A novel human leucocyte surface membrane antigen defined by murine monoclonal antibody

A murine monoclonal antibody has been produced which identifies a novel human leucocyte differentiation antigen. The antibody, designated WM-66, of IgM subclass, was cytolytic with human complement. WM-66 was shown to react with virtually all normal T and B lymphocytes from peripheral blood and lymphoid tissues, as well as blood monocytes and approximately 40% of bone marrow mononuclear cells. The antibody also bound to the majority of cases of chronic B-cell malignancies, including chronic lymphatic leukaemia and non-Hodgkin's lymphoma, but not to cases of acute leukaemia or to the majority of leukaemic and lymphoblastoid cell lines. WM-66 also reacted with epithelium of bronchus and salivary gland ducts. A single band of relative molecular mass 65,000 Daltons was immunoprecipitated from membrane extracts of normal lymphocytes and the B-cell line Daudi. Treatment of a number of WM-66-negative B-cell lines with neuraminidase resulted in WM-66 binding, indicating that the antigen exists in a covert form masked by sialic acid residues on a wider spectrum of cell types than was initially apparent. The reactivity pattern of WM-66 indicates that it recognises a previously undescribed surface membrane molecule with broad non-lineage-specific distribution on leucocytes. This has recently been confirmed at the Fourth International Workshop on Human Leucocyte Differentiation Antigens. Although the biological function of the molecule recognised by WM-66 is unknown, the lytic properties of the antibody suggest a possible in vivo therapeutic role as an immunosuppressant or for treatment of lymphoid malignancy.     

Mycoplasma hyorhinis GDL surface protein antigen p120 defined by monoclonal antibody.

Four antigens of Mycoplasma hyorhinis GDL were defined by murine monoclonal antibodies. Components of broth-grown mycoplasmas were separated under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent protein blots were stained with individual antibodies. Each antibody reacted with a distinct component with relative molecular weights of 120,000, 73,000, 51,000, and 38,000, respectively (termed p120, p73, p51, and p38). Trypsin treatment of protein blots specifically abrogated binding of antibodies, suggesting that the epitopes recognized were associated with proteins. By using indirect immunofluorescence and immunoferritin techniques, mycoplasmas colonizing the surface of chronically infected BW5147 murine T-lymphoblastoid cells were selectively stained with antibody to p120, indicating the localization of the corresponding epitope at the mycoplasma surface. Protein blots of mycoplasmas derived from BW5147 cell cultures were stained with antibody to p120, revealing a component identical to that observed with broth-grown organisms. These results establish the identity of a surface protein antigen of M. hyorhinis GDL expressed at the surface of organisms during their colonization of host cells.     

A rat cytotrophoblast antigen defined by a monoclonal antibody.

A monoclonal antibody that recognizes specifically a cytotrophoblast antigen was obtained. The monoclonal antibody 22H6 was tested on rat choriocarcinoma (in vivo, in vitro), normal placenta, ectoplacental cone, blastocysts, and several normal organs. The antigen was detected on frozen sections and on tissue culture by indirect immunofluorescence. The monoclonal antibody 22H6 reacts with the cytotrophoblasts of rat choriocarcinoma. The giant cells do not display a positive reaction. It is not expressed on other tumors than choriocarcinoma. In adult rats the only cells revealing a positive reaction are the hepatocytes and the epithelial cells lining the small intestine. In the pregnant rat, the antigen is expressed on the cytotrophoblasts of the junctional zone in the placenta, but not on the giant cells. The mab reacts only with the small trophoblast cells of the ectoplacental cone, but not with trophectoderm of blastocyst. The mab has an IgG2b isotype and is not cytotoxic for choriocarcinoma cells in a complement-dependent cytotoxicity test. The described monoclonal antibody is to our knowledge the only known marker of rat benign and malignant cytotrophoblast.     

Decreased specific CD8+ T cell cross-reactivity of antigen recognition following vaccination with Melan-A peptide

The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8(+) T cells following vaccination of HLA-A2(+) melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8(+) T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8(+) T cell clones. While Melan-A-reactive CD8(+) T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.     

