T cell activation by monoclonal antibodies bound to tumor cells by a cell surface displayed single-chain antibody.

Tumor cells often lack the costimulatory molecules necessary for T cell activation. However, the transformation of cells with more than one stimulatory molecule is a difficult procedure. We therefore developed a retroviral vector for the expression of a cell membrane anchored single-chain antibody fragment (scFv) directed against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). Proteins and peptides can be readily modified with this hapten, thus, enabling them to be bound to cells with the cell surface displayed anti-phOx scFv. To test combinations of surface-bound stimulatory molecules on T cell activation, SK-Mel63 human melanoma cells expressing the membrane anchored anti-phOx scFv were incubated with phOx-labeled mAbs against CD3, CD28 and CD5. Cells presenting a given mixture of modified anti-CD3 and anti-CD28 molecules stimulated T cell activation better than any single antibody and a given mixture of anti-CD3, anti-CD28 and anti-CD5 provided a stimulatory response higher than the best double combination. However, the relative concentrations are very important and must be carefully chosen. Concentrations of antibodies giving good T cell responses when used alone can block synergistic effects.     

Stimulation of interleukin-3 dependent cell growth by specific antibody.

We are attempting to raise antibodies against interleukin-3 receptors by immunizing mice with one cloned line of IL-3 dependent cells. In syngeneic mice immunized with a basophil cell line AC-2, serum contains stimulatory activity preferential for the immunizing cells but largely inactive against an alternative IL-3 dependent line of different phenotype. The serum activity corresponds to a fraction containing a cell agglutinin. Supernatants from hybridomas constructed from immune spleen cells raised by direct spleen immunization also contain binding antibodies, of which a select number show phenotype-restricted proliferative activity. We suggest that such antibodies may be recognizing receptors for IL-3, and that phenotypically different IL-3 dependent cells may express different membrane epitopes concerned with interacting with IL-3.     

Immunoreactivity to a monoclonal antibody (OS-3) is shared by osteoclasts and bicarbonate-secreting cells

OS-3, a monoclonal antibody raised against macrophagic cells derived from cultured rat glomeruli, reacts with the plasma membrane of various bicarbonate-secreting cells such as epithelial cells of the pancreatic excretory duct and type B intercalated cells of the kidney, suggesting that the antigenic molecule of OS-3 is involved in bicarbonate production and/or secretion. Since osteoclasts must vigorously extrude bicarbonate to maintain cytoplasmic pH in a physiologic range during proton secretion, we examined the localization of OS-3 immunoreactivity in the bone tissue to determine the involvement of the detected molecule in the transmembrane transport of bicarbonate in osteoclasts. The OS-3 selectively stained the basolateral plasma membrane of osteoclasts. Ultrastructurally, the immunoreactivity with OS-3 was associated with small cytoplasmic projections and microplicae of the basolateral plasma membrane. This finding suggests that osteoclasts express the molecule common to bicarbonate-secreting cells to utilize it for bicarbonate transport during bone resorption.     

The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface.

We generated monoclonal antibody (mAb) DCGM4 by immunization with human dendritic cells (DC) from CD34+ progenitors cultured with granulocyte-macrophage colony-stimulating factor and TNF-alpha. mAb DCGM4 was selected for its reactivity with a cell surface epitope present only on a subset of DC. Reactivity was strongly enhanced by the Langerhans cell (LC) differentiation factor TGF-beta and down-regulated by CD40 ligation. mAb DCGM4 selectively stained LC, hence we propose that the antigen be termed Langerin. mAb DCGM4 also stained intracytoplasmically, but neither colocalized with MHC class II nor with lysosomal LAMP-1 markers. Notably, mAb DCGM4 was rapidly internalized at 37 degrees C, but did not gain access to MHC class II compartments. Finally, Langerin was immunoprecipitated as a 40-kDa protein with a pI of 5.2 - 5.5. mAb DCGM4 will be useful to further characterize Langerin, an LC-restricted molecule involved in routing of cell surface material in immature DC.     

Tritium (3H) radiolabeling of protein A and antibody to high specific activity: application to cell surface antigen radioimmunoassays.

Staphylococcal protein A and several different immunoglobulins have been radiolabeled to high specific activities (greater than 10(6) cpm/microgram) by reductive methylation with tritiated (3H) sodium borohydride. The proteins retain excellent functional and antigenic properties. The utility of these reagents in a variety of assays for cell surface antigens is illustrated. The results indicate that this radiolabeling procedure may become the method of choice for many cell surface and solution immunoassays.     

