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1.
A monoclonal antibody (mAb), 2D1(IgM), was identified for its anti-proliferative effect on human T leukemia cell line, SUP-T13. The cells bound with 2D1 showed DNA ladder patterns of oligonucleosomes, demonstrating apoptosis. Peripheral mononuclear cells activated by phytohemagglutinin or OKT3 induced expression of 2D1 antigen and were growth-inhibited by the antibodies. Amsong the cell lines tested, T cell lines tended to be growth-inhibited by the antibodies. Epstein-Barr virus-transformed B cells were reactive with 2D1, but were not growth-inhibited by the antibodies. We established stable 2D1-resistant variants LAC2D1R and JKT2D1R from the original SUP-T13 and Jurkat T cell lines, respectively. These variant cells demonstrated phenotypes identical to the original cells, including reactivity to 2D1 and expression of cytoplasmic Bc1-2 protein. The 2D1-resistant cells were as sensitive as the original cells to the other apoptosis-inducing stimuli, such as γ-irradiation or calcium ionophore A23187. However, the 2D1-resistant variants were also insensitive to anti-Fas, another apoptosis-inducing mAb. Binding of 2D1 was blocked by anti-Fas mAb, suggesting that 2D1 reacts with an epitope of human Fas molecules. The present results demonstrate that a 2D1-reactive, but not 2D 1-sensitive, population may exist in highly 2D1-sensitive human leukemia T cells and that pairs of 2D 1-sensitive and 2D1-resistant cells are useful in the biochemical analysis of Fas-mediated apoptosis in human T cells.  相似文献   

2.
Responses to Thy-1 were used as a model system to examine parameters which affect the production of antibody-secreting lines derived from somatic cell hybridization. Experiments with the Thy-1.1 response revealed that the frequency of clones producing Thy-1.1 antibodies is a constant of 4 to 6% for each 10000 plaque-forming cells (PFC) input in the fusion cell mixture, regardless of the maturational stage of the response. Therefore, PFC responses to Thy-1 were optimized by studying variables in the choice and dose of antigen, the response kinetics and in the fusion procedures. Thus, to produce Thy-1.1 antibody-secreting cell lines, we used (a) spleen cells at the peak of the PFC response, (b) xenogeneic (rat) rather than allogeneic donors, (c) secondary rather than primary responses and (d) high ratios of NS-1 to spleen cells. For the reproducible production of Thy-1.2 antibody-secreting hybridomas, PFC responses to Thy-1.2 were similarly optimized in AKR mice. Response kinetics and antigen dose were shown to be very critical parameters. By varying the number of cells used for priming, it was revealed that doses only slightly higher than optimal produced a dramatic hyporesponsiveness in the subsequent secondary response. Using the above information, hybrid lines secreting antibody to Thy-1.2 were obtained reproducibly and one line, F7D 5, which secretes a cytotoxic IgM antibody was characterized in detail since a monoclonal antibody may differ from conventional antisera for immunochemical and genetic reasons. Serologically, F7D 5 Thy-1.2 antibody was found to behave as a conventional Thy 1.2 alloantiserum, At high dilutions however, the antibody can be used to discriminate long-lived T cells (adult thymectomized mice) from newly produced T cells (antilymphocyte antiserum-treated mice). Functionally, in numerous T cell-dependent assays both in vivo and in vitro, including helper, suppressor and cytotoxic T cell functions as well as responses to mitogens and antigens, the F7 D 5 antibody behaved as a potent and absolute T cell-depleting agent. This cell line and some anti-Thy-l.1 producing lines are available for research purposes.  相似文献   

3.
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.  相似文献   

4.
目的;构建分泌人源性单允隆抗体(McAb)的永生化B淋巴细胞系。方法:用正丁醇法提取的人卵巢癌细胞系aol10/17抗原在含免疫活化剂的无血清培基中免疫人淋巴细胞,将制备ao 10/17细胞DNA转染经体外免疫的淋巴细胞。采用ELISA筛选和有限稀释法克隆化。  相似文献   

