The role of lymphokine-activated cell-associated antigen. II. Distribution and correlation with cell cycle

It has previously been shown that killer-blocking monoclonal antibody (KBA MAb) recognizes lymphokine-activated cell-associated antigen (LAA) involved in broad-reactive killer. (BRK) cell-mediated cytotoxicity. We now report that LAA is expressed on all lymphoid cells, though the amount of LAA on unstimulated lymphocytes is low. In contrast, lymphocytes activated in vitro with either concanavalin A, alloantigens, lipopolysaccharide, or recombinant interleukin 2 express high levels of LAA. In addition, in vivo activated lymphocytes, such as OK-432-activated lymphocytes and tumor-infiltrating lymphocytes express higher levels of LAA than unstimulated lymphocytes. We also demonstrate that the expression of LAA is restricted in T-cell lymphomas and a M phi cell line, while myelomas, fibrosarcomas, and carcinomas do not express LAA. Cell cycle analysis using propidium iodide and KBA MAb showed that LAA expression was closely correlated with the transition of cells from G1a to G1b phase.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.     

Dual parameter flow cytometric analysis of DNA content, activation antigen expression, and T cell subset proliferation in the human mixed lymphocyte reaction

This study provides direct correlation via dual parameter flow cytometry (simultaneous assessment of immunofluorescence and DNA content) between mixed lymphocyte reaction (MLR) responder cell entry into the S/G2/M phases of the cell cycle with the kinetics of expression of two activation-associated cell surface proteins, Tac (IL 2 receptor) and 4F2 (unknown metabolic function). A small population of activated cells was identifiable by expression of both Tac and 4F2 antigens before peak DNA synthesis in the MLR. This population of activation antigen-positive cells expanded linearly in size from days 3 to 7 of culture. Treatment of immature MLR cultures with anti-4F2 Mab and complement (C) before DNA synthesis (treatment on day 3, peak DNA synthesis on days 5 to 6) resulted in blunted proliferation and activation antigen expression when the same culture was analyzed after maturation on day 6, indicating that the activated population had been previously detected and removed by anti-4F2 Mab + C. The 4F2 antigen was expressed on a greater percentage of cells in the MLR at all times (days 3 to 9) than was Tac, was present on virtually all S/G2/M phase responder cells, and a large fraction of cells remained intensely 4F2+ subsequent to peak DNA synthesis. In contrast, after initially preceding responder cell entry into the S phase of the cell cycle, the kinetics of Tac antigen expression closely paralleled the kinetics of responder cell proliferation. A subpopulation of cycling responder cells was noted in all MLR cultures studied that expressed Tac antigen weakly or not at all. Cells within both T4 and T8 cell subsets proliferate with similar kinetics in response to alloantigen. The possibility that activation antigens can be utilized to study effector cell generation in the MLR and that this flow cytometric technique may be utilized to analyze the response to various alloantigens is discussed.     

Inhibition of lymphocyte proliferation by monoclonal antibody directed against the T3 antigen on human T cells

Peripheral blood mononuclear cells from 40% of normal donors are mitogenically unresponsive to UCHT1, a monoclonal antibody reactive to the T3 surface molecule on human T lymphocytes. Cell preparations from non-UCHT1 responders were used to examine whether and how interaction of UCHT1 with the T3 molecule affects T-cell functionality. It was found that UCHT1 profoundly (greater than 85%) suppressed lymphocyte proliferation induced by plant mitogens (phytohemagglutinin (PHA) and concanavalin A (Con A], recall antigen (candidin), and allogeneic non-T cells. The antibody abrogated both the production of interleukin 2 (IL-2) by and the expression of IL-2-specific receptors on T lymphocytes stimulated by PHA or allogeneic non-T cells. UCHT1 was maximally suppressive when added to cells within 2 hr (PHA stimulation) or 1 day (allogeneic non-T cell activation) after the initiation of the culture period. The inhibiting activity of UCHT1 could be related to its ability to modulate T3 molecules from the T-cell surface: both actions displayed the same antibody concentration dependence and had a comparable time dependence. Moreover, after modulation, unresponsive lymphocytes regained responsiveness to PHA in parallel with reexpression of surface T3 molecules. These findings are consistent with the idea that the human T3 molecule functions as an essential signal transducer during the early phases of T-cell activation.     

