To see the other types of publications on this topic, follow the link: Hybridoma cell.
Journal articles on the topic 'Hybridoma cell'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Hybridoma cell.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Ozaki, S., S. K. Durum, K. Muegge, J. York-Jolley, and J. A. Berzofsky. "Production of T-T hybrids from T cell clones. Direct comparison between cloned T cells and T hybridoma cells derived from them." Journal of Immunology 141, no. 1 (July 1, 1988): 71–78. http://dx.doi.org/10.4049/jimmunol.141.1.71.
Full textAbstract:
Abstract It has been assumed, without direct evidence, that T cell hybridomas and non-transformed T cell clones are both good models of normal Ag-specific T cells. To compare directly the difference in activation of cloned normal T cells and T hybridoma cells with the same TCR, cloned T hybridoma cells were obtained by fusing pre-established, myoglobin-specific, Iad-restricted T cell clones (14.5 and 9.27) with BW5147 cells. T cell clones were pre-activated with IL-2 as well as specific Ag before fusion. Cloned T hybridoma A3.4C6 was derived from Lys 140-specific and I-Ed-restricted clone 14.5. The other cloned T hybridoma, C7R14, was a fusion product of Glu 109-specific and I-Ad-restricted clone 9.27. Both T hybridomas showed the same Ag specificity and Ia restriction as the parental cloned T cells. However, C7R14 showed higher apparent affinity and broader cross-reactivity than 9.27. Clone 14.5, but not hybridoma A3.4C6, appeared to stimulate splenic cells to secrete cytokines inhibiting HT-2A cell proliferation. The most striking difference between the clones and hybridomas was that both clones, but neither of the matched hybridomas, were induced to synthesize IL-1 on stimulation with Ag. Finally, both cloned T cells and T hybridomas killed Ag-pulsed Iad-bearing B lymphoma target cells. This evidence suggests that killing function can be inherited from clones to hybridomas. However, the clones were much more efficient at killing than the hybridomas, and the hybridomas were more efficient at IL-2 production than the clones. Thus, matched pairs of clones and hybridomas differ in their capacity to mediate the two functions or may tend to be selected differently during cloning. Thus, although our results generally support the validity of T cell hybridomas as faithful models of the corresponding T cell clones, a number of subtle and not-so-subtle differences indicate that caution must be used in such an extrapolation.
2
Hawksworth, David, Jeff Moore, and Isaac Larkin. "Diversity and affinity from plasma vs. B cells for monoclonal antibody development." Journal of Immunology 212, no. 1_Supplement (May 1, 2024): 0234_4543. http://dx.doi.org/10.4049/jimmunol.212.supp.0234.4543.
Full textAbstract:
Abstract Diagnostic assay development requires a diverse panel of high affinity antibodies. Historically, our lab has used splenic CD19+ B-cells to generate mouse hybridoma cell lines. However, literature suggests that, as a group, terminally differentiated, CD138+ plasma cells secrete the highest affinity antibodies, making these cells an attractive target for hybridoma development. To compare the use of these two lymphocyte populations for generating mouse hybridomas, three fusion experiments were completed whereby cells were separately isolated and fused. Hybridomas were plated in semi-solid media, expanded, and selected based on size and antibody secretion. An average of 246 (+ 73) plasma cell hybridoma colonies were available for selection, per 1x106 specific lymphocytes used for fusion, compared to 19 (+ 13) B cell hybridoma colonies. Cells from selected colonies were transferred to 96-well plates, grown for 7 days and the antibodies screened for antigen binding by enzyme immunoassay. Across the three fusion experiments, 40 (+19)% of the wells with growth from the plasma cell hybridomas were antigen reactive, vs. 6 (+ 0.5)% from the B cell hybridomas. Our data suggests a higher fusion efficiency from the plasma cell population, which allows for a greater sampling of the mouse immune repertoire, leading to identification of more antigen specific antibodies with a wider range of affinities and greater sequence diversity.
3
Iwata, M., K. Katamura, R. T. Kubo, and K. Ishizaka. "Relationship between T cell receptors and antigen-binding factors. I. Specificity of functional T cell receptors on mouse T cell hybridomas that produce antigen-binding T cell factors." Journal of Immunology 143, no. 12 (December 15, 1989): 3909–16. http://dx.doi.org/10.4049/jimmunol.143.12.3909.
Full textAbstract:
Abstract Upon antigenic stimulation with OVA-pulsed syngeneic macrophages, the mouse T cell hybridoma 231F1 produced glycosylation inhibiting factor (GIF) having affinity for OVA and IgE-suppressive factors, whereas another T cell hybridoma, 12H5, cells produced OVA-binding glycosylation enhancing factor (GEF) and IgE-potentiating factor. The OVA-binding GIF from the 231F1 cells is an Ag-specific Ts cell factor, whereas OVA-binding GEF from the 12H5 cells is an Ag-specific augmenting factor. Both hybridomas express CD3 complex and functional TCR-alpha beta. Cross-linking of TCR-alpha beta or CD3 molecules on the hybridomas by anti-TCR-alpha beta mAb or anti-CD3 mAb and protein A resulted in the formation of the same factors as those obtained by the stimulation of the cells with OVA-pulsed syngeneic macrophages. It was also found that both the 231F1 cells and 12H5 cells formed IgE-binding factors upon incubation with H-2d and H-2b APC, respectively, with a synthetic peptide corresponding to residues 307-317 in the OVA molecules (P307-317). Six other synthetic peptides, including those containing the major immunogenic epitope, i.e., P323-339, failed to stimulate the hybridomas in the presence of APC. Indeed, all of the 10 T cell hybridoma clones, which could produce either OVA-binding GIF or OVA-binding GEF, responded to P307-317 and APC for the formation of IgE-binding factors. In contrast, GIF/GEF derived from six other hybridoma clones, whose TCR recognized P323-339 in the context of a MHC product, failed to bind to OVA-coupled Sepharose. The results indicate the correlation between the fine specificity of TCR and the affinity of GIF/GEF to the nominal Ag. The amino acid sequence of P307-317 suggested that TCR on the cell sources of Ag-binding factors are specific for an external structure of the Ag molecules.
4
Tiebout, R. F., F. van Boxtel-Oosterhof, E. A. Stricker, and W. P. Zeijlemaker. "A human hybrid hybridoma." Journal of Immunology 139, no. 10 (November 15, 1987): 3402–5. http://dx.doi.org/10.4049/jimmunol.139.10.3402.
Full textAbstract:
Abstract Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. We have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. We fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1 kappa antibody directed against tetanus toxoid and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1 lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture medium containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassays. Our results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies.
5
Iwata, M., M. Adachi, and K. Ishizaka. "Antigen-specific T cells that form IgE-potentiating factor, IgG-potentiating factor, and antigen-specific glycosylation-enhancing factor on antigenic stimulation." Journal of Immunology 140, no. 8 (April 15, 1988): 2534–42. http://dx.doi.org/10.4049/jimmunol.140.8.2534.
Full textAbstract:
Abstract BDF1 mice were immunized with alum-absorbed OVA and T cell hybridomas were constructed from their splenic T cells. Many of the hybridomas constitutively produced glycosylation enhancing factor (GEF), which could switch the T cell hybridoma 23A4 cells from the formation of IgE-suppressive factors to the formation of IgE-potentiating factors. When one of the hybridoma clones, 12H5, was incubated with OVA-pulsed syngeneic or semi-syngeneic (H-2b) macrophages, the hybridoma produced GEF that have affinity for OVA, but not for either keyhole limpet hemocyanin or BSA. However, the same hybridoma constitutively produced nonspecific GEF, that lacked affinity for OVA. Upon incubation with OVA-pulsed macrophages, the same hybridoma produced both IgE-potentiating factors and IgG-potentiating factors which selectively enhance the IgE response and IgG response, respectively. Both Ag-specific GEF and nonspecific GEF from the hybridoma bind to p-aminobenzamidine-agarose, and are recovered by elution with benzamidine. It was also found that both OVA-specific GEF and nonspecific GEF from the hybridoma induced the release of arachidonic acid from phospholipids of mouse fibrosarcoma cell line, HSDM1C1 cells. GEF formed by the 12H5 hybridoma bound to alloantibodies reactive to the product(s) of the I-Ab subregion of major histocompatibility complex. The Ag-specific GEF consisted of two Mr species, of 70 to 90 kDa and 50 to 60 kDa, whereas nonspecific GEF consisted of 50 to 60 kDa and 25 to 30 kDa molecules. Reduction and alkylation treatment of the OVA-specific GEF resulted in the formation of nonspecific GEF, suggesting that Ag-specific GEF is composed of Ag-binding polypeptide chain and nonspecific GEF.
6
Kawasaki, H., C. A. Martin, T. Uchida, M. Usui, T. Noma, M. Minami, and M. E. Dorf. "Functional analysis of cloned macrophage hybridomas. V. Induction of suppressor T cell responses." Journal of Immunology 137, no. 7 (October 1, 1986): 2145–51. http://dx.doi.org/10.4049/jimmunol.137.7.2145.
