T-cell surface antigens defined by monoclonal antibodies, involved in the induction of human interferon-gamma and interleukin 2

Monoclonal antibodies specific for human T-cell-differentiation antigens were used to investigate the mechanism of induction of interferon-gamma (IFN-gamma) and interleukin 2 (IL-2). High levels of IFN-gamma, accompanied by IL-2 production, were detected in the lymphocyte cultures stimulated by pan T monoclonal antibodies that were mitogenic. These antibodies recognize an antigen complex Tp 19-29 (a complex of T-cell proteins of 19-29 kDa). However, it was possible to induce IL-2 without concomitant production of IFN-gamma using some antibodies specific for other T-cell surface antigens, e.g., Tp 32-45, Tp 41, and Tp 100-120. These antibodies were not mitogenic. The production of lymphokines, therefore, appears to be regulated at the cell surface by receptors or interaction molecules involved in cell triggering. Binding of antibodies to T3 receptor was obligatory for both IFN-gamma induction and mitogenesis but was not required in the induction of IL-2 activity.     

Storage and reconstruction of hybridomas producing monoclonal antibodies against viral antigens

The conditions were optimized for freezing storage, restoring and further cultivation of hybridoma cells producing antibodies to viral antigens. The effect of density of cellular suspension frozen,concentration of calf embryo serum in cryoprotected medium and mild conditions of the restoring of hybridoma were studied. To restore deeply frozen hybridoma 24 hole plastic panels with a layer of feeding cells of the HEPES and insulin containing medium were used. The fulfilling of these requirements makes possible restoration of intact antibody-producing hybridoma from 10(2)-10(3) frozen cells.     

Human monoclonal antibodies against surface antigens of Langerhans cells from lymphocytes of diabetics transformed by Epstein-Barr virus

Human monoclonal antibody against islet cell surface antigens was generated from a pre-diabetic patient's peripheral blood lymphocytes transformed with Epstein-Barr virus. Reactivity of these transformed lymphocytes was evaluated using indirect immunofluorescence on rat islet cell suspensions and frozen sections of human pancreas. Several lymphoblastoid cell lines that react with islet cell surface were obtained. Preliminary immunoblots with enriched rat islet cell membrane antigens suggest a reactivity toward a 64 kdalton antigen.     

Analyses of epididymal sperm surface antigens by monoclonal antibodies against hamster spermatozoa

BALB/c mice were immunized with spermatozoa from cauda epididymides of hamsters and the immune spleen cells were fused with mouse myeloma cells (P3U1). Seven hybridomas (GHS-1,-2,-3,-4,-5,-6, and -7) that produced monoclonal antibodies (Mabs) binding to the epididymal spermatozoa were established. Three Mabs (GHS-3,-4, and -6) were IgM and the other four were IgG1. All Mabs reacted to hamster spermatozoa from cauda epididymides but none of the Mabs except GHS-5 and -7 reacted to spermatozoa in testis. GHS-5 and -7 Mabs bound to the acrosome region of spermatozoa in both testis and epididymis. The antigens corresponding to GHS-2, -4, and -6 Mabs appeared to be excreted from epithelial cells of caput epididymis, while those to GHS-1 and -3 Mabs seemed to be produced in cauda epididymis. Both groups of the antigens bound to the surface of spermatozoa during their epididymal transit. Immunoblotting analyses of epididymal fluid showed that the antigen epitopes corresponding to GHS-1,-2,-3,-4, and -6 Mabs were distributed to multiple components with different molecular weights ranging from over 100 to 25 kd. The distribution patterns of the epitopes corresponding to GHS-1 and -3 Mabs and GHS-2,-4, and -6 Mabs were very similar, respectively, but each group pattern was quite different from each other. GHS-5 Mab reacted to a component of sperm extract with a molecular weight of around 94 kd, while GHS-7 Mab failed to recognize any components transblotted.     