Monoclonal anti-actin antibody recognizes a surface molecule on normal and transformed human B lymphocytes: expression varies with phase of cell cycle

A monoclonal anti-actin antibody, 2C9, was used to study the distribution of an actin-like cell-surface antigen (hereafter termed actin) on a lymphoblastoid cell line LA350 and on human peripheral blood lymphocytes. It was determined that 8-40% of LA350 cells and 3-15% of peripheral blood lymphocytes from healthy donors stain specifically with 2C9, almost exclusively on IgM-positive cells. Treatment of cells with 2C9 prior to incubation caused cell-surface actin to first patch and then to cap. Treatment of cells with nonspecific protease caused a loss of surface actin, with reexpression of the marker after 8-12 hr. The expression of LA350 surface actin also increased with DNA synthesis and was demonstrated to be maximal during late G1/early S phase. Thus, this antigen may be a sensitive marker for activated lymphocytes. These studies contribute to our understanding of the expression and distribution of actin-like membrane proteins that may participate in regulatory signals mediated by anti-actin antibody.     

Identification of an early activation antigen (Bac-1) on human B cells

We have produced a monoclonal antibody, Bac-1, that appears to identify a novel antigen on activated human B cells. The Bac-1 antigen can be detected between 8 to 16 hr, as well as transferrin receptors (T9), after activation of small resting B cells with phorbol myristic acetate, anti-IgM antibody, Staphylococcus aureus Cowan I, or Epstein-Barr virus. The expression of the Bac-1 antigen precedes that of IL 2 receptors (Tac-1). Peak expression of the Bac-1 antigen was observed on day 3 after activation, and decreased thereafter. The Bac-1 antigen was present on a minor subpopulation of relatively large B cells isolated from blood samples, and on "preactivated" B cells of heterogeneous size isolated from spleens and tonsils. It was not detected on bone marrow pre-B cells, blood small B cells, or plasma cells, nor was it expressed by resting or activated T cells or nonlymphoid cells. Certain B cell neoplasms and B lymphoblastoid cell lines were Bac-1+, but neoplastic cells of non-B lineage were Bac-1-. With immunoperoxidase staining, Bac-1+ cells were detected predominantly in the germinal centers of tonsil sections. The Bac-1 antigen on activated B cells was destroyed by protease treatment and was enhanced by neuraminidase treatment, suggesting that the Bac-1 antibody detects a cell surface molecule via an antigenic determinant which is partially obscured by neighboring sialic acid residues. The reactivity pattern of Bac-1 differs from the patterns of cellular reactivity reported for other monoclonal antibodies with specificity for activated human B cells.     

The expression,regulation and adhesion function of a novel CD molecule,CD226, on human endothelial cells

CD226 is a 67 kDa type I transmembrane glycoprotein mainly expressed on activated T cells, NK cells and platelets, and involved in the differentiation of cytotoxic T lymphocytes (CTL) and NK, as well as platelet activation and aggregation. Here we found that the expression of CD226 protein and CD226mRNA were very weak in resting HUVEC and ECV304 cells, whereas high level expression could be observed when these cells were stimulated. The binding activities between activated endothelial cells and activated Jurkat cells could be partly blocked by CD226/Ig fusion protein. Similarly, CD226/Ig could also partly block the adhesion between activated endothelial cells and some leukocytes or colo205 cells. These data provided the evidence that activated endothelial cells could express high level of CD226, and CD226 was involved in the endothelial cells' adhesion. The above findings suggested that CD226 is a novel inducible adhesion molecule on human endothelial cells.     

Expression of glycolytic isoenzymes in activated human peripheral lymphocytes: cell cycle analysis using flow cytometry

Human peripheral lymphocytes activated with concanavalin A and phorbol myristate ester exhibit an increase in glycolysis on a time-course similar to that for DNA synthesis. Elevated glycolysis is accompanied by increased specific activities of the glycolytic enzymes. Increased enzyme activities are accounted for by the appearance of specific isoenzyme forms (muscle forms) normally expressed in rapidly growing tumor cells or in growth-stimulated cells. In the present study we analyzed the expression of the glycolytic isoenzymes during cell cycle progression of activated human lymphocytes using two-parameter (DNA and protein) flow cytometry. Time-course studies and analysis of subpopulations prepared by elutriation centrifugation showed that the inducible isoenzymes are expressed at low levels or not at all in G0 cells. They are expressed first during the G0 to G1 transition or in early G1. However, expression increases throughout G1, reaching a maximum in S-phase. Thus, induction of glycolytic isoenzymes provides an excellent marker of T-cell activation and progression toward DNA synthesis.     

