Transplantable Marek's disease lymphomas. III. Induction of MHC-restricted tumor immunity by lymphoblastoid cells in F1 hosts

F1 chickens, which were a cross between birds of the related inbred lines G-B1 and G-B2, were immunized with in vitro cultured virus-non-producer lymphoblastoid cells that were either syngeneic or allogeneic with the challenge tumor cells. The lymphoblastoid cells were derived from Marek's disease herpesvirus (MDV)-induced transplantable tumors. Previous findings showed that such immunization of G-B1 and G-B2 chickens prevented early mortality caused by the tumors. Lymphoblastoid cells syngeneic with the tumors were more effective than allogeneic cells, suggesting that an MHC-restricted immune response was induced, or, alternatively, that immune elimination of allogeneic cells prevented them from initiating strong immunity to MDV-associated tumor antigens. Immune elimination of the immunizing cells should not occur in F1 birds heterozygous for the 2 different parental MHC haplotypes (B6 and B13). Early mortality among F1 chickens immunized with lymphoblastoid cells that were syngeneic with the challenge tumor cells was significantly lower than for non-immunized control chickens or for birds immunized with lymphoblastoid cells that were allogeneic with the challenge tumor cells. Our results suggest that MDV-induced tumor antigens may be recognized by the host as altered-self MHC antigens.     

Development of cell-mediated immunity to Marek's disease tumor cells in chickens inoculated with Marek's disease vaccines.

Chickens inoculated with herpesvirus of turkeys or with apathogenic or attenuated vaccine strains of Marek's disease virus (MDV) developed a T-cell-mediated immune response to Marek's disease (MD) tumor cells. This immune response was detected in a 4-hour 51Cr-release assay in which effector cells obtained from spleens of vaccinated chickens were reacted with 51Cr-labeled target cells of an MD lymphoblastoid cell line (MSB-1). The cytotoxic effector cells generated by the vaccine viruses had characteristics similar to those noted previously for anti-MSB-1 effector cells generated by MDV. The immune response was specific to MSB-1 cells, because another target cell line (TLT) antigenically unrelated to MSB-1 cells was not lysed by the effector cells nor did the unrelated target cells inhibit the cytotoxicity of effector cells against MSB-1 target in a cold-target inhibition assay. Because MSB-1 cells contain MD tumor-associated surface antigen, we postulated that the immune response detected in the vaccinated chickens may be directed against this antigen and that the antitumor antigen immunity may play a role in the mechanism of vaccine protection against lymphoma development by pathogenic MDV.     

Establishment of Marek's disease lymphoblastoid cell lines from transplantable versus primary lymphomas

Six new Marek's disease (MD) lymphoblastoid cell lines were established in vitro by cultivation in a medium containing 2-mercaptoethanol (2-ME). Attempts using primary lymphoma cells were generally unsuccessful; only one of 28 lymphomas yielded a cell line and that one came from an experimentally immunosuppressed chicken. In contrast, two of seven low-passage, and two of two established MD transplantable lymphomas grew readily in vitro. A sixth line was obtained using buffy coat cells from a leukemic chicken. It was concluded that the use of transplantable tumor cells and a medium containing 2-ME provided a combination highly suited to the establishment of cell lines from MD.     

Properties of a chicken lymphoblastoid cell line from Marek's disease tumor.

Properties of a chicken lymphoblastoid cell line (MSB-1) from a Marek's disease tumor were studied. The cell line grew well at 41 degrees C in medium RPMI-1640 supplemented with 10% bovine fetal serum and had a doubling time of 8-12 hours. Cells grown in stationary suspension culture did not attach to the vessel and had the morphology of typical lymphoblasts. At 37 degrees C, the cell line grew initially but ceased to divide after several subcultures. In the subcultures maintained for 48-72 hours, 1-2% of the cells produced Marek's disease virus (MDV)-specific intracellular and mambrane antigens and contained herpesvirus particles when examined by the electron microscope. Cocultivation of these cells with duck or chicken embryo fibroblast cultures resulted in transfer of infection and production of microplaques typical of MDV. Peripheral nerve lesions and lymphoid tumors characteristic of Marek's disease were caused by inoculation of susceptible chicks with MSB-1 cells or duck cells infected with strain BC-1 of MDV recovered from the MSB-1 cell line. No specific tumors were produced at the site of inoculation, and infection was readily transmitted to cagemates. Tumors were also produced in the skeletal muscles and seemed to be largely virus induced. MSB-1 cell line was free of C-type virus particles.     

The ultrastructure of lymphoblastoid cell lines from Marek's disease lymphomata.