A highly restricted antigen for renal cell carcinoma defined by a monoclonal antibody

Monoclonal antibodies against renal cell carcinoma (RCC) antigens were generated by immunizing Balb/c mice with the human RCC cell line 7860. After cloning many RCC reactive monoclonal antibodies, one antibody (D5D), was of special interest. Specificity testing against 33 human tumor cell lines revealed D5D to have no specific reactivity with 17 specimens of normal adult or fetal kidney. Reactivity with 67 specimens of nonrenal tissue demonstrated no reactivity. The reactivity of D5D with 15 different RCC tissues has been variable, being present in 60% of cases. Preliminary data, however, suggest that the antigen may be reexpressed for some of the remaining 40% during in vitro propagation.     

A human X-linked antigen defined by a monoclonal antibody

We have constructed hybrids between human thymocytes and the mouse thymoma BW5147. These hybrids, and others, have been used to show that the expression of a thymocyte antigen is controlled by an X-linked gene.     

A human thymocyte antigen defined by a hybrid myeloma monoclonal antibody.

Spleen cells from a BALB/c mouse that had been immunized with human thymocytes were fused with the myeloma line P3-NS 1/1 Ag 4.1. One of the resulting hybrid clones (NA 1/34) secreted an antibody that was highly specific for human thymocytes. Eighty-five % of thymocytes expressed the antigen designated HTA1. There were an estimated 15 x 10(4) molecules of HTA 1 per cell, and it is therefore a major surface molecule. The expression of this antigen on thymocytes appears to be reciprocal to HLA, as recognized by another monoclonal antibody W6/32. Immunoprecipitated material from [125I]-labeled thymocyte membranes was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate which disclosed a single component of 45,000 molecular weight.     

A 55,000 Mr surface antigen on activated human T lymphocytes defined by a monoclonal antibody

A T cell growth factor-dependent alloreactive human T cell line has been used to generate a monoclonal antibody B1.49.9 that reacts with an antigen present on most if not all mitogen or alloantigen activated T cells but not on resting T cells. The T lymphoblastoid cell line HUT-102 is also strongly reactive with B1.49.9 but all other T and non-T leukemia-lymphoma cell lines tested were negative. The B1.49.9 antigen is a glycoprotein of 55,000 Mr on mitogen or alloantigen activated T cells and 50,000 Mr on the cell line HUT-102. Pulse labeling experiments showed that a 40,000 Mr precursor (at approximately 0.7 h) which does not bind to ricin lectin precedes the appearance of the ricin-binding 55,000 Mr form. Comparisons of the monoclonal antibody anti-Tac, which recognizes the IL-2 receptor, to B1.49.9 suggest that B1.49.9 also recognizes a structure similar or identical to the IL-2 receptor.     

Characterization of an antigen defined by monoclonal antibody KMO1.

We have isolated an antigen defined by the monoclonal antibody KMO1 from tissue culture supernatant as a glycoprotein and from cancer cells as a glycolipid. The antigenic determinant was a carbohydrate. Antibody KMO1 inhibited the binding activity of 19-9 to CA19-9, but 19-9 antibody did not inhibited the binding activity of KMO1 to KMO1 antigen. The glycolipid antigen of KMO1 was a monosialoganglioside and was separated into 3 components on thin layer chromatography. The 19-9 antibody reacted with one of the components. These results suggest that the KMO1 antigen is similar to but not identical to CA19-9.     

识别胸腺髓质上皮细胞和胸腺树突状细胞的单链抗体的筛选

目的　利用噬菌体展示技术寻找胸腺基质细胞表面的新抗原。方法　用培养的胸腺基质细胞系MTDC作为靶抗原富集初级噬菌体抗体库 ,从经过富集后的次级抗体库中挑选克隆 ,制备单链抗体 ,并在冰冻切片及培养的细胞系上检测抗体的特异性。结果　从经 4轮富集的次级抗体库中挑选到一个新的克隆。用此克隆制备的单链抗体可同时辨认胸腺髓质上皮细胞亚群和胸腺树突状细胞亚群。结论　胸腺髓质上皮细胞和胸腺树突状细胞均具有异质性 ,同时噬菌体展示技术可以作为寻找细胞表面新分子的强有力工具。     

A novel human B-lymphocyte antigen shared with lymphoid dendritic cells: characterization by monoclonal antibody.

