin (absorbed) - ATG anti-human thymocyte globulin (not absorbed) - Bm-C bone marrow cells - CFA complete Freund's adjuvant - CLL chronic lymphatic leukemia - EBV Epstein-Barr virus - GPC' guinea pig complement - HBSS Hanks' balanced salt solution - Ig immunoglobulin - PBS phosphate buffered saline - Per-Ly peripheral blood lymphocytes (normal) - Spl-Ly spleen lymphocytes - SRBC sheep red blood cells - VBS veronal buffered saline - Thy-Ly thymus lymphocytes - Ton-Ly tonsil lymphocytes     

Effects of nonleukemogenic and wild-type Moloney murine leukemia virus on lymphoid cells in vivo: identification of a preleukemic shift in thymocyte subpopulations.

Infection of mice with Moloney murine leukemia virus (M-MuLV) as well as with a nonpathogenic variant, Mo+PyF101 M-MuLV, was studied. Mo+PyF101 M-MuLV differs from wild-type M-MuLV by the addition of enhancer sequences from polyomavirus in the long terminal repeat. Previous experiments indicated that Mo+PyF101 establishes infection in animals, even though it does not induce disease. In vivo infection studies with particular attention to the thymus were performed, since the thymus is the target organ for M-MuLV leukemogenesis. Mice inoculated at birth with wild-type M-MuLV developed maximal levels of thymic infection by 2 to 3 weeks. Animals inoculated with Mo+PyF101 M-MuLV showed considerably less thymic infection at early times (2 to 4 weeks); nevertheless, by 5 to 6 weeks infection equivalent to wild-type M-MuLV-inoculated animals developed. Therefore the nonpathogenicity of Mo+PyF101 M-MuLV did not simply reflect a lack of thymotropism. Furthermore, thymic infection by itself may not be sufficient to induce leukemia. The relative deficit of Mo+PyF101 M-MuLV thymic infection at early versus late times did not reflect a change in the nature of the cells in the thymus, since in vitro infection of primary thymocytes from 2- and 6-week-old animals was equally efficient. One possible explanation is that infected thymocytes normally arise from progenitor cells which were infected in the bone marrow or spleen, and the cells restricted for Mo+PyF101 M-MuLV are located in those organs. Comparison of wild-type and Mo+PyF101 M-MuLV also allowed identification of important preleukemic changes in the thymus of wild-type M-MuLV-inoculated mice. Flow cytometry with monoclonal antibodies specific for thymocyte subpopulations was used. Staining of cells for Thy-1 or Thy-1.2 antigens indicated a shift toward low or negative cells. A concomitant increase in cells positive for antigen Pgp-1 was also observed. This is consistent with an increase in the relative frequency of immature blastlike cells. Importantly, thymuses from mice inoculated with Mo+PyF101 M-MuLV did not show these shifts in thymocyte subpopulations.     

I-region linked complementing loci in resistance to viral leukemogenesis in the mouse

A-RadLV, a variant of the radiation leukemia virus, inoculated intrathymically into adult mice, causes a high frequency of leukemia in haplotypesb, f, k, d, p andj on the B10 background, whereas H-2^S mice are resistant. Resistance is dominant and segregates withH-2 ^S in the offspring of (b×s)b and (b×t ₂)b backcrosses. Analysis of recombinant strains revealed that resistance is associated withI-A andI-B. B10.A(5R), a recombinant of two sensitive haplotypes, was found to be resistant, suggesting intra-H-2-gene complementation. The resistance of such complementing loci was demonstrated also in thetrans position by testing F₁ mice bred from sensitive parents. These data are taken to suggest thatI-region linked complementing loci, similar to classicalIr genes, may be involved in resistance to murine leukemia.     

Association of endogenous retroviruses with radiation-induced leukemias of BALB/c mice.

X-irradiation of BALB/c mice in the second month of life induced a high incidence of generalized lymphatic leukemia of T-cell origin, beginning at 7 months of age. Infectious ecotropic murine leukemia virus (B-tropic predominant over N-tropic) was isolable from all tumor extracts but exhibited a wide titer range among individual leukemias. Detection of infectious xenotropic virus usually required extensive amplification on indicator cells. Dual-tropic (mink cell focus-forming) virus has not been found in the leukemias. Expression of ecotropic virus in tail extracts prepared at 6.5 months of age, although greatly enhanced compared with unirradiated controls, was not found to be prognostic of tumor development in individual mice. We conclude that leukemogenesis does not show a simple dependence on infectious murine leukemia virus expression in these mice.     

Cytogenetic characteristics of the cells of different organs in AKR mice during the development of transplanted lympholeukemia

The process of cell generalization of lymphatic leukemia transplanted clone of AKR mice was studied by the routine and differential methods of metaphase chromosome staining. In 99.5% cases, the cells have an additional small chromosome specific for this type of leukemia, the chromosome being comparable in size with 18-19 pairs of chromosomes of mouse karyotype. Generalization process within 7 days' experiment (from the moment of transplantation up to the moment of animals' death from lymphatic leukemia) appeared to be slower in thymus and bone marrow of AKR mice than in spleen, lymphatic nodes and liver of the same animals with nearly the same generalization rate. A change in the frequency of marked leukemic cells in various organs at different time intervals after transplantation of lymphatic leukemia correlated with intensive cell division of an undulating character in all organs. The data obtained show that hyperdiploid cells carrying the specific additional small chromosome are responsible for the generalization process, this chromosome being also present in spontaneous strain of AKR mice, from which this clone was obtained.     

