Chicken thymocyte-specific antigens identified by monoclonal antibodies: characterization and distribution in normal tissues and in tumoral tissues from Marek's disease chicken

Monoclonal antibodies (MAbs) were obtained against purified thymocyte membrane extracts. Five MAbs TA3, TB1, TB6 (IgG1), TC4, and TA1 (IgG2a), were tested by immunofluorescence and by immunoperoxidase tests against normal cells from different organs, Marek's disease (MD) cell lines, and MD tumoral cells from chickens. Three of them, TA3, TB1, and TB6, reacted exclusively with lymphoid cells in both cortical and medullary areas of the thymus and with less than 8% bursa cells. They identified a protein of apparently 40 kD. The other two revealed antigenic determinants on most medullar thymocytes and some cortical thymocytes, and on some splenic and peripheral blood lymphocytes. They were positive with MD cell lines and cells deriving from MD tumors. TC4 and TA1 detected molecular masses of about 110 kD and 16 kD, respectively. No MAbs reacted with erythrocytes, bone marrow, liver, brain, and skin cells. Not all of the tested cells were stained after contact with an anti-chicken immunoglobulin serum. In this paper, we determine a specific antigen restricted to T cells from thymus and different markers belonging to the mature T cells. The latter are also present on MD cell lines and MD tumoral cells.     

A pomona serogroup-specific, agglutinating antigen in Leptospira, identified by monoclonal antibodies

Monoclonal antibodies were produced by hybridoma cell lines derived by fusion of mouse NS-1 myeloma cells with splenocytes from mice immunized with Leptospira interrogans serovar pomona. One hybridoma (A3) produced an IgG2a antibody which agglutinated all leptospires of the Pomona serogroup but not leptospires representative of serovars of any other serogroup. The antibody precipitated in immunodiffusions with either alkali- or phenol-extracted lipopolysaccharide and also with the TM antigen of Yanagawa et al., indicating a common determinant on both antigens. A3 antibody opsonized viable leptospires for phagocytosis by mouse macrophages in vitro.     

Functional and biochemical characterizations of avian T lymphocyte antigens identified by monoclonal antibodies

Seven monoclonal antibodies (mAb) were used to characterize antigens present on chicken T lymphocytes and on natural killer cells by flow cytometry, radioimmunoprecipitation and by effects on cell-mediated cytotoxicity and mitogen-induced proliferation. mAb CTLA8 and 5 stained 73% of thymus, 44% of spleen and 51% of peripheral blood lymphocytes (PBL), respectively, and immunoprecipitated 65- and 45-kDa proteins from detergent extracts of ¹²⁵I surface-labeled thymocytes. Pretreatment of splenic lymphocytes with mAb CTLA5 and 8 in the presence of rabbit complement (C) eliminated the concanavalin A (Con A)-induced T cell proliferative responses. mAb CTLA3, 4 and 9 stained 43% of thymus, 36% of spleen and 18% of PBL, and immunoprecipitated 33–35-kDa proteins. Pretreatment of spleen cells with mAb 4 or 9 plus C reduced, but did not eliminate, the Con A-induced proliferative response and significantly reduced both major histocompatibility complex (MHC)-restricted and non-MHC-restricted cellular cytotoxicity. mAb CTLA1 and 6 stained 58% of thymus, 13% of spleen and 19% of PBL. mAb CTLA 1 and 6 immunoprecipitated a 65-kDa protein. mAb CTLA l and 6 had no effect on the Con A-induced blastogenesis and CTLA 6 caused no decrease in virus-specific cytotoxic T lymphocyte and natural killer activity. These results indicate that (a) mAb CTLA 5 and 8 identify antigens on mature T lymphocytes that are similar in tissue distribution, molecular mass and function to the mammalian CD5 antigen; (b) mAb CTLA 3, 4 and 9 detect the avian homologue of CD8 antigen; and (c) mAb CTLA l and 6 identify the avian homologue of CD4 antigen.     