Full textAbstract:
Abstract It has been suggested that macrophage-like accessory cells are involved in suppressor T cell (Ts) induction. To further analyze this issue, we obtained several cloned macrophage hybridoma cell lines by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serological and functional characteristics of macrophages. We evaluated the ability of these hybridomas to induce third order or effector Ts (Ts3) to suppress the contact sensitivity response against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). In contrast to the parental P388D1 and two other macrophage hybridomas, one macrophage hybridoma clone, termed 63, when conjugated with NP, induced Ts3, which suppressed contact sensitivity responses against NP but not DNFB, showing that the Ts3 were antigen specific. Macrophage hybridoma 63 could specifically induce Ts3 activity in either H-2k, H-2d, or H-2k/H-2d heterozygous hosts. Thus, macrophage hybridoma 63 functionally expressed major histocompatibility complex-related restricting determinants, and the fusion with cells from a H-2k macrophage donor caused a functional complementation of H-2d-related, Ts-inducing elements. The genetic restriction governing induction of Ts3 was controlled by genes that mapped to I-J region. Furthermore, NP-conjugated macrophage hybridoma 63 could serve as a target for elicitation of suppressor responses after administration of I-Jk, but not I-Jb, restricted suppressor factor. The data suggest that macrophage hybridomas represent a means to dissect heterogeneity within the macrophage population. The data also imply that the I-J determinants expressed on macrophages represent a ligand for the antigen receptor of Ts.
7
Lanier, L. L., J. J. Ruitenberg, and J. H. Phillips. "Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes." Journal of Immunology 141, no. 10 (November 15, 1988): 3478–85. http://dx.doi.org/10.4049/jimmunol.141.10.3478.
Full textAbstract:
Abstract CD16 Ag is associated with the low affinity FcR for IgG expressed on human NK cells and granulocytes. In this study, we demonstrate that NK cells specifically lyse murine anti-CD16 hybridoma cell lines, but do not lyse hybridomas against other cell surface differentiation Ag expressed on NK cells. Moreover, the CD18 structure is involved in the CD16-specific xenogeneic interaction between human effector cells and murine hybridoma target cells. Although interaction with anti-CD16 hybridomas or antibodies triggers the cytolytic mechanism of NK cells, this interaction does not induce cellular proliferation. In contrast to NK cells, CD16+ granulocytes do not lyse anti-CD16 hybridoma cell targets and do not mediate ADCC against antibody-coated human tumor cell targets. These findings indicate a fundamental difference in the antibody-dependent cellular cytotoxicity mechanisms of NK cells and granulocytes. Comparative biochemical analysis of CD16 on NK cells and granulocytes revealed significant differences in the size of the polypeptides obtained after removal of N-linked carbohydrate residues with endo-F and N-glycanase digestion.
8
Kuchroo, V. K., M. C. Byrne, E. Greenfield, M. J. Whitters, E. A. Nalefsky, A. Rao, M. Collins, and M. E. Dorf. "Transfection of TCR alpha-chains into suppressor and T helper cell hybridomas. Production of suppressor factors with predicted antigen specificity." Journal of Immunology 154, no. 10 (May 15, 1995): 5030–38. http://dx.doi.org/10.4049/jimmunol.154.10.5030.
Full textAbstract:
Abstract Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.
9
Takahashi, M., and S. A. Fuller. "Production of murine hybrid-hybridomas secreting bispecific monoclonal antibodies for use in urease-based immunoassays." Clinical Chemistry 34, no. 9 (September 1, 1988): 1693–96. http://dx.doi.org/10.1093/clinchem/34.9.1890.
Full textAbstract:
Abstract To produce bispecific antibodies (BiAbs) for enzyme immunoassay (EIA) to replace antibody-enzyme conjugates, we developed a panel of 8-azaguanine/ouabain-resistant anti-urease variant hybridoma cell lines for use in hybridoma-hybridoma fusions. These variants represent mouse immunoglobulin subclasses IgM, IgG1, IgG2a, and IgG2b and have growth rates equal to those of the parental hybridomas. We fused an anti-urease-secreting variant hybridoma with an anti-human choriogonadotropin (hCG)-secreting hybridoma (both of IgG1 subclass) and selected the desired product with growth media containing hypoxanthine-aminopterin-thymidine (HAT) and ouabain. Over 95% of the resulting hybrids secreted anti-urease, and 60% of these secreted anti-hCG. The bispecific nature of secreted antibodies was demonstrated in a simultaneous EIA where BiAbs, hCG, and urease (EC 3.5.1.5) were incubated together in anti-hCG-coated microwells. As little as 25 int. units of hCG per liter could be reliably detected, which is equivalent to that for antibody-enzyme conjugates in EIA.
10
Dzhambazov, Balik, Tsvetelina Batsalova, Patrick Merky, Franziska Lange, and Rikard Holmdahl. "NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II." International Journal of Molecular Sciences 24, no. 13 (June 28, 2023): 10811. http://dx.doi.org/10.3390/ijms241310811.
Full textAbstract:
It has been shown that synovial fibroblasts (SF) play a key role in the initiation of inflammation and joint destruction, leading to arthritis progression. Fibroblasts may express major histocompatibility complex class II region (MHCII) molecules, and thus, they could be able to process and present antigens to immunocompetent cells. Here we examine whether different types of fibroblasts (synovial, dermal, and thymic murine fibroblasts, destructive LS48 fibroblasts, and noninvasive NIH/3T3 fibroblasts) may be involved in the initiation of rheumatoid arthritis (RA) pathogenesis and can process and present type II collagen (COL2)—an autoantigen associated with RA. Using a panel of MHCII/Aq-restricted T-cell hybridoma lines that specifically recognize an immunodominant COL2 epitope (COL2259–273), we found that NIH/3T3 fibroblasts activate several T-cell clones that recognize the posttranslationally glycosylated or hydroxylated COL2259–273 epitope. The HCQ.3 hybridoma, which is specific for the glycosylated immunodominant COL2 epitope 259–273 (Gal264), showed the strongest response. Interestingly, NIH/3T3 cells, but not destructive LS48 fibroblasts, synovial, dermal, or thymic fibroblasts, were able to stimulate the HCQ.3 hybridoma and other COL2-specific T-cell hybridomas. Our experiments revealed that NIH/3T3 fibroblasts are able to activate COL2-specific T-cell hybridomas even in the absence of COL2 or a posttranslationally modified COL2 peptide. The mechanism of this unusual activation is contact-dependent and involves the T-cell receptor (TCR) complex.
11
Blanckmeister, C. A., K. Yamamoto, M. M. Davis, and G. J. Hämmerling. "Antigen-specific, I-A-restricted suppressor hybridomas with spontaneous cytolytic activity. Functional properties and lack of rearrangement of the T cell receptor beta chain genes." Journal of Experimental Medicine 162, no. 3 (September 1, 1985): 851–63. http://dx.doi.org/10.1084/jem.162.3.851.
Full textAbstract:
T suppressor (Ts) hybridomas were produced by fusion of the III/4 T cell hybridoma with splenic T cells from CBA mice tolerized with subimmunogenic doses of bovine serum albumin (BSA). Both the Ts hybridoma cells and a suppressor factor (TsF) inhibited in an antigen-specific and I-Ak-restricted fashion the in vitro proliferative response of BSA-immunized lymph node cells. In addition to the suppressive activity, the hybridoma lines displayed spontaneous cytotoxicity against various tumor targets. The isolation of Ts subclones that are suppressive but not cytolytic, as well as the existence of the noncytolytic TsF, indicates that suppression of antigen-specific T cell proliferation is not dependent on cytolytic activity. The Ts hybridomas were I-A restricted, as are many T helper cells. Therefore, a potential similarity with respect to antigen receptor genes was expected. Southern blot analysis with a probe specific for genes encoding the beta chain of the T cell receptor on T helper and T killer cells revealed no rearrangement of the beta genes in the Ts cells. The data imply that neither the antigen receptor on the I-A-restricted Ts cells nor the receptor involved in the cytolytic interaction with tumor targets use the same beta chain constant region as T helper and T killer cells.
12
Sherr, D. H., J. Braun, and M. E. Dorf. "Idiotype-specific Ly-1 B cell-mediated helper activity: hybridomas that produce anti-idiotype antibody and nonimmunoglobulin lymphokine(s)1." Journal of Immunology 138, no. 7 (April 1, 1987): 2057–62. http://dx.doi.org/10.4049/jimmunol.138.7.2057.
Full textAbstract:
Abstract Previous work has shown that the expression of a predominant family of idiotypic determinants (NPb) in the in vitro response to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten is dependent on helper activity provided by Ly-1- and Ig-bearing B cells called BH. The ability of these BH cells to perform this idiotype-specific, genetically controlled helper function is related to the NPb idiotype specificity of their cell surface receptors. However, the means by which BH cells communicate with and stimulate NPb idiotypic B cell subsets is unknown. In this paper, an Ly-1- and immunoglobulin-bearing B helper cell hybridoma is described. Supernatants from the hybridoma or its subclones were shown to specifically help the response of NPb idiotypic PFC to NP-Ficoll when added to responder cell cultures depleted of Thy-1 and Ly-1 regulatory cell populations. Under these experimental conditions hybridoma supernatants functioned in much the same fashion as populations of Ly-1- and Ig-bearing BH helper populations described previously. NPb idiotype-specific helper activity was mediated by two separable activities elaborated by the hybridoma, an anti-NPb idiotype antibody and a non-Ig (lymphokine) activity. It was shown that both the Ig and the lymphokine components were required for helper activity. Kinetics experiments showed that the anti-idiotype antibody must be added early in the response to NP-Ficoll, whereas the lymphokine fraction could be added at least as late as day 3 of a 4-day culture in order to observe NPb idiotype-specific help. The data suggest that Ly-1 B cell hybridomas may affect the responsiveness of B cell subsets initially by interaction of anti-idiotype antibody with NPb idiotypic B cell surface receptors, followed by growth or maturation signals mediated by non-Ig lymphokine(s). The possibility that the helper activity of these Ly-1 B cell hybridomas represents the combined effects of an idiotype-specific network system and nonspecific growth or maturation factor activity in direct B cell-B cell interactions is discussed.
