Interleukin-12-induced cytotoxicity against syngeneic B cell lymphomas of SJL/J mice

The B cell lymphomas (RCS) that develop spontaneously in 90% of aging SJL/J mice stimulate syngeneic CD4+ Vbeta16+ Th2 cells to produce cytokines, such as IL-4 and IL-5, which promote lymphoma growth. Although RCS cells express a unique superantigen (vSAg) encoded by an endogenous MMTV (Mtv-29) provirus that also elicits IFN-gamma production from na?ve syngeneic lymphoid cells, there is no development of RCS-specific cytotoxicity. However, addition of IL-12 to co-cultures of SJL spleen and irradiated (gamma-)RCS cells resulted in the appearance of effector cells that killed RCS and NK-susceptible target cells. Antibody depletion studies revealed at least two types of RCS/IL-12-induced cytotoxic cells: (1) NK cells (Asialo GM1+) and (2) CD8+ CTL. Despite high titers of IFN-gamma in the SN of co-culture of SJL spleen and gamma-RCS cells, cytotoxicity only developed if IL-12 was also included in the co-cultures. The results of RNAse protection assays and multi-parameter FACS analysis demonstrated an upregulation of IFN-gamma and decrease in IL-4 by activated Th cells in co-cultures with IL-12. These results indicate that inclusion of IL-12 in primary co-cultures of SJL spleen and gamma-RCS cells influences the qualitative nature of the response to favor use of RCS-responsive Th1 rather than Th2 cells to facilitate the production of cytotoxic effector cells. Results of in vivo experiments support this hypothesis, as judged by tumor growth assays and FACS analysis of the tumor cell content of lymphoid tissues. Inhibition of lymphoma growth was observed in mice given gamma-RCS/IL-12-induced effector cells prior to injection of viable RCS cells. These results demonstrate that IL-12 can be used to alter the host immune response leading to induction of cytotoxic effector cells that inhibit the development and/or progressive growth of otherwise resistant B cell lymphomas in SJL/J mice.     

Regulation of reticulum cell sarcoma tumor growth in SJL/J mice by a serum inhibitor affecting T-cell proliferation

The reticulum cell sarcoma (RCS) tumor in SJL/J mice has been shown to stimulate a strong syngeneic proliferative response. A unique characteristic of the RCS tumor is that it requires host T-cells for growth. Consequently, factors that inhibit host T-cell proliferation may have a profound effect on tumor cell growth in vivo. This report investigates the relationship between a serum inhibitor of T-cell proliferation and tumor growth. Inhibition was measured in an interleukin 2-dependent proliferation of the CTLL-2 line. Sera from mice with RCS transplantable lines or from mice with spontaneous tumors were much less inhibitory than were sera from normal syngeneic SJL/J mice or from allogeneic mice. The inhibitory activity appears to follow a circadian rhythm, because serum derived in the morning was more inhibitory than was serum derived in the evening. Serum from female mice was less inhibitory than serum from male mice. In contrast to male mice, serum from 35-week-old female mice was as inhibitory as was that from young (8- to 12-week-old) mice. The mechanism of serum inhibition in tumor-bearing mice was examined. The serum was tested for the presence of interleukin 2 which could decrease inhibitory activity, and no interleukin was found. Furthermore, absorption of the serum with CTLL-2 cells did not enhance the inhibitory effect to the level of normal mouse serum. These results suggest that tumor growth in vivo coincides with less serum inhibitor, providing an adequate T-cell response requisite for tumor growth. This corroborates the notion of RCS-host T-cell interaction necessary for tumor growth which is in part regulated by a serum inhibitor.     

Inhibition of IA positive reticulum cell sarcoma tumor cell growth in syngeneic SJL/J mice by passive administration of monoclonal anti-IA antibody

SJL/J (H-2s) mice develop spontaneous reticulum cell sarcoma (RCS) tumors at the age of 8-11 months. The RCS tumor expresses IA antigens on the cell surface, stimulates syngeneic T-cell proliferation, and appears to depend on host cells' participation for its own growth. The present study investigates the role of passively administered monoclonal anti-IA antibody on RCS tumor growth. The administration of monoclonal anti-IAs antibody into SJL/J mice prior to tumor inoculation or at the same time as tumor transplantation resulted in a significant inhibition of tumor growth. Furthermore, pretreatment of RCS tumor with antibody prior to inoculation also resulted in tumor growth inhibition. The inhibition seen in all cases studied was tumor specific, since the use of normal ascites on antibody directed against unrelated antigens resulted in no inhibition of tumor growth. Examination of tumor cells derived from spleen and lymph nodes of antibody treated mice demonstrated that the observed inhibition of tumor growth was the result of a significant depletion of IA positive tumor cells. In contrast to other tumor systems studied to date whereby anti-IA antibody promotes tumor growth, the present findings demonstrate that passive administration of anti-IA antibodies inhibit RCS tumor growth in syngeneic mice. The possible mechanisms involved are discussed.     

