Biomedical subjects

L H Glimcher

Publications and source records attributed to L H Glimcher.

At least 55 records · Page 3Linked to original sources

PubMed1997

Thymic stromal cell specialization and the T-cell receptor repertoire.

Ten years ago, we proposed a model for thymus function in which thymic epithelial cells are primarily responsible for imprinting major histocompatibility complex (MHC)-restricted specificity, and bone marrow-derived macrophages or dendritic cells are responsible for the induction of self-tolerance. Since then, transgenic and knockout models have allowed for a dissection of thymic stromal components in vivo, leading to a new understanding of their specialized functions. We have determined that with regard to class II-restricted CD4 T-cell development, two distinct subsets of thymic epithelium help shape the repertoire: Cortical epithelium appears solely responsible for positive selection, whereas a fucose-bearing subset of medullary epithelium is specialized for negative selection. This absolute separation of positive and negative selection into two distinct spatial and temporal compartments leads to a much simpler view of the process of repertoire selection. Finally, a novel view of the function of the thymic medulla is discussed.

Animals↗

PubMed1997

Genes that regulate interleukin-4 expression in T cells.

Interleukin-4 is an immunomodulatory cytokine which plays a central role in the regulation of allergic and atopic immune responses. Significant progress has been made in gaining a detailed understanding of the transcriptional regulation of the interleukin-4 gene. The recent identification and characterization of several key transcription factors has helped to elucidate the molecular mechanisms of T helper cell cytokine gene expression.

Animals↗

PubMed1996

NF-AT-Driven interleukin-4 transcription potentiated by NIP45.

The induction of cytokine gene transcription is mediated in part by the nuclear factor of activated T cells (NF-AT). Factors involved in the mechanisms of NF-AT-mediated transcription are not well understood. A nuclear factor that interacted with the Rel homology domain (RHD) of NF-ATp was identified with the use of a two-hybrid interaction trap. Designated NIP45 (NF-AT interacting protein), it has minimal similarity to any known genes. Transcripts encoding this factor were enriched in lymphoid tissues and testes. NIP45 synergized with NF-ATp and the proto-oncogene c-Maf to activate the interleukin-4 (IL-4) cytokine promoter; transient overexpression of NIP45 with NF-ATp and c-maf in B lymphoma cells induced measurable endogenous IL-4 protein production. The identification of NIP45 advances our understanding of gene activation of cytokines, critical mediators of the immune response.

Amino Acid Sequence↗

PubMed1996

Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex.

The normal development of T cells in the thymus requires both positive and negative selection. During positive selection, thymocytes mature only if their T-cell receptors react with some specificity to host major histocompatibility complex (MHC) and host peptides. During negative selection, thymocytes die if their T-cell receptors react with too high an affinity to the presenting cell, self MHC, and peptides to which they are exposed. These two processes are important for the development of the T-cell repertoire and the acquisition of self-tolerance, but their precise location and temporal relationship are not known. We have used the keratin 14 (K14) promoter to re-express a class II MHC antigen (I-Ab) in class II-negative mice. The transgenic I-A molecule is expressed only on thymic cortical epithelium; thymic medullary epithelium and bone-marrow-derived cells are I-A negative. CD4+ cells are positively selected in K14 mice, but clonal deletion does not ocur in K14 mice or in relB-negative mice, which lack a thymic medulla. The K14 CD4 cells are autoreactive, as they proliferate extensively to and specifically lyse I-Ab-positive target cells. These autoreactive cells make up 5% of the peripheral CD4 T cells, providing and estimate of the minimal frequency of positively selected cells that must subsequently undergo negative selection for self-tolerance to be preserved. Thus positive and negative selection occur in anatomically distinct sites.

Animals↗

PubMed1996

The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4.

The molecular basis for the distinctive cytokine expression of CD4+ T helper 1 (Th1) and T helper 2 (Th2) subsets remains elusive. Here, we report that the proto-oncogene c-maf, a basic region/leucine zipper transcription factor, controls tissue-specific expression of IL-4. c-Maf is expressed in Th2 but not Th1 clones and is induced during normal precursor cell differentiation along a Th2 but not Th1 lineage. c-Maf binds to a c-Maf response element (MARE) in the proximal IL-4 promoter adjacent to a site footprinted by extracts from Th2 but not Th1 clones. Ectopic expression of c-Maf transactivates the IL-4 promoter in Th1 cells, B cells, and nonlymphoid cells, a function that maps to the MARE and Th2-specific footprint. Furthermore, c-Maf acts in synergy with the nuclear factor of activated T cells (NF-ATp) to initiate endogeneous IL-4 production by B cells. Manipulation of c-Maf may alter Th subset ratios in human disease.