The ultrastructure of two lymphoblastoid cell lines derived from Marek's disease lymphomata has been studied. The cells varied from 5 to 12 mum in diameter and had large round or oval nuclei. A nucleolus was occasionally present and about 3% of cells showed projections of the nuclear envelope. The cytoplasm contained many ribosomes and several mitochondria but endoplasmic reticulum was sparse. A small number of cells contained annulate lamellae and crystalline structures were occasionally seen. Cells with immature intranuclear herpesvirus particles were rarely present. The cells had many ultrastructural features in common with Burkitt's lymphoma-derived cell lines.     

Transplantable mouse tumor line induced by injection of SV40-transformed mouse kidney cells

A transplantable tumor of inbred mice was obtained by inoculating BALB/c mice subcutaneously with SV40-transformed mouse kidney (mKS-A) cells. Tumors were produced by mKS-A cells in the 71st cell culture passage, but not by cells in the 26th passage. The tumor line has been serially passed in BALB/c mice 14 times. In vitro cell culture lines were derived from tumors after 1, 2, 8, 10 and 12 passages in mice. The tumors, as well as the In vitro tumor cell lines, contained SV40 T-antigen, and sera from the tumor-bearing mice contained antibodies to the SV40 T-antigen. SV40 was rescued from the In vitro tumor cell lines after fusion with green monkey kidney (CV-1) cells in the presence of UV-irradiated Sendai virus. The In vitro tumor cell lines derived from mouse passages 8, 10 and 12 were used as SV40 virus; 2) SV40-transformed cell lines; 3) primary mouse (BALB/c or Yale Swiss) kidney cells, or 4) primary mouse (BALB/c or Yale Swiss) embryo cells. These results showed that the tumor line and the In vitro tumor cell lines have the transplantation antigen.     

Suppression of NK activity of spleen cells by chicken fetal antigen present on Marek's disease lymphoblastoid cell line cells

Chicken fetal antigen (CFA) expressed on the cell surface of Marek's disease (MD) lymphoblastoid cell line (MDCC-MSBI) cells was purified by affinity chromatography using a monoclonal antibody (2H3). A 4-hr 51Cr release assay was performed using spleen cells as effector cells to investigate the role of the CFA in the immune response of chickens, especially in relation to natural killer (NK) activity. Spleen cells of specific-pathogen-free (SPF) chickens showed reduced NK activity in the presence of CFA in vitro, as did those cells treated with MSBI soluble antigen. NK activity of spleen cells from chickens treated with CFA was also suppressed when compared to the cells from untreated or ovalbumin-treated chickens. In addition, MSBI clo. 18 transplantable tumors grew progressively in some of the 14-day-old chickens treated with CFA, whereas the tumors regressed in age-matched chickens with or without treatment by ovalbumin.     

Reduced incidence of Marek's disease gross lymphomas in T-cell-depleted chickens.

Chickens of line 7, highly susceptible to Marek's disease (MD), were depleted of T-cells by neonatal thymectomy, total-body gamma-irradiation, and multiple injections with antithymocyte serum. In two replicate experiments, significantly fewer gross lymphomas were present in T-cell-depleted chickens than in intact or in T-cell-depleted, reconstituted hatchmates; these findings provided evidence that T-cells may be the principal target for MD virus (MDV) transformation, T-cell depletion was not complete, and the presence of microscopic lesions in T-cell-depleted chickens was attributed to residual T-cells. Ten lymphomas from intact chickens and 2 lymphomas from a T-cell-depleted chicken were examined for cellular composition. All lymphomas consisted predominantly of T-cells. The results of this and other published studies indicated that T-cells may have a dual role in MD; They may serve as a target for lymphoma formation by MDV and also may participate in immune surveillance against the disease in resistant chickens.     

Studies on Marek's disease. II. Pathogenesis

The development of Marek's disease (fowl paralysis; neurolymphomatosis) has been studied by examination of peripheral nerves and other tissues at different times after infection of young chicks with the HPRS-B14 strain of Marek's disease. Three types of nerve lesions were found: 1) A-type, characterized by proliferation of lymphoid cells, presence of Marek's disease cells, and sometimes demyelination and Schwann cell proliferation; 2) B-type, characterized by diffuse infiltration by plasma cells and mainly small lymphocytes, usually interneuritic edema, sometimes demyelination and Schwann cell proliferation; 3) C-type, characterized by light infiltration by plasma cells and small lymphocytes. A mixed A- and B-type lesion was also found. Serial killing experiments and grouping of lesions from transmission experiments according to the time elapsed since infection indicate that the nerve lesion follows the progression: A-type--> mixed A- and B-type-->B-type. The C-type lesion is believed to be a mild form of the B-type. The study indicates that Marek's disease is characterized by a neoplastic-like proliferation of lymphoid cells in the nerves and in other organs, notably the ovary. In some birds the proliferation is progressive and they succumb early in the course of the disease with tumor-like infiltration of the nerves and often other organs. Demyelination and other nerve tissue changes appear to be secondary to the lymphoid proliferation. In other birds the proliferation of lymphoid cells in the nerves is arrested, and the lesion changes into one of a more inflammatory appearance.     

