Ag-B-linked analogue of Ia antigens in the rat.

The reactions of Lewis rat lymphocyte membrane antigens with two alloantisera, BN anti-Lewis and BN anti-Fischer have been studied. Three lines of evidence indicated that these antisera reacted with cell surface antigens homologous to Ia antigens of the mouse. 1) After absorption with Lewis platelets, the antisera killed only 40 to 50% of Lewis spleen cells. The majority of such cells were shown to be Ig-positive B cells by the examination of reaction patterns on lymphocytes after separation on nylon wool into T cell- and B cell-enriched subpopulations. 2) SDS-PAGE analysis of solubilized Lewis spleen cell antigens precipitated with these antisera revealed that the platelet-absorbed antisera reacted with molecules comparable in size to mouse Ia antigens (mw approximately equals 35,000 and 28,000). The unabsorbed sera reacted with these molecules and with additional molecules corresponding in size to mouse K and D antigens (m.w. = 45,000). 3) Neither of these antisera killed significant numbers of spleen cells from the partially congenic strain F.BN (seventh backcross homozygotes), a Fischer rat to which the Ag-B.3 allele is being transferred by repetitive backcrossing, indicating that the genes coding for these Ia-like antigens in the rat are linked to the Ag-B locus.     

Accessory cell function of thoracic duct nonlymphoid cells, dendritic cells, and splenic adherent cells in the Brown-Norway rat

Thoracic duct lymph of lymphadenectomized Brown-Norway (BN) rats is highly enriched for nonlymphoid cells (NLC) which share several characteristics with splenic dendritic cells (DC), e.g., the binding of monoclonal antibody OX2. The accessory cell activity of NLC was analyzed by comparing these cells with DC and splenic adherent cells (SAC). In concanavalin A (Con A)-induced T-cell proliferation NLC, like DC, were very effective accessory cells at low cell numbers, as a consequence of an efficient induction of interleukin 2 (IL-2) production and IL-2 responsiveness. Responses in the presence of SAC were poor, even after the addition of excess IL-2. A fourfold enhancement of accessory cell activity of SAC was achieved by the depletion of FcR-positive cells, which were responsible for suppression of the Con A response. Low responsiveness of BN rats with respect to Lewis rats can in part be explained by a higher suppressive activity of macrophages in the BN rat.     

Membrane-bound and water-soluble nonclassical class I MHC antigens on rat placenta

We have used mouse monoclonal antibodies to different determinants on rat class I major histocompatibility complex (MHC) antgiens in order to identify water-soluble and membrane-bound nonclassical (i.e., non-RT1.A) class I MHC antigens on the spongiotrophoblast and labyrinthine trophoblast of rat placenta. Initial immunohistological studies with monoclonal antibodies reacting with determinant restricted to classical (RT1.A) rat class I antigens confirmed the presence of these antigens on spongiothrophoblast, but not on labyrinthine trphoblast. Staining with another monoclonal antibody, which reacts with both classical and at least some nonclassical rat class I antigens, gave strong staining of both the labyrinthine and spongiotrophoblast. To distinguish membrane-bound from water-soluble class I molecules, quantitative adsorption analyses were carried out using both placental cell membranes and ultracentrifuged aqueous extracts of placenta. The aqueous placental extract had no absorptive capacity for the RT1.A-specific antibodies, but it had very strong absorptive capacity for the more broadly reactive antibody. This strongly suggests the presence of large quantities of a soluble nonclassical class I MHC antigen in rat placenta. The placental cell membranes had four to fivefold greater absorptive capacity for the broadly reactive antibody when compared to the antibodies to classical class I antigens, a result that was consistent with the presence of membrane-bound non-classical class I antigens on rat placenta. The membrane-bound nonclassical class I antigen was purified from detergent extracts of DA rat placental membranes using monoclonal antibody affinity and lentil lectin affinity chromatography. The putative nonclassical class I antigen had a heavy chain of M _r 43 000, which is 2000 smaller than the amino acid sequence analysis demonstrated that the nonclassical placental antigen differed at three amino acid residues from the classical RT1.A class I molecule and also from the Q10-like class I molecule of the DA strain. It differed also from the pAR 1.5 cDNA sequence, the only full-length rat class I DNA sequence available so far. Address correspondence and offprint requests to: J. Fabre.     

