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H Band

Publications and source records attributed to H Band.

At least 55 records · Page 3Linked to original sources
PubMed1994

Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein.

Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.

Amino Acid Sequence↗
PubMed1994

New monoclonal antibody (23D12) recognizing three different V gamma elements of the human gamma delta T cell receptor. 23D12+ cells comprise a major subpopulation of gamma delta T cells in postnatal thymus.

The gamma delta TCR is expressed on 1 to 5% of CD3+ human peripheral blood T lymphocytes. The majority of peripheral blood gamma delta T cells expresses V gamma 9 paired with V delta 2; this subset strongly responds to certain microbial ligands. Other gamma delta T cell subsets with unknown Ag specificity expressing different V gamma elements are present in peripheral blood and lymphoid tissue. We describe a new anti-human V gamma mAb termed 23D12 with unusual specificity. As revealed by analysis of a large number of T cell clones and transfectants expressing molecularly well-defined gamma delta TCR, mAb 23D12 recognized several, but not all, members of the human V gamma 1 family, specifically V gamma 2, V gamma 3, and V gamma 4 but not V gamma 5 or V gamma 8. In combination with available mAb against V gamma 4, mAb 23D12 was used to identify V gamma 2- or V gamma 3-bearing cells. On average, 23D12+ cells accounted for 18% of peripheral blood gamma delta T cells and 56% of postnatal gamma delta thymocytes. In combination with anti-V gamma 9 mAb, mAb 23D12 23D12 identified gamma delta cells expressing V elements other than V gamma 2, V gamma 3, V gamma 4, or V gamma 9. Such cells are detectable in peripheral blood and postnatal thymus. Using mAb 23D12, we also confirmed the appearance of two distinct TCR gamma-chains on the surface of some gamma delta T cells.

Animals↗
PubMed1994

The phosphopeptide-binding specificity of Src family SH2 domains.

BACKGROUND: Src homology 2 (SH2) domains mediate protein/protein interactions by binding phosphotyrosyl proteins with high specificity. The protein Lck, a Src-like lymphocyte-specific tyrosine kinase which is important in signals involved in T-cell development, contains one such domain. The crystal structure of a complex of the Lck SH2 domain with a high-affinity ligand, pY324, is known. This ligand has the sequence EPQpYEEIPIYL. RESULTS: We designed and synthesized a series of phosphopeptides with single amino-acid changes in the four residues C-terminal to the phosphotyrosine (pTyr) in pY324. Surprisingly, the Glu one residue C-terminal to the phosphotyrosine (at position pY + 1) is sensitive to substitution, whereas the Ile at position pY + 3 is much less sensitive, accommodating a Glu with only modest loss of binding affinity. Replacement of the Glu and Pro on either side of the Ile had little effect, as predicted. Truncated phosphopeptides that end at position pY + 5 and have only an acetyl group N-terminal to the pTyr bound with only slightly lower affinity than pY324. In addition, naturally occurring phosphopeptide sequences that span a 1,000-fold range in binding affinity for the Lck SH2 domain have been identified. CONCLUSIONS: The Lck SH2 domain is highly selective for phosphotyrosyl-peptide binding; its specificity is dictated by the first and third residues C-terminal to the pTyr. The unexpected effects of some amino-acid substitutions indicate that the interactions seen between SH2 domains and ligand in the crystal structure may not be identical to those that occur in solution.

Crystallography, X-Ray↗
PubMed1994

Tumor cell-specific loss of p53 protein in a unique in vitro model of human breast tumor progression.

Mutations of the p53 gene are the most frequent genetic lesion in breast cancer. Here, we examined p53 expression in a unique in vitro model of tumor progression derived from a single breast cancer patient (21T series). While the normal mammary epithelial, fibroblast and mesothelial cells derived from this patient expressed easily detectable functional p53 protein, the primary as well as metastatic tumor cell lines demonstrated a lack of p53 protein synthesis. 21T tumor cells failed to exhibit G1 cell cycle arrest upon exposure to gamma-irradiation, and their growth was suppressed by transfection of a normal p53 cDNA, demonstrating a lack of p53-mediated function in these cells. No p53 gene deletion or rearrangements were detectable. PCR and sequence analysis of the entire coding region of p53 gene revealed a novel mutation, an insertion of a single T within codon 33, which resulted in a frame-shift and early termination. The same mutation was observed in all 21T tumor cell lines. These results demonstrate a tumor cell-specific loss of p53 protein due to a frame-shift mutation, and suggest that p53 loss may occur at a relatively early step in breast tumorigenesis before metastatic seeding or emergence of tumor heterogeneity. In addition, the availability of normal and tumor-derived epithelial cells with known p53 sequences from a single breast cancer patient should facilitate understanding of the p53 regulation in mammary cells.

