Biomedical subjects

E L Reinherz

Publications and source records attributed to E L Reinherz.

At least 19 recordsLinked to original sources

PubMed1992

Differential regulation of T-cell receptor processing and surface expression affected by CD3 theta, an alternatively spliced product of the CD3 zeta/eta gene locus.

The T-cell receptor (TCR) is a multisubunit complex consisting of the clonotypic Ti alpha and beta (or Ti gamma and delta) subunits and the invariant CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, and CD3 eta subunits. Herein, we describe an additional product from the CD3 zeta/eta gene locus which we have termed CD3 theta. The cDNA derives from the first seven exons common to CD3 zeta and CD3 eta, 94 base pairs (bp) of the CD3 eta-specific exon 9 and an additional exon 10 encoding the carboxyl-terminal 15 amino acids and the 3'-untranslated region. The expression of CD3 theta is equivalent to that of CD3 eta in tissue distribution and level of expression as judged by RNase protection analysis. Despite the identity of the amino-terminal 121 amino acids of CD3 zeta, CD3 eta, and CD3 theta and an additional 31 amino acids shared between CD3 eta and CD3 theta, transfection of CD3 theta into the CD3 zeta- eta- T-cell hybridoma, MA5.8, failed to restore detectable surface TCR expression in contrast to transfection with CD3 zeta or CD3 eta. Analysis of the CD3 theta protein in transfectants indicated that CD3 theta is associated with the TCR intracellularly. However, unlike with CD3 zeta, Ti alpha-beta chains remain endoglycosidase H sensitive, suggesting a role for the unique COOH-terminal segment of CD3 theta in mediating TCR retention and/or degradation in a pre-Golgi compartment.

Alternative Splicing↗

PubMed1992

Human immunodeficiency virus gp120 binding C'C" ridge of CD4 domain 1 is also involved in interaction with class II major histocompatibility complex molecules.

Using site-directed mutagenesis informed by high-resolution CD4 structural data, we have investigated the role of residues of the C'C'' ridge region of human CD4 on class II major histocompatibility complex (MHC) binding. This C'C'' ridge is homologous to the CDR2 loop of an immunoglobulin variable domain and is known to contain the binding site for human immunodeficiency virus (HIV) coat glycoprotein gp120. Here we report that this region is also involved in interaction with class II MHC. Exposed positively charged residues Lys-35, Lys-46, and Arg-59 and the exposed hydrophobic residue Phe-43 contribute significantly to class II MHC binding. Moreover, mutations in the buried residues Trp-62 and Ser-49, which support the top and bottom of the C'C'' ridge, respectively, disrupt class II MHC interaction. The HIV binding region appears to involve a restricted area of the larger class II MHC binding site on CD4. Strategies of drug design aimed at interrupting CD4-HIV interaction will need to consider the extensive overlap between class II MHC and HIV gp120 binding surfaces in this region of CD4.

Animals↗

PubMed1992

Similarity between fluorescein-specific T-cell receptor and antibody in chemical details of antigen recognition.

A computer-generated model of the single-chain variable V alpha V beta fragment of the RFL3.8 T-cell receptor (TCR) specific for fluorescein served as a starting point for mutagenesis aimed at identification of its antigen-contacting residues. Selected backbone segments of the model representing regions of prominent sequence similarity between antibodies and TCRs were least-squares superimposed onto the corresponding segments of the crystallographically resolved 4-4-20 antibody complexed with its antigen, fluorescein. The superimposition placed the antibody-bound fluorescein molecule close to a cavity on the surface of the TCR model formed by the complementarity-determining region (CDR) loops. Some of the TCR cavity forming loops displayed sequence motifs related to canonical CDR loops previously found in antibodies. Six putative amino acid contacts were identified and single-chain TCRs with mutations at each of these positions were expressed in Escherichia coli, purified, refolded, and assayed for fluorescein binding. Five of the six mutations resulted in a loss of detectable binding. These RFL3.8 antigen combining site residues are distributed among the beta 3, alpha 1, and alpha 2 CDR loops and show striking chemical similarity to the known fluorescein contact residues on 4-4-20. Thus, antibodies and TCRs are similar both in their overall architecture and in the chemical details of specific antigen recognition.

Amino Acid Sequence↗

PubMed1992

Negative selection of thymocytes. A novel polymerase chain reaction-based molecular analysis detects requirements for macromolecular synthesis.