Production of an anti-mouse MHC class II monoclonal antibody with biological activity in transgenic tobacco

To produce a monoclonal antibody specific to a mouse major histocompatibility complex (MHC) class II protein, we synthesized the complementary DNAs for the heavy and light chains of a monoclonal antibody by PCR amplification. These cDNAs were then introduced separately into tobacco plant cells. After performing Northern blot analysis to confirm the expression of each of the chain genes in the transformed plants, we constructed transgenic plants expressing both the heavy and light chains by sexual crossing. The expression of the heavy and light chain genes in the sexually crossed plant was confirmed by Northern and Western blot analyses, respectively. Fluorocytometric analysis showed that the plant-derived antibodies, which we purified using a protein G affinity column, bound specifically to target cells that expressed the cognate MHC class II molecules on their cell surfaces. The results of this study demonstrate that a monoclonal antibody against mouse MHC class II proteins can be expressed in transgenic plants. They also show the specific binding activity of plant-derived antibodies to cognate antigens.     

Accessory cell function of cells isolated from Mycobacterium leprae-induced granulomas

The large cells from Mycobacterium leprae-induced granulomas in guinea pig lymph nodes were separated by Percoll discontinuous density gradient centrifugation and on a fluorescence-activated cell sorter (FACS) using cross-reacting monoclonal antibody to human MHC Class II antigens. Large Percoll-separated cells (83% Class II antigen positive and 52% macrophage-specific antigen positive) and FACS-separated cells are able to act as antigen-presenting cells for T-cell proliferation to PPD. In previous studies, macrophage antigen-positive cells consistently failed to act as accessory cells. This indicates that there is a population of accessory cells which are macrophage antigen negative and MHC Class II antigen positive present in these M. leprae-induced granulomas.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. II. Monoclonal H-2-specific antibodies obtained from an unimmunized C57BL/KaLwRij (H-2b) mouse

A monoclonal 'natural' anti-H-2 IgM antibody produced by a hybridoma cell line OL-3.17 (H-2 m. 209) is described. The OL-3.17 monoclonal antibody was obtained by hybridization of spleen B cells from an unimmunized C57BL/Ka (H-2b) mouse in the serum of which simultaneously an IgM kappa paraprotein of high concentration and a natural H-2-specific antibody of high titer was detected. The monoclonal antibody OL-3.17 reacted strongly with H-2d and H-2s and weakly with H-2k,q,r lymphocytes, thereby detecting a hitherto unknown H-2 public determinant. The target molecules for OL-3.17 cocapped with class-I H-2 antigens, but immunoprecipitation of H-2 antigens was not achieved. This is the first monoclonal H-2-specific antibody obtained from a mouse without intentional immunization and, with high probability, was derived from a B-cell clone which produced natural H-2-specific antibodies detectable in the serum of the original mouse.     

Quantitative estimation of major histocompatibility complex antigens on live tumour cells

Quantitation of major histocompatibility complex (MHC) antigens on cells can accurately be done by using a flowcytometer. Since flowcytometer is not freely accessable an alternate, simple method for relative quantitation of MHC antigens has been devised. In this procedure, YAC lymphoma cells were first treated with a monoclonal anticlass I MHC antibody and then with a rabbit anti mouse Ig-antibody coupled to peroxidase, followed by colour development using a substrate of peroxidase enzyme. Various assay parameters have been optimized. The validity of the procedure was examined by assessing the enhanced MHC expression on YAC cells treated with a soluble rat spleen derived factor, by the new procedure as well as by the flowcytometer. Comparable results were obtained by using both techniques.     

Characterization of a CD4-positive T-cell line derived from an athymic (nu/nu) mouse.

We have isolated a Thy-1+, CD3+, CD4+ T-cell line from the spleen of a 12-week-old nu/nu (nude) BALB/c mouse. The cell line is clonal, and it expresses an alpha beta T-cell antigen receptor. Upon activation, these cells secrete IL-2 but not IL-4, putting them in the Th1 category. The cells can be triggered to proliferate and secrete lymphokines in the presence of irradiated syngeneic or allogeneic splenic feeder cells that express a variety of MHC haplotypes. This response is MHC class II-specific, because it can be blocked by either anti-Ia or anti-CD4 antibodies. From the response pattern of this T-cell line, we conclude that it recognizes a common determinant on class II MHC antigens. This nude mouse T-lymphocyte presumably has not undergone thymic selection. Therefore its unique specificity may reflect both the bias of T-cell antigen receptor genes for encoding receptors that recognize MHC molecules and the requirement for functional thymic epithelial cells for the efficient education of a self-MHC-restricted repertoire.     

Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans

Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses.     

Macrophage antigens associated with adhesion: identification by a monoclonal antibody specific for Lewis lung carcinoma cells

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11-12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.     