Antitumor immunity induced by hybrid tumor cells: comparison between hybrids and the parental tumor

The ability of hybrid tumor cells to induce antitumor immunity has been evaluated in the line 1 alveolar cell carcinoma (L1) model of BALB/c mice. Hybrid tumor cells were produced by fusing freshly dissociated L1 cells isolated from in vivo tumors with the hypoxanthine:aminopterin:thymidine-sensitive cell line, GM 347, derived from C3H mice. Each hybrid was characterized by DNA content and expression of H-2 antigens using a fluorescence-activated cell sorter. Irradiated L1 cells in the presence or absence of Corynebacterium parvum were capable of immunizing BALB/c mice against a challenge of live L1 cells, provided the challenge dose was small (50% lethal dose was between 6 X 10(4) and 1.2 X 10(5) L1 cells). Testing of five hybrid clones and 1 uncloned hybrid line for their immunizing ability demonstrated a range in immunizing ability with none showing a statistically significant improvement in survival (p less than 0.0018) when compared to untreated controls. However, one hybrid clone, MoHb-L1-C2, was selected in which the survival of mice immunized with it compared to controls had a p value of 0.0255. A tumor (labeled L1/A) which grew in one of the mice immunized with this clone was removed and hybridized with GM 347 to yield a second set of hybrids. Both this variant of L1 cells and a hybrid clone made from it (MoHb-L1A-C18) were capable of immunizing mice against a challenge of live L1/A (p values of 0.0000 and 0.0028, respectively, when compared to controls). However, L1 cells were not able to immunize effectively against L1/A, and MoHb-L1A-C18 did not immunize against L1. This suggests that L1/A is a subpopulation of L1 cells with a different antigenic composition. The limited success of MoHb-L1A-C18 against L1/A is thought to be due to the narrower range of antigenic specificities in L1/A and the ability of MoHb-L1A-C18 to represent an important antigenic subpopulation of L1/A.     

Dissociation of antiviral and antitumor immunity in resistance to Marek's disease.

Immunization of chickens either with gluteral-dehyde-inactivated chicken kidney cells infected with Marek's disease (MD) virus or with glutaraldehyde-inactivated cells of MD lymphoma-derived continuous lymphoblastoid cell lines protected against MD. The former type of immunity was associated with an immunologic suppression of virus replication and virus antigen production after challenge with virulent virus, but lymphocytes specifically cytotoxic to cells bearing MD tumor antigens were not detected. In the latter type of immunity, virus multiplication was not affected; some evidence of the stimulation of cell-mediated antitumor immunity was found. The results supported the view that immunity to MD may be directed against either virus-specific or tumor-specific antigens and that in natural resistance to MD both mechanisms may be operative.     

alpha-Fetoprotein-specific tumor immunity induced by plasmid prime-adenovirus boost genetic vaccination.

alpha-Fetoprotein (AFP) is a potential target for immunotherapy in hepatocellular carcinoma; both the murine and human T-cell repertoires can recognize AFP-derived epitopes in the context of the MHC. Protective immunity can be generated with AFP-engineered dendritic cell-based vaccines. We now report a DNA-based immunization strategy using a prime-boost approach: coadministration of plasmid DNA encoding murine AFP and murine granulocyte-macrophage colony-stimulating factor followed by boosting with an AFP-expressing nonreplicating adenoviral vector. This immunization strategy can elicit a high frequency of Th1-type AFP-specific cells leading to tumor protective immunity in mice at levels comparable with AFP-engineered dendritic cells. This cell-free mode of immunization is better suited for large-scale vaccine efforts for patients with hepatocellular carcinoma.     

