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D Y Loh

Publications and source records attributed to D Y Loh.

At least 55 records · Page 3Linked to original sources
PubMed1992

Organ injury associated with extrathymic induction of immune tolerance in doubly transgenic mice.

The developmental fate of autoreactive T cells encountering extrathymically expressed self-antigen was studied in a doubly transgenic mouse model system where pancreatic acinar cells expressed H-2Ld and T cells expressed an antigen receptor (2C TCR) specific for H-2Ld. Thymocytes bearing 2C TCR differentiated normally. They were positively selected without evidence of intrathymic clonal deletion. Survival of H-2Ld-bearing skin allografts was significantly prolonged in pancreatic H-2Ld singly and doubly transgenic mice, consistent with an in vivo state of T-cell tolerance. The mechanism of tolerance induction was determined and found to have two components. First, up to 80% of peripheral CD8+2C TCR+ T cells were eliminated. Second, those T cells which escaped elimination had a significantly reduced proliferative response to H-2Ld. Thus, autoreactive T cells can be made self-tolerant through interaction with self-antigen located extrathymically. This is accomplished by a reduction in the percentage of autoreactive T cells as well as by a reduction in the functional capacity of residual T cells. Surprisingly, although pancreatic lymphocytic infiltration and organ injury were absent in exocrine tissue of singly transgenic mice, it was present in doubly transgenic mice. This suggests that when the percentage of autoreactive T cells is high, tolerance induction can be associated with an inflammatory infiltrate in extrathymic tissue where self-antigen is presented.

Animals↗
PubMed1992

CD8 surface levels alter the fate of alpha/beta T cell receptor-expressing thymocytes in transgenic mice.

The mature T cell receptor (TCR) repertoire is established on the basis of discriminative events involving binding of the TCR alpha and beta chains and CD4 or CD8 on immature thymocytes to major histocompatibility complex (MHC)/self-peptide complexes expressed in the thymus. To ask whether the strength of the interaction between a CD8/TCR complex and a MHC/self-peptide ligand plays a pivotal role in deciding the fate of a maturing thymocyte, we generated lines of transgenic mice that express distinct and elevated levels of CD8 alpha, approximately 2, 3, and 6-10 times. These lines were then crossed to a transgenic line expressing the class I-restricted TCR, 2C. We found that thymocytes expressing the 2C TCR in combination with the highest levels of CD8 were deleted on the H-2 Kb background that is normally positively selecting for the 2C TCR. In contrast, thymocytes coexpressing the 2C TCR and moderately elevated levels of CD8 were selected for maturation. These results demonstrate directly that CD8 levels can affect the developmental fate of a maturing thymocyte and argue in support of an affinity model for thymocyte selection.

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PubMed1991

Identification of a T cell receptor beta chain variable region, V beta 20, that is differentially expressed in various strains of mice.

A cDNA library of TCR beta chain transcripts from BALB/c thymocytes was constructed using anchored polymerase chain reaction (PCR). Screening of this library led to the identification of a V beta gene segment, V beta 20, structurally related to V beta 3 and V beta 17. Genomic analysis of mice displaying deletions in their V beta loci, together with mapping of cosmid clones, situated V beta 20 2.5 kb beside V beta 17. The expression of V beta 20 was estimated by PCR in mice of different H-2 and Mls types. Peripheral T cells from H-2k and H-2d mice did not express V beta 20, whereas in I-E-negative mice (C57Bl/6 and SJL), V beta 20 transcripts were detected. The lack of V beta 20 transcripts in (C57Bl/6 x CBA/J)F1, (C57Bl/6 x BALB/c)F1, and in congenic B6.H-2k mice suggests that the differential use of V beta 20 is due to an I-E-mediated clonal deletion process. The involvement of the Mls super antigens was excluded by analysis of all Mls type combinations. The nature of the V beta 20-deleting element(s) is discussed in the context of the I-E/superantigen systems controlling the expression of V beta 11 and V beta 17.

