Membrane associated antigens of human malignant melanoma V: Serological typing of cell lines using antisera from nonhuman primates

Summary Antisera against various melanoma cell lines were raised in nonhuman primates (Cercopithecus aethiop.). After exhaustive absorption with AB Rh + red blood cells and pooled platelets from about 200 donors the sera were still reactive to various degrees in the microimmune adherence test with other melanoma lines, with embryonic fibroblasts, and with non-melanoma lines. As proven by absorption experiments, the main-specificity of the antisera was not directed against components of the fetal calf serum used for cell culture or against mycoplasma grown from commercial fetal calf serum. In addition, no cross-reactivity was observed with Bacillus Calmette-Gérin, and in blocking experiments no reactivity against extracts of common bacterial antigens or mixed molds was detected. Absorption with embryonic fibroblasts or embryonic tissue showed that the reactivity of most antisera was directed against melanoma-associated antigens expressed also on fetal tissue. It was not possible to determine whether the remaining reactivity on some cell lines was melanoma-specific or directed against fetal antigens not contained in the fetal material used for absorption. Cross-absorption of antisera with other melanoma cells revealed that various cell lines express different patterns of tumor-associated antigens with no, or only partial, overlap. The cross-absorption experiments made it possible to type the cell lines according to their surface antigens and arrange the cell lines in order according to the degree of mutual antigenic relationship.     

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.     

Preparation of fibroblast-free cytotrophoblast cultures utilizing differential expression of the CD9 antigen

Summary The leukemia-associated antigen CD9 is present on a variety of normal cells, with apparent variable expression on normal human fibroblasts. In this study, we demonstrate by immunoperoxidase staining and direct binding studies that the CD9 antigen is uniformly expressed on normal human fibroblasts grown from first trimester and term placenta, embryonic fetal fibroblasts, and from human adult and fetal skin fibroblasts. Higher CD9 expression was present on fetal cells. CD9 antigen was not present on trophoblast. Over 99% of fibroblasts could be absorbed onto antibody to the CD9 antigen conjugated to magnetic beads. By applying this selective immunoadsorption of fibroblasts to term placental cytotrophoblast preparations, we demonstrated that fibroblast contamination could be nearly completely eliminated. This is a novel technique for purifying primary trophoblast cultures and may have wider applicability in cell culture of other cell types.     

Serum antibodies to human fetal antigens in patients with systemic lupus erythematosus (SLE)

Serum antibodies to human fetal antigens were measured by a radiolabeled anti-immunoglobulin binding assay by using human fetal fibroblasts (Flow cell line No. 1000) as target cells. High titers of IgG antibody to the fetal cells were found in sera of patients with systemic lupus erythematosus (SLE). The antibody reacted with surface membrane antigens shared by various fetal tissues of human and murine origin but not by adult tissues. The reaction of the SLE antibody to the fetal cells was inhibited by heterologous antiserum to the Flow 1000 cells and antiserum to murine embryonic fibroblasts, but not by antiserum to human alpha-fetoprotein or human fibronectin. Absorption of SLE serum with isolated nuclei did not abolish the reaction indicating that these were not anti-nuclear antibodies. The antibody activity was found to reside in the F(ab')2 fragment. The serum titer of the anti-fetal antibody was higher in SLE patients with active disease than those in clinical remission.     

Immunohistochemical localization of the immunodominant differentiation antigen lacto-N-fucopentaose III in normal adult and fetal tissues