13
Kuchroo, V. K., P. R. Billings, C. Levine, C. A. Martin, R. T. Kubo, and M. E. Dorf. "Antigen-specific and antibody-mediated growth inhibition of suppressor T cell hybridomas. Roles of H-2 and the CD3-T cell receptor-alpha/beta complex." Journal of Immunology 145, no. 4 (August 15, 1990): 1059–65. http://dx.doi.org/10.4049/jimmunol.145.4.1059.
Full textAbstract:
Abstract Eight different Ts cell hybridomas (including inducer (Ts1) and effector (Ts3) suppressor cells) specific for the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were tested for their ability to respond to Ag or anti-CD3 antibody in a growth-inhibition assay. Results suggest that the expression of the TCR-CD3 complex on Ts hybridomas is required for the Ag or anti-CD3-mediated growth inhibition. One of the CD3+, Ts hybridomas (CKB-Ts3-9.H3) was tested in detail; this CD4- effector suppressor cell hybridoma showed specific inhibition of growth in the presence of NP or NIP-coupled protein conjugates but not in the presence of other irrelevant hapten-protein conjugates. In addition, growth of this hybridoma was specifically inhibited by anti-CD3 and anti-TCR-alpha/beta antibodies but not by control hamster antibodies. In order to study the role of MHC molecules in Ag-mediated growth inhibition, Ts cell hybridomas were incubated with Ag (NP-keyhole limpet hemocyanin) in the presence of spleen cells from various H-2 congenic strains. The results suggest that the Ts hybridomas that express donor Ts-derived TCR beta-chain recognize Ag in an MHC-restricted manner, whereas the two Ts3 hybridomas that utilize BW5147-derived TCR-beta recognize Ag in H-2 unrestricted way. Co-incubation of anti-CD3 and anti-TCR-alpha/beta antibodies with specific Ag enhanced the Ag-mediated growth inhibition, whereas anti-LFA-1 antibody completely blocked the Ag-mediated effect. The combined data suggest that, like Th hybridomas, expression of CD3-associated-TCR complex is essential for the Ag responsiveness of Ts cell hybridomas.
14
Mathur, A., B. G. Van Ness, and R. G. Lynch. "In vivo and in vitro regulation of IgE production in murine hybridomas." Journal of Immunology 145, no. 11 (December 1, 1990): 3610–17. http://dx.doi.org/10.4049/jimmunol.145.11.3610.
Full textAbstract:
Abstract Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.
15
Sulfîanti, A., V. T. Sopandi, and F. N. Ningsih. "Growth capacity of hybridoma clones producing monoclonal antibody against SARS-CoV-2 in low-serum media." IOP Conference Series: Earth and Environmental Science 1271, no. 1 (December 1, 2023): 012074. http://dx.doi.org/10.1088/1755-1315/1271/1/012074.
Full textAbstract:
Abstract Monoclonal antibodies (mAbs) are biorecognition molecules in the COVID-19 detection. Previously, we used the hybridoma technique to generate anti-Spike SARS-CoV-2 monoclonal antibodies. The resulting mAbs captured the commercial Spike protein of SARS-CoV-2 on an indirect ELISA platform, and afterward, the hybridomas were adapted to live in low-serum media. However, a validated hybridoma cell and a specific mAb should be established due to its critical role in large-scale production. Hence, this study aimed to observe the hybridoma cell’s growth capacity and its mAbs production in low-serum media. Two hybridoma clones, SF2 and RF10, were grown in RPMI media supplemented with 3% FBS for 11 days. Their viable cell density, growth rate, and doubling time were measured and calculated. According to the data, the SF2 clone grew slower than the RF10 clone. However, SF2 had shown greater cell density on the logarithmic phase. This finding is linear to the ELISA result, showing that SF2 produces higher absorbance levels. These findings demonstrated that the SF2 clone had the potential to survive under low-serum circumstances and still produce mAbs. This clone might be used to produce mAbs against Spike protein SARS-CoV-2 at a low cost.
16
Hurwitz, J. L., J. Samaridis, and J. Pelkonen. "Immature and advanced patterns of T cell receptor gene rearrangement among lymphocytes in splenic culture." Journal of Immunology 142, no. 7 (April 1, 1989): 2533–39. http://dx.doi.org/10.4049/jimmunol.142.7.2533.
Full textAbstract:
Abstract Bulk populations and 39 hybridomas from splenic Con A cultures were analyzed for rearrangements among TCR genes: alpha, beta, gamma, and delta. Patterns were categorized to reveal general rules governing gene rearrangement within the activated adult peripheral population. Many patterns of gene rearrangement were consistent with previous studies of T cell lines. Additional points of interest were the following: 1) A large proportion of Con A-stimulated splenic cells bore no TCR gene rearrangements. 2) One splenic hybridoma exhibited an unusual gene pattern, with rearrangements, at alpha and beta, but not J gamma 1 or J gamma 2 loci. 3) Multiple gamma rearrangements were noted other than V1.2-J2 and V2-J1. 4) One hybridoma exhibited TCR gene rearrangements typical of day 14 to 15 fetal thymocytes, as well as rearrangements at immunoglobulin gene loci. 5) Among hybridomas with J alpha rearrangements, homologous chromosomes exhibited rearrangements at similar positions along the J alpha locus.
17
Greenfield, Edward A. "Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with Permeabilized Cells to Detect Intracellular Binding." Cold Spring Harbor Protocols 2022, no. 6 (June 2022): pdb.prot103085. http://dx.doi.org/10.1101/pdb.prot103085.
Full textAbstract:
Flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to internal cellular proteins, but the cells must be permeabilized before the hybridoma supernatants are applied. In using this technique, useful controls are positive and negative cell lines with primary and secondary antibodies as well as positive and negative cell lines with secondary antibody alone.
18
Gu, J. J., J. V. Harriss, K. Ozato, and P. D. Gottlieb. "Induction by concanavalin A of specific mRNAs and cytolytic function in a CD8-positive T cell hybridoma." Journal of Immunology 153, no. 10 (November 15, 1994): 4408–17. http://dx.doi.org/10.4049/jimmunol.153.10.4408.
Full textAbstract:
Abstract A previous report from this laboratory described the production of CD8+, class I-specific T cell hybridomas which developed specific cytolytic activity and the ability to secrete IL-2 upon Con A or specific Ag stimulation. Unlike normal lymphocytes or long-term CTL lines for which exposure to Ag triggers both differentiation and proliferation, T cell hybridoma lines can be activated functionally against a background of continuous proliferation. They therefore provide a unique system with which to study the molecular events involved in the induction of cytolytic function. The expression of mRNA from a series of genes was evaluated by Northern hybridization at various times after Con A stimulation of the H-2Ld-specific CD8+ 3D9 hybridoma. Induction of the c-fos proto-oncogene by 45 min poststimulation was followed shortly by c-myc induction. Perforin mRNA was expressed at a low level in the unstimulated hybridomas, but was down-regulated upon Con A stimulation to levels undetectable by PCR. Interestingly, production of granzyme A mRNA was strongly induced by 45 min after Con A stimulation. In the CD8+ RT-1.3G3 hybridoma, which is nonlytic and specific for the HIV-1 envelope glycoprotein, c-fos but not granzyme A mRNA was induced by 45 min poststimulation, and no granzyme A mRNA was detectable at any time. Thus, a significant role for granzyme A in the induction of cytolytic activity is suggested. Cytolysis by the 3D9 hybridoma involved both target cell membrane damage and DNA fragmentation, and both Ca(2+)-dependent and Ca(2+)-independent cytolysis were observed. Although TNF-alpha mRNA was induced by 4 h poststimulation, Ab to TNF-alpha failed to inhibit the Ca(2+)-independent lysis observed, leaving the basis for the observed Ca(2+)-independent lysis unexplained.
19
Kulczycki, A., J. Trial, J. M. Connolly, S. Sharp, and J. A. Kapp. "Structure and expression of Fc gamma receptors on mouse suppressor T cell hybridomas." Journal of Immunology 137, no. 7 (October 1, 1986): 2325–30. http://dx.doi.org/10.4049/jimmunol.137.7.2325.
Full textAbstract:
Abstract Antigen-specific and idiotype-specific mouse suppressor T cell hybridomas were analyzed for the presence and specificity of Fc gamma receptors (Fc gamma R) by EA rosetting and by flow microfluorometry with the use of monoclonal antibodies. We found that four hybridomas expressed Fc gamma R specific for IgG1 and IgG2b, one of which became Fc gamma R- during prolonged culture. Four other hybridomas and the fusion parent, BW5147, consistently lacked Fc gamma R. The 125I-labeled Fc gamma R were isolated from surface radioiodinated hybridoma cells solubilized with 1% Nonidet P-40, were purified by using single or repetitive chromatography on mouse IgG-Sepharose columns, and were analyzed by SDS-PAGE. An 125I-labeled 56,000 to 61,000 Mr macromolecule was isolated from each of the Fc gamma R+ hybridomas, but from none of the Fc gamma R- hybridomas nor from BW5147 cells. This macromolecule rebound to insolubilized mouse IgG1, IgG2b, and human Fc fragments, but not to insolubilized mouse IgG2a, IgG3, or IgA or human F(ab')2 fragments, consistent with the specificity observed for Fc gamma R on intact hybridoma cells. The mouse suppressor T cell Fc gamma R differs in size and specificity from mouse B cell Fc gamma R. A 70,000 Mr protein expressed on all hybridomas and on BW5147 cells was radiolabeled and, despite preclearing with ovalbumin-Sepharose, bound to the mouse IgG-Sepharose columns, presumably due to mouse antibodies to gp-70. This macromolecule was completely and specifically removed by using goat antiserum to gp-70.
