Identification of a novel T cell surface disulfide-bonded dimer distinct from the alpha/beta antigen receptor

Hybridomas were prepared from the spleen of a BALB/c mouse immunized with EL-4 T lymphoma cells. One, designated A1, was found to secrete a monoclonal antibody that reacted with two T lymphoma cells of C57BL origin, EL-4 and C6VLB, but not with normal C57BL/6 splenocytes or thymocytes, C57BL/6 T cell clones, or other T or B lymphomas by complement-mediated cytotoxicity or indirect immunofluorescent staining. Monoclonal antibody (MAb) A1 precipitated a protein that migrated at 85 kD under nonreducing and 43 kD under reducing conditions. The fact that the antigen defined by MAb A1 was a disulfide-linked dimer, together with the essentially clone-specific distribution of the reactive epitope, raised the possibility that the antibody defined an epitope of the antigen receptor. However, several additional observations revealed that the antibody defined a distinct and novel T cell surface structure. MAb 124-40, previously shown to react with the antigen receptor of C6VLB cells, reacted with variants of C6VLB that failed to express the A1 epitope. Sequential immunoprecipitation indicated that MAb A1 and MAb 124-40 reacted with distinct molecular species on C6VLB cells. Endoglycosidase digestion showed that the structure reactive with MAb A1 was not derived from that reactive with MAb 124-40 by addition of N-linked oligosaccharide residues. Two-dimensional gel electrophoretic analysis of precipitates obtained from radioiodinated C6VLB cells with MAb 124-40 resolved the alpha and beta subunits of the antigen receptor. Similar analysis of precipitates obtained with MAb A1 revealed only a single basic chain under reducing conditions, although anomalous mobility suggestive of a second, more acidic chain was observed under nonreducing conditions. Two-dimensional maps of tyrosine-containing chymotryptic peptides of the proteins isolated with MAb A1 and MAb 124-40 were completely different, suggesting that the molecules shared no peptides and were distinct in primary structure. Finally, cross-linking studies performed with a cleavable reagent indicated that the A1 molecule, unlike the antigen receptor defined with MAb 124-40, was not associated with additional, T3-like structures on the surface of C6VLB cells. Although the MAb A1 was unreactive with normal cells in cytotoxicity or staining assays, a molecule of the appropriate size was immunoprecipitated in small amounts from lysates of radioiodinated normal spleen and thymus cells. These data indicate that MAb A1 defines a novel disulfide-linked T cell surface molecule distinct from the antigen receptor.     

Immunophenotyping of Mesothelial Cells and Carcinoma Cells With Monoclonal Antibodies to Cytokeratins, Vimentin, Cea and Ema Improves the Cytodiagnosis of Serous Effusions

This paper presents an immunocytochemical study performed on cytocentrifuged deposits from 109 peritoneal and pleural effusions including 20 transudates, 43 malignant metastatic effusions and 46 effusions containing atypical cells, unidentifiable as reactive mesothelial or malignant epithelial cells on the classical morphological criteria. A panel of four monoclonal antibodies (MAb) was used, including KL1 directed to cytokeratins (KER), V9 to vimentin (VIM), NEO 723 to carcinoembryonic antigen (CEA) and E29 to epithelial membrane antigen (EMA). In most transudates the reactive mesothelial cells coexpressed VIM and KER with a ring-like pattern for the latter proteins. In contrast, they were unreactive to anti-CEA and weakly and inconsistently reactive to anti-EMA. In malignant effusions, most carcinoma cells coexpressed EMA, CEA and KER with a predominant diffuse cytoplasmic pattern for the latter. Only a few malignant epithelial cells from five metastatic adenocarcinomas weakly expressed VIM. When used on the 46 effusions with unidentifiable cells, the panel of MAb allowed reactive mesothelial cells and malignant epithelial cells to be distinguished from each other in 39 of 46 cases (85%).     