A B cell-restricted activation antigen (B8.7) functionally related to the low molecular weight B cell growth factor receptor

A novel monoclonal antibody (anti-B8.7) is reported which recognizes an epitope expressed either on in vitro activated B cells or on a fraction of fresh large B cells (putatively in vivo preactivated). B8.7 antigen is also present on two out of eight B cell lines tested and is characterized as a membrane component displaying an approximate molecular weight of 55,000 to 60,000. By contrast, B8.7 is absent from resting B cells, monocytes, resting or activated T cells, and from the eight non-B cell lines tested. After in vitro activation, B8.7 antigen appears later than the transferrin receptor and its expression increases until day 3. The anti-B8.7 monoclonal antibody induces a dose-related inhibition of the low molecular weight B cell growth factor-dependent proliferation of activated B cells, whereas it does not affect their response to interleukin 2. This strongly suggests that the B8.7 epitope is present on a molecule selectively involved in the interaction between B cells and a B cell growth factor.     

Interleukin 2-driven T lymphocyte proliferation is dependent upon a surface antigen distinct from the interleukin 2 receptor: requirements for inhibition of T-cell proliferation by monoclonal antibody 5C3

A monoclonal antibody (5C3) to an antigen expressed on activated guinea pig T lymphocytes that did not react with the interleukin 2 (IL-2) receptor, but inhibited IL-2-driven proliferative responses has been previously characterized. The present study provides further analysis of the inhibitory capacity of 5C3 for T-cell proliferation and of the relationship between the expression of the antigen defined by 5C3 and the capacity of cells to respond to IL-2. 5C3 inhibited proliferation of T-cell blasts to IL-2-containing fluids when added as late as 8 hr prior to termination of a 26-hr culture. 5C3 pretreatment of the IL-2-responsive blast cells was also sufficient to detect significant inhibition of proliferation. FACS analysis of these blasts indicated that maximal 5C3 binding was required for pretreatment to result in inhibition of IL-2-driven proliferation. Delayed addition of 5C3 to culture or pretreatment with 5C3 of responding cells also resulted in inhibition of proliferation of immune T lymphocytes to antigen-pulsed-presenting cells. Lastly, although modulated 5C3- blasts failed to proliferate to IL-2, induction of the 5C3-bearing molecule on these 5C3- blasts correlated with restoration of the ability of these cells to proliferate to IL-2. Collectively, these results further support the hypothesis that monoclonal antibody 5C3 interferes with a critical signal in the IL-2 growth pathway.     

An alternative pathway of T-cell activation: A functional role for the 50 kd T11 sheep erythrocyte receptor protein

A series of seven monoclonal antibodies was produced against the T-lineage-specific 50 kd T11 sheep erythrocyte rosette (SRBC) receptor protein in order to define the function of the molecule. Three distinct epitopes were detected: T11₁, the SRBC binding site expressed on all T lymphocytes and thymocytes; T11₂, an epitope unrelated to the SRBC binding site but with a similar distribution; and T11₃, a neo-epitope expressed only upon T-cell activation. Simultaneous triggering of T11₂ and T11₃ epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen and/or antigen-presenting cells. This antigen-independent mode of triggering is distinct from that involving the T3-Ti antigen receptor complex and represents an alternate pathway of T-cell activation. Given that T11 is the earliest T-lineage surface glycoprotein to appear in thymic ontogeny and is thus expressed before T3-Ti, the former may be involved in clonal expansion and/or differentiation during early development.     

