Studies on the recovery from tolerance to tumor antigens

Summary The present study investigates the potential of bone marrow cells from mice tolerant to tumor antigens to repopulate tumor-specific effector T cells. C3H/He mice were inoculated i.v. with 10⁶ 10000 R X-irradiated syngeneic X5563 plasmacytoma tumor cells three times at 4-day intervals. This regimen abrogated the ability of spleen cells from these mice to develop anti-X5563 cytotoxic and in vivo protective (tumor-neutralizing) T cell-mediated immunity as induced by i.d. inoculation of viable X5563 cells followed by surgical resection of the tumor. Since such suppression was induced in a tumor-specific way, this represented a state of antitumor tolerance. When bone marrow cells from normal or X5563-tolerant mice were transferred i.v. into 950 R X-irradiated syngeneic C3H/He mice, both groups of recipient mice generated anti-X5563 tumor immunity over a similar time course and to almost the same degree. Anti-X5563 tumor immunity induced in (C3H/He×C57BL/6) F1 mice which had been transferred with bone marrow cells from normal or X5563-tolerant C3H/He mice were mediated by T cells expressing the Ly phenotype of C3H/He, but not of C57BL/6, excluding the possibility that the antitumor effector cells were derived from recipient mice. It was also demonstrated that C3H/He mice which had been reconstituted with normal marrow were rendered tolerant when the tolerance regimen was started 7 weeks, but not 1 week after the bone marrow reconstitution. These results indicate that bone marrow cells from antitumor tolerant mice are not rendered tolerant to the tumor but can provide the potential to repopulate antitumor CTL and in vivo protective effector T cells.This work was supported by the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan Abbreviations used: MHC, major histocompatibility complex; CTL, cytotoxic T lymphocytes; TNP, trinitrophenyl; C, complement; TNBS; trinitrobenzene sulfonate; MMC, mitomycin C     

Studies on the recovery from tolerance to tumor antigens

C3H/He mice were injected i.v. with heavily X-irradiated syngeneic X5563 tumor cells three times at 4-day intervals. This regimen resulted in the abrogation of the potential to generate X5563 tumor-specific T cell-mediated immunity as induced by i.d. inoculation of viable X5563 tumor cells followed by surgical resection of the tumor, representing the tolerance induction. Although such a tumor-specific tolerant state was long-lasting, the recovery of anti-X5563 effector T cell responses was observed when the above ordinary immunization procedure was performed 6 months after the tolerance induction. The present study investigated whether the recovery from the tolerance can be accelerated by applying a helper-effector T-T cell interaction model in which enhanced anti-X5563 immunity is obtained by priming mice with BCG and by immunizing X5563 tumor cells modified with BCG cross-reactive MDP hapten (designated as L4-MDP) in the presence of anti-L4-MDP helper T cells preinduced with BCG. The results demonstrated that BCG-primed mice which received the tolerance regimen failed to generate anti-X5563 immunity when the ordinary immunization was performed 2 or 3 months after the tolerance induction. In contrast, the immunization of BCG-primed and X5563-tolerant mice with L4-MDP-coupled X5563 tumor cells at comparable timing to that of the ordinary immunization were capable of generating potent X5563-specific in vivo protective T cell-mediated immunity. As control groups, BCG-primed or unprimed tolerant mice did not develop anti-X5563 immunity when immunized with L4-MDP-uncoupled or L4-MDP-coupled tumor cells, respectively. These results indicate that immunization of BCG-primed, tumor-tolerant mice with L4-MDP-modified tumor cells results in accelerated recovery from the tumor tolerance.     

Human liver nucleolar antigens

In an extension of previous studies on the antigens in rat liver nucleoli (R. K. Busch, R. C. Reddy, D. H. Henning, and H. Busch, Proc. Soc. Exp. Biol. Med. 160, 185 (1979); R. K. Busch and H. Busch, Tumori 63, 347 (1977); F. M. Davis, R. K. Busch, L. C. Yeoman, and H. Busch, Cancer Res. 38, 1906 (1978), rabbit antibodies were elicited to human liver nucleoli isolated by the sucrose--Mg2+ method (10). Fluorescent nucleoli were found in liver cryostat sections treated with rabbit anti-human liver nucleolar antibodies followed by fluorescein-conjugated goat anti-rabbit antibodies. In HeLa cells, fluorescence was distributed throughout the nucleus and in a nuclear network but was not localized to the nucleolus. In placental cryostat sections, an overall nuclear fluorescence was observed with some localization to nucleoli. Immunodiffusion analysis revealed two immunoprecipitin bands which appeared to be liver specific. Other immunoprecipitin bands were common to liver, placenta, and HeLa nuclear extracts. Rocket immunoelectrophoresis revealed two liver-specific antigens, one migrating to the cathode and the other to the anode Other rockets exhibited identity to antigens of other nuclear extracts. These results demonstrate the presence of human liver nucleolar-specific antigens which were not found in the HeLa and placental cells.     