20
Kisaki, T., T. F. Huff, D. H. Conrad, J. Yodoi, and K. Ishizaka. "Monoclonal antibody specific for T cell-derived human IgE binding factors." Journal of Immunology 138, no. 10 (May 15, 1987): 3345–51. http://dx.doi.org/10.4049/jimmunol.138.10.3345.
Full textAbstract:
Abstract A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.
21
Bierer, B. E., A. Peterson, J. C. Gorga, S. H. Herrmann, and S. J. Burakoff. "Synergistic T cell activation via the physiological ligands for CD2 and the T cell receptor." Journal of Experimental Medicine 168, no. 3 (September 1, 1988): 1145–56. http://dx.doi.org/10.1084/jem.168.3.1145.
Full textAbstract:
T cells may be activated either by the antigen-specific T cell receptor (TCR)-CD3 complex or the cell surface receptor CD2. A natural ligand for CD2 has been found to be lymphocyte function-associated antigen 3 (LFA-3), a widely distributed cell surface glycoprotein. To investigate the interaction of these two pathways, we have expressed the cDNA encoding the human CD2 molecule in a murine T cell hybridoma that produces IL-2 in response to HLA-DR antigens. Expression of the CD2 molecule markedly enhances IL-2 production in response to LFA-3+ antigen-bearing stimulator cells, and this stimulation is inhibited by anti-CD2 and anti-LFA-3 mAb. To further define the role of LFA-3 in antigen-dependent T cell activation, we have studied the ability of the purified ligands of CD2 and the TCR to stimulate the hybridoma. Neither liposomes containing purified HLA-DR antigens nor liposomes containing purified LFA-3 were able to stimulate the parent or the CD2+ hybridoma. However, liposomes containing both purified LFA-3 and HLA-DR, the physiological ligands for CD2 and the TCR, respectively, stimulate IL-2 production by the CD2+ but not the parent hybridoma, suggesting that complementary interactions between the TCR-CD3 complex and the CD2 pathway may regulate lymphocyte activation. To determine whether the CD2/LFA-3 interaction participates in cell-cell adhesion and provides an activation signal, we have constructed a cytoplasmic deletion mutant of CD2, CD2 delta B, in which the COOH-terminal 100 amino acids of CD2 have been replaced with a serine. Hybridomas expressing the CD2 delta B molecule were examined. Deletion of the cytoplasmic domain of CD2 did not alter binding of LFA-3 but eliminated the ability of CD2 to increase the response of the hybridoma to liposomes containing both HLA-DR and LFA-3, demonstrating that adhesion of LFA-3 to CD2 alone was insufficient for activation, and that the cytoplasmic domain was required for LFA-3 stimulation through the CD2 molecule. T cells may be activated by purified LFA-3 binding to CD2 and the TCR interacting with its ligand, and these signals appear to be synergistic for the T cell. These results suggest that the CD2/LFA-3 interaction not only plays a role in cell-cell adhesion but provides a stimulatory signal for T cell activation.
22
Yoo, J., H. Chen, T. Kraus, D. Hirsch, S. Polyak, I. George, and K. Sperber. "Altered cytokine production and accessory cell function after HIV-1 infection." Journal of Immunology 157, no. 3 (August 1, 1996): 1313–20. http://dx.doi.org/10.4049/jimmunol.157.3.1313.
Full textAbstract:
Abstract We investigated cytokine production and accessory cell function in human macrophage hybridoma cell lines and primary monocytes after infection with HIV-1. HIV-1 infection induced IL-10 production in the macrophage hybridoma cell line with loss of IL-12 1 wk after infection. There were also significant increases in production of IL-10 (537 +/- 521 vs 687 +/- 625 pg/ml) while there was a reduction in IL-12 (6.3 +/- 3.1 vs 1.2 +/- 1.0 pg/ml, p = 0.021) in the primary monocytes 5 days after HIV-1 infection. In addition, the hybridoma cell lines and primary monocytes failed to support PHA, Con A, PWM, or anti-CD3- induced T cell proliferation 1 wk after infection. The viability of the T cells cocultured with the HIV-1-infected macrophage cell lines or the primary monocytes as determined by propidium iodide staining was unaltered and there was no increase in apoptosis-specific DNA strand breaks or increased expression of Bcl-2 in the T cells. No soluble suppressor factor was present, since UV-inactivated supernatants from the hybridoma cell line and primary monocytes failed to inhibit mitogen- and anti-CD3-induced T cell proliferation. Early events in T cell activation, including calcium flux and phosphotyrosine kinase activity, were intact in the T cells cocultured with the HIV-1- infected hybridomas and monocytes but there was reduced IL-2 production. Addition of exogenous IL-2 restored the proliferative responses. Taken together, these data suggest that alteration of cytokine production and accessory cell function for mitogens and anti-CD3-induced T cell proliferation independent of induction of apoptosis, suppressor factor production, or inhibition of T cell signaling occurs very early after HIV-1 infection and may contribute to the global immunosuppression observed in AIDS.
23
Hagiwara, H., T. Yokota, J. Luh, F. Lee, K. Arai, N. Arai, and A. Zlotnik. "The AKR thymoma BW5147 is able to produce lymphokines when stimulated with calcium ionophore and phorbol ester." Journal of Immunology 140, no. 5 (March 1, 1988): 1561–65. http://dx.doi.org/10.4049/jimmunol.140.5.1561.
Full textAbstract:
Abstract We produced the T cell hybridoma D9C1.12.17 by fusing an IL-4-producing T cell clone D9.1Hi with the AKR thymoma BW5147. The resulting hybridoma produced IL-2 as well as IL-4 even though none of the parental cells produced IL-2 after stimulation with Con A. The production of IL-2 was confirmed at the mRNA level by using an S1 nuclease protection assay. Further analysis indicated that Con A-induced IL-2 production was a common phenomenon among T cell hybridomas derived from this fusion. Although BW5147 does not produce detectable lymphokines after Con A stimulation, this line was able to produce IL-2, granulocyte-macrophage colony stimulating factor, and small amounts of IL-3 and IFN-gamma when stimulated with calcium ionophore and phorbol ester. The latter agents are thought to mimic the activating signal(s) delivered through the Ag:MHC TCR. This observation indicates that BW5147 has the ability to produce lymphokines but may lack component(s) which couple the extracellular signal to lymphokine production, and suggests that in T cell hybridomas, part of the spectrum of lymphokines produced may be contributed by BW5147.
24
Jaffredo, T., A. F. Horwitz, C. A. Buck, P. M. Rong, and F. Dieterlen-Lievre. "Myoblast migration specifically inhibited in the chick embryo by grafted CSAT hybridoma cells secreting an anti-integrin antibody." Development 103, no. 3 (July 1, 1988): 431–46. http://dx.doi.org/10.1242/dev.103.3.431.
Full textAbstract:
We report a teratological method in which mouse hybridoma cells are grafted into a chick host. CSAT (Cell Substratum ATtachment) hybridoma was used. It produces an antibody directed against the avian integrin complex. The grafts were performed during the second and third days of incubation either at the level of the somites or in the coelom of the chick embryo. The anomalies were revealed by means of a monoclonal antibody that recognizes myogenic cells as soon as they become committed in the myotome. When embryos were grafted at the level of the somites, body wall muscles failed to develop on the side of the graft only. After coelomic grafting, total agenesis of abdominal muscles was induced. The anomalies were specific since the engraftment of three control hybridoma clones induced no change in muscle formation. These control hybridomas produce antibodies directed against the same molecular complex but not against the same epitope as CSAT. The injection of hybridoma cells in an embryo appears as a method of general interest to determine the long-term consequences of perturbing a specific developmental process.
25
Gorczynski, R. M., Z. Chen, H. Zeng, and X. M. Fu. "Specificity for in vivo graft prolongation in gamma delta T cell receptor+ hybridomas derived from mice given portal vein donor-specific preimmunization and skin allografts." Journal of Immunology 159, no. 8 (October 15, 1997): 3698–706. http://dx.doi.org/10.4049/jimmunol.159.8.3698.
Full textAbstract:
Abstract gamma delta TCR+ hybridoma cells prepared from mesenteric lymph node cells of animals receiving donor-specific immunization via the portal vein can adoptively transfer this increased graft survival to naive animals. Analysis of TCR gamma-chain junctional sequence diversity suggested that some 40 to 50% of the hybridomas expressed gamma-chain junctional sequence diversity and were stimulated to produce cytokines both by heat shock proteins and by minor histocompatibility Ag-specific irradiated peritoneal cells. The remaining gamma delta TCR+ hybridoma cells expressed TCR with a common gamma-chain junctional sequence and were stimulated to cytokine production by MHC-matched, but minor histocompatibility Ag-mismatched (as well as matched), peritoneal cells, but not by heat shock proteins. We have compared the effectiveness of representative hybridomas expressing unique gamma-chain junctional sequences or common gamma-chain junctional sequences for prolongation of donor-specific or third-party (MHC-matched or MHC-mismatched) skin grafts. Our data show a good correlation between the specificity for stimulation for cytokine production in vitro and efficacy in graft prolongation assays in vivo. Hybridoma cells expressing unique gamma-chain junctional sequences that showed Ag-specific stimulation of cytokine production in vitro and skin graft survival in vivo augmented survival of third-party skin grafts if simultaneously transplanted with both Ag-specific and third-party skin grafts. Graft prolongation in vivo using cells from either population of gamma delta TCR+ hybridomas was decreased by infusion of anti-IL-10 mAb and abolished when both anti-IL-10 and anti-TGF-beta Abs were used together.