Effects of monoclonal antibodies directed against murine T lymphocyte cell surface antigens on lymphokine production by cloned T lymphocytes reactive with class I MHC or Mls alloantigens

Monoclonal antibodies recognizing murine T lymphocyte cell surface structures implicated in T lymphocyte-mediated cytolysis, including Lyt-2, L3T4, LFA-1, and a cytolytic T lymphocyte (CTL) clonotypic determinant, were used as probes to investigate the role of these structures in lymphokine production by T cell clones induced by antigen or lectin. The clone-specific antibody 384.5 bound to and inhibited antigen-induced lymphokine production by the L3 CTL clone, but did not affect lymphokine production by other T cell clones. Antibodies against the T cell surface structures Lyt-2 or L3T4, which are expressed by mutually exclusive T cell subsets, inhibited antigen-induced lymphokine production by class I major histocompatibility complex (MHC) antigen-reactive CTL clones or an M1s-reactive helper T lymphocyte (HTL) clone, respectively. Antibody against the broadly distributed LFA-1 molecule inhibited antigen-induced lymphokine production by all of the clones tested. Lectin-induced lymphokine production by cloned T cells was not inhibited by the clonotypic antibody, anti-Lyt-2, or anti-LFA-1; slight inhibition of the HTL clone was observed with the anti-L3T4 antibody. None of these structures appear to be uniquely involved with a particular functional response. Our results suggest that each of these structures is involved with the interactions between the effector cell and the stimulating cell leading to lymphokine production.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.     

Immunochemical characterization of two antigens recognized by new monoclonal antibodies against human colon carcinoma

Two different monoclonal antibodies (MAb), called L-D1 and L-C5, were produced after immunization with either intact cells or the methanol phase of glycolipid extracts, respectively, from the same human colon carcinoma line, LoVo. As determined by an antibody-binding radioimmunoassay (RIA) on intact cells, MAb L-D1 and MAb L-C5 were highly reactive with all five colon carcinoma lines tested and with only one out of the 21 cell lines of various tissue origin tested. No reactivity of either MAb was observed with peripheral blood lymphocytes, granulocytes, or erythrocytes from healthy donors of various blood groups. Both MAb were tested in competitive binding experiments with an anti-CEA MAb from our laboratory (CEA 35) and with two previously described anti-colon carcinoma MAb from the Wistar Institute called 1083-17-1A (17-1A) and NS-19.9. In competitive binding experiments, MAb L-D1 was inhibited by MAb 17-1A and reciprocally, whereas MAb L-C5 was not inhibited by any of the other MAb tested. MAb L-D1 precipitated a major protein band with an apparent molecular weight (MW) of 41 kilodaltons (kD); interestingly, MAb 17-1A, which was reported to react with an uncharacterized antigen, precipitated the same protein band of 41 kD. This was confirmed with immunodepletion experiments. Furthermore, after treatment of the colon carcinoma cell line with tunicamycin, both MAb L-D1 and 17-1A precipitated a protein band of 35 kD. This shift of 6 kD suggests that the glycoprotein recognized by these 2 MAb contains two to three N-linked carbohydrate side chains. MAb L-C5 precipitated a group of three to four protein bands ranging from 43 to 53 kD that were not modified by tunicamycin treatment. A preliminary study conducted by using immunoperoxidase labeling on frozen sections of primary colon carcinoma showed that the two new MAb react strongly with these tumors, but also weakly with the normal adjacent mucosa, as did the other anti-colon carcinoma MAb tested.     

The detection of two surface antigens on human lung macrophages using monoclonal antibodies

Monoclonal antibodies (McAb), designated AMH1 (IgM, lambda) and AMH2 (IgG1, Kappa), against specific surface antigens of human lung macrophages were produced by the fusion of the NS-1 plasmacytoma cell line with spleen cells from BALB/c mice immunized with bronchoalveolar lavaged (BAL) cells obtained from selected smoking subjects. The screening and characterization of these McAb were carried out employing cellular radioimmunoassay, flow cytofluorography, and immunohistochemical methods. These two antibodies specifically reacted with macrophages in the alveolar spaces and BAL fluids. AMH1 did not react with peripheral blood cells including freshly separated monocytes, cultured monocytes, lymphocytes, granulocytes, and platelets. In addition, AMH1 did not react with peritoneal exudate cells or pleural exudate cells. On the other hand AMH2 showed the dull-positive reaction with some monocytes and pleural exudate cells among above-mentioned cells. These two McAb seemed to detect cell surface antigens that are expressed by highly differentiated or mature macrophages compared to OKM1. These antibodies will allow not only better characterization of immune cells but also assessment of maturity of lung macrophages.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

