Biomedical subjects

D Y Loh

Publications and source records attributed to D Y Loh.

At least 37 records · Page 2Linked to original sources

PubMed1995

Antigen-driven differentiation of naive Ig-transgenic B cells in vitro.

We have established a culture system in which naive B cells bearing a transgenic, chicken OVA (cOVA)-specific Ig differentiate to plasma cells in vitro after interaction with cOVA plus cOVA-specific helper T cells. B cell-enriched populations from Ig-transgenic mice, but not from nontransgenic mice, proliferated after presenting nanomolar concentrations of cross-linked cOVA to DO11.10 (cOVA plus IAd-specific) T cells. After 6 to 9 days of culture with Ag and specific T cells, the B cells acquired a plasma cell phenotype and secreted the transgene-derived Ig at high levels. Engagement of B cell surface Ig was not essential for primary B cell differentiation. Differentiating B cells enlarged, clustered, and acquired two plasma cell markers, Syndecan and CD43. B cell CD45 isoform expression changed: the B220 isoform was lost in a T cell-dependent manner, whereas the CD45RB isoform was gained in a T-independent manner. Although unstimulated B cells survived less than 72 h in vitro, those in Ag-stimulated cultures showed reduced early death, a surge of proliferation at 3 to 5 days, and increased death late in the culture. Using a large population of naive B cells of defined antigenic specificity permits us to study a primary immune response to an Ag, rather than to less physiologic polyclonal stimuli. Because all steps of differentiation occurred in vitro, they are easily accessible for study. This coculture system provides an opportunity to observe Ag-specific T cell-B cell collaboration.

Animals↗

PubMed1995

Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia.

The cell-positional incidence of both spontaneous and damage-induced apoptosis of epithelial cells was assessed in longitudinal sections of the crypts of small intestine and colon of BDF1 mice. This was compared, using immunohistochemistry, with the pattern of expression of bcl-2, a suppressor of apoptosis. In the small intestine, apoptosis was maximal around cell position 4 from the base of the crypt; this closely corresponds to the position considered to contain the stem cells. In the colon, however, apoptosis was not confined to the area considered to harbour the stem cells (position 1 and 2). Instead, apoptosis was attenuated and distributed along the length of the crypt. Some cells at the base of murine colonic crypts expressed bcl-2 protein, whereas bcl-2 was absent in the crypts of the small intestine. Most pertinently, bcl-2 was absent from small intestinal crypt cells at positions 4-5 (the stem cell region). The importance of the expression of bcl-2 to the attenuation of apoptosis in stem cells was confirmed by analysis of the levels of both spontaneous and induced apoptosis in homozygously bcl-2 null C57BL/6 mice: in colonic crypts the level of spontaneous apoptosis rose significantly, and selectively at the base of the crypt, in comparison with crypts from wild-type animals. In contrast, there was no rise in spontaneous apoptosis in the small intestinal crypts from the bcl-2 null animals. Analysis of sections of human colon and small intestine also showed that expression of bcl-2 was confined to the base of the colonic crypt. The attenuation of apoptosis by bcl-2 in the region of the stem cells of the colonic crypts may dispose these to neoplastic transformation. Indeed, analysis of human carcinomas revealed expression of bcl-2, which in some samples was reciprocal with the expression of p53.

Animals↗

PubMed1995

Superantigens and conventional antigens induce different responses in alpha beta T-cell receptor transgenic mice.

While superantigens such as staphylococcal enterotoxin B (SEB) have been shown to induce both clonal deletion and clonal anergy, it is still not known why tolerance rather than memory is induced. To address this issue, we tested the proliferative capacity of T cells from ovalbumin (OVA)-specific alpha beta T-cell receptor transgenic mice primed with either SEB emulsified in complete Freund's adjuvant (CFA) or with OVA peptide, the specific antigen, in CFA. By contrast cells from mice primed with SEB in CFA appeared to be anergic in that they were hyporesponsive to OVA peptide as well as to SEB. The anergic cells could respond to phorbol myristate acetate (PMA) and ionomycin, suggesting that a proximal signal transduction step was affected. Cells from transgenic mice primed with OVA peptide and CFA were not anergic and in fact displayed an enhanced response when they were challenged with OVA in vitro. Thus, when the two antigens are emulsified in CFA and then injected subcutaneously, they behave very differently: the superantigen SEB induces anergy whereas the conventional antigen OVA induces a memory type of response.

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PubMed1994

Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia.

