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J I Bell

Publications and source records attributed to J I Bell.

At least 73 records · Page 4Linked to original sources
PubMed1995

Sequence analysis of the human alpha beta T-cell receptor CDR3 region.

Although the three-dimensional structure of the T-cell receptor (TCR) has not yet been determined, several groups have proposed that the outline structure of the TCR will closely resemble that of immunoglobulin (Ig). Hypervariable regions can be identified within the TCR variable (V) domains, and by analogy to similar regions in the Ig molecule which together form the antigen combining site these have been termed the complementarity determining regions (CDR) 1, 2, and 3. By far the greatest extent of variability occurs at CDR3 and this has led to the proposal that CDR3 is involved in interaction with the peptide bound within the cleft of a major histocompatibility complex (MHC) molecule. We have cloned and sequenced the CDR3 region of several hundred human TCRA and TCRB transcripts from different T-cell populations and studied the amino acid usage in this region. Results show that the average length of the CDR3 region is 10 amino acids with less variation in length than is seen for the Ig heavy chain. There is no difference in CDR3 length between fetal and adult T cells or between CD4 and CD8 populations. The pattern of amino acid usage in the CDR3 region is dissimilar between TCRA and TCRB transcripts. In particular there is a predominance of charged and polar residues in the region of the TCRA transcript thought to interact with peptide. These data provide information on the general pattern of CDR3 length and composition for both TCRA and TCRB.

Adult↗
PubMed1995

Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon.

The CD44 cell surface glycoprotein is expressed on a broad range of different tissues as multiple isoforms containing from one to ten alternatively spliced exons v1-v10 inserted within the extracellular domain. Differential glycosylation generates still further variability, yielding both N- and O-glycan-modified forms of CD44 in addition to proteoglycan-like variants containing chondroitin sulphate and heparan sulphate. These high molecular mass proteoglycan-like variants, previously identified in lymphocytes, melanomas, and keratinocytes have been implicated in cell-matrix adhesion, cell motility, and invasiveness. More recently, monocyte CD44 molecules presumed to carry glycosaminoglycan chains were shown to bind the chemokine MIP-1 beta (Tanaka, Y.,D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. 1993. Nature (Lond). 361:79-82.) raising the intriguing possibility that proteoglycan-like CD44 variants might play a role in regulating inflammatory responses. Here we have investigated the molecular identity of these proteoglycan-like CD44 variants by generating a panel of recombinant CD44 isoforms using a novel cassette cloning strategy. We show that both chondroitin and heparan sulphate modifications are associated specifically with isoforms (CD44v3-10 and CD44v3,8-10) containing the v3 alternative exon which encodes a consensus motif SGXG for GAG addition. Other isoforms (CD44v10, CD44v8-10, CD44v7-10, and CD44v6-10) are shown to lack these GAG chains but to carry extensive O-glycan modifications, most likely within the mucin-like alternative exon inserts. We also demonstrate that the majority of endogenous GAG-modified CD44 isoforms present in epithelial cells constitute v3 isoforms thus establishing that in these cells the majority of proteoglycan-like CD44 variants are generated by alternative splicing. Finally we present evidence using transfected B lymphoma cells that the GAG-modified CD44 isoforms CD44v3-10 and CD44v3,8-10, unlike CD44H, bind only weakly to hyaluronan. Together with the demonstration in the accompanying paper (Bennett, K., D. G. Jackson, J.C. Simon, E. Tanczos, R. Peach, B. Modrell, I. Stamenkovic, G. Plowman, and A. Aruffo. 1995. J. Cell Biol. 128:687-698.), that CD44 molecules containing the v3 exon bind growth factors, these results highlight a new and potentially important role for CD44 alternative splicing in the control of cell-surface proteoglycan expression.

Alternative Splicing↗
PubMed1995

Regulation of insulin gene expression by the IDDM associated, insulin locus haplotype.

