Extrathymic acquisition of tolerance by T lymphocytes.
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Animals↗
Biomedical subjects
G Morahan
Publications and source records attributed to G Morahan.
T-cell tolerance in transgenic mice expressing major histocompatibility class I molecules in defined tissues.
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Animals↗
Transgenic models of tolerance and autoimmunity.
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Animals↗
Expression of class I histocompatibility antigens in defined tissues: effects on T cell function.
We have generated transgenic mice by introducing into fertilized eggs the major histocompatibility complex class I gene, H-2Kb, linked to various promoters that target gene expression to particular tissues. In one system the gene was expressed in the medullary epithelial cells of the thymus; these were unable to impose tolerance on differentiating T lymphocytes. These findings, together with those of other workers who showed that epithelial thymus grafts can impose tolerance, provide clear evidence of functional heterogeneity among thymus epithelial cells. In other transgenic models the gene was expressed in nonlymphoid tissues, such as the pancreatic islet beta cells or the hepatocytes, exocrine pancreas and kidney tubules. In all these cases the tissues were not subjected to autoimmune attack and the animals were specifically tolerant of H-2Kb-bearing cells in vivo, although some could generate cytotoxic T lymphocytes in vitro. Hence, although the intrathymic environment may be the dominant site for negative selection, it is clear that ancillary mechanisms exist in the periphery to ensure that tolerance is achieved to antigens not synthesized in the thymus.
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Diabetes in transgenic mice resulting from over-expression of class I histocompatibility molecules in pancreatic beta cells.
A class I histocompatibility gene, H-2Kb, linked to the rat insulin promoter, is overexpressed in the pancreatic beta cells of transgenic mice. The mice, whether syngeneic or allogeneic to the transgene, develop insulin dependent diabetes without detectable T cell infiltration, suggesting a direct, non-immune role for the transgenic class I molecules in the disease process.
Animals↗
Expression of T cell antigen receptor and immunoglobulin genes in lymphoid organs visualized by in situ hybridization.
In situ hybridization techniques were used to detect expression within lymphoid tissues of genes encoding T cell receptor (TCR) alpha, beta and gamma chains, as well as immunoglobulin kappa light chain. Transcripts of these genes were specifically detected in frozen sections of thymus, spleen and lymph node but not in non-lymphoid tissues. Differences in the level of beta chain gene transcription were observed within and between the thymus cortex and medulla, with approximately 60% and 34% of cells in these areas labelled, respectively. Expression of the TCR alpha chain genes was more homogeneous, while amongst the cells transcribing the gamma chain gene, there was a subpopulation of 0.2% of heavily labelled cortical thymocytes. Labelling of T cell dependent areas of spleen and lymph nodes was observed with each of the TCR probes, but neither alpha nor beta gene expression was seen in lymphoid tissues of athymic nude mice. The gamma chain gene was, however, expressed in both spleen and lymph nodes of these mice. These results indicate that TCR gene expression is not limited to a small subpopulation of T cell precursors. They are discussed in relation to T cell differentiation within the thymus.
Animals↗
Assays to detect monoclonal anti-idiotypic antibodies.
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Antibodies, Anti-Idiotypic↗
Responses in mice to Sj26, a glutathione S-transferase of Schistosoma japonicum worms.
The genetic variation in antibody responses of mice to glutathione S-transferase (GST) enzymes of Schistosoma japonicum worms, and in particular to a Mr 26,000 species termed Sj26, was analysed. Sera from infected mice, or mice immunized with adjuvant and an Sj26 beta-galactosidase fusion protein produced in Escherichia coli (Sj26FP), or purified near-native recombinant Sj26 produced in E. coli (rSj26), were assayed by enzyme-linked immunosorbent assay (ELISA) for antibody titres to GST purified from adult worms. Anti-GST antibody levels are high in a mouse strain, WEHI 129/J, that is genetically resistant to infection with S. japonicum. Antibody responses to GST are low in BALB/c mice and intermediate in most other mouse strains analysed such as CBA/H and C57B1/6. Responsiveness to Sj26 in adjuvant is dominant in (BALB/c x WEHI 129/J)F1 hybrids. BALB/c.H-2b and BALB/c.H-2k mice are higher responders than BALB/c. One feature of antibody responses to Sj26 is the variability within a group of mice. When rSj26 conjugated to the hapten azobenzenearsonate was used as immunogen, BALB/c mice produced substantial amounts of anti-Sj26 antibodies. In an attempt to correlate antibody levels with resistance in infected mice, a new functional assay was devised to measure the ability of sera to inhibit the binding of rSj26 to glutathione. However, there was no correlation between inhibitory titre in this assay and the numbers of worms recovered. In regard to the candidacy of GST as a vaccinating antigen in schistosomiasis japonica, the data raise the problem of variable responsiveness to the antigen that will need to be overcome by antigen modification and/or powerful adjuvants.