5.
The monoclonal antibody methodology was used to identify membrane structures involved in T cell functions. To optimize chances to produce and detect relevant antibodies, a xenogeneic sensitization protocol was utilized and hybridoma supernatants were screened, on functional rather than structural grounds, for their ability to inhibit a given function. The test function was T cell-mediated cytolysis. Mouse cytolytic anti-allogeneic cell populations were used to sensitize a rat, the spleen cells of which were fused to produce hybridomas; the supernatants of the latter were screened for their ability to inhibit mouse T cell-mediated cytolysis in vitro. Several inhibitory antibodies were obtained, one of which, H35-89.9 monoclonal antibody, was studied in more detail. It inhibited specific and concanavalin A (Con A)-mediated cytolysis by T cells, by acting on the effector cells. It reversibly inhibited soluble antigen-, alloantigen and Con A-induced T cell proliferation (but not LPS-induced B cell proliferation), after the production of interleukin 2, by acting on the responder cells. It also had a desagglutinating effect on Con A and LPS blasts and on EL4 cells. It immunoprecipitated from thymocyte membrane preparations two structures of 94000 and 180000 apparent molecular weight, and recognized cell surface determinants on both T and B lymphocytes. Our findings suggest that several antibodies directed against distinct effector cell membrane structures inhibit cytolysis. The case of H35-89.9 monoclonal antibody, which exerts multiple functional effects and immunoprecipitates two membrane polypeptides, raises the problem of the various possible relationships between these structures and functions.  相似文献   

6.
A monoclonal antibody blocking human T cell function   总被引:32,自引:0,他引:32  
The possible functional role of T cell surface antigens defined by monoclonal antibodies was investigated. Five monoclonal anti-T cell reagents as well as an anti-Ia and anti-beta 2-microglobulin antibody were examined for their effect on T cell function. It was shown that an antibody termed anti-T3, reactive with all peripheral T cells, blocked T cell proliferative responses to soluble and cell surface antigens. This inhibition was seen when T lymphocytes were treated with as few as 10(4) anti-T3 molecules per cell. Although anti-T3 could block the generation of cytotoxic T cells in mixed lymphocyte culture, once generated, anti-T3 had no effect on cytotoxicity. In addition, anti-T3 abrogated the ability of T cells to provide help to B cells in a pokeweed mitogen-driven immunoglobulin system. More importantly, these functional effects were not seen with the other monoclonal antibodies. Both the appearance of this antigen in intrathymic ontogeny and its critical role in T cell function suggests that the T3 molecule is related to an important antigen recognition receptor or cell-cell interactions molecule.  相似文献   

7.
A monoclonal antibody designated 'antibody 390' (Ab 390) with anti-human Thy-1 reactivity was prepared by the hybridoma technique from the splenocytes of BALB/c mice immunized with human fetal brain. This antibody was shown to have anti-human Thy-1 reactivity because (1) it precipitated a molecule with a molecular weight of about 24,000 daltons, (2) it had a pattern of reactivity similar to that of previously described anti-human Thy-1 antibodies and (3) purified human Thy-1 antigen specifically inhibited binding of Ab 390 to a known antigen-positive cell line. It was the intent of this study to investigate the distribution of Thy-1 on normal and malignant haematopoietic cells in humans and non-human primates. We show here that Ab 390 did not react with human peripheral blood leucocytes, bone marrow cells or splenocytes by immunofluorescence but did react with subcapsular and cortical fetal thymocytes by peroxidase-antiperoxidase immunohistology. A section of fetal spleen demonstrated staining of connective tissue and blood vessels and rare reactive lymphocytes. Adult spleen contained Thy-1-positive cells surrounding the white pulp and in the marginal zone, but single-cell suspensions of splenocytes did not react with Ab 390. Ab 390 was tested against a variety of fresh human leukaemia cells and human cell lines and was shown to react with only the acute lymphoblastic leukaemia T cell lines RPMI 8402 and HPB-MLT. Non-human primate studies revealed reactivity with a number of T cell lines from New World primates (cotton-topped and red-bellied marmosets) and peripheral blood granulocytes (owl monkey). Our studies support previous findings that suggest that human Thy-1 may be a marker for early T lymphocytes in man, and its distribution on non-human primate T cell lines suggests the same for certain species of non-human primates. Not consistent with the distribution on human cells was the demonstration of Ab 390 reactivity with owl monkey granulocytes.  相似文献   