Immunological and biochemical characterization of the nonspecific cross-reacting antigen epitopes using twenty-three monoclonal antibodies.

Twenty-three monoclonal antibodies (MAbs) reactive with nonspecific cross-reacting antigen (NCA) were prepared and used for constructing a serological map of the NCA molecule. The MAbs were generated using purified NCA or carcinoembryonic antigen (CEA) as immunogen. The MAbs could be divided into two groups: Group X, 10 clones reactive with NCA and CEA; and Group Y, 13 clones specific for NCA. Cross-competition enzyme immunoassays between MAbs of the individual groups revealed that at least 8 different subgroups can be defined i.e., 5 and 3 subgroups in Groups X and Y, respectively. The chemical nature of the epitopes recognized by those MAbs was tested using chemically or enzymatically treated antigens; all MAbs reacted with periodate-treated NCA and deglycosylated NCA, indicating that all the epitopes identified appeared to be protein in nature. Reduction and alkylation, pepsin digestion or pronase treatment of NCA, however, gave some differential results with respect to MAb binding. The serologic mapping and biochemical studies reported here thus provide information as to the range and nature of the epitopes on the NCA molecule and help form the basis for selecting the anti-NCA MAbs for use in biological and immunological study of NCA.     

Cancer-associated carbohydrates identified by monoclonal antibodies

"New" carbohydrate structures on the surface of or secreted by cancer cells, identified as epitopes by monoclonal antibodies, are reviewed. These structures may represent the accumulation of precursor chains because of decreased activity of synthesizing enzymes, the production of new oligosaccharides due to increased or aberrant glycosylation of carbohydrate chains, a change in density of carbohydrates on the cell surface, or exposure of chains usually covered by other structures. Alterations in glycolipid synthesis include aberrant fucosylation and/or sialyation of the lacto series, sialylation or fucosylation of the globo series, and sialyation of the ganglio series. Many of these carbohydrate epitopes have become useful for the diagnosis, prognosis, and monitoring of patients with cancer. Some of the important markers include CA 15.3, CA 19.9, CA 50, CA 125, CA 242, MCA, SLEX, etc. Incomplete glycosylation of O-linked mucin oligosaccharide is recognized as the important "cancer antigen" B72.3, which is sialyated Tn. The oligosaccharide components of alpha-fetoprotein, carcinoembryonic antigen, and epidermal growth factor receptor are also reviewed. In many instances the glycosylation seen in cancer cells or their products reflects patterns seen during normal development. Thus, cancer-associated oligosaccharides are oncodevelopmental in nature. The biologic significance of carbohydrates on cell surfaces is not known, but several possibilities include a role in cell to cell recognition, intracellular processing of glycoproteins, cell activation, and ability of cancer cells to metastasize.     

Human tumor-associated antigens identified by monoclonal antibodies

Conclusions Monoclonal antibodies have been obtained that are specific for cell surface antigens of human tumors. Some of these antigens are tissue type specific tumor antigens, most strongly expressed on tumors of the same histological type. The antigens identified by the monoclonal antibodies are primarily normal differentiation antigens that are expressed more strongly on neoplastic cells than on most normal adult cells. For several antigens, the higher degree of antigen expression on neoplastic cells appear to be adequate for diagnostic and therapeutic purposes. Future work in this area is likely to have far-reaching consequences for the clinical handling of human cancer patients.This work was supported by grants CA 14135, 19148, 19149, CA 25558, CA 27841 from the National Institutes of Health, and IM 241A from the American Cancer Society. The authors wish to acknowledge collaboration with Drs. M.-Y. Yeh, R. G. Woodbury, S. M. Larson, and K. Nishiyama     

Immunocytochemical distribution of a breast carcinoma associated glycoprotein identified by monoclonal antibodies.