Analysis of Qa-2 antigen expression by preimplantation mouse embryos: possible relationship to the preimplantation-embryo-development (Ped) gene product

The preimplantation-embryo-development (Ped) gene, a gene that controls the cleavage rate of preimplantation mouse embryos, maps to the Qa-2 subregion of the mouse major histocompatibility complex (MHC). A highly sensitive enzyme-linked immunosorbent assay (ELISA) procedure was used to detect Qa-2 antigens on mouse embryos. The use of a monoclonal antibody specific for Qa-2 antigens showed that Qa-2 antigens were present on oocytes, 2-cell, 8-cell, and blastocyst-stage embryos, with the greatest expression found on blastocysts. Expression of Qa-2 antigens by the embryos correlated completely with Ped gene phenotype. Those embryos expressing the fast Ped allele showed the presence of Qa-2 antigens (Qa-2a mice), whereas those embryos expressing the slow Ped allele showed the absence of Qa-2 antigens (Qa-2b mice). It is hypothesized that the Qa-2 antigen may be the Ped gene product.     

Existence of MHC class I-restricted alloreactive CD4+ T cells reacting with peptide transporter-deficient cells

It is generally accepted that as the result of positive thymic selection, CD8-expressing T cells recognize peptide antigens presented in the context of MHC class I molecules and CD4-expressing T cells interact with peptide antigens presented by MHC class II molecules. Here we report the generation of TCRalpha/beta(+), CD3(+), CD4(+), CD8(-), MHC class I-restricted alloreactive T-cell clones which were induced using peripheral blood mononuclear cells from healthy individuals following in vitro stimulation with transporter associated with antigen processing (TAP)-deficient cell lines T2. The CD4(+) T-cell clones showed an HLA-A2.1-specific proliferative response against T2 cells which was inhibited by anti-CD3 and anti-CD4 monoclonal antibodies. These results suggest that interaction of the TCR with peptide-bound HLA class I molecules contributes to antigen-specific activation of these co-receptor-mismatched T-cell clones. Antigen recognition by alloreactive MHC class I-restricted CD4(+) T cells was inhibited by removing peptides bound to HLA molecules on T2 cells suggesting that the alloreactive CD4(+) T cells recognize peptides that bind in a TAP-independent manner to HLA-A2 molecules. The existence of such MHC class I-restricted CD4(+) T cells which can recognize HLA-A2 molecules in the absence of TAP function may provide a basis for the development of immunotherapy against TAP-deficient tumor variants which would be tolerant to immunosurveillance by conventional MHC class I-restricted cytotoxic lymphocytes.     

Molecular nature of the calcium-dependent cell-cell adhesion system in mouse teratocarcinoma and embryonic cells studied with a monoclonal antibody

The molecular nature of the Ca2+-dependent cell-cell adhesion system in mouse teratocarcinoma (t-CDS) was studied using a monoclonal antibody recognizing t-CDS. We isolated a hybridoma clone producing a monoclonal antibody (ECCD-1) able to disrupt cell-cell adhesion when added to monolayer cultures of teratocarcinoma cells. This antibody bound to the cells with intact t-CDS, resulting in an inhibition of their aggregation, but did not bind to cells from which t-CDS was removed by trypsin treatment in the absence of Ca2+. The binding of ECCD-1 to cell surfaces required Ca2+ but not other ions. Western blot analysis showed that ECCD-1 recognizes multiple cell surface proteins, the major one of which is a component with a molecular weight of 124,000. The binding of ECCD-1 to these antigens was Ca2+-dependent even in cell-free systems, suggesting that the molecules involved in t-CDS undergo conformational changes by binding with Ca2+, leading to conversion of their molecular structure into an active form. ECCD-1 also reacted with 8-cell stage mouse embryos and with certain types of epithelial cells (excluding fibroblastic cells) in various differentiated tissues collected from mouse fetuses, again affecting their cell-cell adhesion. We also showed that a monoclonal antibody (DE1) raised against gp84 (F. Hyafil et al., 1981, Cell 26, 447-454) recognizes the same antigens as ECCD-1.     

Analysis of nondominant idiotypes expressed during alloimmune responses

The antibody response of Lewis rats (RT1.A¹) to class I MHC antigens of the Brown Norway rat (RT1.Aⁿ) was studied. Diversity of the serum alloimmune response was analyzed using syngeneic anti-idiotype raised against monoclonal antibodies of the same specificity. Cross-reactive idiotypes were detected on approximately one in one thousand Lewis anti-RT1.Aⁿ serum antibodies, at concentrations ranging from 20 to 600 ng/ml. The kinetics of idiotype expression coincided with that of total anti-BN antibody production, suggesting that both were regulated by the same mechanism. To determine whether humoral anti-idiotype was involved in such regulation, sera from these animals were screened for anti-idiotype content. Using an RIA sensitive to 20 ng/ml, no humoral anti-idiotype could be detected during any phase of the alloimmune response.     