8.
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.  相似文献   

9.
抗人全T细胞单克隆抗体大鼠杂交瘤细胞株的建立   总被引:1,自引:0,他引:1  
通过融合大鼠免疫细胞株IR983F和经T细胞株HPB—ALL免疫大鼠脾细胞,获得分泌单克隆抗体LO—CD6a的杂交瘤细胞株。实验证实,LO—CD6a是人全T细胞特异性的。  相似文献   

10.
The monoclonal antibody, H207 , raised against the human T-cell-derived acute lymphoblastic leukaemia line HSB-2 , recognizes an antigen of apparent molecular weight 125000 which is coded for by the gene, MIC6 , on chromosome 17. Assignment was based on the pattern of reactivity with a panel of somatic cell hybrids. In particular, two hybrids containing only human chromosome 17 or a human translocation chromosome t( 3;17 ) (3pter-3p11::17p11–17qter) respectively bound H207 , whilst their revertands did not. The H207 antigen has a general tissue distribution but is not found on B-cell lines and is lacking from some T-cell lines.  相似文献   

11.
We have previously reported that the murine T cell line EL-4 has an aggregating phenotype, displaying homotypic aggregation (HTA) when exposed to monoclonal antibodies targeting specific cell surface molecules such as leukocyte function-associated antigen-1 (LFA-1). We have used this property of EL-4 cells to isolate additional HTA-inducing MAb by screening a panel of hybridomas that were generated from rats immunized with EL-4 cells. We have isolated a novel anti-Thy-1 MAb (termed FF-10) that is a powerful inducer of HTA in EL-4 cells. In addition to induction of HTA, FF-10 also induces splenocyte proliferation but inhibits anti-CD3-driven T cell proliferation. Thy-1-induced HTA cannot be blocked with MAb targeting intercellular adhesion molecule-1 and -2 (ICAM-1, ICAM-2) or LFA1. Thus, the FF-10 MAb represents a novel and unique tool to investigate the diverse roles of the murine Thy-1 molecule in T cell activation, proliferation and apoptosis.  相似文献   

12.
Conjugates of ricin A-chain with monoclonal anti-light chain antibodies specifically killed cells hearing kappa or lambda immunoglobulin (Ig) light chains. Exposure of cells from B-lymphoblastoid cell lines (B-LCL) to conjugate for less than 30 h had only a slight effect on cell growth, but on 48 h exposure a marked killing effect was achieved. After recovery of growth, cells were re-exposed to conjugate for 9-14 days. Treatment of cells from the EB4 line (sIgG lambda) in this way yielded 4 variants which showed a marked reduction in levels of surface Ig lambda and secreted Ig lambda with slight, or no, reduction in MHC class II expression and similar growth rates to the parent line. Variant lines retained their phenotype over long periods of culture.  相似文献   