A glycoprotein, BCA-225 (Mr 225,000-250,000), has been identified in cells and spent medium of clone 11 T47D breast carcinoma cells by three murine monoclonal antibodies, CU18, CU26, and CU46. The antigen was localized in paraffin sections of 167/178 (94%) Bouin's-fixed human breast carcinoma tissues and few other carcinomas (1/8 lung [squamous], 4/4 uterine cervix) in an intracellular pattern, whereas an apical or glycocalyx distribution was seen in several normal tissues, benign lesions, and malignant tumors. Although the immunocytochemical staining patterns observed with these antibodies have many similarities to those described with other previously reported monoclonal antibodies, notable differences include the lack of reactivity of CU18, CU26, and CU46 with lactating mammary gland and with gastrointestinal malignancies. BCA-225 binds to wheat germ lectin, not to concanavalin A, but monoclonal antibody binding does not appear to involve the carbohydrate component of the molecule. The frequency of the immunocytochemical detection of BCA-225 in breast carcinomas and its restricted distribution in other human tissues suggest considerable clinical potential for this antigen and its corresponding monoclonal antibodies.     

Different epitopes of the CD31 antigen identified by monoclonal antibodies: cell type-specific patterns of expression

3 mAb-5A2.G5, B2B1 and 2BD4-all of IgG1 isotype were identified as belonging to the CD31 cluster by their binding to transfected murine cell lines expressing the CD31 antigen and by sequential immunoprecipitation experiments. Competitive binding experiments were carried out using the human myelomonocytic cell line RC-2A. mAb B2B1 and 2BD4 did not cross block. mAb 5A2.G5 partly inhibited binding of 2BD4 but not B2B1. Thus the epitopes identified by 5A2.G5 and 2BD4 appear to overlap but to be quite separate from that recognized by B2B1. All 3 antibodies bound to a 130 kDa species on Western blots after nonreducing polyacrylamide gel electrophoresis of platelet lysates. Culture of RC-2A cells in the presence of tunicamycin (after removal of surface antigens by pronase) blocked re-expression of the epitopes recognised by all 3 mAb suggesting that they involve N-linked glycosylation. Furthermore, treatment of platelet lysates with Endoglycosidase F prior to electrophoresis and Western blotting abolished the binding of the mAb but not a rabbit polyclonal antiserum to CD31. Nevertheless, neuraminidase treatment of RC-2A cells failed to affect mAb binding. The antibodies displayed typical properties of CD31 antibodies in that all 3 precipitated at 130 kDa cell surface protein and bound strongly to platelets, monocytes, neutrophils and vascular endothelium. All 3 antibodies were positive on hemopoietic progenitor cells which give rise to colonies in the CFU-C assay. However, differences in binding to peripheral blood lymphocytes and to certain human leukemic cell lines were noted. In particular, mAb 5A2.G5 bound weakly to lymphocytes and to the lymphoid cell line HSB-2 compared with the other 2 antibodies.     

Human monocyte-histiocyte differentiation antigens identified by monoclonal antibodies

Two distinct differentiation antigens of human myelomonocytic cells are defined using murine monoclonal antibodies. The antigens recognized by antibodies 20.2 and 20.3 are expressed by all cells of the monocyte lineage in both peripheral blood and bone marrow. Cell-sorting experiments demonstrated that histiocytes and immature bone marrow cells with detectable alpha-naphthyl butyrate esterase activity also express both antigens. Within cells of other lineages, the antigens had distinct patterns of expression. Immature myeloid cells were 20.2 negative, but 20.3 positive; whereas mature myeloid cells were 20.2 positive, but 20.3 negative. Nucleated erythroid cells and platelets expressed only the 20.3 antigen. These results indicate that myeloid and monocytic cells share common differentiation antigens with cells of the erythroid and megakaryocytic lineages. The 20.2 and 20.3 antibodies reacted with the leukemic cells from some patients with acute nonlymphocytic leukemia (FAB, M1-M5) and with some cell lines derived from patients with nonlymphocytic leukemia, but not with blast cells from patients with lymphoid leukemia or with lymphoid leukemic cell lines. These antibodies may prove useful in studying the differentiation of bone marrow stem cells, in defining the cellular origins and classification of leukemias, and in the identification of distinct prognostic subgroups of acute nonlymphocytic leukemia.     