A 4.1 kb genomic region, spanning the insulin (INS) gene, confers genetic susceptibility to Type 1 or insulin-dependent diabetes mellitus (IDDM). Ten polymorphisms within this region form two predominant, complementary haplotypes. We have been studying the effects of these polymorphisms on the levels of insulin mRNA. Cloned genomic DNA fragments representing these two separate haplotypes were transiently transfected into a rodent pancreatic beta cell line, HIT-T15. These studies revealed that insulin mRNA levels were consistently higher in the transfectants expressing the diabetic haplotype. Over-expression of insulin mRNA may provide the basic mechanism for the diabetic susceptibility encoded at the INS locus.

Animals↗
PubMed1994

Binding of soluble natural ligands to a soluble human T-cell receptor fragment produced in Escherichia coli.

An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.

Amino Acid Sequence↗
PubMed1994

Expression of alternatively spliced forms of the CD44 extracellular-matrix receptor on human lung carcinomas.

Expression of isoforms of the CD44 hyaluronan receptor/lymph-node endothelial receptor by human tumour cells is thought to play a role in tumour growth and metastasis. These isoforms which vary in the length of the extracellular domain are generated by differential RNA splicing that involves the 10 alternative exons (v1 to v10) encoding the membrane proximal region of the molecule. Several tumours have been shown to over-express CD44 containing the v6 exon, and this, together with other evidence, has led to the suggestion that v6 may play a causative role in tumour metastasis. In this report we have compared the expression of CD44 isoforms between different lung tumour lines, including SCLC, squamous-cell carcinoma, adenocarcinoma and mesothelioma, using both RT-PCR and fluorescent antibody staining with a panel of CD44 exon-specific monoclonal antibodies (MAbs). Our results show large differences in vCD44 expression between individual tumour lines. Little or no vCD44 containing the metastasis-associated v6 exon was detected in most tumours, including the highly metastatic SCLC lines. Indeed, the SCLC lines and some squamous-cell carcinomas contained only very low levels of either vCD44 or CD44H, indicating that CD44 expression may not always correlate with tumour development or dissemination. One of the squamous-cell carcinomas studied (HOTZ) was found to express a complex mixture of CD44 splice variants similar to the immortalized normal bronchial epithelial line BEAS-2B. Cloning and sequencing of vCD44 from the HOTZ cell line yielded several splice variants that have also been identified on leukaemic cells, normal keratinocytes and activated peripheral-blood lymphocytes.

Alternative Splicing↗
PubMed1994

CD44 isoform expression mediated by alternative splicing: tissue-specific regulation in mice.

CD44 is a widely distributed cell surface glycoprotein which shows heterogeneity in molecular expression as a result of post-translational modification as well as alternative splicing of CD44 mRNA. Functional studies have indicated that CD44 plays a role as an adhesion molecule and that different CD44-expressing cells differ in their capacities for CD44-dependent ligand binding. These observations have raised the possibility that structural modifications of CD44, including those resulting from alternatively spliced mRNA isoforms, are involved in the functional heterogeneity of CD44. To assess the expression of CD44 isoforms in the mouse, we examined CD44 cDNA by reverse transcription polymerase chain reaction (RT-PCR). Southern blotting of PCR products with a CD44 cDNA probe or with internal oligonucleotides revealed the expression in mouse tumor cell lines and normal tissues of multiple CD44 mRNA products which are larger than that observed in the absence of variable exon expression. Interestingly, different mouse tissues, including lymphoid cells, showed unique patterns of alternative CD44 mRNA in Southern blotting analysis. The use of exon-specific primers allowed detection of multiple alternatively spliced mRNA species involving expression of at least seven variable exons. Cloning and sequencing of these PCR products revealed sequence identity with recently identified genomic CD44 sequences and confirmed that the PCR products correspond to mature mRNA expressing alternatively spliced CD44 exons. Taken together, these findings demonstrate that the mouse expresses multiple variably spliced CD44 isoforms and that expression is regulated in a tissue- and cell-type specific manner.

Alternative Splicing↗
PubMed1994

HLA-DR and HLA-DP genotypes and immunoglobulin E responses to common major allergens.