13.
Lectin-resistant mutants with specific defects in glycosylation have been selected from the mouse lymphoma cell line, BW5147 (Thy-1+). The quantitative expression of cell surface glycoproteins on the mutant cells has been studied. The results show that some glycosylation defects that confer resistance to the cytotoxic effects of concanavalin A block the expression of Thy-1 glycoprotein on the cell surface. However, some changes in the oligosaccharides of Thy-1 glycoprotein generated by glycosylation defects found in PHAR mutant cells and restricted to the termini of complex-type oligosaccharides have no effect on the ability of Thy-1 to reach the cell surface. No glycosylation defects were found that interfered with the expression of either gp 69,71 or H-2 on the surface of the mutant cells. It is concluded that aberrant biosynthesis of Thy-1 oligosaccharides can interfere with its expression on the cell surface, but that specific changes in oligosaccharide structure are necessary to block transport to the cell surface and integration into the plasma membrane.  相似文献   

14.
15.
Monoclonal antibody (MoAb) KT38 raised against a human T leukaemic cell line TALL-1, reacted with another T leukaemic cell line Jurkat, but not with any other cell lines tested. The co-modulation of CD3 and KT38 antigen was observed with stimulations of either MoAb T3 or KT38 on both TALL-1 and Jurkat. Upon radioimmunoprecipitation and SDS-PAGE analysis, MoAb KT38 precipitated the heterodimer of 40-60 kD from Jurkat and TALL-1 under reducing conditions. Thus, MoAb KT38 is considered to be an anti-T idiotype (Ti) antibody to TALL-1 and Jurkat cells. MoAb KT38 was also shown to react with a minor population of peripheral blood lymphocytes (PBL) and with very few cells (0.5-2.0%) in the paracortical area of the lymph node. When PBL were stimulated in a KT38-coated culture flask for 5 days, the percentage of KT38-positive PBL was markedly increased. The CD3 antigen on these cultured PBL in the flask was modulated by the stimulation with MoAb KT38. Thus, it is suggested that a common idiotope exists on the T cell receptor of Jurkat, TALL-1 and a small percentage (1.9-6.1%) of PBL.  相似文献   

16.
The role of perforin in cytotoxicity is controversial. This paper characterizes a novel monoclonal antibody (anti-Phu) against human perforin, using murine cell lines transfected with human perforin cDNA. The antibody specifically stains human perforin in transfected mouse CTLL-2. Anti-Phu blocked granule-mediated haemolysis in an in vitro assay using intact granules isolated from the natural killer (NK)-like human cell line YT, indicating that perforin is a major granule component causing lysis of red blood cells (RBC) in this assay. Inhibition of haemolysis by anti-Phu demonstrated that the antibody binds to undenatured protein as well as fixed perforin molecules. However, the antibody did not inhibit lysis by an allospecific T-cell clone or by YT cells. This could be due to an extremely tight contact between effector and target cell, preventing the antibody from interfering with perforin function by steric hindrance. Physiologically this may reduce lysis of bystander cells. The anti-Phu antibody is a useful tool for further studies of perforin-induced cytotoxicity in vitro and in vivo.  相似文献   

17.
Seventeen monoclonal anti-Thy-1 antibodies (mAb) derived from LOU/M rats immunized with mouse T cell clones were used to study the role of Thy-1 in antigen-independent T cell activation. These mAb identified Thy-1.2 or monomorphic determinants and immunoprecipitated a molecule of 25-28 kDa from detergent-solubilized, 125I-labeled T cell surface proteins. Competitive cross-inhibition binding assays demonstrated that these reagents defined 3 epitope groups including either Thy-1.2 (group A) or Thy-1 monomorphic (groups B and C) determinants. Experiments using high titered culture supernatants revealed that all 6 IgG mAb defining the epitope group C, and one IgG2c mAb directed at a determinant in group A were capable of stimulating the terpolymer-L-glutamic acid60-L-alanine33-Ltyrosine10 (GAT) plus I-Ad-reactive BALB/c T cell hybridoma T14-117.9 to produce interleukin 2 (IL2) in the absence of accessory cells. Cross-linking of cell-bound rat mAb by a BALB/c anti-rat kappa chain mAb, or the presence of B cell lymphomas in the culture resulted in an increase of the Thy-1-mediated IL2 responses of this hybridoma. Some mAb from group B required antibody doses exceeding 80 micrograms/ml in order to activate T cells, while others remained nonstimulatory at any dose tested. Striking synergy in mAb-mediated T cell activation was observed when nonmitogenic doses of mAb group groups A and C were mixed in the same culture. Analysis of a panel of GAT plus I-Ad-specific T cell hybridomas revealed that these cells markedly differed in the magnitude of their IL2 responses induced by a given amount of stimulating anti-Thy-1 mAb. Such reagents also stimulated normal thymocytes to express IL2 receptor on their surface. These studies show that the epitopic specificity and the amount of anti-Thy-1 mAb, and the susceptibility of the T cell examined represent important parameters for the triggering of the Thy-1 pathway of T cell activation.  相似文献   