Amino Acid Sequence↗
PubMed1991

Receptor-stimulated death pathway is opened by antigen in mature T cells.

Clonal deletion provides an important mechanism for the elimination of autoreactive T cells. Deletion is accomplished by programmed cell death directed by interaction of the T-cell receptor (TCR) of the developing thymocyte with major histocompatibility complex elements in the thymic environment. In this report we present evidence to support the hypothesis that the activation and the maturation state of the T cell may be important in coupling the TCR to the "death program." We show that coupling of the TCR to the death program is open during maturation but closed in naive mature T cells. However, during primary antigenic stimulation, coupling between the TCR and the death program is reopened, as demonstrated by the stimulation of the death of these cells by immobilized anti-TCR. Our results suggest that further examination of mature cells that are either resistant or sensitive to receptor-stimulated death may lead to the identification of the components of the death pathway and may provide clues to the regulation of their coupling to TCR signals.

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PubMed1991

Molecular requirements for cell fate determination during T-lymphocyte development.

Antigen-specific T lymphocytes recognize peptide antigens in conjunction with the products of the self major histocompatibility complex (MHC). In addition, they are immunologically self-tolerant. To acquire these characteristics, thymocytes undergo a stringent cellular selection process during development. The study of thymocyte development at the molecular level is impeded in mammalian systems by the heterogeneity of the thymocyte population in each individual. However, the use of mice transgenic for the T-cell receptor successfully circumvented this problem and made it possible to elucidate some of the requirements for positive selection, which leads to thymocyte differentiation, survival, and MHC restriction, and negative selection, which leads to programmed cell death, clonal deletion, and self-tolerance. T-cell fate is determined primarily by the nature of the interaction between a complex composed of the T-cell receptor and CD4 or CD8 molecules on the T-cell surface and the peptide antigens that are bound to MHC products and are displayed by other nonlymphoid cells present in the thymus. The molecular analysis of the receptor-ligand interactions involved in this process in transgenic mice provides opportunities to dissect cell fate determination in an intact mammalian system and to understand the molecular basis for immunological self-tolerance and MHC-restriction.

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PubMed1990

Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo.

In order to examine the mechanisms by which clonal deletion of autoreactive T cells occurs, a peptide antigen was used to induce deletion of antigen-reactive thymocytes in vivo. Mice transgenic for a T cell receptor (TCR) that reacts to this peptide contain thymocytes that progress from the immature to the mature phenotype. Intraperitoneal administration of the peptide antigen to transgenic mice results in a rapid deletion of the immature CD4+ CD8+ TCRlo thymocytes. Apoptosis of cortical thymocytes can be seen within 20 hours of treatment. These results provide direct evidence for the in vivo role of apoptosis in the development of antigen-induced tolerance.

Amino Acid Sequence↗
PubMed1990

Evidence for CD8-independent T cell maturation in transgenic mice.

Double-negative (CD4-/CD8-) T cells expressing the alpha/beta transgenic TCR from the 2C cell line (anti-H-2Ld) were examined in the periphery of animals whose MHC type produces positive, negative, or no selection for differentiation of the TCR on single positive (CD8+) cells. Regardless of the selection haplotype the CD4-/CD8- cells are capable of activation by anticlonotypic mAb indicating that negative selection does not inactivate the "forbidden" TCR. Rather, the lack of response to H-2Ld in the negative haplotype is likely to absence of CD8 required to produce a functional response to H-2Ld. The similarity of surface phenotype and functional activity of these CD4-/CD8- cells maturing in different haplotypes suggests they may arise by an alternative lineage which, unlike the dominant TCR alpha/beta pathway, does not require coexpression of CD4/CD8.

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PubMed1990

Positive selection of transgenic receptor-bearing thymocytes by Kb antigen is altered by Kb mutations that involve peptide binding.

