Graft versus leukemia without fatal graft-versus-host disease in AKR mice.

BW 5147 leukemia in AKR mice has been successfully treated by adoptive immunotherapy using allogeneic spleen cells from C57BL/6J mice. Graft-versus-host reaction was prevented by treatment with spleen cells from a second allogeneic strain (CBA, H-2 identical with AKR), followed by cycloposphamide and syngeneic spleen cells. Successful treatment of leukemia without graft-versus-host reaction is dependent upon a close relationship at the H-2 locus between the second allogeneic donor and the host AKR mice, since cells from a non-H-2 identical donor (DBA/2) do not increase survival. The doses of cyclophosphamide and of C57BL/6J spleen cells are also parameters of critical importance in successful treatment.     

Detection of a unique antigen on radiation leukemia virus-induced leukemia B6RV2

Radiation leukemia virus-induced leukemia of a male C57BL/6 mouse, B6RV2, is immunogenic to female BALB/c X C57BL/6 F1 mice. In these mice, B6RV2 tumors regressed after initial growth, and after tumor regression the mice were resistant to repeated inocula of up to 10(8) B6RV2 cells. Serum from these mice reacted with B6RV2 in mixed hemadsorption or protein A assays, and absorption analysis indicated that the antigen was restricted to B6RV2; it could not be detected in normal thymocytes or spleen concanavalin A blasts from different inbred strains, nor in 16 C57BL/6 or BALB/c leukemias. Spleen cells from mice in which the tumor had regressed were cytotoxic to B6RV2 after in vitro stimulation with B6RV2, as shown by 51-chromium release assay. This cytotoxicity was eliminated by pretreatment of the cells with anti-Thy-1.2, anti-Lyt-2.2, anti-Lyt-3.2, and complement, indicating that the effector cells were T-cells. The specificity of T-cell killing of B6RV2 was examined by competitive inhibition assays with unlabeled cells; only B6RV2 inhibited killing, while eight other C57BL/6 leukemias did not inhibit. Thus, the antigen on B6RV2 defined serologically and by cytotoxic T-cells is a unique antigen. However, it was not revealed by antibody-blocking test whether the unique determinant defined serologically was related to that recognized by T-cells; B6RV2 antiserum did not block lytic activity in the absence of added complement, irrespective of whether the target cells were untreated or anti-H-2b-treated B6RV2. H-2Kb antisera, but not H-2Db antisera, blocked lysis. This indicated that the H-2Kb molecule was exclusively involved in recognition of B6RV2 by cytotoxic T-cell.     

Immunotherapy of murine leukemia. VII. Prevention of friend leukemia virus-induced immunosuppression by passive serum therapy

Previous studies have suggested that the passive therapy of Friend leukemia virus (FLV)-induced disease with chimpanzee anti-FLV serum operates by reducing the level of infectious virus in the treated animal below the immunosuppressive threshold, thereby allowing the host to mount anti-viral immune responses which are responsible for long-term protection. The present study was undertaken to examine directly the effect of passive serum therapy on the marked immunosuppression induced by FLV in progressively infected mice, as well as to determine whether virus-specific host cellular immune effector functions are augmented in serum-protected animals. Using a variety of assays of host immunocompetence, including natural killing (NK), antibody-dependent cellular cytotoxicity (ADCC) in vivo and in vitro induction of allogeneic killers, and mitogen blastogenesis, a marked compartmentalization of FLV immunodepression was observed in progressively infected DBA/2 mice, possibly reflecting the distribution of FLV target cells in various host lymphoid populations. Thus, spleen-cell functions were suppressed most rapidly and to the greatest degree, followed by peritoneal cells and peripheral blood lymphocytes, while lymph node cells and thymocytes maintained normal levels of activity. In contrast, serum-protected mice demonstrated no sign of FLV-induced immunosuppression regardless of the host effector-cell population or immune function examined. However, we were not able to identify host anti-viral cellular immune functions which are significanly enhanced in serum-protected animals; thus the specific role of the host immune system in the passive serum therapy of FLV-induced disease remains undefined at the present time.     

Autogenous immunity to mouse mammary tumor virus in mouse strains of high and low mammary tumor incidence.