Mice carrying ablated coding regions of the bcl-2 alpha and bcl-2 beta transcripts have been made. bcl-2-/- mutants are smaller but viable, although about half of them die by 6 weeks of age. As shown earlier with somatic bcl-2 gene-targeted mice, the number of lymphocytes markedly decreased within few weeks after birth while other hematopoietic lineages remained unaffected. Among lymphocytes, CD8+ T cells disappeared most quickly followed by CD4+ T cells, whereas B cells were least affected. bcl-2-/- lymphocytes, however, could respond normally to various stimuli including anti-CD3, Con A, phorbol 12-myristate 13-acetate plus ionomycin, interleukin 2, lipopolysaccharide, and anti-IgM antibody. Abnormalities among nonlymphoid organs include smaller auricles, hair color turning gray at 4-5 weeks of age, and polycystic kidney disease-like change of renal tubules. These results suggest that Bcl-2 may be involved during morphogenesis where inductive interactions between epithelium and mesenchyme are important such as in the kidneys, hair follicles, and perichondrium of auricles. Surprisingly, the nervous system, intestines, and skin appear normal despite the fact that these organs show high levels of endogenous Bcl-2 expression in normal mice.

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PubMed1994

Requirement for CD8 beta chain in positive selection of CD8-lineage T cells.

CD8 is either an alpha alpha homodimer or an alpha beta heterodimer, although most peripheral CD8-lineage T cells express only the CD8 alpha beta heterodimer. The physiological function of CD8 beta was elucidated with mice that were chimeric for the homozygous disruption of the CD8 beta gene. The CD8 beta-1- T cells developed normally to CD4+CD8+ stage, but did not efficiently differentiate further, which resulted in few peripheral CD8+ T cells. The number of peripheral CD8+ T cells was restored by transfer of an exogenous CD8 beta gene into CD8 beta-deficient T cells. Thus, CD8 beta is necessary for the maturation of CD8+ T cells.

Animals↗

PubMed1994

Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo.

An adoptive transfer system was used to monitor physically the behavior of a trace population of TCR transgenic T cells in vivo. After subcutaneous injection of antigen in adjuvant, the antigen-specific cells accumulated first in the paracortical region of the draining lymph nodes, proliferated there for several days, and then moved into lymph node follicles, where they accounted for most of the T cells. They then disappeared slowly from the draining nodes, and the remaining cells were hypersensitive to antigenic stimulation in vitro. In contrast, when the antigen was introduced into the blood, the antigen-specific cells rapidly accumulated in the paracortical regions of all lymph nodes, proliferated there for a short time, but never entered follicles. Most of the cells then rapidly disappeared, leaving behind a population that was hyporesponsive to antigenic stimulation. These results provide a physical basis for the classical finding that antigen-specific memory and tolerance can be influenced by the form of antigen administration.

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PubMed1993

Distinct requirements of positive and negative selection for selecting cell type and CD8 interaction.

We investigated the requirements of positive and negative selection in the thymus for CD8 interaction and the selecting cell type. Thymic epithelial cells are known to mediate positive selection, whereas thymocytes fail to do so. The reason for this failure could be either the low amount of MHC class I molecules on thymocytes or the lack of other properties required for positive selection. To address this question a CD2.Kb transgenic mouse was prepared in which the expression of the Kb gene is under control of the CD2 promoter. In these mice the thymocytes exhibited very high levels of Kb. The mice were crossed with two TCR transgenic mice expressing either the Des.TCR (anti-Kb), which is positively selected on Kk and negatively on Kb, and the 2C.TCR (anti-Ld) with positive selection on Kb and negative on Ld. Despite the high Kb expression on thymocytes in CD2.Kb x 2C.TCR mice no positive selection was observed, whereas efficient negative selection was found in CD2.Kb x Des.TCR F1 mice. Thus, although thymocytes can negatively select, even a strong increase of MHC class I expression cannot convert them into positively selecting cells. This is consistent with the notion that thymic epithelium is specialized for positive selection, but the respective difference between thymocytes and thymic epithelium is not clear. The influence of CD8 was investigated using transgenic mice expressing a Kk/A2 or a Kb/A2 hybrid gene in which the promoter, the alpha 1, alpha 2 domains were of mouse origin and the alpha 3 domain of human origin. Because the murine CD8 molecule does not efficiently bind to human HLA class I, in these mice the CD8 interaction was impaired. In Kb/A2 x 2C.TCR and Kk/A2 x Des.TCR mice no positive selection of the respective TCR was found, whereas in Kb/A2 x Des.TCR mice negative selection was still functional. Altogether, the results indicate that positive selection depends more strictly on CD8 interaction and cell type than negative selection.