Xenopus NK cells identified by novel monoclonal antibodies

Early-thymectomized (Tx) Xenopus frogs, which are permanently deficient in T cells, are used as a model sytem for the characterization of novel monoclonal antibodies (mAb) which identify candidate NK cells at the amphibian level of evolution. Hybridomas, generated from mice immunized with splenocytes from Tx Xenopus following B cell and thrombocyte depletion, were screened by flow cytometry. Three mAb (1F8, 4D4 and 1G5) were identified that stained increased proportions of splenocytes from Tx compared with control frogs. These mAb identified lymphoid populations from Xenopus spleen, liver and gut which, after 48 h culture in growth factor-rich medium, exhibited spontanous killing of MHC-deficient allotumor targets. mAb-defined splenocytes also rapidly induced apoptosis of such tumor targets. Dual color analysis confirmed that NK cells are neither T nor B cells. Cytospins of splenocytes isolated with anti-NK mAb revealed large lymphoid cells with distinct pseudopodia. Immunohistology indicated each anti-NK mAb routinely labeled cells within the gut epithelium but NK cells were difficult to visualize in spleen sections. Western blotting of spleen, liver and intestinal lysates subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that 1G5 reacted strongly with protein bands of approximately 70 - 85 kDa, whereas mAb 1F8 and 4D4 stained less intensely, but identified similar protein bands.     

Identification and tissue distribution of rabbit leucocyte antigens recognized by monoclonal antibodies.

Three monoclonal antibodies which recognize rabbit leucocytes have been characterized by immunofluorescence staining of a variety of cell populations and also by immunochemical techniques. The evidence obtained suggests that these antibodies recognize the rabbit equivalents of the CD58/LFA-3 (VC21), CD43/leukosialin (L11/135) and CD9 (MM2/57) antigens. A fourth antibody, RPN3/57, recognizes an antigen expressed strongly on T cells, thymocytes and neutrophils and at lower levels on platelets. It has not, however been possible to characterize the antigen recognized by RPN3/57 in molecular terms. Both L11/135 and RPN3/57 are useful reagents for the detection of T cells both by flow cytometry and by immunohistochemistry.     

Generation of monoclonal murine anti-DNP-IgE, IgM and IgG1 antibodies: biochemical and biological characterization.

Monoclonal DNP-specific IgG (lambda 2 epsilon 2), IgM (kappa 2 mu2) and IgG [kappa 2 (gamma 1)2] were isolated fom the culture supernatant of hybridomas by affinity chromatography with 2,4-dinitrophenol bovine serum albumin (DNP-BSA) sepharose and characterized by biochemical and biological methods. The molecular weights were 84,200 for the epsilon chain, 55,400 for the gamma chain and 77,500 for the mu chain as determined by sodium dodecylsulphate polyacrylamide del electrophoresis (SDS-PAGE). The association constants for [3H]-DNP-lysine determined by equilibrium dialysis were 0 . 87 X 10(7) l/mol for IgE and 1 . 91 X 10(8) 1/mol for IgG1. The isoelectric focusing of the purified monoclonal antibodies revealed for IgG1 seven bands at a pH range of 6 . 3 - 7 . 2 and for IgE sixteen bands at a pH range of 4 . 5 to 6 . 8. the binding of 125I-anti-IgE to rat basophilic leukaemia (RBL) and rat mast cells which had been preincubated with various amounts of monoclonal IgE was studied. At saturation conditions of IgE, about 2 . 14 X 10(5) molecules of anti-IgE were bound per rat mast cell. Rat mast cells coated with monoclonal anti-DNP IgE were triggered for the release of histamine in the presence of either the antigen or guinea-pig anti-mouse IgE. A mutual inhibition of the passive cutaneous anaphylaxis (PCA) reaction in the rat by either mixing mouse reaginic serum directed against ovalbumin or rat reaginic serum directed against Nippostrongylus brasiliensis with monoclonal mouse anti-DNP IgE was demonstrated.     