Breast↗
PubMed1994

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.

Amino Acid Sequence↗
PubMed1993

Expression and stability of p53 protein in normal human mammary epithelial cells.

To examine the p53 protein expressed in human mammary epithelial cell strains grown in vitro we first established the presence of only wild-type p53 and lack of any missense mutations in a representative normal mammary epithelial cell strain, 76N, by cloning and sequencing the entire coding region of the p53 mRNA. The p53 protein expressed in this cell strain and a number of similarly derived normal mammary epithelial cell strains was compared with mammary epithelial tumor cell lines having known p53 mutations and with fibroblasts derived from normal mammary tissue. In all cell strains, immunoprecipitation from metabolically labelled cells using monoclonal antibodies (mAb) PAb 1801 and PAb 122 revealed easily detectable p53 protein. Surprisingly, mAb PAb 1620 (wild type-specific) and PAb 240 (mutant-specific) each immunoprecipitated p53 protein from both normal and tumor cells. Furthermore, p53 protein in normal mammary epithelial cells was shown to be markedly more stable (half-life of approximately 3 h) compared to that in mammary fibroblasts or rodent fibroblasts (half-life < 30 min). Immunocytochemistry with PAb 1801 showed detectable p53 protein in normal mammary epithelial cells with a predominantly nuclear staining; however, p53 protein was undetectable in fibroblasts by immunocytochemistry. Together, these results reveal unusual features of wild-type p53 protein in normal human mammary epithelial cells, suggesting a cell type-specific regulation of its expression and function.

Antibodies, Monoclonal↗
PubMed1992

Gamma delta T lymphocytes in human tuberculosis.

The manifestations of tuberculous infection reflect the immune response to infection. Most healthy tuberculin reactors develop protective immunity; tuberculous pleuritis reflects a resistant response manifest by mild disease, whereas advanced pulmonary and miliary tuberculosis reflect ineffective immunity. The role of gamma delta T cells was assessed in tuberculous infection by evaluating expansion of these cells from blood mononuclear cells after stimulation with Mycobacterium tuberculosis. After culture in vitro, the percentages of gamma delta+ cells were significantly greater in patients with protective and resistant immunity (tuberculin reactors, 25% +/- 4%; tuberculous pleuritis, 30% +/- 7%) than in those with ineffective immunity (advanced pulmonary tuberculosis, 9% +/- 3%; miliary tuberculosis, 2% +/- 1%). In leprosy, expansion of gamma delta+ cells was greater in immunologically resistant tuberculoid patients (32% +/- 4%) than in Mycobacterium leprae-unresponsive lepromatous patients (9% +/- 2%). M. tuberculosis-reactive gamma delta T cell lines produced interferon-gamma, granulocyte-macrophage colony-stimulating factor, interleukin-3, and tumor necrosis factor-alpha, cytokines that activate macrophages and may contribute to mycobacterial elimination. These findings suggest that gamma delta T cells contribute to immune resistance against M. tuberculosis.

Cell Line↗
PubMed1991

A predominance of the T cell receptor V gamma 2/V delta 2 subset in human mycobacteria-responsive T cells suggests germline gene encoded recognition.

Little is known about the nature of Ag recognition by the TCR-gamma delta. The recent observation that gamma delta T cells preferentially recognize mycobacterial Ag provides a model to examine the molecular basis of gamma delta-TCR recognition. Here, examination of the Mycobacteria-stimulated peripheral blood T cells with TCR-specific mAb revealed a predominance of T cells bearing V gamma 2/V delta 2 gene products. PCR cloning and sequence analysis of the TCR chains demonstrated extensive junctional diversity indicating that the response was polyclonal. The marked in vitro gamma delta T cell response to Mycobacteria was also detected in newborns before encounters with foreign Ag and exclusively involved the same V-gene usage observed in adults. Together, these results suggest that a major mechanism of gamma delta T cell reactivity involves recognition mediated by germline-encoded segments of the TCR.