Self-tolerance is mainly established through clonal deletion of autoreactive T cells during thymic differentiation. The mechanisms by which deletion is achieved are poorly understood. Here we use a specific polymerase chain reaction-based system to characterize DNA fragmentation and show that after in vivo treatment of neonatal mice with staphylococcus enterotoxin B, selective apoptosis of V beta 8+ thymocytes occurs. This process precedes detectable deletion of V beta 8+ cells as determined by phenotypic analysis. Moreover, in vivo administration of cycloheximide and, to a lesser extent, actinomycin D, inhibits apoptosis of staphylococcus enterotoxin B specific thymocytes. Thus, macromolecular synthesis is a requirement for negative selection.

Animals↗

PubMed1992

CD10/NEP is expressed on Thy-1low B220+ murine B-cell progenitors and functions to regulate stromal cell-dependent lymphopoiesis.

To further characterize the function of the common acute lymphoblastic leukemia antigen (CALLA; CD10, neutral endopeptidase 24.11, NEP) in early lymphoid development, we have identified murine lymphoid progenitors expressing CD10/NEP and analyzed the effects of inhibiting the enzyme in in vitro assays of murine lymphoid differentiation. CD10/NEP transcripts and enzymatic activity were primarily restricted to the subpopulation of murine lymphoid progenitors, termed pro-B cells, which were isolated from bone marrow (BM) and modified Whitlock-Witte cultures and defined by coexpression of B220 and low levels of Thy-1. CD10/NEP transcripts and cell surface enzymatic activity were also detected in BM stromal cells known to support the development of B-lymphoid progenitors. In contrast, Abelson and H-ras transformed pre-B-cell lines were CD10/NEP- as were Thy-1-B220+ pre-B cells from BM and modified Whitlock-Witte cultures and Thy-1lowLin- (B220-Mac-1-GR-1-Ly-2/3-) uncommitted hematopoietic progenitors from BM. The expression of CD10/NEP on murine pro-B cells and BM stromal cells suggests a role for the enzyme in early B-cell ontogeny. In modified Whitlock-Witte cultures in which Thy-1lowLin- progenitors plated on BM stromal cells differentiate into Thy-1lowB220+ pro-B and Thy-1-B220+ pre-B cells, the addition of specific CD10/NEP inhibitors increased the number of lymphoid colonies at days 5 through 7 by 34% (P < .001). The results suggest that CD10/NEP participates in the regulation of the earliest stages of stromal cell-dependent B lymphopoiesis.

Animals↗

PubMed1992

The human immunodeficiency virus gp120 binding site on CD4: delineation by quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high-resolution CD4 atomic structure.

The first immunoglobulin V-like domain of CD4 contains the binding site for human immunodeficiency virus gp120. Guided by the atomic structure of a two-domain CD4 fragment, we have examined gp120 interaction with informative CD4 mutants, both by equilibrium and kinetic analysis. The binding site on CD4 appears to be a surface region of about 900 A2 on the C" edge of the domain. It contains an exposed hydrophobic residue, Phe43, on the C" strand and four positively charged residues, Lys29, Lys35, Lys46, and Arg59, on the C, C', C", and D strands, respectively. Replacement of Phe43 with Ala or Ile reduces affinity for gp120 by more than 500-fold; Tyr, Trp, and Leu substitutions have smaller effects. The four positively charged side chains each make significant contributions (7-50-fold). This CD4 site may dock into a conserved hydrophobic pocket bordered by several negatively charged residues in gp120. Class II major histocompatibility complex binding includes the same region on CD4; this overlap needs to be considered in the design of inhibitors of the CD4-gp120 interaction.

Antibodies, Monoclonal↗

PubMed1992

The CD3 zeta cytoplasmic domain mediates CD2-induced T cell activation.

