Monoclonal antibodies as probes for differentiation and tumor-associated antigens: a Forssman specificity on teratocarcinoma stem cells

A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel. One of these antibodies was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested, including differentiated derivatives of teratocarcinomas. In the adult mouse, this antigen is expressed by subpopulations of cells in the spleen, bone marrow, lymph node, brain, kidney and testes, although not in liver and thymus. This antigen has a species and tissue distribution consistent with that of Forssman antigen. The molecules which carry this specificity on the embryonal carcinoma cells appear to be glycolipids.     

Murine T lymphoma cells express a novel membrane-associated antigen with unique features

A novel membrane-associated antigen expressed on various murine T lymphoma cells has been detected by a rat monoclonal antibody. The antibody YE6/6 initially produced against Moloney leukemia virus-transformed T lymphoma line MBL-2, reacted with several other lymphoma lines including non-T lymphoma lines as well as thymocytes from leukemic AKR mice, but it did not show significant reactivities with resting or mitogen-activated normal lymphocytes by flow cytometric analysis. The antibody did not bind to some Abelson leukemia-transformed cells, which express Moloney virus antigens, suggesting that the antigen is unlikely to be encoded by Moloney virus genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the antigen molecules immunoprecipitated by the antibody revealed three major polypeptides. Two of the polypeptides, with approximate m.w. of 95,000 and 35,000, can be labeled by the cell surface iodination and, therefore, seem to be exposed on the cell surface. The third polypeptide, with approximate m.w. of 65,000, is not labeled by the surface iodination but it is readily detected by [35S]methionine labeling. The third polypeptide was labeled with [32P]orthophosphate indicating that it is a phosphoprotein. Western blot analysis showed that YE6/6 antibody primarily reacts with 35,000 m.w. polypeptide. Furthermore, the same 35,000 m.w. protein was also detected in concanavalin A-activated spleen cells at a low level by Western blot, but normal resting lymphocytes were negative. These results suggest that the antigen detected by YE6/6 antibody may be a cell proliferation-associated antigen and its expression is highly elevated on transformed lymphoma cells as compared to normal mitogen-activated lymphocytes.     

Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane.

To analyze cell surface murine leukemia virus gag protein expression, we have prepared monoclonal antibodies against the spontaneous AKR T lymphoma KKT-2. One of these antibodies, 43-13, detects an AKR-specific viral p12 determinant. A second monoclonal antibody, 43-17, detects a novel murine leukemia virus-related antigen found on glycosylated gag polyproteins (gp95gag, gp85gag, and gp55gag) on the surface of cells infected with and producing ecotropic endogenous viruses, but does not detect antigens within these virions. The 43-17 antibody immunoprecipitates the precursor of the cell surface gag protein whether in its glycosylated or unglycosylated state, but does not detect the cytoplasmic precursor of the virion gag proteins (Pr65gag). Based on these findings, we have localized the 43-17 determinant to the unique amino-terminal part of the glycosylated gag polyprotein (the L domain). We have determined that gp95gag contains L-p15-p12-p30-p10 determinants, whereas gp85gag lacks the carboxyterminal p10 determinant, and gp55gag lacks both p30 and p10 carboxy terminal determinants. Analysis of cell surface gag expression with the 43-17 antibody leads us to propose that the L domain plays a crucial role in (i) the insertion and orientation of murine leukemia virus gag polyproteins in the cell membrane and (ii) the relative abundance of expression of AKR leukemia virus versus Moloney murine leukemia virus glycosylated gag polyproteins in infected cells.     