Metastatic dissemination of 3LL variants after treatment with monoclonal antibody to a tumor-associated antigen

Two tumor lines derived from 3LL (Lewis lung carcinoma) endowed with different metastatic potential and stable for their metastatic phenotype during serial in vivo passages, have been analysed for growth and dissemination following treatment with a monoclonal antibody. We have used a recently developed MoAb 135-13C to a tumor-associated antigen of murine lung carcinoma having an apparent molecular weight of 180000 (TSP-180). The metastatic dissemination of the 3LL variants before and after treatment with the MoAb has been correlated with the expression on the cell surface of the MHC antigens (Db, Kb) and of the TSP-180 protein. The results of this study indicate that cell with high TSP-180 protein expression and MHC antigen expression have the greatest metastatic potential. Administration of MoAb 135-13C to tumor-bearing mice or i.v. injection of cells preincubated with the MoAb 135-13C increase the dissemination capacity of the variant endowed with lower metastatic potential while inducing a reverse effect on the high metastatic one. Studies on the MHC expression demonstrate that MoAb 135-13C treatment induces changes in the D^b and K^b expression at level of secondary neoplasms. The results are discussed in view of the importance of the use of the metastatic variants to study therapeutic effect of specific targeting agent.     

In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In vitro immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections. In this paper, a method for the detection of the labeling index of S-phase cells in normal and neoplastic tissues with in vitro BrdU labeling and standard immunohistochemical techniques using anti-BrdU-MAb and avidin-biotin peroxidase complex is described. We have employed this method in 47 human solid tumor samples, including squamous cell carcinomas of head and neck and cervix uteri, adenocarcinomas and malignant lymphomas, and also evaluated the possible application of the BrdU labeling index to estimate the cycling S-phase cells in neoplastic cell populations. In our data, the in vitro labeling index varied greatly in an individual case (3.56-29.2%) and from an area to an area within the same case. Squamous cell carcinomas of the head and neck showed higher LI than those of the cervix uteri. A case of metastatic carcinoma to the lung from ductal carcinoma of the breast had the highest LI (29.2%), in contrast to the low LI (3.6%) in the primary ductal carcinoma of breast.     

Murine monoclonal anti-idiotype antibody (alpha) as a probe to detect human monoclonal antibody bound to human tumor tissues

A new immunohistochemical assay was developed for the detection of human monoclonal antibody (HuMAb) bound to human biopsied tumor tissues. A murine anti-idiotype monoclonal antibody, alpha type, 18C6 (IgGl), was raised against an IgM HuMAb, L612, defining a tumor-associated ganglioside antigen (GM3) and used as a probe in a three step cell-binding assay (HuMAb + anti-id + biotinylated anti-mouse Ig). Anti-id 18C6 has an exclusive binding specificity for HuMAb L612, but does not interfere with the binding of L612 to antigen positive melanoma cell lines or to a purified antigen, GM3. The applicability of 18C6 in the three step cell-binding assay was tested first using a melanoma cell line, UCLASO-M12. L612 bound to M12 cells was specifically detected by 18C6 without any background reactivity in ELISA. When this assay was compared with the standard two-step cell-binding assay (HuMAb + peroxidase-conjugated anti-human IgM) using various cultured tumor cell lines, parallel reactivity was observed. The three-step cell-binding assay was then applied to various fresh-frozen human tumor sections. Positive reactivity was demonstrated on various histologic types of human tumor tissues: primary melanoma (10/10), metastatic melanoma (4/4), nevus (10/10), lung cancer (3/6), breast cancer (2/6), and colon cancer (1/1). Adjacent normal tissues were unstained. Control experiments included the cell-binding assay with L612 alone, 18C6 alone. L612 + unrelated mouse IgG, and unrelated IgM HuMAb (L72) + 18C6; but biotinylated anti-mouse IgG did not react with these control preparations. The results indicate that anti-id 18C6 is a highly specific probe to assess the expression of the ganglioside antigenic epitope recognized by the L612 HuMAb on biopsied human tumor tissues.     

A cell surface monoclonal antibody (H366) helps to discriminate human cytotoxic from suppressor T cells.