Influence of T-helper cell specific monoclonal antibody on progressive growth of B-cell lymphomas in SJL/J mice: correlation of acute treatment dosage with tumor dormancy or complete remission in long-term survivors

Transplantable follicular center cell lymphomas of SJL/J mice (SJL/FCC) are B-cell-derived tumors that stimulate host T-helper (TH) cells and grow progressively in the lymphoid tissues of immunocompetent, syngeneic recipients. The host TH cells that respond to the I-As determinants on SJL/FCC lymphoma cells produce a variety of lymphokines, some of which (e.g., IL-5) promote in vitro tumor growth. The results presented here demonstrate that removal of the cellular source of tumor growth-promoting lymphokines by treatment of lymphoma-injected mice with TH cell specific monoclonal antibodies (anti-L3T4a) inhibits progressive tumor growth and prolongs survival significantly. However, long-term survival is mediated by different mechanisms, depending on the dosage of L3T4a mAb used. Tumor cells are present, but dormant, in mice that receive low-dose (less than 200 micrograms/injection) treatment. In contrast, tumor cells are undetectable in mice that receive high dose (1200 micrograms/injection) treatment.     

Initiation and characterization of cultured tumor lines from spontaneous reticulum cell sarcoma of SJL/J mice.

Three reticulum cell sarcoma lines (LA1, LA6, and LA8) have been established from SJL/J spontaneous "Hodgkin's-like" reticulum cell sarcoma. All cultures are lymphoid with blast-like morphology by light and electron microscopy and produce the type B neoplasm (Dunn classification) when injected into young SJL/J mice. Identification of these tumors as lymphocytic and monocytic was investigated by surface markers, histochemical staining, and phagocytic function. LA6 and LA8 bear receptors for the Fc portion of immunoglobulin complement receptors; Thy 1.2 antigens and surface immunoglobulin were not detected on any of the three lines. No lines were able to synthesize immunoglobulin or phagocytose Degalon beads. Histochemical staining was presumptive of lymphocytes or lymphoblasts with slight differences between the lines. Although a T- or B-cell classification cannot definitively be made for these tumors lines because of their lack of specific markers, the results are consistent with an immature B-cell origin for the SJL/J reticulum cell sarcoma.     

Treatment of reticulum cell sarcoma in SJL/J mice with Tween 80

Tween 80 (polyoxyethylenesorbitan monooleate) was found to have anticancer activity in the SJL/J tumor system. Investigations to evaluate variables of importance in treatment revealed a loss of inhibitory influence when Tween 80 was administered by the subcutaneous rather than the intraperitoneal route of injection. Age at the onset of treatment was also a factor with significant reductions in incidences of reticulum cell sarcoma when treatment was initiated at 30 or at 40 weeks, but not at 20 weeks of age. Optimal inhibitory effects were achieved with concentrations of 5% Tween 80 (0.2 ml/mouse) or 10% Tween 80 (0.1 ml/mouse). No differences in inhibitory influence on SJL/J tumorigenesis were observed with variation in the frequency of treatment (biweekly, weekly or twice weekly injections).     

Non-H-2-linked control of in vivo growth of SJL/J-derived reticulum cell sarcoma in recombinant inbred strains between BALB/cKe and SJL/J mice

This report investigated the growth of reticulum cell sarcoma (RCS) in several congenic and recombinant inbred strains between BALB/cKe female (H-2d) and SJL/J male (H-2s) mice. SJA20 mice congenic with SJL/J except at the Igh locus supported RCS growth. Recombinant mice of the H-2d haplotype did not support RCS growth. However, recombinant inbred mice of the H-2s haplotype varied in their susceptibility to permit RCS growth in vivo. These results supported the role of non-H-2 gene(s) controlling the growth of RCS. Since the recombinant strains of mice exhibited different immunologic characteristics and since RCS tumor growth depended on the ability of the mice to develop a strong antitumor proliferative response, the findings reported here suggested that non-H-2 genes control the magnitude of the syngeneic proliferative response and consequently regulate RCS growth in vivo.     