Adult↗
PubMed1991

Increased frequency of T cell receptor V alpha 12.1 expression on CD8+ T cells: evidence that V alpha participates in shaping the peripheral T cell repertoire.

The T cell receptor repertoire has a potential for vast diversity. However, this diversity is limited by the fact that the majority of thymocytes die as the repertoire is shaped by positive and negative selection events during development. Such thymic selection affecting TCR V beta gene segment usage has been demonstrated in the mouse. However, similar data has not been forthcoming in man, and little is known about the role of the TCR alpha chain in antigen/major histocompatibility complex (MHC) recognition in any species. Here, we used a monoclonal antibody recognizing the TCR V alpha 12.1 gene product to assess the expression of this gene in the peripheral blood of man. In most individuals tested, the percentage of cells expressing V alpha 12.1 was significantly higher in CD8+ T cells than in CD4+ T cells. That the V alpha gene product itself was responsible for this increased expression in CD8+ T cells was underscored by the lack of substantial skewing of V beta usage in the V alpha 12.1-bearing T cells. Moreover, the skewed expression of V alpha 12.1 was already present at birth, indicating that it was likely to be due to a developmental process rather than the result of exposure to environmental antigens. Based on the established role for CD8 in binding to class I MHC molecules, we suggest that increased expression of V alpha 12.1 on CD8+ T cells points to a role for TCR's using V alpha 12.1 in class I MHC/Ag recognition. These results indicate that V alpha gene usage in the peripheral blood of man is not random, and they support a role for V alpha as a participant in the self-MHC recognition process that shapes the TCR repertoire.

Antibodies, Monoclonal↗
PubMed1991

Evidence for clonal selection of gamma/delta T cells in response to a human pathogen.

T cells bearing gamma/delta antigen receptors comprise a resident population of intraepithelial lymphocytes in organs such as skin, gut, and lungs, where they are strategically located to contribute to the initial defense against infection. An important unsolved question about antigen-driven gamma/delta T cell responses regards the breadth of their T cell receptor (TCR) repertoire, since many specific epithelial compartments in mice display limited diversity. We have examined the diversity of TCR delta gene expression among human gamma/delta T cells from skin lesions induced by intradermal challenge with Mycobacterium leprae. We show that the vast majority of gamma/delta cells from M. leprae lesions use either V delta 1-J delta 1 or V delta 2-J delta 1 gene rearrangements and, within a given region of the lesion, display limited junctional diversity. This contrasts markedly with the extensive diversity of gamma/delta T cells from peripheral blood of these same individuals, as well as skin from normal donors. These results indicate that the gamma/delta response to M. leprae involves the selection of a limited number of clones from among a diverse repertoire, probably in response to specific mycobacterial and/or host antigens.

Amino Acid Sequence↗
PubMed1990

Evidence for extrathymic changes in the T cell receptor gamma/delta repertoire.