26
Cole, B. C., B. A. Araneo, and G. J. Sullivan. "Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. IV. Murine T hybridoma cells exhibit differential accessory cell requirements for activation by M. arthritidis T cell mitogen, concanavalin A, or hen egg-white lysozyme." Journal of Immunology 136, no. 10 (May 15, 1986): 3572–78. http://dx.doi.org/10.4049/jimmunol.136.10.3572.
Full textAbstract:
Abstract A T-cell mitogen present in culture supernatants of Mycoplasma arthritidis (MAS) is known to exhibit an absolute dependence on E alpha-bearing accessory cells (AC), which appear to function by binding the mitogen. We therefore compared the specificity and nature of the AC requirements for MAS and antigen-induced production of IL 2 in T hybridoma cell lines originating from a fusion by using hen egg-white lysozyme (HEL)-specific, H-2d-restricted T blasts. A marked specificity was noted in the ability of the hybridoma lines to become activated by Con A, MAS, or HEL antigen. Thus all three lines produced IL 2 in response to Con A without the addition of B lymphoma AC. Two lines responded to MAS, but only in the presence of AC, and only one line responded to HEL antigen in the presence of AC. Using the HEL responsive T hybridoma line, we demonstrated that disrupted AC and AC membranes could present MAS but not HEL. MAS rapidly associated with AC at 4 degrees C, whereas HEL failed to do so. Paraformaldehyde-fixed AC could absorb the mitogen in MAS and present it to T hybridoma cells within several minutes, whereas HEL antigen could only be presented by fixed AC if there was a prolonged period of incubation (greater than 30 min) at 37 degrees C before fixation. The combined data indicate that metabolically active cells are not required for the association of MAS with AC or for presentation of MAS to T hybridomas. In contrast, HEL antigen requires metabolically active cells for both of these processes. Thus, the mitogen in MAS can bind to AC without any processing requirements, and it is likely that the resulting complex of mitogen and Ia molecules can directly activate T hybridoma cells.
27
Colic, Miodrag, Dragana Vucevic, Petar Popovic, and Aleksandar Dujic. "Bidirectional Interactions Between Thymocytes and Thymic Epithelial Cell Lines in Vitro." Developmental Immunology 6, no. 1-2 (1998): 71–79. http://dx.doi.org/10.1155/1998/80391.
Full textAbstract:
In vitrointeractions of thymocytes and thymocyte hybridomas with cortical (R-TNC.1) and medullary (TE-R 2.5) rat thymic epithelial-cell (TEC) lines were studied. It was found that the cortical line had better adhesion capability. It bound exclusively immature CD4+CD8+αβTCR10thymocytes, induced apoptosis of a subset of these cells, and stimulhted proliferation of the BWRT (CD4-CD8-αβTCR-) hybridoma. The medullary line bound both immature and mature thymocytes, decreased their apoptosis, and induced apoptosis of the BWRT 8 (CD4+CD810αβTCRhi) hybridoma. Thymocyte differently modulated cytokine production by TEC lines, upregulating the secretion of IL-1 by R-TNC.1 and IL-6 by TE-R 2.5 cells. Finally, coculture of thymocytes with TEC lines resulted in different patterns of protein-tyrosine phosphorylation in thymocytes. These results show the existence of mutual bidirectional interactions between thymocytes and TEC linesin vitro, but these processes differed depending on phenotypic characteristics and origin of TEC lines used.
28
Goldfien, R. D., P. J. Chen, T. J. Kipps, G. Starkebaum, J. G. Heitzmann, V. Radoux, S. Fong, and D. A. Carson. "Genetic analysis of human B cell hybridomas expressing a cross-reactive idiotype." Journal of Immunology 138, no. 3 (February 1, 1987): 940–44. http://dx.doi.org/10.4049/jimmunol.138.3.940.
Full textAbstract:
Abstract We have examined the genetic basis for the expression of a human cross-reactive idiotype (CRI) commonly found on monoclonal IgM rheumatoid factors. The CRI was identified with a monoclonal antibody (17.109) and has been localized previously to the kappa-variable region. By using the human lymphoblastoid cell line WI-L2-729-HF2, and mononuclear cells from several sources, a panel of hybridomas was generated that produced 17.109 CRI-positive Ig. A recently cloned human germ-line V kappa III gene, Humkv305, served as a probe to identify genes which were rearranged and expressed in 17.109 CRI-positive and -negative hybridomas. This probe, when hybridized to human genomic DNA under stringent conditions, identified only two to five germ-line bands. In 10 separate 17.109 CRI-positive hybridoma clones, an additional rearranged V kappa band was identified. The probe did not anneal to rearranged V kappa bands in hybridoma clones that produced kappa-chains lacking the CRI. RNA dot-blot studies provided evidence for expression of genes hybridizing to the Humkv305 probe. The results indicate that the 17.109 CRI is a serologic marker for a single V kappa gene, or a small family of closely related V kappa genes, which is identified by the Humkv305 probe.
29
Mikszta, J. A., Y. S. Jang, and B. S. Kim. "Role of a C-terminal residue of an immunodominant epitope in T cell activation and repertoire diversity." Journal of Immunology 158, no. 1 (January 1, 1997): 127–35. http://dx.doi.org/10.4049/jimmunol.158.1.127.
Full textAbstract:
Abstract In the present study, the extent of heterogeneity in the high responder T cell response to the predominant epitope region of hen egg white lysozyme (HEL46-61) was examined. Through analyses of T cell proliferation and precursor frequency, the C3H T cell response is shown not to be limited to peptides containing the previously defined minimal epitope of residues 52-61, but rather is quite heterogeneous, encompassing much of the 46-61 sequence. Further characterization using a panel of T cell hybridoma clones revealed T cell recognition of diverse minimal epitopes within this region. Interestingly, these T hybridomas could be grouped into three distinct categories based on the ability to respond to peptides with or without the native arginine residue at position 61 (61-required, 61-inhibitory, dual responders). Using analogue peptides containing single amino acid substitutions at position 61, further heterogeneity within these hybridoma groups was identified, suggesting the presence of an extremely diverse T cell repertoire for the epitope region. The charge and/or size of the C-terminal residue appears to be a critical factor for certain clones; replacement of the native arginine residue with aspartic acid or glutamic acid enabled a nonstimulatory ligand to specifically antagonize a T cell hybridoma response. Collectively, these results strongly suggest that the C-terminal residue of the predominant epitope in high responder mice plays a critical role in T cell diversity and activation.
30
Yang, Y., M. Merćep, C. F. Ware, and J. D. Ashwell. "Fas and activation-induced Fas ligand mediate apoptosis of T cell hybridomas: inhibition of Fas ligand expression by retinoic acid and glucocorticoids." Journal of Experimental Medicine 181, no. 5 (May 1, 1995): 1673–82. http://dx.doi.org/10.1084/jem.181.5.1673.
Full textAbstract:
Activation of T cell hybridomas induces a G1/S cell cycle block and apoptosis. We isolated a variant of the 2B4.11 T cell hybridoma that, when activated via the TCR, produced IL-2 and underwent growth inhibition but did not die. Analysis of a variety of cell surface molecules revealed that the variant cell line, termed VD1, expressed very low levels of Fas compared to the wild type cells. Unlike 2B4.11 cells, VD1 cells were not killed by Fas ligand (FasL)-bearing effector cells. To determine if Fas is involved in activation-induced apoptosis, two different reagents that specifically bind Fas without killing the T cell hybridomas, a monoclonal antibody and a soluble Fas:Fc chimeric molecule, were added to activated T cell hybridomas. Both treatments prevented activation-induced apoptosis in a dose-dependent manner, but had no effect on IL-2 production or growth inhibition. Northern blot analysis revealed that unactivated 2B4.11 cells expressed negligible levels of FasL mRNA, but transcripts were detectable as early as 2 h after activation and continued to increase up to 4-6 h after activation. Anti-TCR induced activation of 2B4.11 cells in the presence of a TCR- 2B4.11 variant resulted in death of the unactivated "bystander" cells, which was inhibited by anti-Fas antibodies. Finally, treatment of T hybridoma cells with 9-cis retinoic acid or glucocorticoids, which are known to prevent activation-induced T cell apoptosis, inhibited the up-regulation of FasL. We conclude that up-regulated expression of FasL and its subsequent interaction with Fas accounts for the apoptotic response of T cell hybridomas to activation, and that retinoic acid and corticosteroids inhibit activation-induced apoptosis by preventing up-regulation of FasL.
31
Kim, D. T., J. B. Rothbard, D. D. Bloom, and C. G. Fathman. "Quantitative analysis of T cell activation: role of TCR/ligand density and TCR affinity." Journal of Immunology 156, no. 8 (April 15, 1996): 2737–42. http://dx.doi.org/10.4049/jimmunol.156.8.2737.
Full textAbstract:
Abstract (B6 X A)F1 mice were immunized with sperm whale myoglobin, and T cell clones and hybridomas were generated. Hybridoma 74a.e9 was specific for the sperm whale myoglobin 67-79 peptide and could be partially activated by a peptide analogue, equine myoglobin with a natural 74G substitution. Using this hybridoma in T cell activation assays, we studied the effects of varying the avidity of the TCR for its ligand, the concentration of MHC:peptide complex on the APC, and the density of TCR on the surface. Varying ligand concentration on the surface of the APC, the TCR avidity, or the density of TCR on the T cell were equally important parameters in driving T cell activation. The mouse myoglobin (74T) analogue, however, acted as an antagonist to the T cell response. Its effectiveness was also partially determined by its ability to bind to MHC. By independently altering each of these variables and following T cell activation, we describe the interrelationships among these three components (MHC:peptide:TCR) that control the activation of the T cell.