Specific radioimmune precipitation assays were utilized to demonstrate the presence of precipitating antibodies to mouse mammary tumor virus (MMTV) in the high-spontaneous mammary tumor strains of mice: C3H/HeN+, GR/N, BALB/cfC3H, and C57BL/6 X C3H F1 (hereafter called B6C3F1). Antibody titers in C3H/HeN+ mice increased with age, with highest titers observed in tumor-bearing animals. MMTV-precipitating antibodies were not detectable by radioimmune precipitation assay in low-mammary tumor strains (AKR, BALB/c C57BL/6, and C3H/HeN-) but were detectable in MMTV-inoculated BALB/c mice. Appearance of antibodies preceded palpable tumor formation, and antibody titers were directly correlated to virus dose. Natural antibody to MMTV in C3H/HeN+ and B6C3F1 mice coexists with the murine leukemia virus natural antibody as determined by competition radioimmunoassays.     

Significance of strain and sex differences in the development of 252Cf neutron-induced liver tumors in mice.

Mouse liver tumors occurring in C3H/HeN, C57BL/6N and C3B6F1 hybrid (C3H x C57BL) were studied following 252Cf fission neutron irradiation. Three strains of mice of both sexes (about 30 mice/group) were irradiated once with 252Cf at doses of 0, 12.5, 50 and 200 cGy. The groups were observed for 13 months after irradiation. The incidence of liver tumors in the non-irradiated controls was 0% in both sexes of C57BL/6N, 11.7% in males and 0% in females of C3B6F1 and 39.5% in males and 11.4% in females of C3H/HeN mice. In the four strains of mice thus far studied, including B6C3F1 hybrid (C57BL x C3H) which was previously studied, 252Cf irradiation has increased the tumor incidence dose-dependently in males and in females, but less effectively in females. The mean number and size of liver tumors were clearly correlated with tumor incidence. The incidence was always highest in C3H/HeN mice of both sexes, followed by B6C3F1, C3B6F1 and C57BL/6N mice. The influence of sex hormones was studied in B6C3F1 mice of both sexes after 200 cGy of 252Cf irradiation. In males, the incidence of liver tumors was significantly decreased from 55.2% to 23.3% and 25.9% after orchidectomy, and in females it was slightly decreased from 27.6% to 14.8% and 18.8% after ovariectomy. Supplementation of testosterone in orchidectomized mice did not restore the occurrence of liver tumors.     

Significance of Strain and Sex Differences in the Development of 252Cf Neutron-induced Liver Tumors in Mice

Mouse liver tumors occurring in C3H/HeN, C57BL/6N and C3B6F1 hybrid (C3H × C57BL) were studied following ²⁵²Cf fission neutron irradiation. Three strains of mice of both sexes (about 30 mice/group) were irradiated once with ²⁵²Cf at doses of 0,12.5, 50 and 200 cGy. The groups were observed for 13 months after irradiation. The incidence of liver tumors in the non-irradiated controls was 0% in both sexes of CS7BL/6N, 11.7% in males and 0% in females of C3B6F1 and 39.5% in males and 11.4% in females of C3H/HeN mice. In the four strains of mice thus far studied, including B6C3F1 hybrid (CS7BL × C3H) which was previously studied, ²⁵²Cf irradiation has increased the tumor incidence dose-dependently in males and in females, but less effectively in females. The mean number and size of liver tumors were clearly correlated with tumor incidence. The incidence was always highest in C3H/HeN mice of both sexes, followed by B6C3F1, C3B6F1 and C57BL/6N mice. The influence of sex hormones was studied in B6C3F1 mice of both sexes after 200 cGy of ²⁵²Cf irradiation. In males, the incidence of liver tumors was significantly decreased from 55.2% to 23.3% and 25.9% after orchidectomy, and in females it was slightly decreased from 27.6% to 14.8% and 18.8% after ovariectomy. Supplementation of testosterone in orchidectomized mice did not restore the occurrence of liver tumors.     

Immunochemotherapy of transplantable Moloney leukemia with cyclophosphamide and allogeneic spleen lymphocytes and reversal of graft-versus-host disease with alloantiserum.