A study has been undertaken to differentiate T cytotoxic (Tc) and T suppressor (Ts) cell subsets using a monoclonal antibody termed H366 (mouse IgG2b) previously reported to phenotype natural killer and killer (NK/K) cells. Mononuclear cell suspensions from 14 normal subjects were depleted of H366+ cells by means of complement dependent cytotoxicity and the remaining cells were phenotyped with CD8 and CD4 monoclonal antibodies. The effects of depletion with H366 plus complement (C1) on the induction and activity of suppressor and cytotoxic T cells was also examined. The results indicate that H366 antibody recognizes in addition to NK/K cells, a population of Tc but not Ts or helper cells. Therefore, H366 antibody can be useful for obtaining Ts enriched lymphocyte subpopulations and this property may also be used for the enumeration of suppressor cells in the peripheral blood in disease states.     

Distribution of cells labelled by a monoclonal antibody (A3) against a cloned cell line derived from a rat malignant fibrous histiocytoma

To pursue the histogenesis of malignant fibrous histiocytoma (MFH), of which the cell of origin is still debated, a monoclonal antibody (A3) was produced against a rat MFH-derived cloned cell line (MT-8). Antigen recognized by A3 was around 80 kDa in molecular weight and was seen on the cytoplasmic membrane of MT-8 cells by immunoelectron microscopy. A3 reacted specifically with MT-8 cells, with another rat MFH-derived cell line (MT-9) and with their induced tumours in syngeneic rats, but not with other rat tumours such as fibrosarcoma, histiocytic sarcoma, malignant meningioma, uterine leiomyosarcoma, endometrial stromal sarcoma, mononuclear cell leukaemia and malignant schwannoma. These findings indicate that A3 has a high specificity for rat MFH cells. In fetuses on gestation days 15, 18 and 20 and in postnatal rats aged 1, 4 and 8 days, A3 reacted with primitive mesenchymal cells in visceral organs and around arteries and bronchi, as well as in the lamina propria of intestinal mucosa, renal interstitium, meninges and perineurium. There were no A3-positive connective tissue cells in organs or other sites in adult rats more than 10 weeks old. It is therefore likely that MFH cells share antigens with primitive mesenchymal cells, which may be multipotent for mesenchymal differentiation. The present study suggests that MFH consists of a population of primitive, undifferentiated mesenchymal cells. A3 also immunolabelled endothelial cells of arteries, venules and pulmonary capillaries in fetal, postnatal and adult rats; vascular endothelial cells in chemically induced hepatic and renal lesions also reacted strongly with A3. However, the significance of endothelial immunoreactivity with A3 remains to be elucidated.     

Detection of Thy-1 on cell surface of human T lymphoid cell lines by a monoclonal antibody

A mouse IgG2b(kappa) monoclonal antibody (MAb) HB-2S-1 against human brain Thy-1 was secreted by a hybridoma clone after fusion of mouse myeloma cells with spleen cells from a mouse that went through a prolonged immunization procedure before fusion. When tested against isolated human Thy-1 by the enzyme-linked immunosorbent assay (ELISA), MAb HB-2S-1 in culture supernatant showed a titer of over 100,000, and a titer of over 10 million in ascites of a mouse injected with the hybrid clone. By immunoblotting, this antibody was found to bind a doublet of protein bands of approximately 25,000 daltons among all proteins solubilized by deoxycholate (DOC) from membrane of human brain cells. When tested on human lymphoid cell lines by immunofluorescence, MAb HB-2S-1 strongly stained four T lymphoma cell lines, C91-Pl, HUT-102, HUT-78, and C5-MJ; and weakly two leukemia cell lines, MOLT-3 and Jurkat(clone E6-1). It did not stain a third T leukemia cell line, CCRF-CEM; a human B cell line, Raji; a plasmacytoma cell line, HMy2; or a myelomonocytic cell line, HL-60. Peripheral blood lymphocytes from ten normal human adults and the viable T cells isolated from another normal individual were also negative.     

Protection against candidiasis by an immunoglobulin G3 (IgG3) monoclonal antibody specific for the same mannotriose as an IgM protective antibody

