Biomedical subjects

R E Hussey

Publications and source records attributed to R E Hussey.

13 recordsLinked to original sources

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

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

CD3 eta and CD3 zeta are alternatively spliced products of a common genetic locus and are transcriptionally and/or post-transcriptionally regulated during T-cell development.

The CD3 eta subunit of the T-cell receptor is thought to subserve an important role in signal transduction and possibly T-cell development. Herein we characterize the organization of the mouse CD3 eta gene and show that it is part of one gene locus that also encodes CD3 zeta on chromosome 1. The NH2-terminal sequence of CD3 zeta and CD3 eta, which share the same leader peptide and are identical through amino acid 122 of each mature protein, is encoded by exons 1-7. However, exons 8 and 9 are differentially spliced to give rise to CD3 zeta and CD3 eta: exons 1-8 encode CD3 zeta and exons 1-7 plus 9 encode CD3 eta. RNase protection analysis with RNA from a variety of fetal, neonatal, and adult cell types indicates that expression of both gene products is T-lineage-restricted. Importantly, expression of CD3 zeta and CD3 eta mRNA appears before or on day 16 of fetal gestation. Expression is apparently coordinate since no cell types tested express CD3 zeta or CD3 eta alone. The steady-state level of CD3 zeta mRNA is greater than or equal to 40-60 times that of CD3 eta mRNA. In immature CD4+CD8+CD3low double-positive thymocytes and CD4+CD8-CD3high or CD4-CD8+CD3high single-positive thymocytes, the respective steady-state CD3 zeta and CD3 eta mRNA levels are equivalent, whereas the amount of receptor-associated CD3 zeta and CD3 eta proteins in double-positive thymocytes is approximately 10 times less than in single-positive thymocytes. Nevertheless, the CD3 zeta/CD3 eta protein ratio remains constant in all populations (40-60:1). Furthermore, discordance between mRNA and protein levels for CD3 zeta and CD3 eta is also observed in splenic T cells. Thus, posttranscriptional and/or transcriptional regulatory mechanisms control CD3 zeta and CD3 eta expression during T-cell development.

Amino Acid Sequence↗

PubMed1989

Structural and binding analysis of a two domain extracellular CD2 molecule.

The 50-kD CD2 (T11) surface glycoprotein on human T lymphocytes and thymocytes plays a critical role in T lineage cell activation and adhesion via its ligand LFA-3. To begin to define structure-function relationships in the extracellular segment of the transmembrane CD2 molecule, we have used a eukaryotic expression system and a CD2 cDNA to produce milligram amounts of recombinant soluble CD2 molecule that corresponds to the two extracellular segment exons. We show that this protein, termed T11ex2, behaves as a monomer in aqueous solution and includes a proteolytically resistant NH2-terminal fragment (domain I) encoded by the first extracellular segment exon. Circular dichroism analysis of T11ex2 demonstrates that its stabilized secondary structure is dependent on the intrachain disulfide bonds present in domain II. The T11ex2 monomer binds directly to the CD2 ligand LFA-3 with a dissociation constant of 0.4 microM. This relatively low affinity implies that cooperative binding resulting from an array of transmembrane CD2 molecules is important to facilitate physiologic T cell adhesion.

Antibodies, Monoclonal↗

PubMed1988

Substitution of murine for human CD4 residues identifies amino acids critical for HIV-gp120 binding.

Human CD4 is the receptor for the gp120 envelope glycoprotein of human immunodeficiency virus and is essential for virus entry into the host cell. Sequence analysis of CD4 has suggested an evolutionary origin from a structure with four immunoglobulin-related domains. Only the two NH2-terminal domains are required to mediate gp120 binding. The extracellular segment of murine CD4 has an overall 50% identity with its human counterpart at the amino-acid level, but fails to bind gp120. To define those residues of human CD4 critical for gp120 binding, we have taken advantage of this species difference and substituted all non-conserved murine for human CD4 residues between amino-acid positions 27-167. We used oligonucleotide-directed mutagenesis to create each of 16 individual mutant human CD4 molecules containing from 1-4 amino-acid substitutions. Introduction of as few as three amino acids into corresponding positions of human CD4 abrogates gp120 binding. Furthermore, these critical residues are located in domain I with a contribution from domain II. Modelling studies using the three-dimensional coordinates of the V kappa Bence-Jones REI homodimer localize the site in domain I to the C" beta strand within CDR2 but projecting away from the homologues of principle antigen-binding regions CDR 1 and 3.

Amino Acid Sequence↗

PubMed1988

A soluble CD4 protein selectively inhibits HIV replication and syncytium formation.

The CD4 (T4) molecule is expressed on a subset of T lymphocytes involved in class II MHC recognition, and is probably the physiological receptor for one or more monomorphic regions of class II MHC (refs 1-3). CD4 also functions as a receptor for the human immunodeficiency virus (HIV) exterior envelope glycoprotein (gp120) (refs 4-9), being essential for virus entry into the host cell and for membrane fusion, which contributes to cell-to-cell transmission of the virus and to its cytopathic effects. We have used a baculovirus expression system to generate mg quantities of a hydrophilic extracellular segment of CD4. Concentrations of soluble CD4 in the nanomolar range, like certain anti-CD4 monoclonal antibodies, inhibit syncytium formation and HIV infection by binding gp120-expressing cells. Perhaps more importantly, class II specific T-cell interactions are uninhibited by soluble CD4 protein, whereas they are virtually abrogated by equivalent amounts of anti-T4 antibody. This may reflect substantial differences in CD4 affinity for gp120 and class II MHC.

Animals↗

PubMed1988

The T 11 (CD 2) cDNA encodes a transmembrane protein which expresses T 11(1), T 11(2) and T 11(3) epitopes but which does not independently mediate calcium influx: analysis by gene transfer in a baculovirus system.