Histoincompatible (H-2) spleen cells from C57BL/6 mice that were sensitized with Moloney sarcoma virus caused fatal graft-versus-host disease in BALB/c mice when the cells were used in conjunction with an immunosuppressive, chemotherapeutic dose of cyclophosphamide to treat transplantable Moloney virus-induced leukemia. When antiserum against the donor spleen cells was administered 2 days after immunochemotherapy, graft-versus-host disease was prevented, but no immunotherapeutic effect was observed. When the antiserum was delayed for 3 days or more, lethal graft-versus-host disease occurred. Substitution of Moloney sarcoma virus-sensitized BALB/c X C57BL/6 F1 (hereafter called CB6F1) spleen cells for C57BL/6 cells, in conjunction with cyclophosphamide, "cured" 70% of the treated mice (accumulated value of two experiments). When anti-CB6F1 serum (alloantiserum) was administered 2 days after immunochemotherapy, the immunotherapeutic effect was abolished. Alloantiserum was not able to reverse the immunotherapeutic effect 3 days postgrafting or later, and there resulted a high percentage of long-term survivors. About two-thirds of the cured mice had positive donor-specific gamma-globulin titer 8 weeks postgrafting.     

Genetic control of hapten-reactive helper T-cell responses and its implications for the generation of augmented antitumor cytotoxic responses

The genetic control of hapten-reactive helper T-cell activity involved in cytotoxic T-lymphocyte (CTL) responses and its implications for augmenting tumor-specific immunity were studied. C57BL/6N mice were immunized to trinitrophenyl (TNP) or N-iodoacetyl-N'-(5-sulfonic l-naphthyl)ethylenediamine (AED) hapten by inoculation of hapten-modified syngeneic spleen cells. Spleen cells from these hapten-immunized mice were tested for hapten-reactive helper T-cell activity for generation of CTL. TNP-primed spleen cells resulted in only marginal help for the generation of anti-TNP-modified syngeneic spleen cell (TNP-self) CTL response when cocultured with normal C57BL/6N spleen cells (responding cells) in the presence of TNP-self. In contrast, AED-primed spleen cells exhibited appreciable help for AED-induced CTL responses. Furthermore, AED-helper, but not TNP-helper, T-cell activity was demonstrated to augment the generation of antitumor (RBL-5 leukemia) CTL responses from normal syngeneic spleen cells when stimulated with the corresponding hapten-self plus RBL-5 tumor cells. These results indicate that the successful augmentation of syngeneic tumor immunity through T-T-cell interaction with the use of hapten-reactive helper T-cells can depend on selection of the appropriate haptenic reagent in an individual expressing a given major histocompatibility haplotype.     

Abelson murine leukemia virus-induced thymic lymphomas: transformation of a primitive lymphoid precursor

For the investigation of whether Abelson murine leukemia virus (A-MuLV) is able to transform in vivo lymphocytes other than those of the B-cell lineage, newborn BALB/c and C57BL/6 mice were given an injection of A-MuLV directly into the thymus. Thymic lymphomas appeared with a short latent period of 4-5 weeks in BALB/c mice and 8 weeks in C57BL/6 mice. Cell lines derived from some thymic lymphomas presented a very immature phenotype and did not express cellular markers of either T-cells (Thy 1.2, Lyt 1.2, and Lyt 2.2) or B-cells (cytoplasmic IgM) even after treatment with several differentiation inducers. Molecular analysis showed that T-cell receptor (TCR) beta chain genes were never rearranged; in one case only, rearrangement of TCR gamma chain genes could be demonstrated, confirming the immaturity of the presumptive T-cell lines studied. Furthermore, the cell lines consistently carried diversity (D)-joining (J) but not variable (V)-D-J rearrangements of the immunoglobulin heavy chain genes. On the whole, these findings suggest that following intrathymic A-MuLV injection neoplastic transformation does involve lymphocytes possibly of T-cell lineage, at a very early stage of differentiation.     