Spontaneous lymphomas in SJL/J-(v+) mice: ecotropic and dualtropic virus expression in normal and lymphomatous tissues

Approximately 60% of inbred SJL/J-(v+) adult mice having high levels of ecotropic endogenous XC+ virus showed virus activation within the first month of life, while the others produced virus at comparable levels later on, in an attempt to correlate the time of virus activation with the incidence and latency of lymphomas, the tails of 57 1- and 2-month-old mice were tested for virus presence, and the mice were then observed for lymphoma appearance. While all 2-month-old mice expressed ecotropic virus, only 63% of the 1-month-old mice were virus-positive. However no relationship existed between early virus production (within 1 month) or late virus production and lymphoma latency, total lymphoma incidence, and histopathology. In contrast with high titers of XC+ virus in tail tissues of diseased mice, a markedly low virus content was found in lymphomatous organs. This difference was not due to selective growth of poor virus-producer cells or to inhibitory factors possibly released by the inflammatory cell component. Viral protein content and XC+ virus titer were not closely correlated in the neoplastic organs. Search for XC- viruses revealed that only 1 of 6 aged normal and 16 of 19 lymphomatous mice produced viruses that grew on mink lung cells. By use of a standard limiting dilution cloning procedure, four isolates were obtained that showed tropism for both murine and heterologous cells. Three of these isolates induced cytopathic changes similar to those induced by MCF viruses on mink lung cells but not on mouse cells. Interference and neutralization assays performed to better characterize the virus envelope properties further indicated that SJL/J isolates had features typical of MCF dualtropic viruses.     

Properties of reticulum-cell sarcomas in SJL/J mice. I. Proliferative response to tumor cells of T-derived lymphoid cells from normal mice

Cells from reticulum-cell sarcomas (RCS), tumors with a probable B-cell origin in the SJL/J mouse strain, induced a high degree of proliferation in O-bearing syngeneic lymph-node and thymus cells obtained from young non-tumours mice. Although of considerably lower magnitude, a proliferation of SJL/J thymus cells to syngeneic normal lymph-node cells was also noted. Sera from normal syngeneic mice did not block either of these proliferative responses. Stimulation by RCS cells also occurred with normal lymph-node cells from F1 hybrids, SJL/J times C57Bl/6, and SJL/J times ASW and from the H-2s identical ASW mouse strain. It is suggested that these results are due either to the presence of viral antigens on the surface of RCS cells or to an exaggerated form of the normal syngeneic response of T to B lymphocytes. Another tumor of B-cell origin, the PU5 tumor of BALB/c mice, failed to induce proliferation in normal syngeneic lymph-node or thymus cells.     

In vivo T-lymphocyte response against spontaneous reticulum cell neoplasms in SJL/J mice

The properties of lymphocytes associated with reticulum cell neoplasms (RCN) (type B) occurring spontaneously in SJL/J mice were examined. The activity of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) was used as a marker for activated T-cells. High levels of this enzyme were found in cell suspensions of tumors taken from 6- to 7-month-old mice. Treatment of the cells derived from tumorous organs with anti-theta serum and complement resulted in a loss of the 20 alpha-SDH activity; this indicated that T-lymphocytes populate the RCN. The activated T-cells in the neoplastic tissue were larger than small lymphocytes. In the more advanced stage of tumor growth seen in 1-year-old mice, the percentage of malignant reticulum cells was low and the neoplastic tissue showed low levels of 20 alpha-SDH activity. Tumor cells irradiated in vitro triggered syngeneic lymphocytes to proliferate in tumor-lymphocyte mixed cultures. The T-cell proliferative response measured by [3H]thymidine incorporation was accompanied by a marked increase in 20 alpha-SDH activity. The spleen cells taken from mice bearing old tumors that showed marked fibrosis did not respond to T- and B-cell mitogens. Histologically, the structure of the spleen was preserved, with few or no tumor cells. Spleen cells from age-matched healthy mice responded to mitogens.     

The CD4 cell dependency of SJL/J B-cell lymphomas as a target for cyclophosphamide therapy

SJL/J B-cell lymphomas induce proliferation of syngeneic CD4 cells, on which the tumors are dependent for growth. We sought to determine whether cyclophosphamide (CY) treatment of tumor-bearing mice would be successful through effects on tumor cells, CD4 cells, or both. The markedly increased survival of treated mice derived predominantly from reduced proliferation of CD4 cells in response to tumor. Reduced proliferation of CD4 cells created a microenvironment that was not conducive to tumor growth, as evidenced by the failure of a subsequent tumor cell challenge to treated mice to significantly increase the rate of the mice. We concluded that CY affected the tumor-stimulated environment of SJL/J mice by killing CD4 cells that had been activated by a pre-existing tumor stimulus and by promoting the appearance of a population of CD8 cells that suppressed the ability of residual or recovering CD4 cells to proliferate in response to tumor. The CD8 population from treated mice was specific, based on the ability to suppress a tumor-stimulated mixed lymphocyte response (MLR) and the related autologous MLR and an inability to suppress an allogeneic- or Con A-induced response. Since CD8 cells from CY treated mice had no demonstrable antitumor activity, the most likely suppressor targets were responder CD4 cells in the tumor-stimulated MLR. The results emphasize that the design of a treatment protocol can take advantage of the immunodependency of a tumor.     