A novel 95-kilodalton antigen of Wuchereria bancrofti infective larvae identified by species-specific monoclonal antibodies.

CBA and BALB/c mice produced polyspecific and monospecific polyclonal antibody responses, respectively, following immunization with Wuchereria bancrofti stage-3 larvae. Two monoclonal antibodies (MAbs) were produced from the immunized BALB/c mouse. These MAbs (both isotype M) recognized a previously undescribed highly expressed W. bancrofti antigen present in stage-3 larvae. The epitopes bound by the MAbs appear to be species specific for W. bancrofti since the MAbs did not bind to antigens of either nine other nematode species or two vector species in Western blots (immunoblots). Phosphorylcholine epitopes, responsible for immunological cross-reactivity among nematodes, were identified only on a 200-kDa antigen and not on the 95-kDa molecule. The targets of these immunoglobulin M MAbs are not carbohydrate epitopes.     

A human leukocyte antigen identified by a monoclonal antibody

Leukocyte antigen of approximate molecular weight 200,000 daltons have beendescribed in mouse, rat, and man. We described here the reactivities of a monoclonal antibody. GAP 8.3, which identified such an antigen on human leukocytes. We found the leukocyte antigen H-T200 on T and B lymphocytes, granulocytes, monocytes, and platelets, but not on erythrocytes or nonhematopoetically derived cells. Resting and activated T cells had more antigen or their surfaces than did resting B lymphocytes and EBV-transformed B cells, respectively. The leukocyte antigen was detected on approximately 75% of bone marrow cells; cells of the erythroid series comprised the negative population. The GAP 8.3 antibody and its F(ab′)₂ fragments had no effect on in vitro stimulation of peripheral blood cells by mitogens or allogeneic cells. Antigen isolated from T cell lines had a higher electrophoretic mobility than did antigen from B cell lines; antigen from the mycloid line U937 comigrated with that from B cell lines. In addition, we detected very small but reproducible differences in the electrophoretic mobility of the antigen on two T cell lines.     

Axolinin localization in the nervous tissue of squid revealed by monoclonal antibodies specific for axolinin: characterization of monoclonal antibodies against axolinin

Monoclonal antibodies against squid axolinin (the 260-kD glycoprotein in squid axoplasm) have been produced using spleen cells immunized with purified axolinin in vivo and in vitro. The produced antibodies can be categorized into two groups according to the nature of their antigenic site. The monoclonal antibodies belonging to group I recognize the protein backbone of axolinin, while those belonging to group II bind to the sugar chains of axolinin. Group II was further divided into two subgroups IIA and IIB; the subgroup IIA antibodies recognize sugar chains to which concanavalin A binds, while the IIB antibodies bind to sugar chains lacking the lectin-binding sites. Only the group I antibodies can be used as specific probes for axolinin since the sugar chains recognized by the group II antibodies are present on other squid glycoproteins in addition to axolinin. It follows from our results that polyclonal antisera against axolinin cannot generally be expected to be specific for this protein.     

Mac-1: a macrophage differentiation antigen identified by monoclonal antibody.

We have previously described the derivation of M1/70, a hybrid myeloma line secreting monoclonal rat anti-mouse cell surface antibody (Springer, T., Galfre, G., Secher, D. S. and Milstein, C, Eur. J. Immunol. 1978. 8: 539). We have now investigated the cellular distribution of this antigen using a ¹²⁵I-labeled anti-rat IgG indirect binding assay, the fluorescence-activated cell sorter, autoradiography and precipitation of cell surface molecules. Screening with a tumor cell panel showed strong reactivity with a macrophage-like line but no reactivity with B or T lymphoma lines. In normal tissues, M1/70 antigen was found to be present in small amounts on spleen and exudate granulocytes and a subpopulation of bone marrow cells, in moderate amounts on spleen and blood monocytes and expressed in much larger amounts on spleen histiocytes and peritoneal exudate macrophages. In contrast, M1/70 antigen was found to be absent from erythroid and lymphoid cells. M1/70 antibody precipitated two polypeptides of 190 000 and 105 000 mol. wt. which were present in much greater amounts on peritoneal exudate macrophages than on spleen cells. The expression on phagocytes of two other antigens identified by monoclonal antibodies M1/69 and M1/9.3 was also examined. Monocytes and granulocytes expressed large amounts of M1/69 and low amounts of M1/70 antigen, while in peritoneal exudate macrophages this pattern was dramatically reversed. M1/70 thus defines a differentiation antigen on mononuclear phagocytes and granulocytes, the expression of which is specifically increased during monocyte maturation. This antibody is the first to be described which recognizes a discrete molecule specific to phagocytes.     