Oncogenicity of friend-virus-infected cells: Determination of origin of spleen colonies by the H-2 antigens as genetic markers

Spleen cells from mice infected with Friend leukemia virus (FLV) inoculated by the intravenous route give rise to macroscopically visible colonies in the spleens of normal F₁ histocompatible hybrid hosts. A study of H-2 antigens as generic markers for identification of strains of origin of cells constituting the spleen colonies was undertaken. The standard cytotoxic test was demonstrated to be suitable for characterizing the H-2 antigens present on the surface of spleen cells from normal or FLV-leukemic parents or F₁ hybrid mice. Individual colonies dissected out of the spleen of (C3H × C57BL/6) F₁ recipients (H-2^k/H-2^b), 10 days after the intravenous graft of FLV-infected spleen cells of C3H origin (H-2^k), were all sensitive to anti-C57BL/6 antibodies. In the same way, colonies obtained from the spleens of (DBA/2 × C57BL/10) F₁ recipients (H-2^d/H-2^b) grafted with DBA/2 leukemic spleen cells (H-2^d) were all sensitive to both anti-H-2^b and anti-H-2^d antibodies. These results directly prove that the main cell population constituing a spleen colony arises from the recipient. The authors conclude that the spleen colonies do not result from the neoplastic proliferation of injected donor cells but rather from the multiplication of host cells transformed by Friend virus produced by the grafted cells.     

In vitro studies of cell-mediated immunity to Moloney murine leukemia virus and Moloney leukemia-associated surface antigens.

Cell-mediated immunity to Moloney murine leukemia virus (M-MuLV) and to tumor-associated surface antigens of leukemia cells induced by the virus was studied with an in vitro migration inhibition factor assay. Spleen cells of C57BL/6N mice at Day 14 following inoculation with Moloney murine sarcoma virus, produced migration inhibition factor in response to M-MuLV. The Moloney murine sarcoma virus-immune spleen cells, however, did not respond to other murine type C viruses, to AKR and Rauscher viruses, or to murine mammary tumor virus. The immune spleen cells also responded specifically to purified glycoprotein with molecular weights of 69,000 and 71,000 and proteins with molecular weights of 30,000 and 12,000, but not to protein with a molecular weight of 10,000, of the homologous M-MuLV. Migration inhibition factor production was also observed in response to soluble 3 M KCl extracts of leukemia cells, MBL-2, induced by M-MuLV. Similarly, the immune spleen cells responded to membrane fractions purified from the MBL-2 cells. Comparable membrane fractions prepared from a Gross virus-induced leukemia, E male G2, and a radiation-induced leukemia, RL male 1, were not active. The tumor-associated surface antigens of MBL-2 membranes could be solubilized by the detergent, Nonident P-40. Thus, C57BL/6N mice inoculated with Moloney murine sarcoma virus developed cell-mediated immunity to envelope and some internal antigens of M-MuLV and also to tumor-associated surface antigens of a tumor induced by this leukemia virus.     

红色荧光蛋白标记的小鼠白血病模型的建立

 目的　利用红色荧光蛋白（DsRed）标记的小鼠淋巴瘤EL4细胞株,建立荧光标记的小鼠白血病模型,并对模型进行分析和鉴定。方法　将EL4／DsRed细胞以低剂量（5×102／只）、中剂量（5×103／只）、高剂量（2.5×104／只）经尾静脉注入经清髓照射的C57BL／6小鼠体内,同时接种骨髓细胞5×106／只,采用流式细胞术（FCM）、反转录聚合酶链反应（RT-PCR）、组织病理等方法鉴定小鼠成模情况。结果　C57BL／6小鼠接种不同剂量EL4／DsRed细胞后白血病发病率达100 %,移植后第2周FCM示受鼠肝、脾、骨髓和外周血中有大量EL4／DsRed细胞;各组组织器官病理检查呈现出不同程度的肿瘤细胞浸润。结论　用DsRed标记的EL4细胞以5×102／只植入C57BL／6小鼠,即可成功建立荧光标记的小鼠白血病模型,可为白血病发病机制、微小残留病检测等研究提供有价值的动物模型。     