HIFU治疗后肿瘤抗原对树突状细胞的活化及其抗肿瘤效应

目的：探讨HIFU治疗小鼠H22抑制性肝癌后产生的肿瘤抗原对机体抗肿瘤免疫功能增强的机制。方法：正常小鼠骨髓中提取骨髓细胞,在rmIL-4、rmGM-CSF奈件下培养7d,制备小鼠骨髓树突状细胞,用HIFU治疗小鼠移植性肝癌后产生的肿瘤抗原活化树突状细胞,再用活化后的树突状细胞激活T淋巴细胞为细胞毒性T细胞,用MTT法检测CTL在体外特异性杀伤肿瘤靶细胞的能力。结果：B16肿瘤HSP70-肽复合物组和H22肿瘤HSP70-肽复合物组的脾淋巴细胞的增殖率均高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组,P〈0．001;但两组之间差异无统计学意义,P〉0．05。H22肿瘤HSP70-肽复合物组CTL对H22肿瘤细胞的杀伤率为70．0％,明显高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组（P〈0．001）,但对非靶细胞B16肿瘤的杀伤率与上述各组的差异无统计学意义;B16肿瘤HSP70-肽复合物组CTL对B16肿瘤细胞的杀伤率为78．5％,对H22细胞的杀伤率为21．4％,表明CTL对肿瘤细胞的杀伤作用具有特异性。结论：HIFU治疗后坏死肿瘤组织中的HSP70-肽复合物作为肿瘤疫苗,通过活化DC和刺激T淋巴细胞增殖为CTL,发挥特异性抗肿瘤免疫功能。     

Resistance to Marek's disease. Effect of Corynebacterium parvum and Marek's tumor cell vaccines on tumorigenesis in chickens.

A study of nonspecific stimulation of the avian immune system with Corynebacterium parvum and specific stimulation with Marek's tumor cell vaccines revealed that nonspecifically stimulated outbred White Leghorn-type cockerels had higher incidences of tumors than did controls. A study of tumor cell cytotoxicity of sera from Marek's disease virus exposed birds indicated that humoral factors may play some role in tumor resistance.     

Inducing tumor immunity by specific tumor antigens.

A handy experimental model for testing anti-tumor agents and for studying tumor immunity is the use of Walker 256 carcinosarcoma in Long-Evans hooded rats. This neoplasm is so easily transplantable and growth is so rapid that a large series of animals can be studied in a relatively short period of time.     

Cell-mediated cytotoxic response to cells bearing Marek's disease tumor-associated surface antigen in chickens infected with Marek's disease virus.

In a microcytotoxicity test in which 51Cr-labeled cells of a Marek's disease (MD) lymphoblastoid line (MSB-1 line) were used, cell-mediated cytotoxicity of spleen cell suspensions prepared from chickens inoculated with MD virus was demonstrated. This cytotoxic response, presumably directed against MD tumor-associated surface antigen, was detectable briefly after virus infection and paralleled the appearance of early lymphoproliferative lesions characteristic of MD.     

Protective tumor immunity induced by potassium chloride extracts of guinea pig hepatomas.

Tumor-specific antigens of cells of the diethylnitrosamine-induced hepatomas in strain-2 guinea pigs were extracted with 3 M KCl. Immunization of normal animals with the extracted tumor antigens in adjuvant protected them against a subsequent challenge with viable tumor cells. Extracted tumor-specific antigens were less effective immunogens than viable tumor cells for both of two antigenically distinct lines.     

Further characterization of Marek's disease virus-infected lymphocytes. II. In vitro infection

Lymphocyte cultures from chicken spleens had been shown to be susceptible to in vitro infection by Marek's disease virus (MDV)4 in an earlier report from this laboratory. In that study, virus infection was evidenced by virus isolation and detection of viral internal antigen (VIA) 2 days post inoculation (DPI), and serial passage was accomplished by adding fresh spleen cells at 2-day intervals. The susceptible cells were identified as bursa-derived lymphocytes (B cells). Using a dual fluorescence technique to identify surface markers for B cells, thymus-derived lymphocytes (T cells) or Ia antigen on VIA-positive cells, we have now shown that a small proportion (generally less than 10%) of VIA-positive cells observed 2 DPI are T cells, and that a low level of infection can be maintained by serial passage of MDV in cultures totally free of B cells. Most infected T cells in this study had Ia antigen. As the incubation period for infected cultures was extended from 2 to 4 or 5 days, the average number of viable cells decreased but the percentage of viable cells infected with MDV (VIA-positive) increased. Also, both the proportion and the actual number of infected T cells increased, significantly more so in cultures from genetically susceptible P-2 donors than from resistant N-2 donors. Spleen-cell cultures from resistant Line 6 chickens were markedly less susceptible than those from susceptible Line 7 chickens to in vitro MDV infection, as assessed by numbers of VIA-positive cells at 5 DPI.