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D Lo

Publications and source records attributed to D Lo.

At least 109 records · Page 6Linked to original sources
PubMed1989

T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E.

T-cell reactivity to the class II major histocompatibility complex I-E antigen is associated with T-cell antigen receptors containing the V beta gene segments V beta 17a and V beta 5. Mice expressing I-E with the normal tissue distribution (on B cells, macrophages, dendritic cells and thymic epithelium) induce tolerance to self I-E by clonal deletion in the thymus. By contrast, we find that transgenic INS-I-E mice that express I-E on pancreatic beta-cells, but not in the thymus or peripheral lymphoid organs, are tolerant to I-E but have not deleted V beta 5- and V beta 17a-bearing T cells. Moreover, whereas T-cell populations from nontransgenic mice proliferate in response to receptor crosslinking with V beta 5- and V beta 17a-specific antibodies, T cells from INS-I-E mice do not. Thus, our experiments provide direct evidence that T-cell tolerance by clonal paralysis does occur during normal T-cell development in vivo.

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PubMed1989

Tolerance in transgenic mice expressing class II major histocompatibility complex on pancreatic acinar cells.

To study the nature of tolerance to antigens not expressed by cells of the lymphoid system, expression of class II MHC I-E was targeted to the acinar cells of the exocrine pancreas in transgenic mice (elastase [EL]-I-E). Despite the absence of detectable I-E in the thymus of EL-I-E transgenic mice, both thymocytes and peripheral T lymphocytes were tolerant to I-E, and the pancreas was free of autoimmune infiltrates. Nontolerant T cells adoptively transferred into irradiated or T-depleted transgenic mice rapidly destroy the I-E+ components of the pancreas; however, adoptive transfer of nontolerant T lymphocytes into nonirradiated transgenic mice do not. These results suggest that tolerance in transgenic mice is maintained by some peripheral tolerance mechanism. However, further studies indicate that tolerance in transgenic mice is not maintained by specific Ts cells. For example, cell mixing experiments both in vitro and in vivo fail to reveal dominant unresponsiveness. Furthermore, nontolerant T cells injected into otherwise unmanipulated EL-I-E mice can be primed in situ (by injections of I-E+ spleen cells) to destroy the I-E+ acinar cells.

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PubMed1989

Inhibition of immunoglobulin gene rearrangement by the expression of a lambda 2 transgene.

The rearrangement of Ig genes is known to be regulated by the production of H and kappa L chains. To determine whether lambda L chains have a similar effect, transgenic mice were produced with a lambda 2 gene. It was necessary to include the H chain enhancer, since a lambda gene without the added enhancer did not result in transgene expression. The lambda 2 transgene with the H enhancer was expressed in lymphoid cells only. The majority of the B cells of newborn transgenic mice produced lambda, whereas kappa + cells were reduced. Concomitantly, serum levels of kappa and kappa mRNA were diminished. By 2 wk after birth the proportion of kappa-expressing cells was dramatically increased. Adults had reduced proportions of B cells that produced lambda only, but the levels of lambda were still higher than in normal littermates. Also, kappa + cells were still lower than in normal mice. Analysis of hybridomas revealed that reduction of kappa gene rearrangement was the basis for the decreased frequency of kappa + cells. Furthermore, many cells also contained an unrearranged H chain allele. It was concluded that feedback inhibition by the lambda 2 together with endogenous H protein may have inhibited recombinase activity in early pre-B cells, leading to inhibition of both H chain and kappa gene rearrangement. Thus, lambda 2 can replace kappa in a feedback complex. The levels of serum lambda 1 and, to a lesser degree, of spleen lambda 1 mRNA were reduced in the lambda 2 transgenic mice. However, the proportion of hybridomas with endogenous lambda gene rearrangement was at least as high as in normal mice. It was therefore concluded that the suppression of functional lambda 1 may be a consequence of decreased selection of endogenous lambda-producing cells because of the excess of transgenic lambda. The escape of kappa-producing cells from feedback inhibition may be the result of several mechanisms that operate to varying degrees, among them: (a) kappa rearrangement during a period in which the recombinase is still active after appearance of a lambda 2/mu stop signal; (b) a B cell lineage that is not feedback inhibited at the pre-B cell stage; (c) subthreshold levels of transgenic lambda 2 in some pre-B cells; and (d) loss of the lambda 2 transgenes in rare pre-B cells.

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PubMed1989

A novel MHC class II epitope expressed in thymic medulla but not cortex.