两种肺纤维化模型小鼠肺组织胶原含量定量分析方法的比较研究

目的利用两种肺纤维化小鼠模型,对肺组织中胶原表达定量分析的两种方法——天狼猩红染色图像分析和羟脯氨酸测定法进行比较。方法采用20Gy^60Coγ射线胸部局部照射C57BL／6J和C3H／HeN两种小鼠分别建立敏感性／抗性肺纤维化模型,照射后3m活杀取肺组织,左肺连续切片,根据均匀系统随机抽样的原则,在连续切片中均匀随机抽取一组切片,进行天狼猩红染色及图像分析;右肺采用碱水解法进行羟脯氨酸含量测定。结果两种分析方法结果均表明,γ射线照射后3mC57BL／6J小鼠肺组织胶原含量较照射前明显增高,且显著高于C3H／HeN小鼠肺组织中胶原含量。天狼猩红染色图像分析法显示,C57BL／6J小鼠肺组织胶原面密度为C3H／HeN小鼠的2．29倍,而羟脯氨酸测定法显示,C57BL／6J小鼠肺组织羟脯氨酸含量为C3H／HeN小鼠的1．34倍。结论天狼猩红染色图像分析法较羟脯氨酸含量测定法能较理想地反映肺泡壁胶原含量的变化,且能更客观反映敏感性／抗性肺纤维化小鼠胶原表达的差异。     

Immunosuppression by the radiation leukemia virus and its relation to lymphatic leukemia development

The immune response of C57BL/6 mice following intra-thymic injection of the radiation leukemia virus was tested. A marked depression in plaque-forming capacity of the 19S type to sheep red cells was observed in adult intact and thymectomized mice inoculated with the virus 5–60 days prior to the immunization. Serum hemagglutinin titers were depressed as well, but to a lesser degree. The radiation leukemia virus did not significantly depress cell-mediated immunity. Tests for prolongation of skin allograft survival, allogeneic tumor takes and graft-versus-host reactivity (Simonsen's test) were used as parameters for the evaluation of defects in cellular immunity. No correlation was found between the immunosuppressive effect of the radiation leukemia virus and lymphatic leukemia development. It is proposed that the decrease observed, with aging, in the immune response of normal C57BL mice, to sheep red cells, is due to the radiation leukemia virus that is present during post-natal life in non-irradiated C57BL mice, and that its titer increases with aging.     

Cellular immune reactivity in vitro and tumor rejection provided by tumor-associated antigens of friend-virus-induced leukemia.

Cell-mediated immunity (CMI) and tumor rejection were studied in the Friend virus leukemia system of C57Bl/6 mice. Mice were immunized with Friend leukemia virus (FLV) or X-irradiated FBL-3 leukemic cells and studied temporally for the development of CMI reactivity by assays of 51Cr release lymphocyte cytotoxicity, lymphocyte transformation, migration inhibition, Winn tumor cell neutralization and transplantation rejection. High levels of specific lymphocyte cytotoxicity were observed by day 7 f0llowing FLV infection; this reactivity reached a peak between 17 and 21 days, and returned to background levels by day 36. Further, positive Winn assays were obtained with spleen cells from mice immunized with FLV at times when the mice resisted live FBL-3 tumor challenge. Positive lymphocyte transformation was obtained with spleen cells from mice immunized with FLV or FBL-3, but not with cells from normal mice or mice immune to a syngeneic methycholanthrene-induced tumor, when cultured with papain-soluble FBL-3 or RBL-5 tumor-cell extracts or mitomycin-C (MMC)-treated FBL-3 or RBL-5 cells. Positive reactivity in the lymphocyte transformation assay occurred after reactivity had peaked in the lymphocyte cytotoxicity test. Similar positive macrophage migration inhibition patterns were also obtained with peritoneal exudate cells (PEC) from FLV-immunized mice using papain-solubilized tumor-associated antigen (TAA) from FBL-3 cells. These data suggest that sequential development and modulation of CMI reactivity occurs as observed in different assays following immunization in this system.     

Strain differences in the susceptibility to azoxymethane and dextran sodium sulfate-induced colon carcinogenesis in mice