CD2-mediated T lymphocyte activation requires surface expression of CD3-Ti, the T cell receptor (TCR) for antigen major histocompatibility complex protein. Given the importance of CD3 zeta in TCR signaling, we have directly examined the ability of the CD3 zeta cytoplasmic domain to couple CD2 to intracellular signal transduction pathways. A cDNA encoding a chimeric protein consisting of the human CD3 zeta cytoplasmic domain (amino acid residues 31-142) fused to the CD8 alpha extracellular and transmembrane domains (amino acid residues 1-187) was transfected into a CD2+CD3-CD8- variant of the human T cell line Jurkat. The resulting transfectants expressed the CD8 alpha/CD3 zeta chimeric receptor at the cell surface in the absence of other TCR subunits. Stimulation of these transfectants with anti-T11(2) + anti-T11(3) monoclonal antibodies (mAbs) initiated both a prompt cytosolic free calcium ([Ca2+]i) rise and protein tyrosine kinase activation. Stimulation with either intact anti-T11(2) + anti-T11(3) mAbs or purified F(ab')2 fragments resulted in interleukin 2 (IL-2) secretion. In contrast, control cell lines transfected with a cDNA encoding wild-type CD8 alpha, and thus lacking surface expression of the CD3 zeta cytoplasmic domain, failed to show any [Ca2+]i rise, protein tyrosine kinase activation, or IL-2 secretion after identical stimulation. These data directly establish the CD3 zeta cytoplasmic domain as a necessary and sufficient component of the CD3-Ti complex involved in T lymphocyte activation through CD2. Moreover, they show that CD2 signaling can function in the absence of Fc receptors.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1992

Murine common acute lymphoblastic leukemia antigen (CD10 neutral endopeptidase 24.11). Molecular characterization, chromosomal localization, and modeling of the active site.

To further analyze CD10/NEP function in lymphoid and nonlymphoid cells using well characterized murine systems, we isolated the murine CD10/NEP homologue, determined its chromosomal location, and modeled the enzyme's active site. The murine CD10/NEP cDNA predicts a 750-amino acid (aa) type II integral membrane protein with 90% identity to the human CD10 sequence and 100% conservation of critical aa and functional motifs. The latter include the pentapeptide consensus sequence required for zinc binding and catalytic activity, additional aa associated with substrate binding, and the extracellular cysteines that participate in disulfide bonds required for enzymatic activity. Like its human homologue, murine CD10/NEP has multiple alternative 5'-untranslated region sequences. The gene is localized on the proximal half of murine chromosome 3. In Northern analysis, murine CD10/NEP transcripts are abundant in bone marrow stromal cells that support pre-B cell differentiation but are undetectable in representative Abelson transformed pre-B cell lines. The murine CD10/NEP active site was modeled by aligning critical conserved CD10/NEP residues with comparable residues in the active site of thermolysin, a bacterial metalloprotease with similar substrate specificity. The model predicts that the two enzymes have similar clefts that comprise the active site and permit zinc-dependent substrate interactions.

Amino Acid Sequence↗

PubMed1992

A population of early fetal thymocytes expressing Fc gamma RII/III contains precursors of T lymphocytes and natural killer cells.

We have identified a dominant fetal thymocyte population at day 14.5 of gestation in the mouse that lacks CD4 and CD8 but expresses Fc gamma RII/III several days prior to acquisition of the T cell receptor (TCR) in vivo. If maintained in a thymic microenvironment, this population of CD4-CD8-TCR-Fc gamma RII/III+ thymocytes differentiates first into CD4+CD8+TCRlowFc gamma RII/III- thymocytes and subsequently CD4+CD8-TCRhighFc gamma RII/III- and CD4-CD8+TCRhighFc gamma RII/III- mature Ti alpha-beta lineage T cells. However, if removed from the thymus, the CD4-CD8-TCR-Fc gamma RII/III+ thymocyte population selectively generates functional natural killer (NK) cells in vivo as well as in vitro. These findings show that a cellular pool of Fc gamma RII/III+ precursors gives rise to T and NK lineages in a microenvironment-dependent manner. Moreover, they suggest a hitherto unrecognized role for Fc receptors on primitive T cells.

Animals↗

PubMed1992

CD3 zeta dependence of the CD2 pathway of activation in T lymphocytes and natural killer cells.