Animals↗

PubMed1993

Thymic selection of cytotoxic T cells independent of CD8 alpha-Lck association.

The CD8 alpha cytoplasmic domain associates with p56lck, a nonreceptor protein-tyrosine kinase. The biological relevance of CD8 alpha-Lck association in T cell development was tested with transgenic mice generated to express a CD8 alpha molecule with two amino acid substitutions in its cytoplasmic domain, which abolishes the association of CD8 alpha with Lck. The CD8 alpha mutant was analyzed in a CD8-/- background and in the context of the transgenic 2C T cell receptor. The development and function of CD8+ T cells in these mice were apparently normal. Thus, CD8 alpha-Lck association is not necessary for positive selection, negative selection, or CD8-dependent cytotoxic function.

Animals↗

PubMed1993

Positive selection of T lymphocytes on fibroblasts.

Thymocytes are selected for expression of alpha beta T-cell antigen receptors (TCR) which recognize antigen in conjunction with self-major histocompatibility complex (MHC) molecules. In the thymus the restriction element is imprinted on radioresistant stromal elements and on cells of haematopoietic origin. In mice negative for beta 2-microglobulin that are devoid of mature cytotoxic T lymphocytes, we find that intrathymic injection of different fibroblasts causes the maturation of CD4-CD8+TCRhigh thymocytes with distinct patterns of TCR V beta distribution. Here we show that in TCR-transgenic mice, intrathymic injection of L cells expressing the selecting H-2Kb molecule (L-Kb cells) reconstitutes the maturation of thymocytes bearing the transgenic TCR, and that in normal B10.BR (H-2k) mice, H-2Kb molecules expressed on L-Kb cells lead to the development of T lymphocytes with recognition restricted to H-2Kb. A class I MHC restriction element can thus be selected by interaction with fibroblasts, that is, cells of other than epithelial or haematopoietic origin.

Animals↗

PubMed1993

Differential activation of antigen-stimulated suicide and cytokine production pathways in CD4+ T cells is regulated by the antigen-presenting cell.

Stimulation of the TCR of activated, mature T cells by an APC produces a spectrum of functional responses. Most of those responses positively promote immunity and inflammation. However, death of the responding T cell, which would limit subsequent inflammation, can also result from such stimulation. We compare the Ag threshold necessary to promote inflammation through cytokine production with the Ag threshold necessary to limit inflammation through T cell death. These two responses are independently regulated by the APC and this regulation is selective for both different APC products and different T cell subsets. The costimulator B7 selectively decreases the Ag threshold for cytokine production in Th1 cells with no effect on the Ag threshold for Th1 death. B7 has no effect on the Th2 Ag threshold for cytokine production or death. In contrast, IFN-gamma negatively modulates a macrophage product that selectively increases the threshold of Ag necessary to stimulate death in Th2 cells, but has no effect on cytokine production by Th2 cells or cytokine production or death of Th1 cells. This selective regulation of cytokine production and T cell death may play an important role in both shaping the quality of a given immune response and in providing a general mechanism for the regulation of the immune response by limiting inflammation and through peripheral deletion of inappropriate T cell specificities.

Animals↗

PubMed1993

Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes.

Introduction of TCR alpha transgene, TCR beta transgene, or both into RAG-2-/-mice differentially rescues T cell development. RAG-2-/- mice have small numbers of TCR-CD4-CD8-(double negative, DN) thymocytes that express CD3 gamma delta epsilon and zeta proteins intracellularly. Introduction of a TCR beta transgene, but not a TCR alpha transgene, into the RAG-2-/- background restored normal numbers of thymocytes. These cells were CD4+CD8+ (double positive, DP) and expressed small amounts of surface TCR beta chain dimers in association with CD3 gamma delta epsilon but not zeta. RAG-2-/- mice that expressed alpha and beta TCR transgenes developed both DP and single positive thymocytes. Thus, the TCR beta subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.

Animals↗

PubMed1993

CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny.