32
Kuchroo, V. K., M. Minami, B. Diamond, and M. E. Dorf. "Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression." Journal of Immunology 141, no. 1 (July 1, 1988): 10–16. http://dx.doi.org/10.4049/jimmunol.141.1.10.
Full textAbstract:
Abstract We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.
33
Van Snick, J., A. Vink, S. Cayphas, and C. Uyttenhove. "Interleukin-HP1, a T cell-derived hybridoma growth factor that supports the in vitro growth of murine plasmacytomas." Journal of Experimental Medicine 165, no. 3 (March 1, 1987): 641–49. http://dx.doi.org/10.1084/jem.165.3.641.
Full textAbstract:
We have recently described the purification and NH2-terminal amino acid sequence of a T cell-derived hybridoma growth factor that was provisionally designated interleukin-HP1 (IL-HP1). Here we report that a T cell supernatant containing high titers of this hybridoma growth factor considerably facilitated the establishment of primary cultures of murine plasmacytomas. Most plasmacytoma cell lines derived from such cultures remained permanently dependent on IL-HP1-containing T cell supernatant for both survival and growth in vitro. These cell lines, however, retained their ability to form tumors in irradiated pristane-treated mice. Analytical fractionation of a T cell supernatant rich in IL-HP1 by either gel filtration, isoelectric focusing, or reversed-phase HPLC revealed the existence of only one plasmacytoma growth factor activity that strictly copurified with IL-HP1, strongly suggesting the identity of both factors. This conclusion was further supported by the finding that IL-HP1 purified to homogeneity supported the growth of both B cell hybridomas and plasmacytomas. For half-maximal growth, plasmacytomas, however, required a concentration of IL-HP1 of approximately 30 pM, which is approximately 200 times higher than that required by B cell hybridomas. A clear difference in the specificity of IL-HP1 and B cell stimulatory factor 1 (BSF-1) was demonstrated by the finding that IL-HP1-dependent plasmacytomas did not survive in the presence of BSF-1, whereas helper T cell lines that proliferated in the presence of BSF-1 failed to respond to IL-HP1.
34
Ashwell, J. D., R. E. Cunningham, P. D. Noguchi, and D. Hernandez. "Cell growth cycle block of T cell hybridomas upon activation with antigen." Journal of Experimental Medicine 165, no. 1 (January 1, 1987): 173–94. http://dx.doi.org/10.1084/jem.165.1.173.
Full textAbstract:
Stimulation of antigen-specific T cell hybridomas with the appropriate antigen/MHC combination, at concentrations that resulted in the secretion of the lymphokine interleukin 2, resulted in a dose-dependent decrease in both [3H]thymidine incorporation and cell growth. Flow cytometric studies demonstrated that stimulation with antigen resulted in a cell cycle block that was most evident at the G1/S border, and mixing studies revealed that bystander T cells of different antigen specificities were not affected. For at least the large majority of T cells, the G1/S cell cycle block appeared to be irreversible after 24 h of exposure to antigen. This cell cycle block may be useful as a rapid and quantitative measure of T cell hybridoma activation, as a means of selecting T cell hybridomas that have functional alterations in the reception of stimulatory signals, and may serve as a model of the induction of tolerance in immature T cells.
35
Nakano, T., Y. Ishii, and K. Ishizaka. "Biochemical characterization of antigen-specific glycosylation-inhibiting factor from antigen-specific suppressor T cells. I. Identification of a 55-kilodalton glycosylation-inhibiting factor peptide with TCR alpha-chain determinant." Journal of Immunology 156, no. 5 (March 1, 1996): 1728–34. http://dx.doi.org/10.4049/jimmunol.156.5.1728.
Full textAbstract:
Abstract Stimulation of OVA-specific suppressor T cell (Ts) hybridoma and bee venom phospholipase A2 (PLA2)-specific Ts hybridoma with Ag-pulsed APC or by cross-linking of CD3 resulted in the formation of Ag-specific glycosylation-inhibiting factor (GIF). Affinity-purified Ag-specific GIF preparations, obtained by using Ag-coupled Sepharose or anti-TCR alpha-chain-coupled Affi-Gel, contained a 55-kDa peptide, which bound both polyclonal anti-GIF Abs and anti-TCR-alpha mAb in immunoblotting. The same hybridomas constitutively secrete 13-kDa bioactive GIF peptide that has no affinity for homologous Ag, but neither the Ag-specific GIF activity nor 55-kDa GIF peptide was detectable in culture supernatants of unstimulated cells. Northern blot analysis of mRNA from the anti-CD3-stimulated hybridoma with 32P-labeled GIF cDNA revealed only 0.6 kb mRNA, which encodes the 13-kDa nonspecific GIF. No mRNA of the 55-kDa GIF was detectable. A representative OVA-specific Th hybridoma, DO 11.10 cells contain the 0.6 kb GIF mRNA and constitutively secrete inactive GIF peptide. However, the Th hybridoma failed to secrete the 55-kDa peptide or any peptide with the TCR-alpha determinant upon stimulation with anti-CD3. It appears that the formation of the 55-kDa peptide with the TCR-alpha determinant is unique for a subset of T cells including Ts cells that form bioactive GIF.
36
Uchida, T., S. Ju, A. Fay, Y. Liu, and M. E. Dorf. "Functional analysis of macrophage hybridomas. I. Production and initial characterization." Journal of Immunology 134, no. 2 (February 1, 1985): 772–78. http://dx.doi.org/10.4049/jimmunol.134.2.772.
Full textAbstract:
Abstract A series of macrophage hybridomas were generated by fusion of splenic adherent cells with P388D1 tumor cells. Forty-two cell lines were established, and each was cloned by limiting dilution. Six clones that exemplified the spectrum of macrophage heterogeneity were selected for further analysis. Qualitative and quantitative differences in phenotype and functional activity were noted. Some clones constitutively expressed Ia antigens, whereas others only expressed detectable levels of Ia after lymphokine activation. The level of antigen-presenting activity generally correlated with the level of Ia expression. Furthermore, interclonal differences were noted in the levels of receptor-mediated phagocytosis and IL 1 secretion. Generally, the hybridoma clones maintained stable phenotypic and functional properties during approximately 1 yr of continuous in vitro culture. These cloned hybridoma cell lines represent a useful resource to analyze macrophage biology and to dissect structure and function relationships.
37
Buzás, E. I., F. R. Brennan, K. Mikecz, M. Garzó, G. Negroiu, K. Holló, G. Cs-Szabó, E. Pintye, and T. T. Glant. "A proteoglycan (aggrecan)-specific T cell hybridoma induces arthritis in BALB/c mice." Journal of Immunology 155, no. 5 (September 1, 1995): 2679–87. http://dx.doi.org/10.4049/jimmunol.155.5.2679.
Full textAbstract:
Abstract Aggrecan, the high buoyant density cartilage proteoglycan (PG), has been shown to induce progressive polyarthritis and ankylosing spondylitis in genetically susceptible BALB/c mice. To further characterize the nature of the autopathogenic effector T cells operating in these mice and to determine the region(s) of the PG molecule recognized by these T cells, we generated PG-specific T cell hybridomas from arthritic mice. One of the PG-specific T cell hybridomas (5/4E8), when injected into naive irradiated BALB/c mice, was capable of inducing clinical and histopathologic signs of arthritis. Massive swelling and redness of the paws dominated the clinical picture. A reactive synovial cell proliferation, the accumulation of hybridoma and inflammatory cells in the enlarged joint space, the loss of PG from the superficial layer of the articular cartilage, and the erosion of articular surface were identical histopathologic signs to those found either in primary or adoptive transfer of PG-induced arthritis. The PG-specific and arthritogenic T cell hybridoma (5/4E8) expressed TCR-alpha beta + (V beta 4), CD4+, and CD8- phenotypes and belonged to the Th1 subset, as the cells secreted IL-2 and IFN-gamma, but not IL-4 upon PG stimulation, and the response was MHC class II (I-Ad)-restricted. These observations provide direct evidence that PG-specific Th cells play crucial roles in autoimmune arthritic processes.
38
Jayaraman, S., C. A. Martin, and M. E. Dorf. "Enhancement of in vivo cell-mediated immune responses by three distinct cytokines." Journal of Immunology 144, no. 3 (February 1, 1990): 942–51. http://dx.doi.org/10.4049/jimmunol.144.3.942.
Full textAbstract:
Abstract The ability of recombinant/purified cytokines to augment delayed-type hypersensitivity (DTH) responses was investigated. Suboptimal doses of haptenized SC were treated in vitro with purified or recombinant derived cytokines and tested for their ability to enhance DTH in vivo. With the use of this protocol, it was shown that both human and mouse rIL-6, as well as mouse rTNF-alpha, potentiated DTH in a dose-dependent manner. In accordance with these data, IL-6/TNF-alpha-containing supernatant from long term nonlymphoid cell lines also possessed the ability to augment DTH. By using the same protocol, we have also identified T cell hybridomas that produce DTH-augmenting activity constitutively. The hybridoma-derived factor, termed the T cell enhancing factor (TCEF), was functionally distinguishable from the defined cytokines IL-1 through IL-6, IFN-gamma, and TNF by bioassay. Furthermore, RNA derived from the hybridoma failed to hybridize with cDNA probes specific for IL-1 to IL-6, IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF. Further characterization of the serum-free conditioned media derived from the hybridoma indicated that the TCEF was a soluble acid labile glycoprotein (Mr greater than 30,000). Finally, we investigated the cellular requirements for DTH augmentation by IL-6, TNF-alpha, and TCEF; all are dependent upon the presence of T cells in the immunizing inoculum. We propose that these cytokines play a critical role in the development of DTH responses in vivo.