The germline repertoire of variable genes for the TCR-gamma/delta is limited. This, together with the availability of several V delta-specific and a C delta-specific mAbs, has made it possible to assess differences in the TCR-gamma/delta repertoire in man. TCR-gamma/delta cells expressing particular V gene segments have been previously shown to be localized in different anatomical sites. In this study, analysis of TCR-gamma/delta V gene segment usage performed on subjects from the time of birth through adulthood revealed striking age-related changes in the TCR-gamma/delta repertoire in peripheral blood. V delta 1+ gamma/delta T cells predominated in thymus as well as in peripheral blood at birth and then persisted as a relatively constant proportion of CD3+ PBL. However, V delta 2+ gamma/delta T cells that constitute a small proportion of the CD3+ cells in thymus and in peripheral blood at birth, then expand and account for the major population of gamma/delta T cells in PBL in adults. No parallel postnatal expansion of V delta 2+ cells in the thymus was observed, even when paired thymus-peripheral blood specimens were obtained on subjects between the ages of 3 d and 8 yr. The subset of V delta 2+ lymphocytes that was expanded in peripheral blood expressed high levels of CD45RO suggesting prior activation of these cells, consistent with the possibility that their expansion might have resulted from exposure to foreign antigens or superantigens. In contrast, V delta 1+ T cells in PBL showed no comparable increase in relative numbers and were either negative or expressed only low levels of CD45RO. Consistent with evidence for extrathymic peripheral expansion of selective TCR-gamma/delta subsets, no link between MHC haplotype and differences in the TCR-gamma/delta V gene usage between individuals was apparent, and identical twins displayed TCR-gamma/delta variable gene segment phenotypes that were strikingly different from one another. The elements that determine the TCR-gamma/delta repertoire in individuals are not known. It is possible that both thymic selection and extrathymic factors may influence the peripheral repertoire. Recently, TCR-gamma/delta+ lymphocytes have been shown to expand markedly in peripheral lymphoid tissues and infectious lesions in response to mycobacterial antigens, and a correlation between mycobacterial responses and TCR-gamma/delta V gene usage has been shown in mice. The data presented here demonstrated peripheral age-related changes in the gamma/delta repertoire and point to the importance of extrathymic expansion of specific gamma/delta subsets in generating the human TCR-gamma/delta repertoire.

Adult↗
PubMed1990

Human T cell receptor-gamma and -delta chain pairing analyzed by transfection of a T cell receptor-delta negative mutant cell line.

Specific TCR V gamma and V delta segments are found to be coordinately used on subpopulations of gamma delta T lymphocytes. The reasons for this phenomenon are unknown, but may include the inability of particular chains expressing unique V delta and V gamma segments to physically associate. V delta 2 is typically used together with V gamma 2 on human peripheral blood gamma delta T lymphocytes. To examine whether V delta 2 can be used in conjunction with distinct V gamma segments, a TCR- mutant of the human gamma delta T cell line MOLT-13, which expresses parental TCR gamma (V gamma 1.3) but not TCR-delta protein, was transfected with plasmids containing full-length TCR-delta cDNA using either V delta 2 or V delta 3. TCR reconstitution was successful in both transfectants and resulted in TCR protein and RNA levels similar to that of the parental MOLT-13 cell line. These cell lines could be activated through their receptors as assessed by increases in cytoplasmic free calcium. These studies imply that physical constraints cannot explain the observed chain pairing preferences. Other possible explanations are discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Expression of human T cell receptor-gamma delta structural forms.

The human TCR-gamma delta occurs in three biochemically distinct forms (forms 1, 2bc, and 2abc). A 40-kDa TCR gamma-chain is disulfide-linked to the TCR delta-chain in form 1, whereas 40-kDa or 55-kDa TCR-gamma polypeptides are noncovalently associated with the TCR delta-chain in forms 2bc and 2abc, respectively. Sequence analysis of TCR-gamma cDNA clones indicates that form 1 utilizes the C gamma 1 gene segment, whereas forms 2bc and 2abc appear to use allelic C gamma 2 gene segments containing either two copies (b and c) or three copies (a, b, and c) of the CII exon, respectively. We transfected TCR-gamma cDNA encoding form 1 or form 2abc into the MOLT-13 cell line that expresses form 2bc. The transfected TCR gamma-chains associate with the resident MOLT-13 TCR-delta, normally part of form 2bc, to yield CD3-associated TCR-gamma delta heterodimers identical to those seen on the donor cell lines (form 1 or 2abc). These transfection experiments show directly that, 1) when a single TCR-delta subunit is available, the presence or absence of disulfide linkage between TCR gamma- and TCR delta-chains is controlled by the TCR gamma-chain, and 2) the difference in the amount of N-linked carbohydrate attached to the transfected TCR-gamma proteins of form 2bc vs form 2abc is influenced by the presence or absence of CII exon copy "a" which appears to alter the secondary and/or tertiary structure of these TCR gamma-chain constant regions, thereby affecting the attachment of N-linked glycans. In contrast to the similar structure and usage of C beta 1 and C beta 2, TCR-gamma delta forms show striking differences in structure and are not equally represented in peripheral blood. Although the role of each form is unknown, it is possible that variable or joining-gene segment selection events or functional differences account for their unequal usage.

Cell Line↗