In order to test for human histocompatibility leucocyte antigens (HLA) class II restriction of IgE responses, 431 subjects from 83 families were genotyped at the HLA-DR and HLA-DP loci and serotyped for IgE responses to six major allergens from common aero-allergen sources. A possible excess of HLA-DR1 was found in subjects who were responsive to Fel d I compared with those who were not (Odds Ratio (OR) = 2, P = 0.002), and a possible excess of HLA-DR4 was found in subjects responsive to Alt a I (OR = 1.9, P = 0.006). Increased sharing of HLA-DR/DP haplotypes was seen in sibling pairs responding to both allergens. Der p I, Der p II, Phl p V and Can f I were not associated with any definite excess of HLA-DR alleles. No significant correlations were seen with HLA-DP genotype and reactivity to any of the allergens. The results suggest class II HLA restriction is insufficient to account for individual differences in reactivity to common allergens.

Alleles↗
PubMed1994

Role of human papillomavirus in determining the HLA associated risk of cervical carcinogenesis.

AIMS: To investigate the role of human papillomavirus (HPV) in the association between HLA DQw3 and squamous cell cancer of the cervix (SCCC). METHODS: Tissue from 194 cervical samples, ranging from normal, through cervical intraepithelial neoplasia, to SCCC, were typed for HPV by amplification of the L1 gene using degenerate consensus primers, followed by oligonucleotide probing. HLA DQw3 typing was undertaken in the same samples using a new PCR amplification system using primers common to all DQ loci, followed by restriction digestion with Mlu 1 to differentiate HLA DQw3 types--null, heterozygous, and homozygous. The data were analysed using chi 2 analysis and by calculating relative risks with the 95% confidence interval. RESULTS: Samples (n = 188) were successfully typed for HPV and 177 were typed for HLA DQw3. There was a nonsignificant rise in the prevalence of HLA DQw3 in SCCC (64.3%) compared with the group with normal histology (53.2%). Analysis of the prevalence of HLA DQw3 on the basis of HPV infection rather than histology showed that 63 of 95 (66.3%) of the HPV positive samples contained HLA DQw3 alleles, compared with 39 of 78 (50.0%) of the HPV negative samples (chi 2 4.06; p < 0.05). CONCLUSIONS: There was a significant association between HLA DQw3 and cervical HPV infection. This may be because people with HLA DQw3 are less able to mount an effective immune response to HPV, which predisposes them to the development of SCCC.

Base Sequence↗
PubMed1993

A method for producing monoclonal antibodies to human T-cell-receptor beta-chain variable regions.

Study of the T-cell repertoire in humans has been hampered by the lack of monoclonal antibodies (mAbs) to the T-cell receptor (TCR) variable region (V) gene products. We describe a method for producing mAbs to the human TCR beta-chain V (V beta) gene products in which mice were immunized with a rat basophil cell line (RBL-2H3) transfected with the extracellular domain of the TCR heterodimer fused to the lambda chain of CD3. These cells acted as excellent immunogens for raising anti-TCR mAb and also formed the basis of a rapid screening assay. We generated mAbs against V beta protein of the TCR, showed that these mAbs stained approximately 1% of peripheral blood T cells, and further showed that the mAbs could stimulate proliferation of these T cells. We then characterized the mAbs by amplifying TCR cDNA derived from mAb-stimulated cells and sequencing the beta chain. All clones sequenced used the V beta 7.1 chain, proving conclusively that the mAbs generated were specific for V beta 7.1 subfamily. This method generates mAbs to human TCR V beta proteins efficiently and might allow production of a complete panel of mAbs directed against human TCR V beta proteins.

Amino Acid Sequence↗
PubMed1993

The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor. Comparison of all 10 variable exons between mouse, human, and rat.

The human CD44 cell surface glycoprotein family which has been implicated in lymphocyte homing, tumor metastasis, and extracellular matrix attachment consists of a large number of related isoforms that derive from the differential splicing of a single CD44 gene transcript. Recent mapping of the CD44 locus in man indicated the presence of at least nine alternative exons within the region of the gene encoding the variable membrane proximal extracellular domain. Here we report the identification of a 10th alternative exon (termed V1) in the mouse, human, and rat CD44 genes. We demonstrate tissue-specific patterns of expression for transcripts containing exons V1-V10 in the mouse and a highly restricted usage of exon V1 in transcripts from mouse gastric tissue. Close sequence homology between exons V1-V10 from mouse rat and human points to a specific functional role rather than a purely structural role for the membrane proximal extracellular domain of the CD44 molecule.