Studies on human brain-thymus cross-reactive antigens.

Antigens shared by human brain and thymocytes and by human and mouse tissues were studied with rabbit anti-human thymocyte antiserum (RAHT). It was found that cytotoxicity of RAHT serum against mouse thymus cells was not absorbed by mouse liver or bone marrow cells. Human brain and thymus cells completely absorbed the anti-thymocyte activity from this antiserum. It was suggested that human brain had antigenic determinants identical or very similar to those found on human thymocytes. Activity of RAHT antiserum against mouse thymus cells was completely removed by an absorption of mouse brain and thymocytes. These results demonstrated that there were shared antigenic determinants between human and mouse tissues.     

Proliferating cell nuclear antigen (PCNA) and human malignant tumor nucleolar antigens (HMTNA) in nucleoli of human hematological malignancies

Lymphoma (Lymphocytic non-Hodgkin's malignant lymphoma) and leukemic (chronic lymphocytic, acute and chronic myeloid, myelomonocytic leukemia) cells were studied by indirect immunofluorescence to evaluate the presence of proliferating cell nuclear antigen (PCNA) and human malignant tumor nuclear antigen (HMTNA) in their nucleoli. Most cells in lymph node smears of lymphocytic non-Hodgkin's malignant lymphoma (NHML) developed a bright nucleolar fluorescence with HMTNA antibodies. PCNA was detected in nucleoli of a limited number of cells which apparently represent the proliferating cell population in these lymphomas. Similarly, in the bone marrow smears of patients with chronic lymphocytic leukemia most cells possessed a nucleolar fluorescence for HMTNA and PCNA was present in nucleoli of a limited number of cells. In the bone marrow smears of patients with myeloid or myelomonocytic leukemias most blastic or monocytoid cells also developed a bright nucleolar fluorescence with HMTNA antibodies and PCNA was present only in a small percentage of these cells. Leukemic cells with PCNA in their nucleoli like thekhuntigen might represent a proliferating cell population in late G1-early S phase.     

Serologic analysis of human tumor antigens

Summary The immune adherence (IA) assay was used to measure serum reactivity of patients with melanoma and osteosarcoma against paired cell lines (tumor cells and normal skin fibroblasts obtained from the same individuals) grown in tissue culture. Sera from 224 patients with various stages of melanoma were compared with sera from 100 normal age- and sex-matched donors. None of the 18 stage I sera (0%), 23 of 166 (14%) stage II sera, 3 of 40 (7%) stage III sera, and 3 of 100 (3%) normal sera were highly reactive to a standard allogeneic melanoma-fibroblast pair. None of the sera exhibited unique activity against melanoma. There was no correlation between stage of melanoma and high serum reactivity, nor was this reactivity predictive of recurrence. Sera from 39 tumor-bearing osteosarcoma patients prior to amputation were compared with sera from 50 normal age-and sex-matched donors. Eight of 39 (21%) patient sera and 1 of 50 (2%) normal sera were highly reactive to an osteosarcoma-fibroblast pair. No sera had reactivity uniquely directed against osteosarcoma. Eight osteosarcoma and two melanoma patients were tested simultaneously against their autologous cultured tumor and skin cells. Only one of these patients exhibited high reactivity towards autologous cells, and this reactivity was equal against both osteosarcoma and normal cells. None of seven highly reactive osteosarcoma or six highly reactive melanoma sera had residual tumor-specific reactivity against allogeneic osteosarcoma or melanoma after absorption with cultured fibroblasts, cultured fetal fibroblasts, or fetal calf serum.     

Cellular distribution of the hamster liver specific nucleolar antigens

The immunochemical localization of hamster liver nucleolar antigens in subcellular fractions (nuclei, 10,000 x g pellet, 100,000 x g pellet and supernatant), nuclear substructures (chromatin, nuclear matrix, nuclear envelope, nucleoli, RNP particles and nucleosomes), and three classes of nonhistone chromosomal proteins with different affinities to DNA (NHCP1, NHCP2 and NHCP3) from nuclease-sensitive and nuclease-resistant chromatin fractions of hamster liver were studied. Six main nucleolar antigens with mol. wts 27,000; 29,000; 30,000; 36,000; 45,000; and 46,000 were found in subcellular fractions, nuclear substructures and classes of non-histone proteins of hamster liver. The antigens with mol.wts of approx. 27,000; 29,000; and 36,000 which were absent in hamster pancreas, spleen and Kirkman--Robbins hepatoma nuclei, seem specific for liver tissue.     