In T lymphocytes, signal transduction through the CD2 adhesion molecule requires surface expression of the CD3-Ti T-cell receptor (TCR) complex. In contrast, in natural killer (NK) cells, CD2 functions in the absence of a TCR. Because the TCR on T lymphocytes and the CD16 low-affinity IgG Fc receptor (Fc gamma receptor type III) complex on NK cells share a common CD3 zeta subunit, we reasoned that CD3 zeta may be critical for CD2 signaling in T lymphocytes and NK cells. Here we show that transfection of a cDNA encoding a transmembrane form of CD16 into TCR- variants of the Jurkat T-cell line results in CD16 expression in association with endogenous CD3 zeta homodimers and restores CD2 signaling function. To test directly the role of CD3 zeta in CD2 triggering, we then transfected human CD2 into each of two murine T-T hybridomas that express TCRs containing either a full-length CD3 zeta subunit or a truncated CD3 zeta subunit incapable of transducing signals. Despite expression of equivalent surface levels of TCR, CD2-mediated signaling is seen only in the T cells containing wild-type CD3 zeta. These findings show that (i) CD16 on NK cells is functionally equivalent to the TCR on T lymphocytes for coupling CD2 to signaling pathways and (ii) CD2 signal transduction depends upon the CD3 zeta subunit of the TCR complex and, by inference, the CD3 zeta subunit of the CD16 complex.

Animals↗

PubMed1992

Phosphorylation of multiple CD3 zeta tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation.

T lymphocyte activation resulting from antigen recognition involves a protein tyrosine kinase pathway which triggers phosphorylation of several cellular substrates including the CD3 zeta subunit of the T cell receptor (TCR) to form pp21. The homologous TCR-associated protein, CD3 eta, is an alternatively spliced product of the same gene locus as CD3 zeta. CD3 eta lacks one of six cytoplasmic tyrosine residues (Tyr-132) found in CD3 zeta and is itself not phosphorylated. Site-directed mutagenesis in conjunction with in vitro and in vivo phosphorylation studies herein demonstrates that Tyr-132 is required for the formation of pp21. Moreover, the differential phosphorylation of CD3 zeta versus CD3 eta is not due to a selective association of the known TCR-associated protein tyrosine kinase, p59fyn; p59fyn but not p56lck or p62yes is associated with each of the three TCR isoforms containing CD3 zeta 2, or CD3 eta 2, or CD3 zeta-eta. This association occurs through components of the TCR complex distinct from CD3 zeta or CD3 eta. In addition, we show that pp21 formation is not only dependent on Tyr-132 but results from concomitant phosphorylation of other CD3 zeta residues including Tyr-121. Mutation of Tyr-90, -121, or -132 does not alter primary signal transduction as shown by the ability of individual CD3 zeta Tyr----Phe mutants to produce interleukin-2 upon TCR stimulation. Thus, the substantial structural changes in CD3 zeta upon TCR stimulation as reflected by alteration in its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis may affect subsequent events such as receptor desensitization, receptor movement, and/or protein associations.

Amino Acid Sequence↗

PubMed1992

T cell receptor complexes containing Fc epsilon RI gamma homodimers in lieu of CD3 zeta and CD3 eta components: a novel isoform expressed on large granular lymphocytes.

CD3 zeta and CD3 eta form disulfide-linked homo- or heterodimers important in targeting partially assembled Ti alpha-beta/CD3 gamma delta epsilon T cell receptor (TCR) complexes to the cell surface and transducing stimulatory signals after antigen recognition. Here we identify a new TCR isoform expressed on splenic CD2+, CD3/Ti alpha-beta+, CD4-, CD8-, CD16+, NK1.1+ mouse large granular lymphocytes (LGL), which are devoid of CD3 zeta and CD3 eta proteins. The TCRs of this subset contain homodimers of the gamma subunit of the high affinity receptor for IgE (Fc epsilon RI gamma) in lieu of CD3 zeta and/or CD3 eta proteins. The LGL display natural killer-like activity and are cytotoxic for B cell hybridomas producing anti-CD3 epsilon and anti-CD16 monoclonal antibodies, demonstrating the signaling capacity of both TCR and CD16 in this cell type. These findings provide evidence for an additional level of complexity of TCR signal transduction isoforms in naturally occurring T cell subsets.

Animals↗

PubMed1992

Human genomic sequences corresponding to murine CD3 eta-related transcripts: lack of conservation or expression of homologous human products.

We have cloned and sequenced human genomic DNA homologous to exons 9 and 10 of the CD3 zeta/eta/theta locus. Although there are open reading frames within the human sequences corresponding to the translated portions of murine exons 9 and 10, we find no evidence of conservation of the encoded polypeptide product. Furthermore, using oligonucleotides derived from these homologous sequences, we are unable to detect human CD3 eta- or CD3 theta-like transcripts by polymerase chain reaction amplification of reverse-transcribed RNA from a variety of human lymphoid tissues. Despite the absence of evidence for conservation of human CD3 eta and CD3 theta, there is a surprising degree of similarity between human and murine nucleotide sequences, not only for exons 9 and 10 (78% and 70%, respectively), but also for the 9/10 intron (71%). A possible mechanism for this conservation is discussed.