Anti-inflammatory effect of spontaneous lymphoma in SJL/J mice.

Histiocytic lymphomas develop spontaneously in about 80% of SJL/J mice between the ages of 8 and 14 months. These animals were used for the determination of whether spontaneously arising cancers compromise monocyte function similar to the monocyte defects described during growth of transplanted tumors. SJL/J mice with tumors accumulated significantly fewer macrophages than did age-matched animals without tumors either on sc implanted filters or in peritoneal exudates induced by phytohemagglutinin. There was no corresponding defect in polymorphonuclear neutrophil responses. Whereas no apparent correlation existed between tumor size and degree of inhibition, animals without demonstrable tumors had no inflammatory defects. Aging did not alter the inflammatory response to implanted filters but increased both the resident peritoneal macrophage population and the total macrophage yield to phytohemagglutinin provocation. When given transplants of histiocytic lymphomas, young SJL/J mice developed similar inflammatory defects. This study represents the first demonstration that spontaneous tumors, in addition to transplanted tumors, produce abnormalities in monocyte inflammatory responses.     

Evaluation of the anticancer activity of CuDIPS in SJL/J mice

A copper complex, Cu(II)2(3,5-diisopropylsalicylate)4 (CuDIPS) was found to have anticancer activity in the SJL/J tumor system. Toxic effects were eliminated by use of the subcutaneous rather than the intraperitoneal route of administration and, unlike Tween 80, polyvinyl alcohol was shown to have no effect on development of reticulum cell sarcoma when used as the carrier. Age at the onset of treatment was a factor of importance with significant reductions in tumor incidences when therapy was initiated at 20 or at 40 weeks, but not at 30 weeks of age. Maximal inhibitory effects on tumor incidence were achieved by CUDIPS concentrations of 0.35 mumol/mouse and 0.50 mumol/mouse. No difference in inhibitory influence was observed when mice were injected weekly rather than biweekly with the complex.     

Studies on leukemia development in the SJL/J strain of mice

Spontaneous reticulum cell neoplasms developed in 70-80% of intact male and female SJL/J mice at a mean age of 380 days. The neoplasms had a basic histological pattern of a multicellular type-B reticulum cell neoplasm, as designated by Dunn. The tumors were transplanted in isogenic mice. Serial passages of cell suspensions of these tumors were carried on through at least 4 tumor-inducing generations, and the growing transplanted tumors maintained the structure of a reticulum cell neoplasm similar to the original tumor. A histological survey of spontaneous lesions in SJL/J mice revealed different characteristics of reticulum cell neoplasms, some similar to Hodgkin's lesions. Five or ten feedings of 7,12-dimethylbenz-[alpha]anthracene (DMBA) in polyethylene glycol 400 (1 mg DMBA per feeding) to SJL/J mice induced lymphosarcomas in 74-83% of the mice at a mean age of 157-188 days. A single feeding of DMBA induced lymphosarcomas in only 27% of the mice at a mean age of 246 days. These DMBA-induced lymphatic leukemias do not appear to depend on the thymus for development--a 60% incidence of lymphosarcomas was obtained in adult thymectomized mice treated with DMBA, and 27% of the thymectomized DMBA-treated mice developed myeloid leukemia at a mean age of 180 days. Urethan was only slightly leukemogenic in SJL/J mice.     