Monoclonal antibodies to the antigens of human HeLa tumor cells

A panel of 6 hybridomas "XEJIMA" producing monoclonal antibodies specific to HeLa cells is prepared. Monoclonal antibodies do not bind to antigens of human diploid fibroblasts, human continuous B- and T-lymphocytes and animal cell lines. The specificity of monoclonal antibodies to cellular antigens of 5 HeLa-like cell lines and 6 human tumour cells lines, not contaminated with HeLa cells, is determined. Antibody containing ascitic fluid and culture media of hybridomas XEJIMA-3, -12, -13, and -22 significantly decrease the attachment of HeLa cells to the surface of culture flasks. Monoclonal antibodies XEJIMA-11, -12 and -13 block the multiplication of HeLa cells. The effect depends on serum concentration in the nutrient medium.     

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.     

Studies on antigens of human lung adenocarcinoma with McAb LC-1]

The soluble antigens extracted from both human lung adenocarcinoma cell line SPC-A-1, and normal adult lung tissue with non-idet P-40 were subjected to 10% SDS-PAGE. The number of bands distinguishable by naked eyes of lung adenocarcinoma are 57, in which 4 bands are more significant. The bands of normal human lung tissue are 52, in which 2 bands are more significant. The molecular weights of these bands mainly are within 30 to 94 KD The thin layer chromatographs of these two antigenic extracts have shown that there is difference in their sugar content, but both of them shown little sialic acid. The Immunoblot pattern of McAb LC-1 reacted with the extracts of SPC-A-1 cells shows that all of 3 bands detected can be stained by alcian blue, indicating that they are glycoproteins. However, of them two bands, M. W. of 70 KD and 51 KD can also be stained by Sudan Black B, indicating that these two bands are glycolipoproteins. The ganglioside and neutral glycolipid can inhibit the binding of LC-1 with the extract of SPC-A-1 cells. The results indicate that the epitopes of SPC-A-1 cell extract reacted with McAb LC-1 are probably located in the polysaccharide.     

Studies on biosynthesis, assembly and expression of human major transplantation antigens

Biosynthesis and regulation of expression of transplantation as detected by a monoclonal antibody to HLA-A,B,C antigens (human leucocytic antigen) and a polyclonal antiserum to beta 2-microglobulin have been investigated using radioactive amino acids and sugars to label human lymphoid cells. We found unbalanced synthesis of HLA heavy chains and beta 2-microglobulin, the latter being in excess and secreted to the extracellular medium. In DAUDI cells, which are defective in beta 2-microglobulin, no HLA-A,B,C could be detected intracellularly even in the presence of added beta 2-microglobulin. Treatment of BRI-8 cells with tunicamycin, an antibiotic which inhibits glycosylation of polypeptides, almost had no effect on the levels of beta 2-microglobulin, while it markedly decreased that of HLA heavy chains, both on the cell surface and intracellularly. Glycosylation of the HLA heavy chains appeared to be an essential requirement for the normal expression of HLA-A,B,C antigens. The translation in vitro in a messenger-dependent reticulocyte system with total polysomes obtained from BRI-8 cells showed that beta 2-microglobulin was synthesized as a precursor. This larger polypeptide was converted into mature beta 2-microglobulin when protein synthesis was performed with microsomes instead of polysomes.     

High frequency antigens of human erythrocyte membrane sialoglycoproteins, III. Studies on the EnaFR, Wrb and Wra antigens

The nature of the common erythrocyte antigens EnaFR and Wrb, that are both absent from En(a-) cells, and the rare Wra receptor, apparently encoded by an allele of Wrb, was investigated. Various modification, fractionation or cleavage products of erythrocyte membranes were used in hemagglutination inhibition assays. The EnaFR and Wrb antigens were shown to represent labile structures within the residues approx. 62-72 of the major (MN) sialoglycoprotein that require lipids, at least for complete expression of antigenic activity. During the course of these experiments, the arrangement of the MN glycoprotein's peptide chain with respect to the lipid bi-layer was also studied, using various proteinases. Furthermore, the MN glycoprotein was found to aggregate with the major membrane protein (band 3) in the presence of Triton X-100. The Wra antigen was shown to exhibit properties that differ considerably from those of the Wrb receptor. Analyses on the MN glycoprotein, isolated from the red cells of the only known Wra homozygote and two WraWrb individuals, did not reveal any amino-acid exchange within the residues 40-96 of the molecule. Therefore, the Wr locus that determines the presence or absence of the Wrb antigen on the MN glycoprotein might influence the post-translational modification of amino-acid residues, the structure of tightly bound lipids or the aggregation of the MN glycoprotein with a different protein such as band 3.