Animals↗

PubMed1996

The basic domain/leucine zipper protein hXBP-1 preferentially binds to and transactivates CRE-like sequences containing an ACGT core.

The transcription factor hXBP-1 belongs to the family of basic region/leucine zipper (bZIP) proteins and interacts with the cAMP responsive element (CRE) of the major histocompatibility complex (MHC) class II A alpha, DR alpha and DP beta genes. However, the developmental expression of hXBP-1 as revealed by in situ hybridization in mouse embryos, has suggested that it interacts with the promoter of additional genes. To identify other potential target genes of this factor, we performed binding site selection experiments with recombinant hXBP-1 protein. The results indicated that hXBP-1 binds preferably to the CRE-like element GAT-GACGTG(T/G)NNN(A/T)T, wherein the core sequence ACGT is highly conserved, and that it also binds to some TPA response elements (TRE). hXBP-1 can transactivate multimers of the target sequences to which it binds in COS cells, and the level of transactivation directly correlates with the extent of binding as observed in gel retardation experiments. One target sequence that is strongly bound by hXBP-1 is the 21 bp repeat in the HTLV-1 LTR, and we demonstrate here that hXBP-1 can transactivate the HTLV-1 LTR. Further, the transactivation domain of hXBP-1 encompasses a large C-terminal region of the protein, containing domains rich in glutamine, serine and threonine, and proline and glutamine residues, as shown in transient transfection experiments using hXBP-1-GAL4 fusion proteins and a reporter gene under the control of GAL4-binding sites.

Animals↗

PubMed1996

Rejection of MHC class II-transfected tumor cells requires induction of tumor-encoded B7-1 and/or B7-2 costimulatory molecules.

Many tumor cells that have been transfected with genes encoding B7 costimulatory molecules become effective cellular vaccines against wild-type tumor. The improved immunity is dependent on newly induced tumor-specific CD8+ and/or CD4+ T cells and presumably occurs because the B7 transfectants provide the requisite second signal for activation of T cells in conjunction with tumor cell-presented MHC class I/tumor peptide and/or MHC class II/tumor peptide complexes, respectively. Since B7 expression is such a potent enhancer of tumor immunity, and yet some tumors are immunogenic in the absence of B7 transfection, we have used class I+ class-II-transfected tumors to investigate whether costimulatory molecules are also involved in rejection of immunogenic, non-B7-transfected tumor. Blocking studies with B7 mAbs demonstrate that induction of tumor immunity in naive mice requires B7-1 and/or B7-2 expression, while experiments with tumor-primed mice indicate that once antitumor immunity is established, expression of B7 is not necessary. Flow cytometry analyses demonstrate that costimulatory molecules are expressed by the tumor cells via an in vivo induction process. Experiments with class II genes with truncated cytoplasmic tails indicate that the cytoplasmic region of the tumor-expressed class II heterodimer is involved in induction of B7. We therefore conclude that for this class I+ class II-transfected tumor, generation of tumor immunity requires induction of tumor cell-encoded B7 molecules that are mediated by the cytoplasmic region of the transfected class II heterodimer.

Animals↗

PubMed1996

Genetic evidence for a new type of major histocompatibility complex class II combined immunodeficiency characterized by a dyscoordinate regulation of HLA-D alpha and beta chains.

Major histocompatibility complex (MHC) class II combined immunodeficiency (CID), also known as type II bare lymphocyte syndrome, is an autosomal recessive genetic disorder characterized by the complete lack of expression of MHC class II antigens. The defect results from a coordinated lack of transcription of all class II genes. Cell fusion studies using many patient- and experimentally derived class II-negative cell lines have identified four distinct genetic complementation groups. In this report, we present genetic evidence that cell lines derived from two newly described MHC class II-deficient patients, KER and KEN, represent a fifth complementation group. In addition, the KER and KEN cell lines display a unique pattern of dyscoordinate regulation of their MHC class II genes, which is reflected in a new phenotype of in vivo promoter occupancy as revealed by in vivo genomic footprinting. These data point to a new defect that can result in the MHC class II-deficient phenotype.

Cell Fusion↗

PubMed1996

A cell type-specific enhancer in the human B7.1 gene regulated by NF-kappaB.