Detection of a transforming gene in spontaneous reticulum cell sarcoma of SJL/J mice: genetically linked and host-dependent neoplasia

Spontaneous reticulum cell sarcoma (RCS) tumor induction occurs in 90% of SJL/J mice of 8-13 months of age. Tumor induction and growth has been shown to be under the influence of both H-2 and non-H-2 genes as well as the presence of an intact host T-cell system. We postulated that cellular oncogenes may play a role in the induction, growth, and characteristics of RCS. DNA-mediated gene transfer protocols were adopted to investigate the presence of transforming genes in DNA from RCS of SJL/J mice. High molecular weight DNA was isolated from these tumors as well as from brains and livers of control tumor-free SJL/J mice and transfected into NIH-3T3 mouse and F2408 rat fibroblast cell lines. Foci of transformed cells with a peculiar round morphology were scored in both rat and mouse cultures given tumor DNA, but not in those receiving DNA from normal tissues. DNA from first-cycle transformants was transfected in further cycles of transfection, giving rise to foci with similar morphological appearances and growth properties. These experiments suggest that a transforming gene, present in RCS spontaneous tumors, is involved in the malignant conversion of the transfected normal fibroblasts. The implication of these results with respect to the induction and growth properties of RCS is discussed.     

Progression of transplanted SJL/J lymphomas attributed to a single aggressive H-2Ds-negative lymphoma

Spontaneously arising, H-2Ds-positive SJL/J lymphomas have been reported to become irreversibly more aggressive and H-2Ds-negative upon successive transplantation in syngeneic mice. In an effort to determine whether this process was one of tumor progression, we sought to: (a) establish whether a clonal relationship exists between readily transplantable aggressive SJL/J lymphomas and their respective indolent predecessors; and (b) identify genetic events critical to the process of acquisition of increased malignancy. Examination of putatively distinct, aggressive, H-2Ds-negative lymphomas, including the long term transplantable line RCS5, revealed them to have the same heavy and light immunoglobulin chain gene rearrangement patterns, a characteristic karyotype marked by nine chromosomal abnormalities, and approximately ten newly acquired ecotropic murine leukemia proviruses at similar genomic sites. Independent, spontaneously arising H-2Ds-positive lymphomas, in early transplant, were found to be genetically distinct from the respective more malignant H-2Ds-negative tumors to which they gave rise during successive transplantation. The data are interpreted as indicating that the aggressive H-2Ds-negative tumors in this study originated from a common source, most likely the RCS5 tumor, rather than through progression of separate spontaneously arising SJL/J lymphomas. It cannot be concluded which of the multiple genetic abnormalities of the H-2Ds-negative tumors were critical to their highly malignant phenotype. However, chromosomal abnormalities and newly acquired murine leukemia proviruses are discussed as to the roles they might play in SJL/J lymphomas.     

High incidence of acute myeloid leukemia in SJL/J mice after x-irradiation and corticosteroids

SJL/J mice which developed a high incidence of spontaneous reticulum cell neoplasms, developed a low rate incidence (20–25%) of myeloid leukemia (ML) after X-irradiation. The possible effect of adrenal steroid imbalance to radiation-induced ML in SJL/J mice was tested. Intact and thymectomized animals were exposed to a single dose of 300 r whole body irradiation and treated with either hydrocortisone acetate, prednisone, metyrapone and adrenocorticotropin as coleukemogenic agents.

Hydrocortisone and prednisone exerted a marked coleukemogenic effect, increasing the ML incidence to a similar rate of about 50–70%, at a mean latent period of 300 days. Prominent leukemic infiltration were observed in the bone marrow, spleen, lymph nodes and liver of the leukemic animals. Results of cytological and histological studies, including cytochemistry and ultrastructure, were all consistent with the diagnosis of acute myeloid leukemia (AML). Since AML is the type of human secondary leukemia which appears increasingly in patients treated with alkylating drugs and/or irradiation and corticosteroids for Hodgkin's disease or other neoplastic diseases, the experimental model of AML induced in SJL/J mice could be used for elucidation of mechanisms of leukemogenesis in secondary leukemia.     

Evaluation of combinations of CuDIPS, Tween 80 and cyclophosphamide as therapy for reticulum cell sarcoma in SJL/J mice

Three agents, shown previously to have anticancer activity in the SJL/J tumor system, were tested in combinations for effects on survival rate and incidence of reticulum cell sarcoma at 52 weeks of age. Combined treatment with Cu(II)2(3,5-diisopropylsalicylate)4 (CuDIPS) and polyoxyethylenesorbitan monooleate (Tween 80) proved of no value. Indeed, inhibitory influence on tumorigenesis was diminished when Tween 80 was injected weekly at 24 hours or at 72 hours after weekly injections of CuDIPS. However, tumor incidence was reduced significantly, 90% (controls) to 7%, in mice treated weekly with cyclophosphamide (CY) at 24 hours after weekly injection of CuDIPS. Further, weekly administration of CY at 72 hours after weekly injection of Tween 80 resulted in a significant decrease in tumor incidence, 85% (controls) to 0%, and an increase in survival rate, 75% (controls) to 100%. Implications of these findings are discussed.