Animals↗
A sensitive radioimmunoassay for the detection of monoclonal anti-idiotope antibodies.
A radioimmunoassay was developed in order to detect anti-idiotypic antibodies in the supernatants of hybrid cells. This assay is both sensitive and specific for anti-idiotypic (but not anti-allotypic) antibodies. Monoclonal antibodies present in test supernatants are bound by an anti-immunoglobulin coated solid phase. Subsequent incubation with a source of mouse immunoglobulin 'blocks' unreacted anti-immunoglobulin antibodies on the solid phase. Anti-idiotypic antibodies are then detected by their ability to bind 125I-labelled idiotype-bearing antibody. This paper describes the use of this assay to detect monoclonal anti-idiotypic antibodies in 2 systems: the cross-reactive idiotype of A/J anti-ABA antibodies, and the idiotype expressed by the myeloma protein HOPC 8. Similarly, 125I-labelled anti-idiotype antibodies may be used in this assay to detect monoclonal idiotype-bearing antibodies. Further modifications are described which would allow the detection of monoclonal anti-allotype antibodies.
Animals↗
An idiotypic determinant formed by both immunoglobulin constant and variable regions.
Immunoglobulin idiotypes are serologically defined determinants associated with the variable (V) region of antibody molecules (reviewed in refs 1-4). One of the best defined idiotype systems is that borne by the phosphorylcholine (PC)-binding IgA proteins TEPC15 (T15) and HOPC8 (H8). The T15 idiotype, defined by sera raised in A strain mice, or in rabbits, is considered identical to that expressed by the majority of BALB/c anti-PC antibodies. To define the idiotypic determinants (idiotopes) of which the T15 idiotype is comprised, monoclonal anti-T15 antibodies were used here to examine both serum and monoclonal anti-PC antibodies. The latter were found to differ from T15 with respect to the idiotope defined by the monoclonal anti-idiotope antibody, 21A5, in that the '21A5 idiotope' was absent from anti-PC sera; of the monoclonal anti-PC antibodies examined, only those which were both T15+ and of the IgA isotype seemed to express this idiotype fully. This result suggests that not only the V region, but also the constant (C) region, of the immunoglobulin molecule can contribute to the formation of an idiotypic determinant. Isotype-restricted idiotopes may be involved in the regulation of antibody responses of particular classes.
Animals↗
Production of RNA for secreted immunoglobulin mu chains does not require transcriptional termination 5' to the microM exons.
The mouse immunoglobulin mu gene encodes both secreted and surface-bound mu heavy chains produced by cells of the B lymphoid series. Transcripts of the mu gene are processed into mu mRNA species which differ at their 3' termini, bearing either 'microsecond' or 'microM' segments, distinguishing secreted and cell-membrane-bound mu polypeptides. During maturation of surface IgM-bearing B cells to IgM-secreting plasma cells, both the total amount of mu mRNA and the ratio of microsecond- to microM-terminated mRNA increase greatly. Two possible mechanisms for the developmental regulation of 3' RNA processing cannot yet be distinguished. One mechanism would yield the microsecond terminus by specific cleavage of a common presursor transcript encompassing both microsecond and the microM exons (Fig. 1), the other by regulated termination of transcription upstream from the microM exons. While the first mechanism would produce, as a by-product, RNA fragments containing microM exons, the second would not. We report here the detection of such microM fragments in cells producing predominantly microsecond-terminated RNA species.