Tissue distribution and biochemical and functional properties of Tp55 (CD27), a novel T cell differentiation antigen

Two monoclonal antibodies (CLB-CD 27/1 and CLB-CD 27/2) were raised against a novel determinant on human T lymphocytes. One of these antibodies, CLB-CD 27/1 (clone 9F4), was grouped by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens together with three other monoclonal antibodies (VIT 14, OKT 18A, and S152) in the new cluster CD27. In this paper we show that antibodies belonging to this cluster recognize an antigen present on a large subset of peripheral T lymphocytes and most medullary thymocytes. At least two different nonoverlapping epitopes were identified with directly labeled monoclonal antibodies. Immunoprecipitation studies indicate that the target antigen of CD27 antibodies is a polypeptide of 55 kDa, which appears in the form of a disulfide-linked homodimer on the T lymphocyte membrane (Tp55). Stimulation of T cells via the T3/T cell antigen-receptor complex, with either phytohemagglutinin or CD3 monoclonal antibodies, resulted in a fivefold increase in the membrane expression of Tp55, whereas activation by phorbol myristate acetate caused a marked down-regulation. Moreover, an additional molecule of 32 kDa was precipitated from the membrane of activated but not of resting T cells. Addition of CD27 antibodies to cultures stimulated with either phytohemagglutinin or CD3 monoclonal antibody led to enhanced proliferation, whereas no effect was observed in phorbol myristate acetate or interleukin 2-stimulated cultures. The possible role of the Tp55 antigen in T cell activation is discussed.     

Association of protein kinase C activation with IL 2 receptor expression

Tac antigen (as a measure of the IL 2 receptor) acquisition and regulation by IL 2, an antigen-receptor agonist (anti-T3), phorbol esters, and phytohemagglutinin (PHA) were studied. Phorbol esters stimulated de novo acquisition of Tac antigen, which was associated with the subcellular redistribution of protein kinase C (PK-C) from cytosol to particulate membranes of human T lymphocytes. PHA and anti-T3 (alpha-T3) antibody also stimulated a transient redistribution and activation of PK-C that reached a maximum within 20 min after stimulation. Both phorbol esters and alpha-T3 could increase Tac expression and stimulate PK-C translocation on 5 and 12 day activated T cells that were at the G0/G1 stage of the cell cycle due to IL 2 deprivation. Tac antigen-specific mRNA was seen in the nucleus within 2 hr after stimulation. In contrast, IL 2 alone could only increase Tac expression and stimulate PK-C translocation on day 5 but not day 12 activated T cells. IL 2 synergizes with alpha-T3 and phorbol ester for the regulation of Tac expression. Although IL 2 increased expression of Tac, the majority if not all of these receptors possessed low affinity for IL 2. These data suggest that the activation of PK-C is a common transmembrane signal shared by IL 2 and antigen stimulation. The results also imply that PK-C activation is necessary for the regulation of Tac antigen expression.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

TH(1)- and TH(2)-TYPE cytokine expression by activated t lymphocytes from the lung and spleen during the inflammatory response to respiratory syncytial virus

RSV is an important cause of lower respiratory tract illness in infants and the elderly worldwide. The components involved in immunity and those that contribute to inflammation of RSV-induced disease are not clearly understood. To address the relationship between activation antigen and cytokine expression, intracellular levels of IL-2, IL-4, IL-5 and IFN-gamma were determined for CD3, CD44, CD49d, CD54, CD62L and CD102 lymphocytes from the bronchoalveolar lavage and spleen. To examine activation at the DNA level, lymphocytes expressing IL-2, IL-4, IL-5 or IFN-gamma were analysed for G2+M DNA content or phosphatidylserine expression (apoptosis). Trafficking of lymphocytes to the BAL was detected at day 5 p.i., peaked day 7 p.i., and predominately involved CD54(+)and CD102(+)lymphocytes expressing high levels of IL-2, IL-4, IL-5 and IFN-gamma. Lymphocytes expressing CD44(+), CD49d(+)and CD62L(lo)were also observed, however they expressed these cytokines to a lesser extent. DNA analysis of lymphocytes expressing IL-2 or IFN-gamma revealed higher G2'M levels compared to lymphocytes expressing IL-4 or IL-5, suggesting greater activation of Th(1)-type lymphocytes in the lung. These data demonstrate that RSV-induced pulmonary inflammation involves extensive cellular activation and cytokine expression, particularly by CD54(+)and CD102(+)lymphocytes in the lung.     

