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.     

Demonstration of Marek's disease tumor-associated surface antigen in chickens infected with nononcogenic Marek's disease virus and herpesvirus of turkeys.

The presence of Marek's disease tumor-associated surface antigen (MATSA) was demonstrated on spleen cells from P-line chickens inoculated 5--6 days earlier with herpesvirus of turkeys and SB-1 (a nononcogenic Marek's disease virus). Antisera against MATSA expressed on five Marek's disease lymphoblastoid cell lines were able to recognize the MATSA present on SB-1-infected spleen cells. No viral membrane antigens and only a low incidence of viral internal antigens could be demonstrated.     

Analysis of Marek's disease tumor-associated surface antigen on MDCC-MSB1-clo.18 cells.

The physico-chemical nature of Marek's disease tumor-associated surface antigen (MATSA) on Marek's disease (MD) lymphoblastoid tumor cell line (MDCC-MSB1-clo.18) was examined by the cellular enzyme-linked immunosorbent assay (CELISA) and the sandwich enzyme-linked immunosorbent assay (ELISA) using an anti-MATSA immune serum or a monoclonal antibody (MAb) 2B9 developed against MATSA. Our results indicate that MATSA is a glycoprotein and 2B9 recognizes an antigenic site in the protein moiety of MATSA. MATSA was solubilized from MSB1-clo.18 cells by treatment with 0.5% Nonidet P-40, and purified by affinity chromatography coupling with 2B9 and further by ion exchange chromatography on diethylaminoethylcellulose (IECD). MATSA was eluted with 0.2 to 0.3 M KCl in IECD and the purity of MATSA was increased about 2,500-fold. The purified MATSA was shown to have a molecular weight (Mr) of 70,000 by SDS-PAGE. The reactivity of purified MATSA with anti-thymus cell serum was examined. MATSA was detectable by anti-thymus cell serum, although 2B9, which was used to purify MATSA from MSB1-clo.18 cells, was not reactive to cells prepared from the thymus. However, MATSA was no longer detectable after the absorption of anti-thymus cell serum by chicken bursa cells. The absorption of anti-thymus cell serum by chicken red blood cells (RBC) had no effect on the reactivity against MATSA. These results suggest that MATSA may be a lymphocyte-specific antigen modified during leukemogenesis by Marek's disease virus (MDV).     

Expression of a putative tumor-associated surface antigen on normal versus Marek's disease virus-transformed lymphocytes

Various avian tumor cell lines and normal spleen cells from 3 genetic strains of specific-pathogen-free (SPF) chickens were examined for expression of Marek's disease (MD) tumor-associated surface antigen (MATSA). Two anti-MATSA monoclonal antibodies (RPH 6 and EB 29) and a rabbit anti-MATSA antiserum were used in indirect fluorescent antibody tests, and cells were examined by fluorescence microscopy and with a fluorescence-activated cell sorter (FACS). Less than 5% MATSA-positive cells were observed in 2 non-MD tumor cell lines (LSCC-RP 9 and RECC-CU 60) with RPH 6, but 7-82% positive cells were observed with EB 29 or the rabbit antiserum. Five MD tumor cell lines (MDCC-CU 2, -CU 14, -CU 25, -CU 32, and -CU 41) had 12-72% positive cells detected with one or both monoclonals and 31-99% positive cells detected with the rabbit antiserum. Over 90% of cells in all MD lines were la and T3 positive, while values for the same parameters in LSCC-RP 9 were 100 and 3% and for RECC-CU 60, 48 and 51%, respectively. Evidence for cell-cycle-dependent expression of MATSA on MDCC-CU 2 was obtained from cell sorting experiments with the FACS and from examination of the MATSA-staining characteristics of 3 clones derived from the parent culture. Less than 5% MATSA-positive cells were observed in uncultured spleen cells from SPF chickens or in spleen cells stimulated for 48 hours with concanavalin A or phytohemagglutinin-M. However, with one exception, 10-53% of normal spleen cells were MATSA positive with RPH 6, after stimulation by mitogen for 24 or 48 hours followed by maintenance in conditioned medium (CM) for various times or after culture directly in CM for 3 days. More limited experiments with rabbit anti-MATSA antiserum yielded 55-85% MATSA-positive cells. From 60 to 97% of these MD virus-free, MATSA-positive cells were la-positive; and, in 2 cases, 89 and 90% were T3 positive.     

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.     

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.     

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.     

Enhanced oncornavirus expression in Marek's disease tumors from specific-pathogen-free chickens.

An oncornavirus was recovered from cell cultures of kidney tumors from specific-pathogen-free chickens inoculated with Marek's disease herpesvirus (MDHV). The MDHV inoculum was free of infectious avian leukosis virus (ALV). Direct examination of a variety of tissues from MDHV-inoculated chickens demonstrated increased levels of ALV-specific RNA compared to tissues from diluent-inoculated (control) chickens. DNA from cultured kidney tumor cells annealed to an ALV complementary DNA probe at the same rate and exhibited the same extent of homology as DNA from cultured control kidney cells. This finding indicated the absence of exogenous ALV proviral sequences. As with vertically transmitted endogenous ALV of subgroup E, the oncornavirus recovered from kidney tumor cell cultures failed to replicate in chicken embryo fibroblast (CEF) cultures of the C/E phenotype, but did replicate in turkey embryo fibroblasts (TEF), which are permissive for replication of endogenous ALV of subgroup E. These oncornavirus particles served as a helper virus to form Rous sarcoma virus pseudotypes, which produced foci in TEF cultures but not in CEF cultures of the C/E phenotype. Whether enhanced expression of endogenous oncornavirus contributes to MDHV-induced tumorigenesis is not known.     

Cutaneous changes associated with Marek's disease of chickens

Intranuclear inclusion bodies were observed in the leather follicle epithelium of chickens with Marek's disease (MD). These inclusions and associated cellular degenerations were shown to be part of the MD process by correlation of their occurrence with that of MD and by determination of their distributional dependence on the presence of the immunofluorescent (IF) antigen of MD. Inclusions and IF antigen appeared in the epithelial layers of the feather follicle, superficial to, but never in, the basal layer. The distributional relationship between cutaneous IF antigen and lymphoid aggregates, typical of MD, was studied; there was an apparent interaction between the aggregates and IF antigen.     

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.     

Oncofetal antigen: a tumor-associated fetal antigen immunogenic in man.

Oncofetal antigen (OFA) has been defined with the use of human natural antibodies as a membrane antigen of human cancer cells that cross-reacts with human fetal brain tissues. The immunogen that elicits the antibody is unknown. The present study was undertaken to examine the immunogenicity of the OFA found on tumor cells. Postoperative melanoma patients were immunized with OFA-positive melanoma cells. Anti-OFA reactivities in the immunized sera were titrated by the immune adherence assay with the use of a known OFA-positive cultured melanoma cell line, M14, as target cell. Alloantibodies were excluded by absorption with lymphoblastoid cells autologous to M14. Anti-OFA antibody then was identified by absorption with fetal brain. In 6 months of immunization, 19 of 23 patients produced increased anti-OFA antibodies. The peak titers ranged from 1:16 to 1:2,048. Sera from 18 patients who were not immunized also were tested for 6 months postoperatively, and none had significant increases in antibody titers. The increase of anti-OFA antibody titer in response to the immunization with OFA-positive tumor cells suggests the immunogenic capability of tumor-related OFA in man.