To determine whether a full-length human T 11 (CD 2) cDNA encodes a protein which expresses all three T 11 epitopes (T 11(1), T 11(2), T 11(3] and independently triggers activation in a cell other than a T lymphocyte, a baculovirus expression system was employed. Here we show that a recombinant T 11 cDNA-containing baculovirus can induce high-level expression of T 11(1), T 11(2) and T 11(3) epitopes on the surface of gut epithelial SF9 cells. However, in this environment the T 11 protein cannot be triggered to transduce a signal resulting in an elevation of the cytosolic free Ca2+ as is known to occur in T lymphocytes. These results support the notion that T 11 functions in a coordinate fashion with other intracellular lymphoid components.

Animals↗

PubMed1988

Adhesion domain of human T11 (CD2) is encoded by a single exon.

The 50-kDa T11 (CD2) T-lymphocyte surface glycoprotein facilitates physical adhesion between T-lineage cells and their cognate cellular counterparts (cytotoxic T-lymphocytes-target cells, helper T lymphocytes-antigen-presenting cells, or thymocytes-thymic epithelium) as well as signaling through the antigen-specific T3-Ti receptor complex. To examine the relationship between the structure and function of the T11 molecule, we have utilized a baculoviral expression system to produce milligram quantities of the hydrophilic extracellular T11 segment. Enzyme cleavage, microsequencing, and HPLC analyses of the expressed protein in conjunction with genomic cloning information show that the domain involved in cellular adhesion is encoded by a single 321-base-pair exon.

Amino Acid Sequence↗

PubMed1988

Binding site for human immunodeficiency virus coat protein gp120 is located in the NH2-terminal region of T4 (CD4) and requires the intact variable-region-like domain.

The external segment of the T4 (CD4) glycoprotein functions as the T-cell surface receptor for human immunodeficiency virus by binding the major viral coat protein (gp120) with relatively high affinity. To more precisely define the region of T4 involved in gp120 interaction, we used purified, soluble forms of T4 anchor-minus polypeptides (produced in a baculovirus system) in conjunction with proteolytic fragmentation, microsequencing, and a specific T4-gp120 binding assay. The results indicate that the NH2-terminal region of T4 including the immunoglobulin variable-region-like domain is required for gp120 interaction. In contrast, the COOH-terminal half of the molecule, containing the two potential N-linked glycosylation sites, is not necessary. Furthermore, reduction of intrachain disulfide bonds in the T4 molecular abrogates gp120 binding, thereby strongly implying that the binding site for gp120 is dependent on the stabilized domain structure of the active binding region.

Amino Acid Sequence↗

PubMed1987

Molecular cloning and expression of T11 cDNAs reveal a receptor-like structure on human T lymphocytes.

The T11 (CD2) sheep-erythrocyte-binding protein is a T-cell surface molecule involved in activation of T lymphocytes and thymocytes, including those lacking the T3-Ti antigen-receptor complex. The primary structure of T11 was deduced from protein microsequencing and cDNA cloning. The mature human protein appears to be divided into three domains: a hydrophilic 185 amino acid external domain bearing only limited homology to the T-cell surface protein T4 and the immunoglobulin kappa light chain variable region, a 25 amino acid hydrophobic transmembrane segment, and a 126 amino acid cytoplasmic domain rich in prolines and basic residues. Transfection of cDNAs encoding either the 1.7- or the 1.3-kilobase T11 mRNA into COS-1 cells resulted in expression of surface T11 epitopes as well as sheep-erythrocyte-binding capacity. The predicted structure is consistent with the possibility that T11 functions in signal transduction.

Amino Acid Sequence↗

PubMed1985

Analysis of T-cell receptor gene rearrangement and expression in human natural killer clones.

A series of clones of human natural killer (NK) cells was characterized with respect to expression of the Ti alpha and Ti beta genes of the T-cell receptor. T11+T3+ NK clones contained Ti alpha and Ti beta RNA transcripts and expressed disulfide-linked heterodimers, demonstrating the presence of a functional T-cell receptor. In contrast, T11+T3- NK clones expressed only 1.0-kilobase truncated Ti beta transcripts, without a Ti alpha transcript and no detectable surface Ti protein. Since previous studies demonstrated that Ti beta gene activation precedes Ti alpha gene activation in thymic ontogeny, the T11+T3- NK cells appear to be derived from T-lineage precursors.

Clone Cells↗

PubMed1985

Molecular analysis of T cell receptor (Ti) variable region (V) gene expression. Evidence that a single Ti beta V gene family can be used in formation of V domains on phenotypically and functionally diverse T cell populations.

We examine the rules governing Ti beta variable (V) gene segment usage in the formation of T cell antigen-MHC receptors in diverse regulatory and effector T lymphoid subpopulations. To this end, a single Ti beta V gene family and its products were analyzed. A monoclonal antibody, termed anti-Ti3A, which was shown to be reactive with an epitope encoded by members of the REX cell line Ti beta V gene family, and which was expressed on 2% of human T lymphocytes was used in selection of clones from unprimed peripheral T lymphocytes. Both T4+, as well as T8+ T cell clones with inducer, suppressor, and/or cytotoxic function were defined. Southern analysis, isoelectric focusing and two-dimensional peptide mapping indicated that individual members of the REX V gene family were linked to different Ti beta diversity and/or joining and constant region segments. Moreover, the Ti alpha chains of such clones were distinct. These results imply that Ti beta V gene usage is not restricted to any functionally or phenotypically defined T cell subsets, and there is presumably little, if any, restriction on the mechanisms that generate combinational, junctional or chain association-mediated diversity.

Antibodies, Monoclonal↗

PubMed1985

Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations.

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.

Antibodies, Monoclonal↗