Simultaneous measurement of transferrin receptor and DNA content of human IL 2 dependent T cells by flow cytometry

This paper describes a method which enables the simultaneous measurement of both the concentration of cell surface receptors and the DNA content of individual lymphoid cells. Cells fixed with PLP (periodate-lysine-paraformaldehyde) were treated with ribonuclease (RNase). Transferrin receptors were then successively bound with monoclonal antibody against them and FITC-labeled antibody against the monoclonal antibody. Cells thus treated were stained with propidium iodide and two-parameter flow cytometric analysis was carried out. Using this method, the expression of transferrin receptors on lymphoid cells was analyzed in relation to the action of T-cell growth factor (IL 2). It was found that cells in the G1 phase were stimulated by IL 2 which increased transferrin receptor concentration after a lag of a few hours. Subsequently, the cells entered the S phase and the receptor levels remained high throughout the S, G2 and M phases of the cell cycle.     

Vitamin D receptor expression in human lymphocytes. Signal requirements and characterization by western blots and DNA sequencing.

The signals controlling the expression of the receptor protein for 1 alpha,25-dihydroxyvitamin D3 in normal human lymphocytes and the relationship of this protein to the classical vitamin D receptor were examined. Lymphocytes activated with the OKT3 antibody to the T-cell antigen receptor expressed fewer binding sites as compared to lymphocytes that were activated by the polyclonal activator phytohemagglutinin (PHA). However, combination of OKT3 and phorbol myristate acetate produced a concentration of binding sites similar to the PHA-activated cells. The receptor from OKT3 and OKT3 + phorbol myristate acetate-activated lymphocytes exhibited decreased binding to DNA-cellulose compared to PHA-activated lymphocytes. In lymphocytes activated either by PHA or OKT3 (but not in resting cells), a 50-kDa species cross-reacting with a monoclonal antibody against the intestinal vitamin D receptor was detected. Finally, RNA from activated lymphocytes was amplified by polymerase chain reaction using oligonucleotide primers flanking the 196 base pair long region encoding the DNA-binding domain of the human intestinal receptor. The amplified product showed an identical nucleotide sequence to the DNA-binding domain of the human intestinal receptor. These findings suggest that expression of the 1,25-(OH)2D3 receptor in lymphocytes is triggered by distinct and contingent signals, and that the protein and the mRNA encoding it are identical to the classical vitamin D receptor.     

Regulation of the expression on mouse T lymphocytes of the epitope identified by monoclonal antibody 3A35

Monoclonal antibody 3A35 (MA 3A35) has previously been shown to be an activation marker of macrophages and T lymphocytes. It immunoprecipitated from macrophages a 200-kDa molecule belonging to the T200 family and from T cells a 85-kDa antigen. In the present work, the factors controlling the expression of the epitope identified by MA 3A35 on polyclonal activated T cells and T-cell clones, as well as the ability of 3A35 alone or together with complement to interfere with T-cell functions, were investigated. Corticoresistant thymocytes unreactive with MA 3A35 became fully reactive after 2 days of in vitro stimulation by PMA and IL-2 and the level of reactivity per cell declined to a low level thereafter. In helper and cytolytic T-cell clones, the expression of the epitope defined by MA 3A35 was also maximal soon after antigenic stimulation then declined. In helper-T-cell clones, the epitope remained detectable during the entire culture period, whereas in cytolytic clones its expression was markedly reduced at the end of the culture. The lineage of cytotoxic T lymphocytes (CTL) as studied in a bulk culture of spleen cells primed in vivo against a syngeneic tumor exhibited similar regulation by antigenic stimulation. The CTL precursors were resistant to lysis by MA 3A35 plus complement; after 3 days of culture with the stimulatory antigen, they became highly sensitive but their sensitivity then diminished and mature CTL were completely resistant. MA 3A35 plus complement also killed the activated T cells which responded to macrophage-presented antigens and were thought to be mainly Lyt-1+. Therefore, the epitope identified by MA 3A35 was expressed predominantly at an early stage of T-cell activation. At a late stage, it persisted almost exclusively on helper and Lyt-1+ cells. In addition, MA 3A35 plus complement lysed NK cells, AK cells, and their precursors present in normal spleen. In the absence of complement, MA 3A35 had no detectable effect on T-cell functions.     

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.     

Syngeneic monoclonal antimelanoma antibodies and their application for analysis of tumor antigens, gene cloning, and in vitro/in vivo diagnosis

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.