During thymic development, immature thymocytes expressing major histocompatibility complex (MHC) class I-restricted T cell receptors (TcR) differentiate into CD8+ T cells with cytolytic functions. To evaluate the role of CD8 in positive and negative selection during thymic ontogeny, mice rendered CD8-null by gene targeting were bred with three lines of transgenic mice expressing unique MHC class I-restricted TcR. In all three instances CD8 was required for positive selection of MHC class I-restricted transgenic T cells. The efficiency of positive selection decreased in accordance with a reduced level of CD8 expression on thymocytes. Surprisingly, there was a differential requirement for CD8 expression in negative selection of MHC class I-restricted thymocytes, depending on the antigen specificity of TcR. These observations show that CD8 is essential for positive selection but is differentially required for negative selection of MHC class I-restricted T cells. Thus thymic selection, at least for negative selection, can occur in the absence of the CD8 accessory molecule.

Animals↗

PubMed1993

The lack of CD8 alpha cytoplasmic domain resulted in a dramatic decrease in efficiency in thymic maturation but only a moderate reduction in cytotoxic function of CD8+ T lymphocytes.

The glycoprotein CD8 is believed to play an important role in the maturation and function of MHC class I-restricted T lymphocytes. CD8 has been proposed to function as a co-receptor of the TcR to participate in signal transduction, possibly through its cytoplasmic domain that binds to protein tyrosine kinase p56lck. A T cell-specific transgene encoding CD8 alpha truncated at the cytoplasmic domain ("tailless CD8 alpha"), was introduced into CD8 alpha-deficient mice. This animal model was used to study the role of the CD8 cytoplasmic domain in T cell ontogeny and function. "Tailless CD8 alpha" was expressed on the cell surface of thymocytes and peripheral T cells. A small population of peripheral CD4- T cells (6% of T lymphocytes) was found to have cell surface expression of "tailless CD8 alpha" and endogenous CD8 beta, indicating that these cells may belong to the CD8+ T cell lineage. A consistent result was obtained from CD8 alpha-deficient mice bearing the "tailless CD8 alpha" and the MHC class I-restricted 2C TcR transgenes. A small population of CD4- T cells expressing CD8 beta, the "tailless CD8 alpha" and the 2C TcR transgenes was present in the periphery of these mice in a selecting background, but was absent in a deleting background. When "tailless CD8 alpha" mice were infected with lymphocytic choriomeningitis virus (LCMV), the peripheral CD8+ CD4- T cell subset expanded dramatically and a significant LCMV-specific cytolytic activity was detected. The results suggest that the cytoplasmic portion of CD8 alpha is not absolutely required but dramatically enhances the efficiency of thymic maturation of CD8+ T cells. The lack of CD8 alpha cytoplasmic domain in peripheral CD8+ T cells does not abolish the generation of cytotoxicity in response to an in vivo LCMV infection, although the cytolytic activity is slightly reduced compared to that in control mice.

Animals↗

PubMed1993

Separation of CD4+ functional responses by peptide dose in Th1 and Th2 subsets expressing the same transgenic antigen receptor.

CD4+ T cells from alpha beta-TCR transgenic mice were used to explore the effect of antigen dose on various functional activities. This transgenic system provides for a source of antigen-specific T cells with the same TCR which can develop into Th1 and Th2 phenotypes under controlled conditions. Of particular interest was to determine if detachment of adherent targets from their substrate could be separated from other functional activities by either antigen dose or functional subset. Using this model, we have found that (a) Th1 and Th2 phenotypes exhibit a similar peptide dose-dependent pattern of detachment and lysis, although Th2 are generally less lytic on both fibroblast and lymphoma targets; (b) detachment and lysis can be dissociated from cytokine production by low peptide doses; (c) detachment and lysis can be physiologically as well as temporally and pharmacologically uncoupled as most targets recovered after detachment by Th2 effectors retain their capacity to proliferate.

Animals↗

PubMed1993

Mutations of the chloramphenicol acetyl transferase transgene driven by the immunoglobulin promoter and intron enhancer.