We previously reported that a liposome-mannan vaccine (L-mann) of Candida albicans induces production of mouse antibodies that protect against disseminated candidiasis and vaginal infection. Immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1, specific for a C. albicans cell surface beta-1,2-mannotriose, protects mice against both infections. Another IgM MAb, termed B6, which is specific for a different cell surface mannan epitope, does not protect against disseminated candidiasis. The B6.1 epitope is displayed homogeneously over the entire cell surface, compared to a patchy distribution of the B6 epitope. To determine if protection is restricted to an IgM class of antibody, we tested an IgG antibody. MAb C3.1 was obtained from L-mann-immunized mice. By results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, capture enzyme-linked immunosorbent assay, and immunodiffusion tests, MAb C3.1 is an IgG3 isotype. By epitope inhibition assays, we determined that MAb C3.1 is specific for same mannotriose as MAb B6. 1. As expected by the results of the inhibition assays, immunofluorescence microscopy showed that the C3.1 epitope is distributed on the yeast cell surface in a pattern identical to that of the B6.1 epitope. Kidney CFU and mean survival times of infected mice pretreated with MAb C3.1 indicated that the antibody enhanced resistance of mice against disseminated candidiasis. Mice in pseudoestrus that were given MAb C3.1 prior to vaginal infection developed fewer vaginal Candida CFU than control animals that received buffered saline instead of the antibody. The finding that an IgG3 antibody is protective is consistent with our hypothesis that epitope specificity and complement activation are related to the ability of an antibody to protect against candidiasis.     

Production and characterization of a monoclonal antibody specific to the human 70-kDa heat shock protein

Heat shock protein 70 (hsp 70) plays major roles in apoptosis prevention and thermotolerance as well as molecular chaperoning. It is also expressed on the surface of human tumor cells, but not on normal cells, suggesting that hsp70 may be some tumor-associated antigen. To investigate the diverse functions of the protein species, various types of transgenic mice or cell models overexpressing human hsp70 have been made. In these models a monoclonal antibody (MAb) specific for the human hsp70 is highly desirable to distinguish the human from the endogenous mouse hsp70. It proved difficult to make this species-specific MAb, because the hsp70 homologues are members of a family of highly conserved, abundant, and ubiquitous proteins expressed in organisms ranging from bacteria to humans. In the present study, we prepared four MAbs against human hsp70. Three, HD 5, HD 7 and HD 11, recognize human and mouse hsp70. One, though, HD 8, recognizes human hsp70, but not mouse hsp70. By Western blot analysis of hsp70 deletion mutants, the epitope of the HD 8 MAb was determined as the 585-616 amino acid region of the human hsp70, a region with relatively low homology to mouse hsp70.     

The effect of skin allograft survival of a monoclonal antibody specific for a polymorphic CD3-like cell surface molecule in rhesus monkeys

FN18, a monoclonal antibody specific for the polymorphic rhesus monkey CD3 antigen on peripheral T cells, has been tested for its immunosuppressive effect in a rhesus monkey skin graft model. Animals were injected i.v. daily with antibody and they received an allogeneic skin graft two or three days after the initial antibody treatment. All animals were carefully monitored regarding levels of the major lymphocyte subsets. In animals in which RhT3 (a CD3-like antigen) is demonstrable (i.e. FN18+ phenotype), all T cells initially disappeared from the circulation. However, T cells without RhT3 on their surface reappeared after several days, indicating that these cells must have been modulated. The survival times of the skin grafts were significantly prolonged in these animals. In monkeys in which RhT3 is not demonstrable (i.e. FN18- phenotype), mainly part of the CD4+ lymphocyte subset was depleted. Although less explicit, skin graft survival was significantly prolonged in these animals as well. In both the FN18+ and FN18- groups a difference in sensitivity between the CD4+ and CD8+ cells for the FN18 antibody could be noticed. From the data it appears that FN18 is immunosuppressive and does not have serious side effects. The data are in agreement with information available for the use of OKT3 in clinical immunosuppression. Thus, extrapolation of data from the rhesus monkey model to the clinical situation seems feasible.     

High reactivity of monoclonal antibody (TO73) with human malignant tumor cell line

The biological characteristics d a new monoclonal antibody (TO73) reacting with a vincristine-resistant human leukemic cell line (KY-VCR) were, evaluated. Immunological and electron-immunological studies showed that TO73 reacted with the surface glycoprotein of KY-VCR. TO73 was found to have no effect on cell growth and intracellular uptake of vincristine. In human neoplastic cell ilnes TO73 was found to react with 11 of 27 (41%) cell lines. With regard to de novo primary tumor with one exception, TO73 did not react with any of the examined primary tumor cells. The patient with TO73-positive leukemia died of induction failure due to drug resistance. Complete remission was achieved in the other leukemic patients. These results indicate that TO73 antigen may be associated with immortalization of tumor cells and poor prognosis in some cases.     