Tumor-specific antigen of murine T-lymphoma defined with monoclonal antibody

A panel of hybridomas was constructed by fusion of P3X63Ag8 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with a C57BL/Ka x-ray-induced lymphoma, C6XL. One of forty-three hybridomas secreting antibodies reactive with the tumor cells was found to be unreactive with normal spleen cells in a radioimmunometric assay. This antibody, designated 124-40, was unreactive with normal adult thymus, spleen, lymph node, or bone marrow cells, or with fetal spleen or thymus cells in radioimmunometric or radioimmunoprecipitation assays. Flow microfluorometric analysis of these nonmalignant lymphoid cells failed to reveal subpopulations reactive with MAb 124-40. The antibody was highly specific for the lymphoma cells used for immunization and did not react with a panel of other spontaneous or x-ray-induced or chemically induced lymphomas. The antigen reactive with MAb 124-40 was isolated by radioimmunoprecipitation and found to be a glycoprotein composed of disulfide-bonded subunits of 39,000 m.w. and 41,000 m.w. A cell surface component of similar structure, but not reactive with MAb 124-40 could be detected by two-dimensional electrophoresis in extracts of purified T cells, but not B cells. These results suggest that the apparently individually specific lymphoma antigen reactive with MAb 124-40 might be a clonally expressed epitope carried by a T cell surface component.     

TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins.

A murine monoclonal antibody was identified by its ability to induce a reversible antiproliferative effect on a human lymphoma cell line. Immunoprecipitation studies revealed that the antibody reacted with a 26-kilodalton cell surface protein (TAPA-1). A diverse group of human cell lines, including hematolymphoid, neuroectodermal, and mesenchymal cells, expressed the TAPA-1 protein. Many of the lymphoid cell lines, in particular those derived from large cell lymphomas, were susceptible to the antiproliferative effects of the antibody. TAPA-1 may therefore play an important role in the regulation of lymphoma cell growth. A cDNA clone coding for TAPA-1 was isolated by using the monoclonal antibody to screen an expression library in COS cells. Analysis of the deduced amino acid sequence indicated that the protein is highly hydrophobic and that it contains four putative transmembrane domains and a potential N-myristoylation site. TAPA-1 showed strong homology with the CD37 leukocyte antigen and with the ME491 melanoma-associated antigen, both of which have been implicated in the regulation of cell growth.     

Characterization of SJL T cell clones responsive to syngeneic lymphoma (RCS): RCS-specific clones are stimulated by activated B cells

To gain insight into the nature of the syngeneic T cell-stimulating molecules on SJL lymphoma cells (RCS), a panel of eight Ly-1+2- T cell clones that are specific for transplantable RCS has been generated. All of these clones proliferate vigorously in response to two independent RCS lines and to LPS-activated syngeneic or F1 B cell blasts, but not to unstimulated SJL spleen cells or to allogeneic B cell blasts. Only one RCS-specific clone displays a proliferative response to (SJL X BALB/c) resting spleen cells, suggesting that I-E molecules are not the source of stimulation of RCS-responsive cells. Responses of the T cell clones to both RCS and syngeneic LPS-activated B cells are inhibited by monoclonal antibodies to I-A antigens, and not by antibody to I-E antigens. These findings suggest that RCS-responsive T cells are stimulated either by syngeneic I-As alone, in a form expressed on activated B cells, or by I-As in combination with X, where X is a cell surface antigen present on B cells at certain stages of differentiation.     

Analysis of the murine sperm surface with monoclonal antibodies

Hybridomas secreting monoclonal antibodies reactive with murine spermatozoa were produced by fusion of myeloma cells with spleen cells from C57BL/6J mice immunized with spermatozoa from mice of the same strain. All antisperm antibodies were of the mu (mu) immunoglobulin heavy chain class; only one (MS-1) bound S. aureus protein A. Antibody MS-1 recognized an antigen present on the sperm acrosomal cap, on the surface of cells from liver and kidney and from some cultured cell lines. The subunit molecular weight (69000) of the polypeptide reactive with MS-1 was determined by SDS-PAGE analysis of sperm membrane proteins followed by their electrophoretic transfer to nitrocellulose.     

Monoclonal antibody analysis of p53 expression in normal and transformed cells.

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.     

Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8

Human herpesvirus 8 (HHV-8; also designated Kaposi's sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA-glutathione S-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.     