Stage-specific, strain-specific, and cross-reactive antigens of Leishmania species identified by monoclonal antibodies.

The fusion of NS-1 myeloma cells with spleen cells from mice chronically infected with Leishmania tropica resulted in nine clones of hybridomas producing monospecific antibodies to membrane antigens of L. tropica. One of the antibodies (L-5-85) bound specifically to the promastigote form of the parasite, and the remaining eight recognized antigens shared by the promastigote and amastigote of L. tropica. Four of the antibodies (L-5-16, L-5-34, L-5-44, and L-2-3F11) detected parasite antigens on the surface of L. tropica-infected macrophages. Common antigens shared by L. tropica, L. mexicana, and L. donovani were identified as well as one antigen apparent on most Leishmania spp. and present also in Crithidia fasciculata. Two monoclonal antibodies (L-5-27 and L-5-41) were found to bind only to strains of L. tropica from simple cutaneous leishmaniasis. A special property shared by these two antibodies was the inhibition of parasite growth in macrophages in vitro.     

Distinct epitopes on Ik gene products identified by monoclonal antibodies

Analysis of reactivity of monoclonal anti-Ia^k alloantibodies, obtained by fusion between NS 1 myeloma and spleen cells from alloimmune A.TH mice, permitted the identification of 9 distinct determinants of the I^k gene products. Competitive binding experiments indicated that 2 private epitopes (defined by H 8–109.13 and H 8–138.4 antibodies) of the I-A^k product could be separated, thereby apparently splitting the Ia.2 specificity. A public determinant of the I-A^k molecule (identified by H 8–15.9 antibody) was found expressed not only on the 1-A products of the H-2^b, H-2^d, H-2^ja, H-2^P and H-2^q murine haplotypes, but also on human HLA-DR antigens. Four determinants of the I-E/C^k antigen (defined by H 7–8.26, H 10–81.10, H 10–93.2 and H 8–86.2 antibodies) had a strain distribution analogous to the Ia.7 specificity. However, competitive binding experiments, and the cross-reactivity pattern with la-like antigens from other species (e.g. human HLA-DR antigens) indicated that these antibodies detected distinct determinants on the I-E/C^k molecule, thereby subdividing the broad Ia.7 specificity. Two other determinants (defined by H9–14.8 and H9–15.4 antibodies) had a strain distribution that did not permit a precise assignment to a given Ia antigen, even though preliminary data suggested that they could detect separate determinants on the I-E/C^k product. All these monoclonal antibodies identified membrane antigens with the expected Ia tissue distribution pattern, and most of them could precipitate a molecule containing two chains of 28 kD and 35 kD, from mouse spleen cell lysates.     

T-lymphocyte subpopulations identified by monoclonal antibodies after human bone-marrow transplantation

Peripheral T-lymphocyte reconstitution after bone-marrow transplantation, 12 allogeneic and 13 autologous, was studied by indirect immunofluorescence assay using mouse monoclonal antibodies. Abnormal counts were detected in the two major sub-populations of T-cells i.e. helper and T cytotoxic-suppressor lymphocytes, defined by monoclonal antibodies (alpha Leu 3a and B 9.2), and in DR antigen-positive T-cells. The pattern of T-lymphocytes replenishment was identical for both types of transplant, and was not affected by Graft Versus Host disease (GVHD).