Detection of at least two distinct mouse I-E antigen molecules by the use of a monoclonal antibody

In an attempt to determine whether the expression of more than a single Ia antigen is determined by the I-E subregion of the mouse major histocompatibility complex (MHC), sequential immunoprecipitation analyses were performed by using a monoclonal antibody and alloantisera reactive with I-E subregion products. 3H-leucine-labeled glycoprotein preparations obtained from H-2d spleen cells were precleared with the monoclonal antibody 14-4-4S and then examined for residual Ia activity precipitable by an alloantiserum detected by SDS-polyacrylamide gel electrophoresis. Residual Ia activity was observed for all three strains of the H-2d haplotype tested. The residual Ia activity could be detected by sera absorbed with B10.A spleen cells, indicating that products of the I-E subregion rather than of the I-C subregion were responsible for this activity. No separable I-Ek molecules were detected in products of B10.A cells with the use of combinations of two monoclonal antibodies (including 14-4-4S) and several appropriate alloantisera. These findings indicate the presence of at least two similar but distinct I-E antigens encoded by the H-2d haplotype.     

A strong,preferential response of mice to polymorphic antigenic determinants of the chicken MHC,analyzed with mouse hybridoma (monoclonal) antibodies

CBA/J mice were immunized with B²/B² chicken RBC's (theB locus is the major histocompatibility complex (MHC) of the chicken). Spleen cells from these mice gave a much higher plaque-forming cell response when tested with RBC's bearing B² alloantigens, than with RBC's bearing any other MHC alloantigens. Similarly, immunization with chicken RBC's of other genotypes also produced responses that were highest when tested on the genotype used for immunization. Spleen cells from mice immunized 3 days previously with B²/B² RBC's were fused with mouse myeloma cells and hybrid clones which secreted anti-B² antibodies were selected. These monoclonal antibodies could be divided into three groups: (1) those which react with all genotypes of RBC's (panreactive); (2) those which react with only certain genotypes of RBC's and detect “public” MHC antigens; and (3) those which react with only B²/B² RBC's and detect “private” MHC antigens. Monoclonal antibodies which detect a private B² alloantigen were shown to be excellent typing reagents, as all birds of an outbred population which possessed the B² allele were easily detected using a simple one-step direct agglutination assay. No “false positives” were seen. The high and preferential response of the mouse to chicken MHC alloantigens suggested that mice might possess preexisting immunity to these antigens. In agreement with this hypothesis, normal mouse serum was found to have high titres of “natural” antibody against chicken MHC antigens.     

Production of Monoclonal Antibody to 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase from Bovine Cerebral White Matter

Lewis rats were immunized with partially purified 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) from bovine cerebral white matter and the spleen cells were fused with cell of a mouse myeloma cell line (SP-2). The production of monoclonal antibody was detected by enzyme-linked immunoadsorbent assay, immunohistochemical staining of bovine cerebrum, Western blotting analysis, and CNPase binding assay. Monoclonal antibody that specifically binds CNPase molecules was obtained. However, the antibody did not suppress the enzyme activity. Western blotting analysis demonstrated that the monoclonal antibody binds both CNa (Wla) and CNb (Wlb). The monoclonal antibody was identified as being of the IgG2c subclass. Immunohistochemical examination revealed that the myelin sheath in the CNS was heavily stained with the monoclonal antibody in several species (bovine, mouse, rat, and human). In contrast, peripheral nervous system myelin was not stained even in bovine tissue. These results suggest that the monoclonal antibody obtained in the present study specifically recognizes the CNPase molecules in the CNS.     

Use of tetrameric antibody complexes to stain cells for flow cytometry

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross-linked by two molecules of a monoclonal rat anti-mouse IgG1. The use of this immunological approach to cross-link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu-3a or Leu-2a antibodies and monoclonal antibodies specific for the fluorochromes B- and R-phycoerythrin. The ability of such cyclic immune complexes to stain T-cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double-staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell-surface antigens.     

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.     

A solubilized T-cell receptor from a human leukemia cell line binds to a ligand in the absence of MHC products

A human T cell antigen receptor from the acute lymphoblastoid leukemia line HPB-ALL (also called HPB-MLT) binds and is precipitated in detergent solubilized form by an antigen present on the surface and secreted by several strains of the gram-positive bacterium Staphylococcus aureus. This binding is completely independent of major histocompatibility complex (MHC) antigens. Receptor/ligand binding is unique to this one cell line (i. e., clonotypic) and furthermore completely blocked by an idiotype-specific monoclonal antibody (mAb) to this receptor, but not by three different nonidiotype-specific mAbs. The nature of this interaction appears more similar to immunoglobulin/antigen binding than to T-cell receptor/antigen/MHC/accessory molecule interactions and would suggest that some T-cell receptors may not require MHC products to interact with antigen.