Inhibition of mixed lymphocyte response by a rat monoclonal antibody to a novel murine lymphocyte activation antigen (MALA-2)

A novel surface antigen expressed on activated and proliferating murine lymphocytes has been identified by a rat monoclonal antibody. The antigen, termed MALA-2, is also expressed on various lymphoid cell lines, but it is absent or present at very low densities on the majorities of unstimulated thymocytes and lymph node cells. Some cells in normal spleen and bone marrow seem to express the antigen at relatively high densities and they may represent proliferating cells in these tissues. MALA-2 has an apparent m.w. of 95,000 to 100,000 under both reducing and nonreducing conditions. The monoclonal antibody YN1/1.7 that reacts with this antigen partially inhibits Con A stimulation of spleen cells, but its inhibition of LPA stimulation is negligible. Furthermore, the antibody profoundly inhibits MLR. The inhibition of MLR by YN1/1.7 antibody is comparable to that caused by anti-transferrin receptor. The time course study suggests that the antibody may directly inhibit proliferating cell populations in MLR.     

Multiple occurrence of spontaneous AKR/J lymphomas with T and B cell characteristics.

Three Thy 1.1-positive and surface IgM-positive (Thy 1+, SIg+) AKR/J lymphoma lines are described. These doubly marked tumors arose spontaneously in the peripheral lymphoid organs of 14- to 16-month-old AKR/J mice that either had spontaneous thymus atrophy or had been thymectomized at 1 month of age. All lines bore surface Thy 1.1, Ly,Ig(micron-chain) and Fc receptor (FcR), detectable by immunofluorescence. Immune response region (Iak) antigen was present on the two lines tested. Persistence of Thy 1.1 antigen and SIg after long-term tissue culture provided evidence that these markers were not passively acquired. One of these tumor lines, AkTB-1 always grows in lymph nodes as Thy 1.1-positive,SIg-negative tumors cells, whereas tumor cells growing in the spleen are initially Thy 1.1 positive, SIg negative, but they rapidly acquire SIg, FcR, and 1a between 18 and 21 days of passage.     

Anti-schistosome monoclonal antibodies of different isotypes--correlation with cytotoxicity

Five monoclonal antibodies specific towards Schistosoma mansoni antigens were prepared by fusion of spleen cells of infected and immunized mouse with the murine myeloma NS-1 cells. Three of the five antibodies belonged to the IgG1 class, one was an IgM and the fifth one was an IgE. The IgE monoclonal antibody designated 54.10, induced antigen-specific degranulation of rat basophilic cell line, a property which served as the basis for the screening assay. Its biological function was demonstrated by a specific macrophage activation that led to killing of schistosomula; no such killing was obtained with anti-schistosome antibodies of other classes or with IgE of different antigenic specificity. The second monoclonal antibody of biological significance was an IgG1, designated 27.21 which is reactive in the immunofluorescence staining of surface antigens on intact schistosomula. All three monoclonal antibodies that belonged to the IgG1 class were effective in mediating killing of schistosomula by complement, with the highest effect exerted by 27.21. It is thus apparent that the 27.21 monoclonal antibody is directed against a densely distributed surface antigen on the schistosomula membrane which is possibly involved in the protective immunity. Preliminary data showed that immunoprecipitation with the 27.21 antibodies results in the isolation of three major protein bands, of 60 kd, 50 kd, 19 kd, respectively.     