Transgenic mice containing the chloramphenicol acetyl transferase (CAT) gene driven by the immunoglobulin (Ig) VH promoter and the heavy chain intron enhancer were prepared to increase our understanding of the mechanism responsible for somatic mutation. The transgene showed similar tissue specificity in terms of expression as endogenous Ig genes. Hybridomas were prepared after multiple immunization of a transgenic mouse with (4-hydroxy-3-nitrophenyl)acetyl-chicken gammaglobulin, a thymus-dependent antigen. We analyzed mutations in the CAT coding region as well as in the region 5' upstream of the promoter after amplification of DNA using the PCR followed by sequencing of cloned DNA. Mutation detection enhancement gel electrophoresis was also used to detect mutations. Only a single band was observed in PCR products from the region 5' upstream of the promoter and from the enhancer, whereas in those from the CAT coding region, three out of 11 hybridomas showed multiple bands. In DNA sequences of the CAT coding region from these three hybridomas, a total of six nucleotide substitutions were observed, but none in the region 5' to the promoter. The results of DNA sequencing and the electrophoresis were in good agreement, suggesting that mutation occurred only in the CAT gene but not in the region 5' to the promoter or the enhancer. The rate of mutation in the CAT gene was estimated to be 1.6 x 10(-5) per base pair per cell division, which was a lower limit of the rate for somatic mutation reported for Ig genes. Location and distribution of mutations were similar to that of the Ig gene, suggesting that (i) mutation in the CAT gene was induced by a mechanism similar to that occurring in the Ig gene and (ii) the mutator mechanism can operate in a non-Ig gene provided it is flanked by the VH promoter and heavy chain intron enhancer.

Animals↗

PubMed1992

CD45RA and CD45RBhigh expression induced by thymic selection events.

CD45 is a protein tyrosine phosphatase involved in T and B cell signaling. While peripheral T cells switch CD45 isoforms upon activation, events leading to exon switching during T cell development in the thymus have not been determined. The expression of high molecular weight isoforms of CD45 was examined on thymocytes from nontransgenic and T cell receptor (TCR) transgenic mice. All thymocytes from nontransgenic mice were CD45RB+ as assessed by staining with MB23G2, an anti-CD45RB-specific monoclonal antibody. Interestingly, there was a small population (1-3%) of thymocytes that displayed a higher intensity of staining with MB23G2, CD45RBhigh. CD45RBhigh thymocytes were found in all subsets defined by CD4 and CD8 expression and were also present within the TCR-alpha/beta high population. To analyze whether or not CD45 expression correlated with thymic selection events, expression of CD45RBhigh and a second isoform, CD45RA, was examined on thymocytes from H-Y and 2C TCR transgenic mice and found to correlate with positive and negative selection events but did not occur in nonselecting backgrounds. CD45RA and CD45RBhigh upregulation was also not observed in transgenic mice backcrossed into CD8-deficient mice, a scenario in which there is no positive selection of transgene-expressing thymocytes. These data suggest that modulation of CD45 isoform expression may be involved in thymic selection events.

Animals↗

PubMed1992

Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes.

Thymocytes from DO10 T-cell-receptor transgenic mice undergo apoptosis, or programmed cell death, when chicken ovalbumin-(323-339) peptide is administered in vivo. Using DO10 mice thymocytes, we have now developed a simple in vitro model system that recapitulates the in vivo clonal-deletion process. When transgenic thymocytes were cocultured with fibroblasts, B cells, or thymic nurse cell lines (all bearing I-Ad) in the presence of chicken ovalbumin-(323-339), deletion of the transgenic TCR+CD4+CD8+ thymocytes was seen within 8-20 hr. Thymocytes designed to bear I-Ad on their surface could mediate the deletion themselves. Thus, thymocyte clonal deletion entirely depends on the stage at which the thymocytes are vulnerable to the onset of apoptosis, rather than on the nature of the peptide antigen-presenting cells. Furthermore, thymic nurse cell line TNC-R3.1 could cause deletion, strongly suggesting that some thymic epithelial/stromal components are potentially capable of participating in negative selection. In all cases examined, little deletion could be induced at a peptide concentration less than 10 nM, thus defining the minimum amount of peptide antigen required for negative selection. The peptide-dependent in vitro negative-selection system will allow further dissection of the molecular and cellular processes involved in clonal deletion due to apoptosis in the thymus.

Amino Acid Sequence↗

PubMed1992

No requirement for p56lck in the antigen-stimulated clonal deletion of thymocytes.

Activation of protein-tyrosine kinases (PTKs) is required for signal transduction during T cell activation, although the pathway used during thymic selection is unknown. An in vitro system was established in which T cell receptor transgenic thymocytes underwent clonal deletion in response to peptide antigen. The effects of two PTK-specific inhibitors, herbimycin A and genistein, on the clonal deletion of immature thymocytes and the activation of mature thymocytes were examined. Clonal deletion occurred while T cell activation was inhibited and when no p56lck activity was evident. Thus, p56lck is not required for the antigen-stimulated step of clonal deletion of immature thymocytes, and negative selection proceeds via a distinct pathway.

Animals↗