Alternative Splicing↗
PubMed1993

CD44 and cancer.

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Humans↗
PubMed1993

Expression and function of HLA-B27 in lipid-linked form: implications for cytotoxic T lymphocyte-induced apoptosis signal transduction.

Major histocompatibility complex (MHC) class I antigen-restricted cytotoxic T lymphocytes (CTL) kill their target cells not only by inducing irreversible membrane damage but also by triggering a programmed suicide cascade (apoptosis) in target cells. Recent evidence suggests that MHC class I antigens are involved in apoptosis signal transduction in T cells. Therefore, it is possible that MHC class I antigens are also responsible for CTL-induced signal transduction in target cells leading to apoptosis. To test this hypothesis, we have expressed HLA-B27 in Chinese hamster ovary (CHO) cells in a phosphatidyl inositol (PI) anchored form. The expressed Pl-anchored HLA-B27 (PI-B27), a 42-kDa molecule which can be cleaved off the cell surface by PI-specific phospholipase C, can function as an MHC restriction and antigen presentation element for specific CTL. Furthermore, PI-B27 transfectant CHO cells undergo rapid DNA fragmentation when pulsed with the appropriate peptide and treated with specific CTL, suggesting that the cytoplasmic and transmembrane domains of the heavy chain of class I MHC molecules are not required in CTL-induced apoptosis signal transduction in target cells.

Amino Acid Sequence↗
PubMed1993

Characterization of the human T cell receptor alpha-chain repertoire and demonstration of a genetic influence on V alpha usage.

Despite considerable knowledge of the determinants and profile of the T cell receptor (TCR) beta-chain repertoire, very little is known about the repertoire of the V alpha and J alpha segments of the TCR in man or mouse. We have used the anchored polymerase chain reaction (PCR) to analyze the human TCR alpha-chain repertoire at the mRNA level in peripheral blood lymphocytes. We find marked differences in the usage of individual V alpha segments. The V alpha repertoire is determined both by differences in the use of individual V alpha gene segments at the time of gene rearrangement and by genetic influences on the selection of the expressed V alpha repertoire, presumably during thymic maturation. Analysis of the J alpha repertoire shows non-random usage of individual J alpha segments but no obvious relationship to chromosomal position. We also report three new V alpha segments, three new J alpha segments and possible allelic variation in V alpha gene segments. Two unusual TCR alpha transcripts are described which add new information on the nature of TCR alpha transcript assembly.

Amino Acid Sequence↗
PubMed1993

Restricted T cell receptor expression by human T cell clones specific for mycobacterial 65-kDa heat-shock protein: selective in vivo expansion of T cells bearing defined receptors.

We have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65-kDa heat-shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigen in vivo. DR3-restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF-derived clones tested expressed either V beta 5.2 or a closely related beta chain, V beta 5.6. The alpha chains expressed by V beta 5.2+ and V beta 5.6+ clones were from different families, V alpha 2.4 and V alpha 23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either V beta 5.2 or V beta 5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of V beta 5. Conservation of the beta chain third complementarity-determining region (CDR3) sequence was not evident, however. Sequencing alpha and beta chains of representative V beta 5.2+ and V beta 5.6+ PB-derived clones revealed TcR which were identical to those utilized by the SF-derived clones, showing that the repertoire for recognition of this antigen is stable over time. Similar studies of TcR expression were carried out on hsp65-specific, DP4-restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of alpha chain usage, since all clones expressed a member of the V alpha 1 family, but again CDR3 sequence conservation was not apparent. beta chain usage was not restricted since different clones expressed V beta 6.7, V beta 22.3 and V beta 12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical alpha and beta TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this defined specificity in the patient's SF.

Amino Acid Sequence↗