A novel cell-surface antigen (L25) expressed on human B cells was identified using a B cell-reactive monoclonal antibody (TB1-4D5). This L25 antigen was expressed on most B-lineage cells but not other cell types including thymocytes, T cells, granulocytes and monocytes. Thus, L25 existed on the majority of normal B cells present in the blood and lymphoid tissues, on cultured cell lines derived from normal and malignant B cells, and on neoplastic cells isolated from patients with B cell-derived malignancies. Though L25 was persistently expressed on B cells until 7 days after their activation with pokeweed mitogen (PWM), neither normal nor neoplastic plasma cells expressed L25. Moreover, L25 was present on cultured as well as freshly isolated leukaemic cells with common acute lymphatic leukaemia (CALL) antigen, which have been thought to correspond to the early B-cell ontogeny. Besides pan-B cell reactivity of TB1-4D5 antibody, it apparently cross-reacted with so-called dendritic or interdigitating cells located in the thymic-dependent areas of peripheral lymphoid organs, which have been presumably ascribed to those associated with accessory-cell function. Functional studies showed that anti-L25 (TB1-4D5) antibody had inhibitory effect on induction of immunoglobulin synthesis by PWM-stimulated B cells.     

Distribution of a guinea pig lymphocyte cell surface antigen of defined function, as detected by the mouse monoclonal antibody MSgp 1

A mouse anti-guinea pig monoclonal antibody, designated MSgp 1, was derived from a fusion between NS-1 myeloma cells and splenocytes hyperimmunised with guinea pig thymocytes. The MSgp 1 determinant is expressed by a subset of small thymocytes and lymph node T cells which participate in mixed leukocyte reactions. The determinant is modulated by antigen in vivo, and MSgp 1 antibody will prevent MHC class II driven proliferation in vitro. In addition, MSgp 1 reacts with a minor population of lymph node B cells, but not with a chronic B cell leukaemic cell line. Resident peritoneal macrophages express the MSgp 1 determinant, whereas chronic oil-induced peritoneal macrophages do not. The role of MSgp 1 defining guinea pig helper T cells is discussed by comparisons with other documented T helper cell reagents.     

A unique human B lymphocyte antigen defined by a monoclonal antibody

We produced a hybridoma designated 4G7 from a mouse immunized with chronic lymphocytic leukemia cells. The 4G7 hybridoma secretes an IgG1 antibody that is specific for normal and malignant B lymphocytes. Using dual color immunofluorescence staining, this antibody reacted with all immunoglobulin-positive cells but no T cells in normal peripheral blood. There was no detectable 4G7 antigen on monocytes, platelets, red cells, granulocytes, or phytohemagglutinin-activated T cells. When PBL were depleted of 4G7 positive cells and stimulated with pokeweed mitogen, secreted immunoglobulin levels fell to less than 10% of control values on Day 5 and less than 1% of control on Day 7. This antibody was reactive with 155 of 176 B lineage neoplasms on which it was screened. Thirty-five cases of myeloid or T-lymphoid malignancy were negative. Our studies show that the 4G7 antigen modulates in the presence of excess antibody. Free 4G7 antigen was not found circulating in human serum. The cell surface antigen identified by 4G7 was sensitive to pronase proteolysis but resistant to trypsin and chymotrypsin digestion. A comparison of 4G7 with other known B-cell antibodies indicates that the 4G7 antigen has not been previously identified. This antibody is of use for the identification of normal B lymphocytes, the study of B-cell differentiation, and the characterization of lymphoid malignancies.     

Uptake of antigen by afferent lymph dendritic cells mediated by antibody.

Dendritic cells isolated from sheep afferent lymph were examined for their ability to bind soluble protein and peptide antigens labeled with fluorescein both in in vitro assays and following intradermal injection of antigen in vivo. Analysis of dendritic cells by flow cytometry revealed weak direct binding of proteins and peptide antigens. However, the degree of uptake was greatly enhanced in the presence of specific antibody in vitro or if antigen was injected intradermally into antigen-primed sheep. About 60% of dendritic cells possessed the ability to take up antigen in both the in vitro and in vivo experiments. The uptake of antigen occurred very rapidly, reaching maximum values in terms of cell numbers and fluorescence intensity in less than 5 min in vitro and 20-40 min following in vivo challenge. Both sheep IgG subclasses could mediate this effect, but F(ab')2 fragments were ineffective. Procedures adopted to remove complement components from the in vitro test mixtures did not result in any reduction in the binding of antigen by dendritic cells. Two-color flow cytometry analysis of the dendritic cell population further showed that 43% of cells taking up the antigen/antibody complexes were CD1+, suggesting a relationship between these cells and Langerhans' cells or other dendritic cells in skin. The results, thus, indicate that approximately two thirds of sheep afferent lymph dendritic cells bind antigen/antibody complexes via an Fc receptor, a mechanism which could be important in the accentuated accessory function of these cells known to occur following secondary antigen challenge.     

Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4⁺ and CD8⁺ populations and assessed for the ability to process and present particulate antigen to CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ DCs both processed exogenous particulate antigen, but CD8⁺ DCs were much more efficient than CD4⁺ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8⁺ DCs, our studies also revealed an important contribution of CD24. CD8⁺ DCs were also more efficient than CD4⁺ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8⁺ T cells and interferon (IFN)-γ-producing CD4⁺ T cells. In summary, CD8⁺ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.     

Identification of a species-specific antigen in Legionella pneumophila by a monoclonal antibody.

A species-specific antigen in Legionella pneumophila was identified by a monoclonal antibody in enzyme-linked immunosorbent and immunofluorescence assays of serogroups 1 through 8. The species-specific antigen was heat stable, and the molecular weight of the major band was 29,000 by immunoblot analysis. In direct immunofluorescence assays, the antigen was cryptic or only partly exposed on the surface of the cells but was effectively exposed by treating the cells with detergent and EDTA. The monoclonal antibody was utilized in direct immunofluorescence assays to specifically identify multiple cultures of L. pneumophila serogroups.     

HBV-SAg特异性靶细胞系的构建

目的　研究特异性细胞免疫反应在病毒性肝炎发生发展中的作用及观察药物等对细胞免疫反应的影响。方法　用ＤＮＡ 重组技术构建逆转录病毒重组质粒ＰＬＸＳＮＳ，以电穿孔方法转入ＰＡ３１７ 细胞，筛选高表达克隆，收集其假病毒颗粒感染ＥＬ４ 细胞，经有限稀释选择高表达克隆。结果　重组质粒转染ＰＡ３１７ 细胞后，５３ 个克隆形成（１∶１０ 传代），在２４ 孔板中扩增后有７ 个克隆细胞上清ＨＢｓＡｇ 阳性。用ＨＢｓＡｇ Ａ值（曾称ＯＤ值） 最高克隆的假病毒感染ＥＬ４ 细胞，再经有限稀释得到稳定、高效表达ＨＢｓＡｇ 的靶细胞系（ＥＬ４Ｓ），在体外传１００ 代以上，ＨＢｓＡｇ 仍能高效表达（４８ 小时上清中ＨＢｓＡｇ 的Ａ值达０－８５）。经检测ＰＬＸＳＮＳ及重组腺病毒ｒＡｄＢ７２Ｓ免疫后小鼠对ＨＢｓＡｇ 特异的细胞免疫反应，得到较为满意的结果。结论　我们成功构建了稳定表达ＨＢｓＡｇ 的靶细胞，对研究ＨＢｓＡｇ 细胞免疫反应及乙肝免疫发病机理等方面有重要意义     

Development of olfactory nerve glia defined by a monoclonal antibody specific for Schwann cells.

Although there is considerable interest in the possible role of olfactory glia in the pathfinding abilities of olfactory nerve axons, the complete development of these glia in vivo has not been described. Using a specific Schwann cell marker, the 1E8 antibody, we have found that olfactory nerve glia can be identified throughout development. These glia appear to originate in the olfactory placode and migrate initially into the periphery of the olfactory nerve, and later into the center of the nerve. Olfactory nerve glia enter the presumptive olfactory bulb with the olfactory receptor neuron axons and distribute themselves along the edge of the olfactory nerve layer. The fact that olfactory nerve glia are specifically immunostained by the 1E8 monoclonal antibody, which recognizes the Schwann cell-specific protein P0, suggests that these cells more closely resemble Schwann cells than astrocytes or enteric glia. These results support and extend previous findings suggesting that olfactory nerve glia have distinctive developmental and anatomical features which may be important to the regenerative capacity of the olfactory system.     

A novel lymphocyte surface antigen recognized by the monoclonal antibody HIS45

A hybridoma producing the monoclonal antibody HIS45 was isolated from a fusion between spleen cells from a Balb/c mouse immunized with rat bone marrow cells and the fusion partner SP2/0. This antibody recognizes a determinant present on the majority of peripheral T and B cells and a small percentage of thymocytes. In a xenogeneic mixed leucocyte reaction HIS45 completely inhibits the proliferation of responding cells. HIS45 does not inhibit natural killer cell-mediated lysis. Comparison with other antibodies which have been reported to effect lymphocyte function fail to reveal any which have similar properties.