Monoclonal antibodies were produced by immunizing rats with human small cell lung carcinoma (SCLC) cell lines. Monoclonal antibodies 600D11 and 624A12 were found to be directed against the ceramide pentasaccharide that contains the lacto-N-fucopentaose III (LNFP III) sequence of sugars, an isomer of the Lewis A blood group antigen. LNFP III is an immunodominant antigen whose reactivity is maintained in formalin-fixed paraffin-embedded sections (PS). LNFP III has been recognized in a number of human tumors including: SCLC; adenocarcinomas of the breast, gastrointestinal tract, genitourinary tract, and lung; renal cell carcinoma; neuroblastoma; and myelogenous leukemia. We now report the normal adult and fetal tissue distribution of the LNFP III antigen by immunoperoxidase staining on PS utilizing 600D11 and 624A12. Binding was demonstrated in bronchial epithelium and bronchial glands; squamous epithelium of the esophagus; gastric crypts, duodenal enterocytes and Brunners glands; argentaffin cells; jejunal and colonic goblet cells; pancreatic acinar cells; salivary glands; endocervical and exocervical cells; skin epidermis; myelinated motor fibers; cells of the adrenal medulla and anterior pituitary gland; polymorphonuclear leukocytes (PMNs); tissue macrophages and renal proximal tubules and loops of Henle. Staining was localized to cell membranes and within the cytoplasm, with greatest intensity at the apical and basal portions of the cells. These staining patterns were noted in adult and neonatal tissues, and initial expression could be traced to approximately the second trimester of fetal development. Knowledge of the normal tissue distribution of this immunodominant antigenic determinant may offer insight into its structural and functional role in benign and malignant tissues.     

Autologous and homologous immunofluorescent antibody to established breast cancer cell lines

Summary Indirect immunofluorescence tests were performed on 14 established human breast cancer cell lines using sera from a variety of subjects. Autologous reactions were studied on 10 cell lines, with positive reactions demonstrable in 8. Tests using sera from a randomly selected population of breast cancer patients showed reactivity in 40 to 66% depending on the target cell line used. Reactivity to other nonbreast cancer cell lines was rare. Several control populations were tested, including normal blood bank donors, persons with benign breast disease, and persons with other forms of cancer; immunofluorescent antibody was detected much less frequently in sera from these populations than those from the breast cancer group. Positive reactions remained in spite of absorption of serum with heterophile antigens, normal human breast tissue, and AB+ red blood cells. Thus established cell lines of human breast cancer possess antigens commonly recognized by sera from breast cancer patients. This work was supported in part by Grant CA 16789 from the National Cancer Institute, NIH, Department of Health, Education and Welfare.     

Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.     

Detection of HLA-DRw (Ia-like) antigens on human T lymphocytes grown in tissue culture.

Human T lymphocytes that proliferate in the presence of conditioned medium from PHA-stimulated allogeneic peripheral blood cells were shown to express IPA antigens after the 8th culture day. Ia antigens as detected by xenogeneic antisera were present on 80 to 90% of the cultured cells which were also strongly reactive with xenogeneic antisera defining a human T cell antigen and formed E rosettes. Control cultures with PHA or no conditioned medium expressed T cell but not Ia antigens. These cultured T cells also express the same HLA-DRw determinants as the B cells of the donor they were derived from. Absorption of xenogeneic Ia, and HLA-DRw alloantisera with cultured T cells completely removed the reactivity of these sera for enriched peripheral blood B lymphocytes from normal donors.     

Humoral immune response to melanoma-associated membrane antigen and fetal brain antigen demonstrated by indirect membrane immunofluorescence. I

Summary A melanoma-associated membrane antigen and a fetal brain antigen were identified on the surface of a human melanoma cell line by indirect membrane immunofluorescence techniques. The target melanoma cells were grown in gamma globulin-depleted human serum. Sera from melanoma patients were used as the source of antimelanoma antibodies. To remove alloantibodies, the allogeneic sera were preabsorbed with cultured lymphoblastoid cells derived from the peripheral lymphocytes of the donor of the target cell line. To further define the antigen responsible for antibody activity, sequential absorption tests were performed with fetal brain cells, cultured sarcomas, and breast carcinomas. Some antibody activity was removed by fetal brain tissues. Further absorption with fetal brain or the cultured sarcoma or breast carcinoma did not remove additional activity. However, antibody activity was completely removed by either cultured or biopsy-derived melanoma cells. A serum autochthonous to the target cell line was also tested. The antibody titer of the serum was completely removed by absorption with either autochthonous biopsied tumor or an allogeneic melanoma cell line, but not with the normal tissues. Thus it appeared that sera from melanoma patients contained antibody to both a melanoma-associated membrane antigen and a fetal brain antigen.     

Cross-reacting antigens in chemically induced sarcomas are fetal determinants.