18.
The present study describes the development of a new IgM monoclonal autoantibody reactive with the Thy-1 antigen. The C16-31 monoclonal antibody (mAb) was considered as autoreactive because it reacted with thymus cells of both the C3H and BALB/c strains, which were involved in the development of the antibody. The antibody was reactive with thymus cells in both immunofluorescent and cytotoxic tests. It also showed a weak immunofluorescent reactivity with peripheral T-lymphocytes. The identification of the specificity detected by the C16-31 mAb as the Thy-1 antigen was based on the following criteria: C16-31 mAB displayed a preferential reactivity with Thy-1.2 bearing thymus cells, rather than with Thy-1.1 bearing thymus cells. The tissue distribution of the antigen detected by the C16-31 antibody by direct tests and by direct tests and by adsorption experiments was in accordance with that characteristic for Thy-1. It was high on brain tissue and on thymus cells, and considerably lower on peripheral T-lymphocytes. Coating of thymocytes with C16-31 antibody blocked their reactivity with other monoclonal Thy-1 antibodies. Conversely, coating of thymus cells with rabbit anti-brain serum (RABR) inhibited the binding of C16-31. The C16-31 mAb differed from the Thy-1 autoantibodies described previously in its relatively strong reactivity with brain tissue and its considerably weaker reactivity with peripheral T-lymphocytes. Moreover, C16-31 mAb showed a preferential allospecificity for Thy-1.2, only in its reactivity with thymocytes. In contrast, it reacted equally well with brain tissue from either Thy-1.2 or Thy-1.1 mice.  相似文献   

19.
A monoclonal antibody (mAb) called 30-3D6 has been raised against the T cell antigen receptor analogue on a human T cell leukemia cell line HPB-ALL. This mAb comodulates the T3 molecule on HPB-ALL and precipitates the heterodimeric structure previously described as a T cell idiotypic receptor analogue on this cell line. 30-3D6 reacts with a variable percentage of normal T cells (up to 6%) depending on the donor and this number is stable on repeated sampling and is not affected by the temperature of the reaction. When normal T cells from a high frequency donor are stimulated with 30-3D6 and interleukin 2 in vitro the idiotype-positive (Id+) population can be expanded. Large numbers of greater than 90% Id+ T cells can be generated. Id cells are present in both the helper and cytotoxic suppressor subsets.  相似文献   

20.
We have produced two monoclonal antibodies, SFR1-Myco 1 and SFR1-Myco 2, that detect Mycoplasma fermentans found to contaminate lymphoblastoid cell lines (LCL). The specificity of these monoclonal antibodies against the M. fermentans was determined by indirect immunofluorescence by demonstrating the reactivity of the monoclonal antibodies with known reference strains of mycoplasma grown on soft agar. The reactivity of these antibodies against LCL in a number of immunoassays correlates completely with the presence of mycoplasma in these cells as determined by a standard mycoplasma detection assay. Because of the potential for widespread contamination of B lymphoblastoid cell lines transformed with Epstein-Barr virus-containing supernatants obtained from marmoset cell lines contaminated with M. fermentans, these monoclonal antibodies have value as screening reagents for this mycoplasma species in LCL.  相似文献   

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