Base Sequence↗
Induction of suppressor T cells by monoclonal anti-idiotope antibody in strains of mice not expressing the idiotope in hyperimmune serum.
A monoclonal antibody, reactive with an idiotope present on some but not all A/J anti-azobenzenearsonate antibodies bearing the cross-reactive idiotype, was used to induce suppressor T cells. These cells suppressed the effector phase of the delayed hypersensitivity response to ABA but not to oxazolone. They could be enriched on dishes coated with ABA but not with trinitrophenylated human gamma-globulin, and the enrichment could be blocked by the anti-idiotope. Suppressor activity was also enhanced by first incubating T cells with the anti-idiotope and then collecting immunoglobulin-coated cells on dishes coated with anti-mouse immunoglobulin. The suppressor cells could be induced in A/J and CBA mice, even though hyperimmune CBA mice did not produce antibodies bearing this idiotope.
Animals↗
A monoclonal anti-idiotope antibody reacting with a determinant of the cross-reactive idiotype on anti-p-azobenzenearsonate antibodies of A/J mice.
A hybrid cell line, 14A1, was produced that secretes antibodies reacting with a monoclonal A/J anti-p-azobenzenearsonate (ABA) antibody, 7.1.3, previously shown to have all of the determinants of the cross-reactive idiotype (CRI) defined by rabbit anti-idiotype sera. 14A1 were detected using a sensitive assay that is specific for antibodies against idiotypic, but not allotypic nor isotypic, determinants. This was confirmed by demonstrating that 14A1 antibodies bind 7.1.3 but not other A/J monoclonal antibodies of the same class or CRI- monoclonal anti-ABA antibodies. The idiotypic determinant recognized by 14A1 is located at or near the hapten-binding site of 7.1.3, as ABA-conjugated tyrosine is capable of blocking this interaction. RAbbit anti-CRI antibodies are prevented from binding to 7.1.3 by the addition of 14A1, demonstrating the spatial proximity of the 14A1 idiotope with determinant(s) of the CRI. All A/J anti-ABA antibodies that have the 14A1 idiotope are also CRI+, while anti-ABA antibodies from other strains of mice to not express this idiotope. Furthermore, antibodies from A/J mice immunologically suppressed for the CRI also fail to interact with 14A1 antibodies. These results have implications both for the nature of the CRI and for the regulation of the immune response by anti-idiotype antibodies.
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Induction of delayed-type hypersensitivity to azobenzenearsonate by a monoclonal anti-idiotype antibody.
Azobenzenearsonate (ABA)-specific sensitivity was induced in A/J mice by injecting a monoclonal anti-idiotype reagent, 14A, directed against a determinant present on a minor subpopulation of immunoglobulin molecules within the anti-ABA antibodies of A/J mice. Sensitivity was transferrable by purified T cells and this was abrogated by treating the cells with 14A, rabbit anti-mouse immunoglobulin and complement, not by treatment with only the last two reagents. The transfer was restricted by the K-end of the major histocompatibility complex.
Animals↗
Monoclonal anti-azobenzenearsonate antibodies expressing the cross-reactive idiotype: immunochemical studies show that all idiotypic determinants reside on a single molecule.
Cell lines that secreted antibodies to the hapten azobenzenearsonate (ABA) were established by hybridization of immune A/J spleen cells to the non-secreting myeloma, NS-1. Solid-phase radioimmunoassays (RIA) were developed for rapid screening of hybridoma supernatants to detect antibodies to ABA and to detect antibodies bearing the ABA cross-reactive idiotype (CRI). Hybrid clones secreting both CRI+ and CRI-- anti-ABA antibodies were obtained. The supernatant from one clone (7-13) strongly inhibited binding of iodinated anti-idiotype serum in a competitive RIA. This clone expressing the CRI produced immunoglobulin of the IgG2a subclass. Solid-phase absorption of anti-idiotype serum followed by competitive radioimmunoassay analyses revealed that all the idiotypic determinants recognized by anti-idiotype serum reside on this monoclonal antibody.
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