The costimulatory molecule B7.1 provides a second signal critical for T cell activation. The distribution of this integral membrane protein is restricted to certain tissues where its level of expression is modulated by multiple exogenous stimuli. To identify the molecular basis for specificity and inducibility, the chromatin configuration of the human B7.1 gene was examined in intact nuclei from various cell types. The identification of a tissue-specific deoxyribonuclease I hypersensitive site approximately 3kb upstream of the transcription start site led to the characterization of a cell type-specific enhancer region. This 183-bp region was both cell type specific and responsive to two distinct stimuli, lipopolysaccharide and dibutyryl cAMP, known to regulate B7.1 expression. Deletional and site-directed mutagenesis revealed the presence of multiple functionally critical cis elements within this region, one of which was a nuclear factor (NF)-kappaB consensus sequence. In B7.1-positive B cells, this element bound several members of the NF-kappaB family, transcription factors already implicated in signal transduction pathways relevant to B7.1 expression. This is the first description, to our knowledge, of regulatory elements that control expression of a gene encoding a B7 costimulatory molecule.

Antigens, CD↗

PubMed1996

Transcription factor B cell lineage-specific activator protein regulates the gene for human X-box binding protein 1.

The transcription factor human X-box binding protein 1 (hXBP-1) is a basic region-leucine zipper protein implicated in the regulation of major histocompatibility complex class II gene expression as well as in exocrine gland and skeletal development. Multiple regulatory elements in the hXBP-1 promoter lie 3' to the transcription start site, including the hX2 site, whose core sequence is an AP-1-like element identical to the hXBP-1 target sequence in the HLA-DRA promoter. One complex identified by electrophoretic mobility shift assay (EMSA), complex 3, was previously shown to protect the hX2 site and more 3' bases. Sequence analysis now shows that this region contains a consensus binding site for transcription factor BSAP (B cell lineage-specific activator protein). Complex 3 and BSAP have identical cell-type specificities, as they are found only in pre-B and mature B cell lines. In EMSAs, BSAP antibody specifically recognized complex 3, and in vitro translated BSAP could bind to an hXBP promoter fragment. Cotransfections using an hXBP-1 reporter construct indicated that BSAP downregulates the hXBP-1 promoter. The highest levels of hXBP-1 mRNA were found when BSAP was not expressed, in pre-Pro-B cells and in plasma cell lines. In addition, hXBP-1 and BSAP levels were inversely correlated along the early stages of B cell development. In the regulation of the hXBP-1 promoter, a strong positive transcriptional influence at the hX2 site is opposed by the downregulatory actions of BSAP.

Antibody Specificity↗

PubMed1996

Two levels of help for B cell alloantibody production.

We have examined whether T cell stimulation by direct or indirect pathways contributes to alloantibody production by B cells after major histocompatibility complex (MHC)-disparate skin graft rejection in mice. Experiments were performed using normal mice, MHC class II-deficient mice, MHC class II-deficient mice with an intact peripheral CD4+ cell population (due to expression of class II antigens only on thymic epithelium), mice lacking the cytoplasmic tail of their MHC class II antigens, and mice depleted of CD4+ cells by anti-CD4 monoclonal antibody treatment. Depletion of recipient CD4+ cells reduced alloantibody production to barely detectable levels. Absence of donor MHC class II antigens did not affect the production of either immunoglobulin (Ig)M or IgG antibodies directed at class I alloantigens. Absence of recipient MHC class II antigens, however, led to production of only IgM but not IgG antibodies, even if the recipients had an intact CD4+ cell population. Absence of the cytoplasmic tail of the recipient's MHC class II antigens led to the production of slightly reduced amounts of IgG antibody. These findings indicate that (a) CD4+ cells are essential helper cells for B cell alloantibody production; (b) production of IgM alloantibody can occur with help from CD4+ cells, which recognize either donor class II antigens or modified recipient class II antigens; (c) isotype switching from IgM to IgG alloantibody requires help from CD4+ cells activated by antigens presented by recipient MHC class II molecules; and (d) the cytoplasmic domain of the recipient MHC class II molecules may be involved in the mechanism that leads to isotype switching by B cells. Thus, there are two levels of CD4-mediated help available for B cells responding to alloantigens: one (involving a noncognate interaction) can produce B cell activation, and a second (involving a cognate interaction) is required for differentiation and IgG alloantibody production.