Animals↗

PubMed1992

Characterization of Fc epsilon RI gamma in human natural killer cells.

We report the characterization of the Fc epsilon RI gamma chain which associates with the transmembrane form of CD16 to form the low affinity receptor for IgG (Fc gamma RIII) expressed on human natural killer (NK) cells. cDNA cloning and sequence analysis of Fc epsilon RI gamma from a polyclonal CD3-CD16+ NK line established that this molecule is identical to Fc epsilon RI gamma previously identified in human basophils as part of a high affinity receptor for IgE. Polymerase chain reaction analysis of Fc epsilon RI gamma gene expression in a series of CD3+CD16- and CD3-CD16+ NK clones reveals that Fc epsilon RI gamma is not directly linked to NK activity since clones of the CD3+CD16- phenotype lack Fc epsilon RI gamma RNA but nevertheless mediate cytotoxicity. Taken together, these results demonstrate that the Fc epsilon RI gamma molecule is expressed in various types within the hematopoietic system as part of multimeric surface receptors involved in different biological functions.

Base Sequence↗

PubMed1992

T-cell-receptor isoforms.

Early work on T-cell hybridomas lacking the T-cell-receptor (TCR) sub-unit CD3 eta had suggested a correlation between the presence of CD3 zeta-eta heterodimers and signalling leading to phosphatidyl-inositol (PI) turnover as well as activation-induced cell death. The cloning of CD3 eta has now allowed thorough and direct analysis of the signal transduction properties of CD3 zeta-zeta-, CD3 zeta-eta- and CD3 eta-eta-containing TCRs. We have found that all forms of the TCR are capable of transducing signals leading to PI turnover, Ca2+ mobilization, IL-2 production and cell-cycle arrest. CD3 zeta and CD3 eta utilize the same promoter which yields coordinate expression of both products, so that restricted CD3 eta expression in a sub-population of thymocytes is unlikely. Immunohistochemical methods employing an anti-CD3 eta-specific monoclonal antibody (MAb) show no detectable staining of thymic sections from adult mice, implying at best a low level of constitutive CD3 eta expression. In contrast, CD3 eta expression is readily detected in the majority of cortical thymocytes of CD3 eta transgenic mice using a Thy-1 promoter construct. However, over-expression of CD3 eta in mice transgenic for this polypeptide does not result in increased negative selection in vivo, consistent with the in vitro findings that induction of cell death is not strictly dependent on CD3 eta. Despite earlier reports of the detection of human CD3 eta protein, we find no CD3 eta message in human thymus or T cells. Cloning of the human CD zeta-eta genomic locus has demonstrated approximately 70% homology between the mouse and human genomic sequence, corresponding to the mouse CD3 eta-specific exon. However, translation of the DNA sequence does not result in a homologous amino acid sequence. Thus, there does not appear to be a CD3 eta protein in humans.

Animals↗

PubMed1991

CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung.

Amino Acid Sequence↗

PubMed1991

A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties.

A strategy for the production of small, soluble, single-chain T-cell receptor (scTCR) fragments that carry an intact TCR antigen-combining site is presented. The rationale is based on structural similarity between TCR and antibody molecules and use of computer modeling methods to derive a model structure of a human scTCR variable (V)-domain dimer. A gene encoding the RFL3.8 TCR protein, specific for the hapten fluorescein in the context of major histocompatibility complex class II and composed of one V alpha and one V beta domain joined via a flexible peptide linker, was assembled in an Escherichia coli plasmid. Subsequently, the protein was produced in a bacterial expression system, purified, refolded, and found to be poorly soluble at neutral pH in aqueous buffers. An inspection of the computer-generated V alpha-V beta domain model showed several surface exposed hydrophobic residues. When these were replaced by water-soluble side chains via site-directed mutagenesis of the corresponding gene, a soluble protein resulted and was shown to have antigen-binding properties equivalent to those of the intact TCR of the RFL3.8 T-cell clone. These results demonstrate the feasibility of obtaining TCR fragments endowed with antigen-combining properties by protein engineering in E. coli.

Amino Acid Sequence↗

PubMed1991

CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide-induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation.

The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, "enkephalinase"). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu-phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.

Antigens, Differentiation↗