Serologic cross-reactivity has been demonstrated among three MCA-induced sarcomas in C57BL/6N female mice (MCA-2, MCA-3, and MCA-12) with a microcytotoxicity assay. Serum from mice bearing MCA-3 tumor was cytotoxic to both MCA-2 and MCA-3 tumor cells at a titer of 1:8. Sequential absorptions of this serum with syngeneic embryo cells completely eliminate cytotoxicity against MCA-2 cells without affecting the cytotoxic titer against MCA-3 cells. Serum hyperimmune to the MCA-3 tumor reacted with MCA-3, MCA-2, and MCA-12 tumors. Absorption of this serum with embryo cells eliminated cytotoxicity against MCA-2 and MCA-12 cells, but was incapable of lowering the titer against MCA-3 cells below 1:40. Similarly, serum hyperimmune to MCA-2 tumor was lytic to MCA-2, MCA-3, and MCA-12 before absorption, but was lytic only to MCA-2 cells after absorption with sygeneic embryo cells. Thus, the in vitro cross-reactivity between MCA-induced sarcomas is due to a common fetal antigen(s), which is distinct from the individual tumor-specific antigens of each tumor. Since these tumors do not exhibit cross-reactivity in in vivo challenge experiments, it appears that this fetal antigen is not responsible for in vivo immune protection.     

Substrate specificity for the detection of autoantibodies to anterior pituitary cells in human sera

Using the indirect immunofluorescence test for the detection of pituitary autoantibodies in human serum, the results obtained with human fetal and non-human pituitary antigens were compared. Of the sera that were positive on human fetal substrate, 4% were recovered with adult baboon, 0% with fetal cymologous, 20% with porcine, 11% with bovine, 11% with ovine, and 7% with rat tissue. The rate of heterophilic antibodies to the above animal substrates was 5% to 14%. In contrast to human adult pituitaries, normal human sera did not bind to Fc receptors on ACTH-cells of human fetal pituitaries. This allowed the demonstration of ACTH-cell antibodies. The specificity of the reaction was proven by absorption studies with purified Fc fragments. These results suggest that human fetal tissue is the best source of antigen for the detection of pituitary autoantibodies. The use of animal tissue including non-human primate pituitary yields results that bear no clinical significance.     

Normal trophoblasts resist induction of class I HLA

Very few types of normal cells fail entirely to express class I human leukocyte antigens (HLA), and many of those cells (sperm, fetal amnion epithelial cells, and fetal trophoblasts) are related to the process of reproduction. Susceptibility of sperm to modulation of class I antigens has not been examined, but it has recently been demonstrated that amnion cells respond to exposure to IFN-gamma with readily detectable levels of class I antigens. In addition, one of two trophoblast cell lines (BeWo) has been shown to exhibit enhanced expression of class I HLA in response to IFN-gamma. Expression by a second trophoblast cell line (Jar) was not inducible. Findings in the present study included demonstration of IFN-gamma-enhanced class I-specific mRNA synthesis in JEG-3 cells, which are derived from BeWo, and failure of synthesis by Jar cells. Those results eliminated trivial explanations for the preceding findings and confirmed the responsiveness of some but not all cells of trophoblast origin to IFN-gamma. When successful modulating conditions for amnion and malignant trophoblast cells were applied to normal tissues, third trimester term chorionic cytotrophoblasts and first trimester villous syncytial and cytotrophoblasts failed to exhibit class I HLA. Neither malignant nor normal trophoblasts expressed class II HLA under any condition of testing. Failure of induction of HLA expression by normal trophoblasts could not be attributed to either loss of viability by tissue explants or failure of modulating reagents to reach the trophoblasts. The results demonstrate that regulation of expression of histocompatibility antigens by major populations of normal trophoblasts and one of two choriocarcinoma cell lines differs markedly from that of other fetal and adult cells. Uncommon regulatory mechanisms may be essential to maintenance of the trophoblast as an immunologically inert barrier between the mother and her antigenically disparate fetus.     

Antibodies to bacterial and tumor-derived antigens in sera from normal guinea pigs.