A specific interaction between the class I major histocompatibility complex molecule Kb and thymocytes expressing the antigen receptor from the cytolytic T lymphocyte 2C enhances maturation of T cells of the CD8 lineage in transgenic mice. By analyzing transgenic mice backcrossed to Kbm mutant strains of mice, we have identified five bm mutations of the Kb antigen-encoding gene that alter the positive selection of thymocytes induced by Kb antigen. Compared with Kb, Kbm10 and Kbm1 did not induce significant maturation of 2C T-cell receptor-bearing thymocytes, and Kbm8 antigen positively selected for transgenic thymocytes only weakly. Altering residue 77 of Kb molecule from aspartic acid to serine made Kbm3 and Kbm11 allogeneic targets for the 2C antigen receptor and caused deletion of transgenic thymocytes. This deletion spared T cells that expressed low levels of CD8, a result differing from the total deletion of CD8-bearing T cells seen in mice that expressed the original target alloantigen Ld. This evidence indicates that (i) self-peptides bound to thymic major histocompatibility complex molecules can influence the positive selection of thymocytes and (ii) thymocytes with apparently weak interaction with self-major histocompatibility complex antigens can escape clonal deletion.

Animals↗
PubMed1990

Genomic organization of mouse Fc gamma receptor genes.

We have isolated and characterized the gene coding for the mouse Fc receptor that is termed Fc gamma RIIa. The gene contains five exons and spans approximately 9 kilobases. Unlike most members of the immunoglobulin gene superfamily, this gene utilizes multiple exons to encode its leader peptide. The first exon encodes the hydrophobic region of the signal sequence; the second exon, which contains only 21 base pairs, encodes a segment of the signal peptidase recognition site; and the beginning of the third exon encodes the predicted site of peptidase cleavage. The third and fourth exons each code for immunoglobulin-like extracellular domains. The fifth exon encodes the hydrophobic transmembrane domain and the cytoplasmic tail. Partial characterization of the Fc gamma RIIb gene indicates that it also contains multiple leader exons, including a 21-base-pair exon and two exons coding for homologous immunoglobulin-like extracellular domains. However, the Fc gamma RIIb gene uses four exons to encode its intracytoplasmic region. Analysis using contour-clamped homogeneous electric field (CHEF) gels indicates that the Fc gamma RIIa and Fc gamma RIIb genes are linked within 160 kilobases on mouse chromosome 1.

Amino Acid Sequence↗
PubMed1990

Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins.

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.

Animals↗
PubMed1989

Identification and characterization of new murine T cell receptor beta chain variable region (V beta) genes.

By screening previously isolated genomic clones spanning the mouse TCR V beta locus with V beta-specific oligonucleotides, we have isolated one new functional V beta gene and six V beta pseudogenes. Because this method of identifying new genes does not depend on expression levels, we conclude that most, if not all, V beta genes in the mouse have been identified. The newly identified pseudogenes increase the frequency of mouse TCR V beta pseudogenes to 28%, a frequency similar to that estimated for mouse Ig VH pseudogenes (24). Three of the newly discovered pseudogenes are clustered in a region around another pseudogene (V beta 17b). The extensive DNA diversity, as reflected in both the nucleotide sequence and the RFLP, indicates that this genomic region is a possible hotspot of recombination. The new functional gene, V beta 19a, is expressed at very low levels, which explains why it has not been isolated earlier. V beta 19 shows expression patterns that correlate with the previously described Va beta and Vb beta haplotypes.

Amino Acid Sequence↗
PubMed1989

Protein kinase C regulation of terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase (TdT) is a nuclear enzyme found in early lymphocytes which is thought to increase junctional diversity of TCR and Ig genes by the addition of N regions. TdT is normally found only in immature lymphoid cells and is turned off in both mature B and T cells. To investigate the regulation of TdT gene expression, pre-B and pre-T cells were treated with PMA or three of its analogs and its effects on steady-state TdT mRNA levels determined. Rapid and reversible decline in steady-state TdT mRNA levels was observed within 6 h with PMA. This rapid decline can be blocked by pretreatment of the cells with a relatively selective protein kinase C inhibitor implicating the role of protein kinase C activation in the decline of TdT mRNA. Nuclear run-off studies demonstrate that TdT transcription is rapidly down-regulated within 45 min after PMA treatment, indicating that this regulation occurs mainly at the level of transcription. Furthermore, the TdT mRNA decline is blocked in the presence of cycloheximide, showing that new protein synthesis is required for inactivation of the gene.