We have recently developed a mouse model for colitis-related colon carcinogenesis by a combined treatment with azoxymethane (AOM) and dextran sodium sulfate (DSS) in male ICR mice. However, strain differences in the sensitivity to AOM/DSS-induced colon carcinogenesis in mice have yet to be elucidated. The aim of this study was to determine the presence of any genetically determined differences in sensitivity to our model of colon carcinogenesis in four inbred strains of mice. Male Balb/c, C3H/HeN, C57BL/6N and DBA/2N mice were given a single intraperitoneal injection of AOM (10 mg/kg body wt), followed by 1% DSS (w/v) in drinking water for 4 days, and thereafter they received no further treatment for up to 16 weeks. At the end of the study (Week 18), all mice were killed and a histopathological analysis of their colon was performed. The incidence of colonic adenocarcinoma was 100% with a multiplicity (no. of tumors/mouse) of 7.7+/-4.3 in the Balb/c mice and 50% with a multiplicity of 1.0+/-1.2 in the C57BL/6N mice. On the other hand, only a few colonic adenomas, but no adenocarcinomas, developed in the C3H/HeN mice (29% incidence with a multiplicity of 0.7+/-1.5) and the DBA/2N mice (20% incidence with a multiplicity of 0.2+/-0.4). The inflammation and immunohistochemical nitrotyrosine-positivity scores of the mice treated with AOM and DSS in the decreasing order were as follows: C3H/HeN>Balb/c>DBA/2N>C57BL/6N and Balb/c>C57BL/6N>C3H/HeN>DBA/2N, respectively. Our results thus indicated the presence of strain differences in the susceptibility to AOM/DSS-induced colonic tumorigenesis. These differences may have been directly influenced by the response to nitrosation stress due to the inflammation caused by DSS.     

Group-specific antigen of murine leukemia viruses in mice of low-leukemic strains

The distribution of group-specific antigen of murine leukemia viruses (MuL V-gs) was studied in mice of low-leukemic strains: C57BL/6, C57BL/10Sn, BALB/c and others. A rabbit monospecific anti-MuLV-gs serum was used for the antigen detection. The analyses were carried out by an indirect immunoradioautography technique which permitted the detection of trace amounts of the antigen. MuLV-gs was found in the spleen of mice of all strains studied. It was present in mouse embryos as well as in adult animals.     

Inheritance of susceptibility to Friend mouse leukemia virus. 8. Effect of a single genetic locus on the pathogenesis of the leukemia

The effect of the Fv locus on the pathogenesis of Friend virus-induced leukemia was studied with two pairs of mouse strains which are congenic except for the locus. The rapid spleen enlargement or spleen focus formation which is characteristic of Friend virus infection occurred in the mice with Fv^s/Fv^s genotype, DDD and C57BL/ 6-Fv^s. These early responses were not seen in the mice with Fv^r/Fv^r genotype, DDD-Fv^r and C57BL/6. When the infected Fv^r/Fv^r mice were observed for 150 days, all of the DDD-Fv^r mice, but none of the C57BL/6, developed splenomegaly. Histological studies of the DDD-Fv^r mice revealed erythroblastic, lymphatic or granulocytic leukemia, or myeloproliferative disorder with a prominent megakaryocyte proliferation. This observation indicates that Friend leukemia virus is able to induce various types of leukemias in mice and the early characteristic responses are completely abrogated in the mice with Fv^r/Fv^r genotype. Involvement of genetic factor (s) other than the Fv locus in the late-occurring leukemias was suggested.     

Cellular immune response induced by the radiation leukemia virus (RadLV)

Studies on the cellular basis involved in the build up of immunity in C57BL/6 mice inoculated intrathymically with the radiation leukemia virus (RadLV) have been carried out. The virus inoculated C57BL/6 mice were resistant to isotransplantation of leukemic cells for two months after immunization. Lymphoid cells from immune mice present in the thymus, lymph nodes, spleen and peritoneal exudate were found to cause tumor growth retardation. RadLV inoculated C57BL/6 mice performed transplantation resistance only to leukemic cells induced by RadLV (127 LC), but not to radiation induced leukemias (XRL-1, XRL-2, XRL-3), to EL₄ or to any other syngeneic tumors tested. The immunological specificity of the lymphoid cells taken from immunized mice (with RadLV) was tested in vivo and in vitro against leukemic cells induced in C57BL/6 mice by various agents (RadLV, chemical carcinogens and X-rays) and were found to be highly specific for the presence of RadLV associated antigen(s) on their cell surface. A cytostatic effect rather than a cytolytic effect was demonstrated, when effector:leukemic cell interaction was tested in vitro. The cytostasis assay was performed on syngeneic leukemic cells (induced by RadLV) in suspension at various leukemic: effector cell ratios. A specific reaction occurred mainly at the ratio of 1:80.