The repertoire of receptors expressed by peripheral T cells is the result of two selective events that occur during intrathymic development. Positive selection expands cells able to recognize foreign peptides presented by self MHC molecules, and negative selection eliminates cells reactive to self MHC molecules and associated self peptides. Chimaera studies suggest that, at least in the case of T cells recognizing MHC class II, interaction with thymic cortical epithelial cells is responsible for the former, whereas thymic medullary cells, of bone marrow origin, mediate the latter. This view of thymic development is supported by recent morphometric analyses, showing that autoreactive cells are found in thymic cortex but not medulla. Although numerous studies have shown that MHC class II molecules are expressed in both sites, none provides any explanation for the differential selection of T cells that is observed. Here, we describe a novel MHC class II epitope which is found on cells in thymic medulla but not cortex. The antibody to this epitope reacts with about 10% of class II molecules on B cells and may be recognizing a self peptide-MHC complex. These results provide the first evidence for differential expression of class II epitopes in different tissues and are compatible with the hypothesis that different ligands, rather than different affinity thresholds for the same ligand, are involved in positive and negative selection of the T-cell repertoire.

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PubMed1989

Selective expression of class II E alpha d gene in transgenic mice.

Class II genes of the MHC must be expressed by APC for activation of CD4+ T cells and efficient delivery of T cell help to B lymphocytes. Class II genes have restricted tissue expression and are under complex regulation. By using various deletion constructs of the class II E alpha d gene in transgenic mice we have mapped different 5' flanking regions which control E alpha d gene expression in distinct cell types. We demonstrate dissociate expression of E alpha d within the macrophage lineage as well as within the B cell lineage, and present evidence for a repressive element operative in B cells and macrophages. We describe the generation of novel transgenic lines with limited constitutive and inducible E alpha mRNA and I-E protein.

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PubMed1989

Targeted correction of a major histocompatibility class II E alpha gene by DNA microinjected into mouse eggs.

DNA molecules containing the 5' end of a functional major histocompatibility class II E alpha gene were injected into mouse eggs bearing E alpha genes with 630-base-pair (bp) deletions encompassing the promoter and first exon. The deletion was corrected by homologous recombination in 1 of about 500 transgenic mice that incorporated the injected DNA. The corrected E alpha gene was transmitted to progeny, which were bred to homozygosity. Southern blot analysis, polymerase chain reaction amplification of the DNA spanning the deletion, and sequence analysis revealed that the corrected allele resembles the wild-type E alpha gene. At sites of single-base-pair polymorphisms, there was apparently random conversion to either the donor or recipient sequence. In addition, many point mutations were introduced. mRNAs were produced from the corrected allele in a tissue-specific manner, but their sizes were different from the wild-type allele, and they did not produce detectable E alpha protein. This experiment demonstrates the feasibility of targeting foreign DNA to a gene that is completely inactive in fertilized mouse eggs.

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PubMed1989

Tolerance to class II MHC in transgenic mice.

Transgenic mice offer new possibilities in experimental techniques for understanding the familiar questions of T cell development and the selection of the T cell repertoire. As a powerful method to manipulate gene expression in the whole animal, precisely defined in vivo models can be developed. In our own studies, we have used transgenic mice with targeted expression of I-E and new monoclonal antibodies defining T cell receptors specific for class II I-E molecules. In the thymus, our results suggest that thymic epithelium has significant tolerance inducing capability, but the mechanism may be different from the clonal deletion induced by bone marrow derived cells. In the periphery, our results suggest that tolerance to tissue restricted antigens is not induced by clonal deletion. Instead, clonal paralysis may be an important mechanism for both inducing and maintaining peripheral tolerance.

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PubMed1988

Antigen presenting function of class II MHC expressing pancreatic beta cells.

Class II major histocompatibility complex (MHC) gene expression in the mouse is generally limited to thymic epithelium and bone marrow-derived cells such as B lymphocytes and cells of the macrophage/dendritic cell lineage (M phi/DC). Class II-bearing B lymphocytes and M phi/DC possess antigen presenting cell (APC) function; that is, they can stimulate T lymphocytes reactive to either antigen plus MHC or foreign MHC alone. To assess whether non-bone-marrow-derived cells can acquire APC function and elicit graft rejection through expression of class II, we studied transgenic pancreatic islet beta cells that express a foreign class II (I-E) molecule. In vivo, grafts of I-E+ transgenic islets into I-E- naive hosts are not rejected unless the host is primed by an injection of I-E+ spleen cells. In vitro, the I-E+ beta cells are unable to stimulate T lymphocytes reactive to I-E plus a peptide antigen. Paradoxically, they induce antigen specific unresponsiveness in the T cells. We propose that expression of class II on non-lymphoid cells may serve as an extrathymic mechanism for maintaining self tolerance.

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PubMed1988

Abnormal differentiation of thymocytes in mice treated with cyclosporin A.

Cyclosporin A (CsA) acts as a powerful immunosuppressive agent, and also, when given in repeated doses, can cause T-cell-dependent graft-versus-host disease and organ-specific autoimmune disease in rodents. This suggests that CsA interferes with the processes governing self-tolerance, either by nullifying the activity of T suppressor cells or by preventing the deletion of autoreactive T cells during ontogeny in the thymus. We report here that irradiated mice given repeated injections of CsA show striking dysfunction of the thymus. There are two different effects, the first of which is that CsA seems to block the differentiation of immature CD4+CD8+ thymocytes into mature CD4+CD8- and CD4-CD8+ cells expressing a high density of T-cell receptors and CD3 molecules. Second, CsA-treated mice show incomplete deletion of T cells expressing T-cell receptor molecules reactive to self H-2 I-E molecules.