Fetomodulin: marker surface protein of fetal development which is modulatable by cyclic AMP

A novel cell surface marker of fetal development was identified in both in vivo and in vitro systems of the mouse using monoclonal antibodies against a glycoprotein of an apparent size of 133,000 Da. Two independent clones of hybridomas were isolated by fusing murine myeloma cells, NS-1, with spleen cells of a rat which was immunized with murine 3T3 fibroblast. The analysis of molecular size and tryptic peptides of the immunoprecipitate indicated that fibroblast and putative parietal endoderm cells, which were derived by induced differentiation of F9 embryonal carcinoma cells with retinoic acid and cyclic AMP, expressed apparently the same protein. Undifferentiated F9 cells and F9 cells which were treated with retinoic acid or cyclic AMP alone had little or no immunoprecipitable proteins. Analogously, parietal endoderm of in vivo embryos tested positive for this protein but visceral endoderm and embryonic ectoderm did not. The amount of this surface protein was increased in fibroblast and differentiated F9 cells by elevation of intracellular cyclic AMP concentrations. These results are consonant with a hypothesis that this surface protein plays a role in fetal development via a quantitative modulation by cyclic AMP.     

Monoclonal antibodies to hamster class II MHC molecules distinguish T and B cells

Monoclonal antibodies were prepared against cell surface antigens present on Syrian hamster lymphocytes and a hamster B cell lymphoma line, GD-36. One of these antibodies, S11, precipitated glycoproteins of 29,000 and 39,000 m.w. These glycoproteins were shown to be identical to or a subset of la-like glycoproteins precipitated by hamster alloantisera; however, molecules identified by S11 differed from the predominant hamster la homologues detected with a cross-reactive monoclonal antibody to murine la.7. The immunofluorescence pattern of both anti-la reagents, S11 and anti-la.7, on hamster lymphoid cells is similar by fluorescence-activated cell sorter analysis. A subpopulation of spleen and lymph node cells stains brightly with these antibodies. By two-color fluorescence, this peripheral lymphocyte subpopulation, identified with monoclonal anti-hamster la, also bears surface immunoglobulin (IgM). These data strongly suggest that hamster resting peripheral B cells, and not T cells, express la antigens and can be identified and isolated differentially by using this marker.     

Pathogenesis of murine cytomegalovirus infection: identification of infected cells in the spleen during acute and latent infections.

Spleen cells which replicate murine cytomegalovirus (MCMV) during acute infection in vivo were identified by electron microscopy and combined immunocytochemical staining and in situ cytohybridization. Most infected cells, as defined by in situ hybridization for viral RNA with MCMV-specific probes, were shown to be positive for factor VIII-related antigen and negative for Ia, Thy-1, and F4/80 antigens. Electron microscopic ultrastructural observations indicated that the infected cells in the spleen are predominantly sinusoidal-lining cells. We also studied reactivation of MCMV from latently infected mice by cocultivation of spleen cells with mouse embryo fibroblasts. Virus was only recovered from cells in preparations of stromal (or reticular) fragments, and not from spleen cell suspensions. Neither removal of immunoglobulin-bearing cells from the stromal fragments by panning nor depletion of Thy-1- and Ia-bearing stromal cells by treatment with monoclonal antibodies and complement reduced the frequency of reactivation of MCMV. These data suggest that T lymphocytes, mature B lymphocytes, and other Ia-bearing cells are not predominant reservoirs of latent MCMV.     

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.     

Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family.

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.     

Medullary epithelial cell lines from murine thymus constitutively secrete IL-1 and hematopoietic growth factors and express class II antigens in response to recombinant interferon-gamma

In this report, we describe the generation of two cloned epithelial cell lines, TE-71 and TE-75, from murine thymus. These cell lines resemble medullary thymic epithelium by a number of criteria, including reactivity with the monoclonal antibodies A2B5 and ER-TR5, the fucose-specific lectin derived from Ulex europeus, and the expression of keratins normally expressed by medullary thymic epithelial cells in situ. Constitutive Class II antigen expression by these cells is not detectable at the light or electron microscopic level or with flow cytometry. Following exposure to recombinant interferon-gamma or supernatants from mitogen-stimulated spleen cells, expression of Class II antigens by these thymic epithelial cell lines is increased, although less than the levels expressed by spleen cells. Medium conditioned by TE-71 and TE-75 cells exhibited colony-stimulating activity for bone marrow cells. In addition, TE-71-conditioned medium exhibited IL-1-like activity which could be neutralized with anti-IL-1 antibodies.     

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.