Antibodies that react with radiolabeled antigens derived from guinea pig line-10 tumor cells and Mycobacterium bovis (BCG) were detected in sera from normal tumor-free strain-2 guinea pigs (NGPS). Binding by NGPS to the two antigens was inhibited by extracts of either line-10 cells or BCG. Binding by NGPS to the line-10 antigen was inhibited by a number of other bacterial extracts. NGPS was tested after absorption with a variety of cells including line-10, line-1, normal guinea pig spleen, normal adult and fetal liver cells. Results indicated that some of the antibodies in NGPS were directed to line-10-specific determinants. The specific stimulating antigen for these antibodies was not identified but because of the antigenic relationship between BCG, line-10 cells and other bacteria, antibodies to line-10-associated antigens might have been induced by exposure to environmental microorganisms.     

Expression of fetal antigens in fetal and adult cells during long-term culture

Summary Expression of fetal antigens in early and late passages of tissue culture cells derived from C3H/HeN and C57BL/KaLw mouse fetal cells and from lung tissue of young C57BL/6N mice was investigated by the isotopic antiglobulin technique. The late passage lines of fetal cells had undergone “spontaneous” neoplastic transformation in culture. The antisera were produced by syngeneic immunization with 5000 R x-irradiated tissues from C3H/HeN and C57BL/6N fetuses of 1 to 2 weeks gestation. Fetal antigens were found to be retained even after 5 years in cell lines derived from fetal tissues In these lines no consistent change in fetal antigen expression could be correlated with neoplastic transformation. In contrast, the early passages of adult cells did not have detectable amounts of fetal antigens. However, fetal antigen(s) was demonstrated in cells of the late passages, and cells of both lines grew as sarcomas when next assayed 55 days later. In addition, fetal antigens were also present in established tumor lines in culture.     

Expression of fetal antigens in fetal and adult cells during long-term culture

Expression of fetal antigens in early and late passages of tissue culture cells derived from C3H/HeN and C57BL/KaLw mouse fetal cells and from lung tissue of young C57BL/6N mice was investigated by the isotopic antiglobulin technique. The late passage lines of fetal cells had undergone "spontaneous" neoplastic transformation in culture. The antisera were produced by syngeneic immunization with 5000 R x-irradiated tissues from C3H/HeN and C57BL/6N fetuses of 1 to 2 weeks gestation. Fetal antigens were found to be retained even after 5 years in cell lines derived from fetal tissues. In these lines no consistent change in fetal antigen expression could be correlated with neoplastic transformation. In contrast, the early passages of adult cells did not have detectable amounts of fetal antigens. However, fetal antigen(s) was demonstrated in cells of the late passages, and cells of both lines grew as sarcomas when next assayed 55 days later. In addition, fetal antigens were also present in established tumor lines in culture.     

Suppression of hamster lymphocyte reactivity to simian virus 40 tumor surface antigens by spleen cells from pregnant hamsters

SV40-transformed tumor cells in hamsters have been found to have cell surface antigens cross-reactive with antigens temporally expressed on fetal tissues. Adoptive transfer assays performed in this laboratory have shown that peritoneal exudate cells from 10-day primiparous hamsters are cytotoxic to SV40-transformed sarcoma cells (WF5-1) carrying fetal antigen, whereas peritoneal exudate cells from multiparous hamsters are less cytotoxic. This suggests a suppressor activity might be present during subsequent pregnancies that reduces the responsiveness of lymphocytes from pregnant hamsters to stimulation by fetal antigens on tumor cells. Using a lymphocyte transformation assay, spleen cells from pregnant hamsters were found to be incapable of responding to preparations of either hamster fetal tissue or SV40-transformed cells. However, a suppressor component can be demonstrated in spleen cell populations of both primi- and multiparous hamsters during pregnancy that is capable of reducing the response of lymphocytes sensitized against SV40 tumor-associated antigens. The degree of suppression is proportional to the ratio of responder cells to spleen cells from pregnant animals. These results suggest there is a subpopulation of spleen cells involved in immunoregulation during pregnancy that has the ability to suppress the reactivity of lymphocytes sensitized against SV40-associated oncofetal antigens.     

Rabbit antiserum against Daudi-cell membrane fractions detects cell surface antigens present on subpopulations of human lymphocytes.