39
Martel, F., R. Bazin, S. Verrette, and R. Lemieux. "Characterization of higher avidity monoclonal antibodies produced by murine B-cell hybridoma variants selected for increased antigen binding of membrane Ig." Journal of Immunology 141, no. 5 (September 1, 1988): 1624–29. http://dx.doi.org/10.4049/jimmunol.141.5.1624.
Full textAbstract:
Abstract Somatic mutation in the Ig genes plays a major role in the increase of antibody affinity observed in secondary immunologic responses. It has been shown that the mechanism responsible for the high rate of somatic mutation in the Ig genes was active not only in normal B lymphocytes but also in B-cell hybridomas secreting mAb. Also, it has been reported that B-cell hybridomas were positive for membrane Ig of the same specificity as the secreted mAb. The presence of membrane Ig suggested that somatic variants secreting mAb of higher affinity could be selected by the increased capacity of these hybridoma cells to bind immobilized Ag. This hypothesis was tested with hybridoma cells secreting an IgM mAb reacting with the A Ag of the ABO blood group system. In two selection experiments, we have isolated several variant cell lines secreting mAb of increased avidity for the A Ag under similar IgM concentrations. Biochemical characterization of one of the variant mAb indicated that the mutation responsible for the increased avidity has occurred in the heavy chain gene. The method developed may have profound implications for the diagnostic and therapeutic use of mAb and will permit the study, in an in vitro system, of the role of somatic mutations in antibody diversity.
40
Yokoyama, W. M., F. Koning, G. Stingl, J. A. Bluestone, J. E. Coligan, and E. M. Shevach. "Production of a T cell hybridoma that expresses the T cell receptor gamma/delta heterodimer." Journal of Experimental Medicine 165, no. 6 (June 1, 1987): 1725–30. http://dx.doi.org/10.1084/jem.165.6.1725.
Full textAbstract:
We have produced a T cell hybridoma line by fusion of an IL-2-dependent, long-term T cell receptor (TCR) gamma/delta+ Thy-1+, bone marrow-derived, dendritic epidermal cell line to the BW5147 tumor line. The resultant hybridoma was rapidly growing, lymphokine independent, and expressed T3 in association with the TCR gamma/delta heterodimer. Several subclones of the hybridoma line produced easily detectable levels of IL-2 after stimulation by anti-T3 or Con A. The availability of these cloned cell lines should greatly facilitate further functional, biochemical, and molecular studies of the TCR delta chain.
41
Vidard, L., K. L. Rock, and B. Benacerraf. "The generation of immunogenic peptides can be selectively increased or decreased by proteolytic enzyme inhibitors." Journal of Immunology 147, no. 6 (September 15, 1991): 1786–91. http://dx.doi.org/10.4049/jimmunol.147.6.1786.
Full textAbstract:
Abstract The ability of splenic APC and a B cell hybridoma (LS.102.9) to process and present OVA to a panel of T-T hybridomas with different fine specificities was investigated. Splenic APC process and present OVA to all the T-T hybrids. The B cell hybridoma could similarly process and present OVA to some T-T hybrids but was very inefficient in stimulating two of the T cell hybridomas. The presentation of native OVA to these two T-T hybrids was significantly increased by leupeptin. Pulsing experiments demonstrated that leupeptin acted on the APC at a step before the processed Ag was displayed on the cell surface in association with MHC molecules. Leupeptin has no effect on the presentation of OVA peptides by LS.102.9 to the T-T hybrids. Leupeptin inhibits the generation of the epitopes of OVA that LS.102.9 produces under basal conditions. We also surveyed the effect of other protease inhibitors and observed similar augmenting and inhibitory effects on the presentation of selected OVA epitopes. The augmentation of processing by a protease inhibitor indicates that in the lysosomal/endosomal compartment proteases have capacity to both generate and destroy immunogenic peptides. Our data suggest that protease inhibitors could potentially be used as immunomodulators and are discussed in terms of physiology of the lysosomal/endosomal compartment.
42
Makoul, G. T., D. R. Robinson, A. K. Bhalla, and L. H. Glimcher. "Prostaglandin E2 inhibits the activation of cloned T cell hybridomas." Journal of Immunology 134, no. 4 (April 1, 1985): 2645–50. http://dx.doi.org/10.4049/jimmunol.134.4.2645.
Full textAbstract:
Abstract To minimize complicating interactions inherent in heterogeneous cell populations, we used a panel of cloned murine autoreactive (E8.A1) and antigen-specific (HEL.C10, HEL.B14) T cell hybridomas to examine the effect of prostaglandin E2 (PGE2) on T cell activation. These T cells secrete interleukin 2 (IL 2) when co-cultured with a cloned population of I region-matched stimulator cells (TA3), or with mitogenic signals in the absence of TA3 stimulator cells. Physiologic concentrations of PGE2 inhibited the induction of IL 2 secretion by the T cell hybridomas tested, when they were activated either by TA3 cells or by mitogenic signals. IL 2 production was inhibited in a dose-dependent manner by concentrations of PGE2 between 10(-7) and 10(-11) M, with 50% inhibition occurring at 10(-10) M. Pretreatment of the T hybridoma cells with 10(-7) M PGE2 for 1 hr before culture also resulted in marked inhibition of IL 2 secretion. Similar pretreatment of the TA3 cells did not affect their ability to activate the T cell hybridomas. PGE2 at 10(-8) M induced a 30-fold increase in cAMP levels within 25 min of addition to culture of the E8.A1 T cell hybridoma, but caused no significant elevation of cAMP levels in TA3 cells. The direct addition of dibutyryl cAMP (dcAMP) to cultures of E8.A1 cells resulted in marked inhibition of IL 2 secretion when stimulated by TA3 or by mitogenic signals, with an average of 80% inhibition occurring at 10(-4) M dcAMP. PGE2 and dcAMP also inhibited the growth of E8.A1 cells. Initially, cell growth was virtually halted, but began to recover between 24 and 48 hr after the addition of either PGE2 or dcAMP. Neither PGE2 nor dcAMP inhibited the division of TA3 cells. High affinity binding sites for PGE2 were detected in the E8.A1 T cell hybridomas with an apparent Kd of 7.6 X 10(-10) M, which is consistent with the functional data. No specific binding was detected in the TA3 stimulator cells. These findings suggest that the immunosuppressive effects of PGE2 are localized to the T cell, are receptor regulated, and may be mediated by the associated increase of cAMP levels in the T cell hybridomas.
43
Maekawa, S., and Z. Ovary. "Studies on immunity in hybridoma-bearing mice. B. Immunity against the hybridoma. II. The phenotype and specificity of the immune cell." Journal of Immunology 136, no. 3 (February 1, 1986): 1131–35. http://dx.doi.org/10.4049/jimmunol.136.3.1131.
Full textAbstract:
Abstract When appropriate numbers of anti-dinitrophenyl (DNP) immunoglobulin (Ig) E-secreting hybridoma (B 53) cells were injected s.c. into normal BALB/c mice, some of the recipients rejected the tumors. These mice were shown to be immune to B 53 as they withstood, without any ill effect, the i.p. injection of lethal doses of B 53 cells. In previous studies, it was shown that the spleen cells of these mice protected against the growth of B 53 cells. In this study, the characteristics and specificity of the immune spleen cells were examined. The cells responsible for this immunity were shown to be T cells that express Ly-2 on their surface. These cells were shown in in vivo and in vitro assays to limit the growth of the immunizing hybridoma, as well as some but not all BALB/c plasmacytomas and hybridomas.
44
Graziano, R. F., and M. W. Fanger. "Human monocyte-mediated cytotoxicity: the use of Ig-bearing hybridomas as target cells to detect trigger molecules on the monocyte cell surface." Journal of Immunology 138, no. 3 (February 1, 1987): 945–50. http://dx.doi.org/10.4049/jimmunol.138.3.945.
Full textAbstract:
Abstract We recently reported the preparation and characterization of a monoclonal antibody, 32.2, specific for the high-affinity Fc receptor (FcR) for IgG on human monocytes. We have utilized the hybridoma cell line producing this antibody as a target for monocyte-mediated cytotoxicity. The hybridoma was selected for stable sublines that expressed high quantities of surface 32.2 immunoglobulin (Ig) through flow cytometry. Monocyte-mediated cytotoxicity, with these sublines used as targets, was evaluated with the use of a 51Cr-release assay. It was found that monocytes could efficiently lyse the hybridoma cells (HC 32.2) bearing surface Ig directed to the high-affinity FcR. Consistent with the specificity of the 32.2 antibody for an epitope on the high-affinity receptor outside of the ligand binding site, human IgG did not block monocyte killing of HC 32.2. In contrast, monocytes could not mediate lysis of hybridoma cells bearing high levels of antibody directed to other monocyte cell surface molecules, in particular, class I MHC molecules, the C3bi receptor, and the My 23 antigen. The effect of IFN-gamma on the ability of monocytes to mediate lysis of the 32.2 Ig-bearing hybridomas was also assessed. Monocytes cultured in the absence of IFN-gamma could lyse the hybridoma line expressing high levels of 32.2 Ig as efficiently as monocytes cultured in the presence of IFN-gamma. However, untreated monocytes were less able than IFN-gamma-treated monocytes to kill HC 32.2 expressing lower levels of Ig. Thus, IFN-gamma may enhance the efficiency of monocyte-mediated antibody-dependent killing under conditions where limited antibody is available on the target. These studies demonstrate that the high-affinity FcR on monocytes can act as a cytotoxic trigger molecule for killing of tumor cell targets and that this trigger does not require specific binding to the Fc binding epitope. These results further encourage possible clinical application of the 32.2 monoclonal antibody in tumor therapy.