广谱抗革兰氏阴性菌单克隆抗体3A8识别表位的初步研究

目的:利用噬菌体肽库技术筛选抗广谱革兰氏阴性菌与LPS单克隆抗体3A8的识别表位,以获得来源于不同LPS的保守表位.方法:用广谱抗G-菌、LPS的单克隆抗体3A8为靶,筛选噬菌体环七肽库,蚴鉴定阳性噬菌体克隆并测序.根据阳性保守序列合成短肽A2及A5,并与KLH载体交联,ELISA鉴定A2-KLH、A5-KLH与3A8单抗的结合.结果:随机挑选的33个噬菌体克隆中有15个可特异性与3A8结合,该结合可被LPS2630所抑制.根据阳性克隆DNA序列推导氨基酸序列,共有7种序列,均以疏水氨基酸为主,且包含保守序列Ser Pro Pro/Pro X Pro;选择所获阳性序列经设计与改造合成延长的两个环肽;经ELISA鉴定表明这些环肽可特异地与3A8结合.结论:得到可被3A8单抗特异性识别并含保守残基Ser Pro Pro/ProX Pro的阳性序列,据此合成的短肽可特异性结合3A8,提示该短肽可能模拟LPs共同表位的抗原性,有望成为疫苗候选表位.     

Homogenous expression of killer cell immunoglobulin-like receptors (KIR) on polyclonal natural killer cells detected by a monoclonal antibody to KIR2D

The activity of human natural killer (NK) cells is in part regulated by the expression of killer cell immunoglobulin (Ig)-like receptors (KIR) that recognize major histocompatibility complex (MHC) class I and can inhibit NK cell cytotoxicity. A monoclonal anti-KIR antibody was established and designated Lig1. Lig1 was shown to be specific for KIR in cell-surface staining and to react with all KIR2D, except KIR2DL4 which lacks a D1 domain, but not with KIR3D molecules in an enzyme-linked immunoadsorbent assay (ELISA) and Western blotting. Unlike other anti-KIR antibodies, Lig1 did not inhibit binding of KIR-Ig-fusion proteins to MHC-class I expressing cells nor did it interfere with KIR-mediated inhibition of NK cell cytotoxicity in a functional assay. Lig1 reacted with all NK cells in polyclonal NK populations from different donors, demonstrating that all NK cells express at least one KIR2D receptor.     

Characterization of monoclonal antibody specific to the Z39Ig protein, a member of immunoglobulin superfamily

Z39Ig, a recently identified immunoglobulin (Ig) superfamily member, is localized in the pericentromeric region of human chromosome X and detectable in all human tissue, but it is predominantly expressed in fetal human tissues as well as in adult lungs and placenta. In the present study, we generated a monoclonal antibody against Z39Ig protein to investigate the immunological role of Z39Ig protein on various immune cells. The anti-Z39Ig mAb that we generated specifically bound to Z39Ig protein on human promonocytic THP-1 cells, monocytes isolated from human peripheral blood mononuclear cells (PBMC) and mature CD14(+) dendritic cells (DC) differentiated from umbilical-cord blood CD34(+) hematopoietic progenitor cells. In addition, a signal through the Z39Ig protein induced an obvious cell surface expression of HLA-DR on THP-1 cells mediated by MHC class II transactivator (CIITA). These data suggest that the Z39Ig protein might be a critical molecule to regulate an immune response mediated by phagocytosis and/or antigen presentation.     

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 human B cell differentiation antigen selectively expressed on mature B cells identified by a monoclonal antibody (HB-2)

A monoclonal IgM antibody (HB-2), produced against a membrane antigen on the Raji, B cell line, reacted by indirect immunofluorescence with 2 to 40% of lymphoblasts from the B cell lines, Raji, Daudi, SN-1036, and SB but not with other types of cell lines, including pre-B, myeloid, melanoma, or T cells. HB-2 antibody reacted with 10 ± 3% of normal blood mononuclear cells, and was unreactive with monocytes, granulocytes, platelets, or erythrocytes. Two-color immunofluorescence revealed that HB-2 antigen expression was confined to cells bearing surface Ig. An interesting finding was the fact that 25% of plasmablasts induced by pokeweed mitogen also expressed the HB-2 antigen. However, pre-B and plasma cells from normal bone marrow did not express the HB-2 antigen either on their membrane surface or in their cytoplasm. Analysis of 85 leukemias revealed that the HB-2 antigen was expressed on acute and chronic B cell leukemias and Burkitt's lymphomas, but not on malignacies of pre-B, T, myelocytic, or plasma cells. The results suggest that expression of the HB-2 antigen is confined to mature B cells.