Animals↗
PubMed1989

Different large deletions of T cell receptor V beta genes in natural populations of mice.

A panel of geographically separate Mus m. domesticus and Swiss mice from several sources was screened for deletions in the T cell receptor variable (V) beta locus. Four out of forty-three strains tested show a deletion identical to or larger than the deletion previously described in SJL mice. To our knowledge, this is the first time that such important V beta deletions are described in inbred or partially inbred wild-derived strains of mice. On the other hand there seems to be very little polymorphism between the remaining V beta genes. Expression of V beta genes in peripheral and intra-thymic T cells was tested using antibodies specific for different V beta polypeptide chains. Flow cytometry analysis revealed a high expression of V beta 6 and V beta 17 genes in the Copacabana Swiss-derived strain and an absence of V beta 17 expression in the WLA wild-derived strain. The three Mus m. domesticus strains (WLA, DDO and WBG) having deleted two to three additional V beta subfamilies compared to SJL present no apparent immune deficiencies or autoimmune disorders. We conclude that relatively few V beta genes may suffice for unimpaired survival of wild mice and that there is a selective pressure for the structural conservation of the remaining V beta genes.

Animals↗
PubMed1989

Identification of T-cell receptor V beta deletion mutant mouse strain AU/ssJ (H-2q) which is resistant to collagen-induced arthritis.

Our laboratory is involved in investigating the role of T-cell receptor (Tcr) in collagen-induced arthritis (CIA). During these studies we found AU/ssJ (H-2q) mice to be resistant to CIA like SWR (H-2q), as compared with other H-2q strains with wild-type Tcr like DBA/1 and B10.Q. Upon screening with monoclonal antibodies F23.1 and KJ23a, AU/ssJ was found to be F23.1 negative (V beta 8 Tcr negative) and KJ23a positive (V beta 17a Tcr positive). Southern blot analysis on liver DNA using specific Tcr-V beta probes confirmed the deletion of V beta 8 gene family and also showed that AU/ssJ mice have deletions of V beta 9, V beta 13, V beta 12, and V beta 11 genes of Tcr. Further, these mice show a restriction fragment length polymorphism pattern with V beta 10, V beta 6, and V beta 17 probes similar to SWR mice as compared with B10 mice. Since SWR and AU/ssJ are from different backgrounds, these studies indicate that specific variable region beta chain genes of Tcr are crucial for susceptibility to CIA in mice. Furthermore, these studies identify an additional inbred strain which has also deleted 50% of its Tcr-V beta genes.

Animals↗
PubMed1989

Peripheral tolerance to allogeneic class II histocompatibility antigens expressed in transgenic mice: evidence against a clonal-deletion mechanism.

To examine the effects of aberrant expression of class II major histocompatibility complex (MHC) proteins on tolerance development, transgenic mice expressing the I-Ad genes under control of the pancreatic elastase promoter were produced. Such transgenic mice express I-Ad exclusively on exocrine pancreas, without expression in thymus or by lymphocytes. No spontaneous development of autoimmune reactivity toward exocrine pancreas was found in transgene-expressing mice of an H-2b background even though such mice could produce in vitro allogeneic responses against I-Ad. When T cells from nontransgenic H-2b mice as well as transgenic H-2b mice were activated in vitro by I-Ad allogeneic stimulator cells and transferred to transgenic mice, an intense, destructive lymphocytic infiltrate specific for exocrine pancreas developed. These findings suggest that aberrant class II MHC expression alone may not trigger autoimmune reactions. Rather, the unresponsiveness to allogenic class II MHC may result from the inability of exocrine pancreas to initiate primary responses by T cells.

Animals↗
PubMed1989

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element.

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

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