45
Sherr, E., D. C. Adelman, A. Saxon, M. Gilly, R. Wall, and N. Sidell. "Retinoic acid induces the differentiation of B cell hybridomas from patients with common variable immunodeficiency." Journal of Experimental Medicine 168, no. 1 (July 1, 1988): 55–71. http://dx.doi.org/10.1084/jem.168.1.55.
Full textAbstract:
Human-human B cell hybridomas constructed from B lymphocytes of common variable immunodeficiency (CVI) patients and the nonsecreting cell line WIL2/729 HF consistently secrete low levels of Ig and appear to retain a defect characteristic of the CVI patient's B cells. We assessed the differentiative capacity of retinoic acid (RA) on these hybridomas, as well as on hybridomas constructed from normal B cells and from patients with selective IgA deficiency. RA at concentrations varying between 10(-5) and 10(-9) M augmented IgM secretion 4-20-fold from four of four CVI hybridomas tested, but did not affect Ig secretion from normal or IgA-deficiency hybridomas. In support of this elevated Ig secretion, RA enhanced the de novo synthesis of biosynthetically labeled light (kappa) and heavy (mu) Ig (up to 4- and 15-fold, respectively) in the CVI hybridoma line JK32.1. The increase in IgM synthesis/secretion could not be accounted for by RA-induced alteration in the cell cycle. In inducing this increase in IgM production, RA was found to affect two aspects of Ig gene expression: (a) the steady-state levels of heavy and light chain mRNAs were enhanced, and (b) the processing of mu heavy chain transcripts to the secreted mRNA form became favored over the membrane mRNA form. We also show that expression of Leu-17 (CD38), a surface marker that is re-expressed in the late pre-plasma stage of B cell development, was increased by RA from less than 20% to greater than 90% of the total cell population, with a concomitant 4-10-fold augmentation in the mean fluorescence intensity. Changes in both Leu-17 expression and de novo Ig synthesis were prominent by 24 h, but could be observed as early as 8 h after induction. Taken together, our study demonstrates that RA affects a marked alteration in the differentiated state of the CVI hybridoma clones. This finding suggests that retinoids can enhance the functional capabilities of B cells with defects in maturation and support further studies to evaluate their clinical potential in CVI.
46
Wolff, H. L., S. J. Burakoff, and B. E. Bierer. "Functional CD2 mutants unable to bind to, or be stimulated by, LFA-3." Journal of Immunology 144, no. 4 (February 15, 1990): 1215–20. http://dx.doi.org/10.4049/jimmunol.144.4.1215.
Full textAbstract:
Abstract To define epitopes on the CD2 (T11, the T cell erythrocyte receptor) molecule that are necessary for interaction with lymphocyte function-associated Ag-3 (LFA-3), we have expressed the human wild-type CD2 cDNA and mutant CD2 cDNA in a murine Ag-specific T cell hybridoma that responds to human HLA-DR Ag. Here we have expressed mutations at amino acid 91 and 92 of CD2 in the T cell hybridoma. The mutated CD2 molecules were functional in that pairs of anti-CD2 mAb that continued to bind were able to stimulate IL-2 production by the hybridomas. However, CD2 mutants with either the 91 or 92 amino acid substitution had lost the ability to bind to or be activated by either SRBC, which bear an LFA-3 homologue, or by murine L cells expressing human LFA-3. Unlike hybridomas expressing the wild-type CD2 molecule, there was no enhanced response to Ag stimulation. Taken together, these data suggest that the mutated CD2 molecules were no longer able to bind to, or to utilize, LFA-3 for activation. We have previously demonstrated that a mutation at amino acid 51 of CD2 results in loss of binding to LFA-3. Whether these two regions of CD2, discrete and separable by amino acid sequence, form one or more binding sites for LFA-3 remains to be determined.
47
Owhashi, M., and E. Heber-Katz. "Protection from experimental allergic encephalomyelitis conferred by a monoclonal antibody directed against a shared idiotype on rat T cell receptors specific for myelin basic protein." Journal of Experimental Medicine 168, no. 6 (December 1, 1988): 2153–64. http://dx.doi.org/10.1084/jem.168.6.2153.
Full textAbstract:
Immunizing Lewis rats with guinea pig myelin basic protein (MBP) yielded an encephalitogen specific, Ia-restricted, rat-mouse T cell hybridoma 5.10, which was used to establish a clonotypic mAb (10.18) that binds to and precipitates the rat TCR. By two-dimensional gel electrophoresis, the rat TCR was shown to consist of two disulfide-linked peptide chains with mol wt of 48,000 and 39,000. 10.18 binds the majority of cells in MBP-specific T cell lines that are capable of transferring experimental allergic encephalomyelitis (EAE) to Lewis rat recipients, but does not bind to either a purified protein derivative of tuberculin-specific cell line or an OVA-specific line. Furthermore, soluble 10.18 can block antigen-specific stimulation of hybridoma 5.10 but cannot control hybridomas, while immobilized 10.18 stimulates 5.10, but cannot control the hybrids. Though 10.18+ cells are very rare in normal rats, increase of 10.18+ cells is observed in MBP-primed paralyzed rats. Finally, when 10.18 is injected into MBP-primed Lewis rats, EAE is abrogated. We have thus characterized EAE as a "mono-idiotypic" autoimmune disease.
48
Minami, M., H. Kawasaki, S. Taira, and H. Nariuchi. "Alloantigen presentation by B cells: two types of alloreactive T cell hybridomas, B cell-reactive and B cell-nonreactive." Journal of Immunology 135, no. 1 (July 1, 1985): 111–16. http://dx.doi.org/10.4049/jimmunol.135.1.111.
Full textAbstract:
Abstract T cell-depleted, Sephadex G-10-passed unstimulated splenic B cells from C57BL/6 mice stimulated splenic T cells from CKB mice to produce IL 2 and to proliferate. The stimulatory ability of the unstimulated B cells was eliminated by 4000 rad irradiation of the unstimulated stimulator B cells. LPS-activated B cells could stimulate responder T cells more efficiently than unstimulated B cells. For further analysis of allostimulation by B cells, we established a series of alloreactive T cell hybridomas. Forty-five percent of these alloreactive T cell hybridomas could be stimulated to produce IL 2 by either macrophage-dendritic cells or unstimulated B cells. Fifty-five percent of these alloreactive T cell hybridomas could be stimulated by macrophage-dendritic cells but not by unstimulated B cells. T cell hybridomas that were not reactive with unstimulated B cells were also nonreactive to LPS-activated B cells. Analysis of two representative I-Ab-reactive T cell hybridoma clones, B cell-reactive clone CB-11.4 and B cell-nonreactive clone HTB-9.3, revealed again that the stimulatory ability of unstimulated B cells was sensitive to 4000 rad irradiation in the activation of CB-11.4 clone and that CB-11.4 could be stimulated more efficiently by LPS-activated B cells than by unstimulated B cells, but HTB-9.3 could not be stimulated by LPS-activated B cells. Thus, there may be two distinct types of T cells in the alloreaction: B-cell-reactive and B cell-nonreactive.
49
Kanagawa, O., M. E. Wiebenga, and B. A. Vaupel. "Defective T cell receptor-mediated signaling and differential induction of T cell functions by murine AIDS virus superantigen." Journal of Immunology 150, no. 5 (March 1, 1993): 1865–72. http://dx.doi.org/10.4049/jimmunol.150.5.1865.
Full textAbstract:
Abstract A B cell line, B6-1710, expressing murine AIDS virus superantigen stimulates T cell hybridomas derived from a Ld-specific CTL clone to produce IL-2 regardless of their CD4/CD8 phenotype. However, B6-1710 cell did not stimulate the original CTL clone, L3, in either proliferation or cytolytic assays. Both B6-1710 and Ld+ P815 cells stimulated a significant Ca2+ influx in the T cell hybridomas. In contrast, only P815 stimulated tyrosine phosphorylation of a 19-kDa protein in the T cell hybridoma. The addition of the nonspecific protein kinase C activator PMA restored the proliferative response of L3 cells to B6-1710. PMA did not induce the lysis of B6-1710 cells by L3. These results suggest that murine AIDS virus encoded superantigen elicits a TCR-mediated signal different from that caused by nominal Ag. This different signaling by viral superantigen may be important for the development of viral pathogenesis in vivo.
50
Alhoderi, Jamila. "Generation of T cell hybridoma as a technique for study the immune response against bacterial infections." Journal of Pure & Applied Sciences 22, no. 3 (October 2, 2023): 185–90. http://dx.doi.org/10.51984/jopas.v22i3.2769.
Full textAbstract:
Study of the acquired immune responses against microbial infection has a high importance, as it is the fundamental basis of designing the vaccines against microbes and production of specific antibodies against infections. Different methods have been established to study the immune responses against infections using different types of immune cells. The main cells of acquired immune system are T cells which generate cellular immune response and B cells that produce humoral immune response. One of the cellular techniques can be generated, as a continues cell system is called a hybridoma. This cell system is used to study the immune responses of T and B cells of the immune system. It can be generated from T cell lines to study cellular immunity, and is called T cell hybridoma, or using B cell lines to study humoral immunity, and is called B cell hybridoma. Generation of T cell hybridoma ( a fusion between antigen-specific primary T cells with an immortal thymoma line) is a significant technique to demonstrate the mechanisms of antigen presenting to T cells. In addition, it is a basic technique for production of monoclonal antibody based on the fusion of B cell lines ( i.e., B cell hybridoma). This article aims to present the procedure of generating T cell hybridoma and its application to study the cellular immune response against M5 protein of Streptococcus pyogenes ( group A Streptococcus - GAS ) as a practical example. It is an important issue to highlight the methods of such successful technique.