Animals↗

PubMed1996

Chondrodysplasia and neurological abnormalities in ATF-2-deficient mice.

Activating transcription factor-2 (ATF-2) is a basic region leucine zipper protein whose DNA target sequence is the widely distributed cAMP response element (CRE). We report here that mice carrying a germline mutation in ATF-2 demonstrated unique actions of ATF-2 not duplicated by other ATF/CREB family members. Mutant mice had decreased postnatal viability and growth, with a defect in endochondral ossification at epiphyseal plates similar to human hypochondroplasia. The animals had ataxic gait, hyperactivity and decreased hearing. In the brain, there were reduced numbers of cerebellar Purkinje cells, atrophic vestibular sense organs and enlarged ventricles. Unlike CREB alpha/delta-deficient mice whose main defect is in long-term potentiation, the widespread abnormalities in ATF-2 mutant mice demonstrate its absolute requirement for skeletal and central nervous system development, and for maximal induction of select genes with CRE sites, such as E-selectin.

Abnormalities, Multiple↗

PubMed1996

Truncation of the class II beta-chain cytoplasmic domain influences the level of class II/invariant chain-derived peptide complexes.

Previous studies have established that antigen presenting cells (APC) expressing major histocompatibility complex class II beta chains with truncated cytoplasmic domains are impaired in their capacity to activate T cells. While it had been widely accepted that this impairment is due to a defect in class II cytoplasmic domain-dependent signal transduction, we recently generated transgenic mice expressing only truncated class II beta chains, and functional analyses of APC from these mice revealed signaling-independent defects in antigen presentation. Here, we demonstrate that T cells primed on such transgenic APC respond better to stimulation by APC expressing truncated beta chains than by wild-type APC. This finding suggests that APC expressing truncated class II beta chains are not inherently defective in their antigen presenting capacity but, rather, may differ from wild-type APC in the peptide antigens that they present. Indeed, analysis of the peptides bound to class II molecules isolated from normal and transgenic spleen cells revealed clear differences. Most notably, the level of class II-associated invariant chain-derived peptides (CLIP) is significantly reduced in cells expressing only truncated beta chains. Prior studies have established that CLIP and antigenic peptides compete for binding to class II molecules. Thus, our results suggest that the cytoplasmic domain of the class II beta chain affects antigen presentation by influencing the level of CLIP/class II complexes.

Amino Acid Sequence↗

PubMed1996

Hyperproliferation and dysregulation of IL-4 expression in NF-ATp-deficient mice.

NF-ATp is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In this study, we show that mice with a null mutation in the NF-ATp gene have splenomegaly with hyperproliferation of both B and T cells. They also display early defects in the transcription of multiple genes encoding cytokines and cell surface receptors, including CD40L and FasL. A striking defect in early IL-4 production was observed after ligation of the TCR complex by treatment with anti-CD3 in vivo. The transcription of other cytokines including IL-13, GM-CSF, and TNF alpha was also affected, though to a lesser degree. Interestingly, the cytokines IL-2 and IFN gamma were minimally affected. Despite this early defect in IL-4 transcription, Th2 development was actually enhanced at later timepoints as evidenced by increased IL-4 production and IgE levels in situations that favor the formation of Th2 cells both in vitro and in vivo. These data suggest that NF-ATp may be involved in cell growth, and that it is important for the balanced transcription of the IL-4 gene during the course of an immune response.

Animals↗

PubMed1996

Blockade of CD86 ameliorates Leishmania major infection by down-regulating the Th2 response.

The costimulatory molecules CD80 and CD86 affect the differentiation of Th1 and Th2 subsets in experimental allergic encephalomyelitis, an autoimmune disorder. It is reported that the CD86 costimulator significantly affects disease outcome in Leishmania major infection, a classic model of Th subset polarization. Treatment of both L. major-resistant (C57BL/6) and susceptible (BALB/c) strains of mice with anti-CD86 substantially decreased parasite burden. This was accompanied, in BALB/c mice, by a decrease in Th2 cytokines. In contrast, anti-CD80 treatment did not affect parasite burden or cytokine levels in either strain. These data illustrate that in L. major infection, anti-CD86 can abrogate Th2 differentiation in a Th2-dominated susceptible mouse and can ameliorate disease in a Th1-dominated resistant strain, although the mechanism involved in the latter is not clear. It is concluded that in L. major infection, Th2 subset differentiation is critically dependent on interaction with the CD86 costimulatory molecule.