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PubMed1988

The effect of thymus environment on T cell development and tolerance.

During development in the thymus, T cells are deleted if their receptors are able to recognize self major histocompatibility complex (MHC) proteins. We show that such clonal deletion can occur because of interaction between receptors on T cells and MHC expressed on bone marrow-derived cells. In addition, development in the thymus picks out T cells to mature if their receptors will be restricted for antigen recognition in association with self MHC alleles expressed on thymus epithelial cells. This process is usually thought to involve positive selection of T cells bearing receptors with high and low affinity for MHC on thymus epithelium, and subsequent deletion of high affinity cells by interaction with bone marrow-derived cells. Our data do not fit such a model, but rather suggest that MHC molecules on thymus epithelium and bone marrow-derived cells may not be seen identically by T cell receptors.

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PubMed1988

Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancreatic beta cells.

Insulin-dependent diabetes is caused by the loss of insulin-producing beta cells in pancreatic islets. It has been proposed that aberrant expression of Class II Major Histocompatibility Complex (MHC) molecules on beta cells stimulates an autoimmune attack against beta cell antigens. To test this hypothesis, we generated transgenic mice that express Class II MHC molecules (E alpha d/E beta b, or I-Eb) on beta cells. Diabetes was found in 100% of transgenic progeny from three expressing transgenic mouse lines, but without evidence for lymphocytic infiltrates. Furthermore, T lymphocytes appeared to be tolerant to the transgene I-Eb molecule, despite the absence of expression of I-Eb in the thymus or any other lymphoid tissue. The results suggest that novel expression of Class II MHC molecules on nonlymphoid cells is by itself insufficient to initiate autoimmune responses against tissue-specific antigens.

Animals↗
PubMed1988

Expression of immunoglobulin genes in transgenic mice and transfected cells.

Immunoglobulin (Ig) genes are expressed sequentially (first H-, then L-chain genes) during the development of B lymphocytes. These studies, performed with transgenic mice and transfected cells, were aimed at the regulation of turning on and off the rearrangement of Ig genes. The specific recombinase is active in pre-B cells, but not in plasma cells. Production of membrane mu, but not secreted mu or gamma-2b, turns off rearrangement of H genes. Feedback inhibition of kappa-gene rearrangement requires kappa and membrane mu. Kappa alone or in combination with secreted mu does not stop recombination. Mouse lambda genes were mapped by deletion analysis and pulsed-field gel electrophoresis. The gene order is V2-C2,4-V1-C3,C1. The distance between V2 and C2 is 74 kb, but that between V1 and C3, 1 is only 20 kb. V2 and C3, 1 are over 190 kb apart. Lambda genes appear to be rearranged in a subset of B cells that do not respond to feedback inhibition at the pre-B cell stage. Lambda and kappa genes are both rearranged and potentially functional in these cells. Kappa genes may then be deleted by recombination of a sequence (described by Selsing and Siminovitch et al.) downstream of C-kappa with sequences upstream of C-kappa. Presumably the recombinase is eventually inactivated in kappa-lambda cells by a mechanism that is different from H-kappa feedback.

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PubMed1986

T cell specificity in twice-irradiated F1----parent bone marrow chimeras: failure to detect a role for immigrant marrow-derived cells in imprinting intrathymic H-2 restriction.

In an attempt to resolve the issue of whether H-2-restricted T cell specificity is controlled by thymic epithelial cells or by cells of the macrophage/dendritic cell (M phi/DC) lineages, long-term F1----parent chimeras were subjected to secondary irradiation and reconstitution with F1 marrow cells. The rationale was that if F1 M phi/DC enter the thymus only quite slowly after irradiation, as claimed by other investigators, leaving F1----parent chimeras for a period of several months before re-irradiation would ensure that the new wave of T cells generated in the thymus of the chimeras would have no difficulty in making contact with donor-derived F1 M phi/DC. According to the view that M phi/DC rather than epithelial cells control H-2 restriction, the T cells differentiating in these chimeras would be expected to show H-2 restriction to both parental strains. In practice, T cells from twice-irradiated (1000 + 800 rad) chimeras showed strong restriction to host (thymic) H-2 determinants, the degree of restriction to host determinants being as marked as with T cells from once-irradiated chimeras. This finding applied both to T proliferative responses to KLH assayed in vitro and to T helper function for sheep erythrocytes measured in vivo. Preliminary experiments established that the initial dose of irradiation used for preparing the chimeras (1000 rad) resulted in almost total replacement of intrathymic M phi/DC by donor-derived cells within 4 wk of irradiation; M phi/DC were typed by determining their capacity to stimulate mixed-lymphocyte reactions. Collectively, the data imply that, at least under the conditions used, H-2-restricted T cell specificity is controlled by epithelial cells rather than by M phi/DC.

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