Leukemogenic activity of thymotropic, ecotropic, and xenotropic radiation leukemia virus isolates.

Thymotropic, ecotropic, and xenotropic oncoviruses were isolated from the C57BL/6 mouse radiation leukemia system and were propagated in culture. The purified viruses were inoculated singly and in various combinations into groups of mice, and leukemia incidence was determined. Only the thymotropic virus was leukemogenic in vivo.     

Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation,differentiation and leukemogenic capacity

Long-term cultures of proliferating hematopoietic stem cells derived from bone marrow permit the study of the interaction between murine leukemia virus (MuLV) infection and the proliferation and differentiation of stem cells. We have used this system to analyze the replication of different biological variants of MuLV in bone marrow cells; the effect of MuLV infection upon pluripotent stem cell (CFU-S) proliferation; and the effect of MuLV on differentiation of CFU-S along different hematopoietic pathways. Two MuLV variants were studied in detail: the Moloney strain of lymphatic leukemia virus (Mol-MuLV) and the erythroleukemic Friend virus complex (FLV) consisting of the lymphoid leukemia helper virus and the defective spleen focus-forming virus (SFFV). Mol-MuLV and its sarcoma virus pseudotype, MSV(Mol-MuLV), replicate efficiently in the bone marrow cultures; however, CFU-S are lost more readily than in uninfected cultures, and the cultures are soon represented by a majority population of mononuclear macrophages. On the other hand, infection with FLV produces a prolonged survival of the spleen colony-forming cells, CFU-S, and CFU-C (the committed granulocytic precursor cells). Production of erythroleukemogenic SFFV is maintained in these cultures for more than 40 weeks. No erythroblastic differentiation was observed in vitro, however, neither erythroblast precursor cells (CFU-E) nor hemoglobin-producing cells could be detected. This suggests that the target cell for FLV is an earlier precursor cell.     

Detection of lymphoid leukemia colony-forming cells in abelson virus infected mice: Differences in inbred strains

BALB/c or DBA/2 mice were infected with Abelson murine leukemia virus (A-MuLV), pseudotype Molony murine leukemia virus (M-MuLV). Infection of these mice with 10⁴ focus-forming units of A-MuLV (M-MuLV) induced overt leukemia, detectable grossly or microscopically in 90% of the mice at 20–38 days. However, these methods did not detect leukemia at 17 days or before. Bone marrow cells from A-MuLV-infected leukemic or preleukemic mice were placed in tissue culture in a soft agarose gel. Cells from leukemic or preleukemic BALB/c mice grew to form colonies of 10³ cells or more, composed of lymphoblasts, whereas marrow cells from normal uninfected mice did not. Cells from these colonies grew to form ascitic tumors after intraperitoneal inoculation into pristane-primed BALB/c recipient. Colony-forming leukemia cells could be detected in the marrow of A-MuLV-infected mice as early as 8 days after virus incoluation. The number of colony-forming leukemia cells increased as a function of time after virus inoculation. Colony-forming leukemia cells require other cells in order to replicate in tissue culture. Normal bone marrow cells, untreated or after treatment with mitomycin-C, provide this “helper” function. Only in the presence of untreated or mitomycin-C treated helper cells was the number of colonies approximately proportional to the number of leukemia cells plated. Marrow cells from leukemic BALB/c mice form more colonies than those from leukemic DBA/2 mice. The number of colonies formed per 10³ microscopically identifiable leukemia cells plated was determined to be 2–3 for leukemic BALB/c mice and 0.3 for DBA/2 mice. Cocultivation of leukemic DBA/2 marrow cells with mitomycin-C treated normal BALB/c cells did not increase the number of colonies formed by the DBA/2 leukemic cells. Thus, the decreased ability of DBA/2 leukemia cells to form colonies appears to be a property of the leukemia cell population.     

Differences in pathogenicity among cloned sublines of a murine leukemia virus.

Five clones of the lymphatic leukemia virus 334C were isolated by a procedure designed to maintain homogeneity of the clones. Three of these induced leukemia in mice with the time course of the uncloned parental virus, one induced leukemia with a delayed time course, and one seemed to be biologically inactive. When the clone inducing leukemia most rapidly and the clone inducing leukemia least rapidly were subcloned, the subclones retained the leukemogenicity of the parental clones. The electrophoretic patterns of purified virion proteins and hybridization of viral RNAs with virus-specific DNA suggest that these clones are two closely related variants, not unrelated viruses. Furthermore, in mice infected with these two clones, viral RNA appears in thymuses and spleens at the same time after infection and at nearly the same concentrations. Thus, variations in leukemogenicity can be determined by a genetic property of an ecotropic leukemia virus, and this property is expressed in some manner more subtle than simple control of replication.