1,25-Dihydroxyvitamin D3 inhibits antigen-induced T cell activation

The proliferative response of murine spleen and thymus cells to antigen but not to lectin was inhibited by the active metabolite of vitamin D3, 1,25-(OH)2D3. To directly examine the effect of 1,25-(OH)2D3 on T cell activation in the absence of other complicating interactions, we utilized a panel of cloned Ia-restricted T cell hybridomas that secrete IL 2 on activation by cloned Ia-bearing stimulator cells (TA3) or when stimulated by mitogen. Physiologic concentrations of 1,25-(OH)2D3 (0.01 to 0.1 nm) inhibited the antigen-induced secretion of IL 2 by several of these T cell hybridomas. This inhibition was dependent on the concentration of the free hormone and could be overcome by increasing the number of Ia-bearing stimulator cells used. Pretreatment of the T hybridoma but not the TA3 stimulator cell with 1,25-(OH)2D3 resulted in inhibition of activation. These results are consistent with the finding that specific 1,25-(OH)2D3 receptors are present on the T cell hybridomas but are lacking in TA3 cells. 1,25-(OH)2D3 failed, however, to inhibit the activation of the T cell hybridomas by lectin or by an anti-Thy-1 antibody. These findings suggest that 1,25-(OH)2D3 may be interfering with early events of antigen-induced T cell activation, perhaps by hindering T cell recognition of the relevant antigen on stimulator cell surfaces. This system should prove useful in studying the molecular mechanisms by which 1,25-(OH)2D3 acts to inhibit T cell activation and subsequent IL 2 production.     

Monoclonal antibodies recognising serum immunoglobulins and surface immunoglobulin-positive cells of puffer fish, torafugu (Takifugu rubripes)

Immunoglobulin of the torafugu, Takifugu rubripes, was purified by a combination of precipitation by low ionic strength dialysis and gel filtration. The Ig was used to immunise mice for the production of monoclonal antibody (MAb). Supernatants of hybridoma cultures were screened by enzyme-linked immunosorbent assay using purified-torafugu Ig-coated plates, and two stable hybridomas producing MAbs against torafugu Ig were obtained. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions and Western blotting indicated that one MAb (16F3) was specific for the deglycosylated heavy chain of torafugu, and the other MAb (4H5) did not bind to the reduced Ig, suggesting that 4H5 recognised the higher-order structure of Ig. Under non-reduced conditions, both MAbs recognised mainly a 750 kDa band and also minor bands of 672, 410 and 205 kDa. MAb 16F3- and 4H5-primed magnetic beads (Dynabeads) adsorbed 84.9+/-3.3% and 63.6+/-4.4% of the torafugu Ig, respectively. The Ig adsorbed by MAb 16F3-primed Dynabeads was reactive to 4H5 on immunoblotting, and vice versa, indicating that the epitopes for both MAbs are held on the same Ig molecule. Both of these MAbs cross-reacted extensively with the Ig of other Takifugu species, but not with other genus. The MAbs were used to identify surface Ig-positive lymphocytes in the spleen, pronephros, peripheral blood and thymocytes of torafugu by flow cytometry. Flow cytometric analysis of the cells in the lymphocyte-enriched fraction revealed that 50.2+/-6.9% in the PBL, 11.8+/-1.7% in the mesonephros, 13.3+/-2.1% in the pronephros, 42.5+/-4.3% in the spleen and 3.2+/-0.6% in thymus were reactive to 4H5 or 16F3.     

Role of the L3T4 antigen in T-cell activation. I. Description of a monoclonal IgM antibody to a distinct epitope (L3T4b) of the L3T4 antigen and its effect on interleukin 1-induced thymocyte proliferation