Animals↗

PubMed1996

Trypanosoma cruzi infection in MHC-deficient mice: further evidence for the role of both class I- and class II-restricted T cells in immune resistance and disease.

The role of T cell population in immune control of Trypanosoma cruzi infection and subsequent development of disease has been examined using gene knockout mice deficient in the expression of either or both class I and Class II MHC. Mice deficient in either class I- or class II-restricted T cell populations show a striking similarity in their mortality rate, parasite load and tissue inflammatory response following infection with the Brazil strain of T. cruzi. In both cases all animals died during the acute phase of the infection with high parasitemias and high parasite loads in the heart and skeletal muscle, but with reduced tissue inflammatory response. Mice deficient in both class I and class II MHC expression demonstrated even higher numbers of circulating and tissue parasites, essentially non-existent tissue inflammatory responses, and succumbed to infection earlier than single-deficient mice. MHC class I-deficient mice which survive into the chronic phase following infection with the M/78 or M/80 clones of T. cruzi have both relatively higher tissue parasite loads and more extensive and severe inflammatory responses than control immunocompetent mice. Immunologically, the acute infection in the double-deficient mice was accompanied by a marked increase in CD4(-)CD8(-)alphabetaTCR+ cells in the spleen. Surprisingly, both class I- and class II-deficient mice produce detectable but sub-normal levels of anti-parasite antibodies while double-deficient mice produced little to no detectable anti-parasite antibody. These results establish the importance of both class I- and class II-restricted T cells in immune control of circulating blood stages and intracellular states of T. cruzi. In addition, this work reinforces the relationship between tissue parasite load and the severity of the inflammatory lesions in chronically infected animals.

Animals↗

PubMed1996

B7-2 (CD86) is essential for the development of IL-4-producing T cells.

The CD28/CTLA-4 ligands, B7-1 (CD80) and B7-2 (CD86), provide a co-stimulatory signal necessary for optimal T cell activation. We have examined the effect of blocking B7-1 and B7-2 in an in vitro system using ovalbumin-specific T cells from alpha beta TCR-transgenic mice. This system allowed us to examine the interaction of B7 co-stimulators on physiologic antigen-presenting cells (APC) with antigen-specific T helper precursor (ThP) cells. We report that blocking Thp/B7-1 or B7-2 interactions in a primary response differentially affects the cytokine profile observed in a secondary stimulation, even in the absence of additional anti-B7 antibody. Engagement of B7-2 in the primary stimulation was found to be essential for production of the Th2 cytokine, IL-4, but not the Th1 cytokines, IL-2 and IFN-gamma, in a secondary stimulation. Conversely, inclusion of the anti-B7-1 mAb in cultures using highly purified naive T cells increased levels of IL-4 and significantly depressed levels of IFN-gamma, upon re-stimulation. The effect of the anti-B7-2 mAb in reducing IL-4 production could be overcome by the addition of recombinant IL-4 in the primary stimulation. The effects of the anti-B7-2 mAb appear to be due to blocking and not cross-linking, as F(ab) fragments mimicked the intact antibody. Taken together, our data demonstrate that the interaction between Thp and B7-2 favors the development of Th2 cells.

Animals↗

PubMed1996

Regulation of the E beta gene in vivo: lessons from E beta d transgenic mice.

We have examined the expression and regulation of the MHC class II E beta d gene in both cell lines and in transgenic mice. In transient transfection assays, as little as 192 bp of the E beta d proximal promoter was sufficient to direct constitutive expression of a reporter gene in a B cell line and to confer inducibility by IFN-gamma in a macrophage cell line. To determine if the same E beta d promoter sequences were also sufficient to direct correct expression in vivo, E beta d transgenes bearing either 4.1 or 0.2 kb of upstream sequence were introduced into an inbred mouse strain with a non-expressed endogenous E beta gene. Expression of both transgenes mirrored the expression of the endogenous I-A protein in thymus, B cells and macrophages with regard to both constitutive and cytokine-inducible expression. These results indicate that for the E beta gene only 200 bp of proximal promoter sequence are required to achieve tissue-specific and cytokine-inducible expression. This is in striking contrast to the E alpha gene, the only other murine class II gene whose promoter has been analyzed in vivo, which has been shown to require 2.0 kb of upstream sequence for appropriate expression. These data demonstrate, therefore, that the location of critical regulatory elements for the E beta and E alpha genes may differ.

Animals↗