An antiserum was produced by immunization of rabbits with membrane preparations of a human lymphoblastoid B cell line, Daudi. After absorption with a human endometrial carcinoma cell line, this antiserum appeared to be specific for antigen(s) present on adult and fetal thymocytes as well as on tonsillar lymphocytes but absent, or present in very small amounts, on normal or phytohemagglutinin- (PHA) stimulated peripheral blood lymphocytes (PBL). When T and B cell-enriched fractions from tonsillar lymphocytes were tested with the anti-Daudi serum, the reactivity was equally distributed in each population. Among 13 human lymphoblastoid cell lines tested, reactivity was demonstrated on three out of four T cell lines, and on four out of nine B cell lines. The positive reacting B cell lines were derived from two African and two American Burkitt lymphomas. The antigen(s) described does not seem to be related either to human Ia-type antigens or to Epstein-Barr virus-associated antigens because these antigens are not present on fetal or adult thymocytes. Reciprocal absorption experiments indicate that this anti-Daudi serum detects the same antigenic structures present on certain subpopulations of T and B lymphocytes.     

Human leukaemia-associated antigens expressed by acute myelocytic leukaemia cells and their detection by heterologous antisera.

Antisera against human acute myelocytic leukaemias were tested in complement-dependent in-vitro cytotocity tests against leukaemia cells and normal cells as targets. After absorption with erythrocytes and spleen cells from allogeneous donors the antisera reacted with leukaemia cells, but not with leukocytes from bone marrow and the peripheral blood of children in remission, lymphocytes from healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-induced blasts and cord blood lymphocytes. Extensive cross reactions were obtained in the tests against leukaemia cells. The antisera reacted not only with AML cells, but also with ALL, CLL, and CML cells. It was possible to remove the cross-reactivity with ALL cells through absorption with ALL cells or with fetal tissue, and to remove the cross reactivity with CLL cells through absorption with CLL. A complete absorption of the anti-AML sera was possible with AML and CML cells. After absorption with fetal tissue and CLL cells the antisera showed exclusively specificity for myelocytic leukaemias. Thus, AML cells contain three leukaemia-associated membrane antigen components: an antigen of fetal origin, a "CLL-specific" antigen, and an antigen that occurs on myelocytic leukaemias.     

Antibodies to an antigen of human thymus epithelial tissue common to the epidermis of the skin in malignant myasthenia]

It has been demonstrated by the indirect immunofluorecent technique that many of the sera of patients with myasthenia gravis react with the anticells of the human thymus epithelial tissue. Sorption of the sera with the suspension of the epidermis cells and the homogenates of the tissues of other human organs showed that the epithelial cell antigen with which the sera of patients with myasthenia reacted were epidermal heteroorganic thymus antigens, i.e. common for the thymus epithelium and skin epidermis. The presence of antibodies to the cells of the epithelial tissue of the thymus in the sera of patients suffering from myasthenia gravis permits to suppose the existence of an immunopathological process against the thymus tissue antigens (including the heteroorganic structures of its epithelium) in this disease.     

Ontogeny of human hematopoietic cells: analysis utilizing monoclonal antibodies

Lymphoid and myeloid cells isolated from second trimester fetal lymphoid organs were characterized by utilizing a panel of monoclonal antibodies that define human lineage-restricted, differentiation, histocompatibility, and activation antigens. At distinct gestational stages, the appearance of morphologically identifiable lymphoid and myeloid cells paralleled the appearance of cells expressing definable lymphoid and myeloid antigens. The proportion of cells in fetal liver, bone marrow, and spleen that expressed histocompatibility, myeloid, and B cell antigens increased with fetal maturation. In contrast, even the earliest fetal thymuses studied were of a phenotype no different than that seen during later stages of ontogeny. Although the cellular lineage of most fetal hematopoietic cells could be identified by this panel of reagents, a considerable number of fetal liver and bone marrow cells did not express any of these antigens, suggesting the possibility that they might represent early hematopoietic progenitor cells. These studies support the notion that the adult cellular phenotype is the result of both an orderly acquisition of differentiation antigens and the migration of these primitive cellular populations to specific fetal organs. Identification of hematopoietic progenitors in fetal tissues may facilitate the identification and isolation of early lymphoid and myeloid progenitor cells in adults.