This report describes a new rat monoclonal IgM/k antibody, monoclonal antibody (MAb) 2B6, which reacts with a cell surface antigen present on a subpopulation of both thymocytes (85%) and peripheral T lymphocytes (55-60%). The antigen recognized by MAb 2B6 has multiple properties in common with the L3T4 antigen, as defined by the recently described MAb GK1.5. Thus, MAb 2B6 and MAb GK1.5 give very similar flow cytometry staining patterns on thymocytes, purified spleen T cells and all tested T-cell hybridomas. Depletion of MAb 2B6-positive cells with antibody and complement led to simultaneous depletion of MAb GK1.5-positive cells, and vice versa. Depletion of Lyt 2-positive cells led to enrichment of both MAb 2B6- and MAb GK1.5-positive cells. Both MAb 2B6 and MAb GK1.5 immunoprecipitate the same pattern of cell surface molecules from detergent extracts of radiolabeled thymocytes, the main components being a 55-kDa and a 115-kDa band. We therefore conclude that MAb 2B6 reacts with the L3T4 antigen. Interestingly, MAb 2B6 and MAb GK1.5 do not cross-block and therefore most probably react with distinct epitopes on the L3T4 molecule. The determinant recognized by MAb GK1.5 is called L3T4a. We suggest that the determinant recognized by MAb 2B6 be named L3T4b. As MAb 2B6 was selected for its ability to inhibit the action of interleukin 1 (IL-1) in the thymocyte costimulator assay, it is likely that the L3T4 molecule is functionally involved in the events taking place during IL-1 induction of thymocyte proliferation.     

Increased antigen binding strengths of hybrid antibodies produced by human hybrid hybridomas

Two cell lines of human hybridomas were fused to generate hybrid antibodies. One human hybridoma cell line was HT2 producing IgM monoclonal antibody (MAb) reactive to carboxy peptidase A (Cpase) and double stranded DNA (ds DNA) and another was SU-1-D2 secreting IgM MAb reactive to ds DNA but not to Cpase. Most hybrid hybridomas obtained by fusion of the two hybridomas secreted hybrid antibodies exhibiting increased antigen binding strengths. All of the hybrid antibodies with increased binding strengths against Cpase and ds DNA contained only the light chains derived from SU-1-D2. These results suggested that increase in the binding strength of the hybrid antibodies resulted from heterogeneous association of H and L chains derived from HT2 and SU-1-D2 cells.     

Production and characterization of a monoclonal antibody against putative T lymphocytes of Catla catla

Catla catla is the fastest growing Indian major carp and one of the major aquaculture species in South Asia. A monoclonal antibody (MAb) designated B8 MAb was produced against nylon wool-enriched thymus mononuclear cells of C. catla. This MAb did not show reactivity with macrophage and epithelial cell lines derived from catla thymus in cellular ELISA. In flow cytometric analysis of gated lymphocytes, the percentage of B8 positive (B8+) cells in thymus (n?=?10, 500?C600?g) was determined to be 77.7?%. Similarly, the percentage of B8+ cells in kidney, spleen and blood (n?=?5) was 15.08, 1.1 and 32.17?%, respectively. Western blotting of reduced membrane proteins showed that B8 MAb reacted with a polypeptide having a molecular weight of 168.2?kDa. In indirect immunoperoxidase test, B8+ cells appeared to be lymphoid cells with a high nucleus to cytoplasmic ratio. B8 reactive cells were densely packed in central region of thymus whereas, a few cells were found to be positive in kidney and spleen sections. B8 MAb also reacted with a significant population of lymphocytes in blood smears. Considering the economic importance of C. catla, this MAb should be a useful tool for studying immune response of this fish species.     

Monoclonal antibody specific for Listeria monocytogenes, Listeria innocua, and Listeria welshimeri

Eight hundred fifty-nine murine hybridomas were produced from eight fusions, and 27 were characterized for secretion of antibodies reactive to Listeria monocytogenes. One monoclonal antibody (MAb), P5C9, reacted with all test strains of L. monocytogenes (31 of 31), L. innocua (3 of 3), and L. welshimeri (1 of 1) but not with any strains of the other four Listeria species or with any of 22 gram-positive or 11 gram-negative species of bacteria when tested in microtiter and dot blot enzyme immunoassays. Of the other 26 antibodies, 20 reacted with either L. monocytogenes Scott A or V7 and with some or all of the other six Listeria species but also cross-reacted with some or all of the non-Listeria bacteria tested. MAb P5C9 is of the immunoglobulin G1 murine subclass. In Western blot (immunoblot) analyses, this MAb reacted with a single antigen with a molecular weight of 18,500, and it is shared in common with all three reactive species, L. monocytogenes, L. innocua, and L. welshimeri. This antigen was extracted with detergent and appeared to be cell bound.     

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.     

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.