Localisation on human chromosome 19 of three genes for cell surface antigens defined by monoclonal antibodies

Expression of three distinct human cell surface antigens defined by monoclonal antibodies (mAbs) was examined in a series of rodent-human somatic cell hybrids retaining different subsets of human chromosomes. Cell surface reactivity with mAbs F8 and G253, detecting a 95 kilodalton (kD) glycoprotein (gp95); with mAbs F10 and A103, detecting a 50 kD glycoprotein (gp50); and with mAb S7 was found to cosegregate with human chromosome 19. However, differential antigen expression was observed with hybrids containing fragments of the 19 and hybrids constructed with different human cell types. Comparison of results from the serological typing with the presence of a number of chromosome 19 DNA markers in hybrid cells and cytogenetic analysis suggests that MSK20, the gene coding for the F10/A103 antigen gp50, is located in chromosome region 19pter----19p13.2. The genes coding for the F8/G253 antigen, gp95 (gene symbol MSK19) and the S7 antigen (MSK37) are located in region 19p13.2----19q13.2. Thus, the cell surface antigens described in this study may be used as selectable markers for specific portions of human chromosome 19.     

Distinct reactivities of four monoclonal antibodies with human interleukin 2 receptor

Two new murine monoclonal IgG1 antibodies, H-31 and H-A26, were characterized in comparison with two previously obtained monoclonal antibodies against human interleukin 2 (IL-2) receptor (IL-2 R), anti-Tac and HIEI. In immunofluorescence assays with various human hematopoietic cells, H-31 and H-A26 antibodies both reacted with only IL-2 R-positive cells, and they precipitated IL-2 R molecules, glycoproteins with molecular weights of 60K and 53K daltons (gp60/gp53), from human T-cell leukemia virus type I (HTLV-I)-carrying MT-2 cells, as demonstrated by sequential immunoprecipitation after absorption of IL-2 R with anti-Tac. Antibody-binding competition assays showed that H-31 and anti-Tac, and H-A26 and HIEI, respectively, competed reciprocally in binding to the cells, and that anti-Tac also inhibited the binding of HIEI but not vice versa. H-31, like anti-Tac, strongly inhibited the IL-2-dependent proliferation of normal activated T-cells, absorption of IL-2 and direct binding of IL-2 to the cells, while H-A26, like HIEI, inhibited those processes only weakly. The spectra of reactivities of these antibodies with various simian cell lines derived by HTLV-I infection were different, as revealed by immunofluorescence studies. Human IL-2 R was shown to express a unique antigenic determinant, detected with HIEI, that was not detectable in IL-2 R molecules of Old and New World monkeys, and also to express determinants common to simian IL-2 R molecules. These observations indicate that H-31 and H-A26 recognize human IL-2 R molecules and that the antigenic sites on the IL-2 R molecule defined by H-31, H-A26, anti-Tac, and HIEI are different.     

The human repertoire of antibody specificities against Thomsen-Friedenreich and Tn-carcinoma-associated antigens as defined by human monoclonal antibodies

Summary Human monoclonal antibodies specific for tumour-associated Thomsen-Friedenreich (TF) [Gal(1–3)GalNAc()-O-] and Tn [GalNAc()-O-] glycoproteins were prepared using peripheral blood lymphocytes from healthy blood donors. The B lymphocytes were either directly transformed with Epstein-Barr virus (EBV) or transformed after an in vitro stimulation period with synthetic glycoproteins. The EBV-transformed lymphocytes were subsequently fused with a mouse-human heteromyeloma to secure antibody production and stability. IgM antibodies exhibiting different patterns of specificity for synthetic TF and Tn antigens were obtained, including antibodies specific for the and forms of different Gal(1–3)GalNAc-O- and GalNAc-O- conjugates and antibodies agglutinating neuraminidase-treated erythrocytes. Several of the human monoclonal antibodies showed an increased binding to cultured carcinoma cells as compared to melanoma cells. This straightforward approach for the production of human monoclonal antibodies demonstrates the possibility of investigating the reactivity pattern of tumour-binding antibodies from peripheral blood lymphocytes. The binding patterns of these monoclonal antibodies show that healthy donors carry different fine specificities against synthetic TF/Tn antigens and that these antibodies react with different tumour cells.     

抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。     

Phylogenic distribution of human renal antigens defined by monoclonal antibodies

The preparation fo five monoclonal antibodies specific of important human renal histologic structures both functionally and organogenetically has permitted to identify the repartition of the corresponding antigens in the vertebrate phylum. For three of them, appeared a clear cut histologic identity in intensity and localization between the mammals studied and man. For the two others a phylogenic and histologic dispersion was observed. It may be supposed, in the latter case, that the evolution and the biotope have acted in different manners on renal function and organogenesis according to the vertebrate classes or species investigated.     

Induction of human T lymphocyte motility by interleukin 2

Interleukin 2 (IL 2) is known to have multiple immunoenhancing activities that are related to its ability to promote the proliferation and the expression of effector functions of human T lymphocytes. We investigated the potential of IL 2 to induce human T lymphocyte migration. Unstimulated T cells did not respond to IL 2, but T cells exposed to dextran or phytohemagglutinin did respond to IL 2 concentrations from 0.01 to 10.0 U/ml, with significantly increased migration. This activity could be specifically blocked with anti-Tac antibody. Analysis of T lymphocyte subsets revealed that OKT4+ but not OKT8+ lymphocytes responded to IL 2 in the chemotaxis assay. Checkerboard analysis demonstrated that the IL 2-induced chemoattractant activity was predominantly chemotactic rather than chemokinetic in nature. The activity of IL 2 was compared with that of another chemoattractant lymphokine, lymphocyte chemoattractant factor, which was found to stimulate lymphocyte migration without prior exposure to mitogen, and which was not inhibited by anti-Tac. Our data suggest that the lymphocyte migratory response to IL 2 is under the control of the inducible receptor recognized by anti-Tac in a manner similar to the proliferative response to IL 2, but differs from proliferation in its OKT4+ cell specificity.     

Stimulation of T lymphocyte proliferation by monoclonal antibodies against GD3 ganglioside

Cell-surface gangliosides are presumed to play a role in cell growth and differentiation. With the use of monoclonal antibodies directed against GD3, a disialoganglioside expressed predominantly by cells of neuroectodermal origin, we have found that GD3 is expressed by a subpopulation of cells of the immune system including: 1) fetal thymocytes in subcortical regions and near vessels, 2) lymph node lymphocytes in interfollicular areas and near vessels, and 3) a small subset of T cells in the peripheral blood. Mouse monoclonal antibodies (two IgGs, one IgM, and F(ab')2 fragments) reacting with GD3 were found to stimulate proliferation of T cells derived from peripheral blood. Proliferation of T cells was observed even in cultures depleted of macrophages, suggesting that activation by anti-GD3 was not dependent on the presence of accessory cells. T cell proliferation was maximum between days 5 and 7 of stimulation and was preceded by expression of interleukin 2 receptors. No stimulation was observed with control antibodies of the identical isotype or with monoclonal antibodies recognizing the gangliosides GD2 or GM2. During stimulation by anti-GD3 monoclonal antibodies, there was an expansion of the GD3+ pool of T cells, but depletion of GD3+ T cells prior to stimulation abrogated the response. Proliferation induced by binding to GD3 could be augmented by exogenous interleukin 2 and phytohemagglutinin. Anti-CD3 (T3) monoclonal antibodies had little or no effect. These results demonstrate that binding to GD3 on the surface of T cells can elicit signals for T cell proliferation.     

Production of human antitetanus antibodies by hybridomas

Peripheral blood lymphocytes from healthy people recently immunized against tetanus toxoid (TT) were fused with human malignant B-cell lines or mouse myeloma cells (X63 Ag8.653) in an attempt to establish stable B cell hybridomas secreting anti TT antibodies. Human-human fusion experiments were not successful. In contrast, the five heterospecific fusion experiments yielded between 60 and 100% of wells that contained growing hybrids. Five of these hybrids repeatedly secreted anti-TT antibodies. One of the hybrids was cloned and secreted 10-20 micrograms/ml of human IgM, lambda anti-TT antibody. Heterospecific hybridization thus appears as an interesting method to obtain human monoclonal antibodies, allowing the study of their properties.