An alternative method for T-cell receptor repertoire analysis: Clustering of human V-beta subfamilies selected in responses to staphylococcal enterotoxins B and E

We have designed a convenient procedure for the analysis of Vβ repertoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucleotidyl transferase and submitted to one-side specificity PCR amplification with a constant r...

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Veröffentlicht in:Molecular immunology 1993-07, Vol.30 (10), p.877-886
Hauptverfasser: Champagne, Eric, Huchenq, Anne, Sevin, Jacqueline, Casteran, Nathalie, Rubin, Bent
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container_end_page 886
container_issue 10
container_start_page 877
container_title Molecular immunology
container_volume 30
creator Champagne, Eric
Huchenq, Anne
Sevin, Jacqueline
Casteran, Nathalie
Rubin, Bent
description We have designed a convenient procedure for the analysis of Vβ repertoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucleotidyl transferase and submitted to one-side specificity PCR amplification with a constant region oligonucleotide primer. The amplified material is then analysed by reverse spot-test hybridization: after 32P-labelling, the amplification product is put to hybridize on a membrane where specially designed Vβ subfamily-specific probes are immobilized. The radioactivity fixed on each probe can then be easily quantified and the signal obtained is directly proportional to the initial amount of homologous RNA. We applied this technique to the study of Vβ gene selection following T-cell stimulation by staphylococcal enterotoxins B and E. We show that with these toxins two almost non-overlapping sets of T-cells are recruited and that this selection is likely to be dependent on specific amino acid residues shaping the fourth complementarity determining region of the TCR-β chain. These residues constitute two tandemly-conserved tripeptide sequences (Asp 39Pro 40Gly 41)-(Val 61)Ser 70Arg 71) and (Arg 66Phe 67Ser 68)-(Asp 88Ser 89Ala 90) in the SEB- and the SEE-responsive Vβ gene clusters respectively.
doi_str_mv 10.1016/0161-5890(93)90011-Y
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Psychology</topic><topic>Genes. Genome</topic><topic>Humans</topic><topic>Immunoblotting</topic><topic>Immunoglobulin Variable Region - chemistry</topic><topic>Immunoglobulin Variable Region - genetics</topic><topic>Immunoglobulin Variable Region - immunology</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Molecular Sequence Data</topic><topic>Multigene Family</topic><topic>Nucleic Acid Hybridization</topic><topic>Oligodeoxyribonucleotides - chemistry</topic><topic>Oligonucleotide Probes</topic><topic>Polymerase Chain Reaction</topic><topic>Receptors, Antigen, T-Cell, alpha-beta - chemistry</topic><topic>Receptors, Antigen, T-Cell, alpha-beta - immunology</topic><topic>Sequence Alignment</topic><topic>Sequence Homology, Amino Acid</topic><topic>Staphylococcus</topic><topic>Staphylococcus aureus - immunology</topic><topic>T-Lymphocytes - immunology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Champagne, Eric</creatorcontrib><creatorcontrib>Huchenq, Anne</creatorcontrib><creatorcontrib>Sevin, Jacqueline</creatorcontrib><creatorcontrib>Casteran, Nathalie</creatorcontrib><creatorcontrib>Rubin, Bent</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Molecular immunology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Champagne, Eric</au><au>Huchenq, Anne</au><au>Sevin, Jacqueline</au><au>Casteran, Nathalie</au><au>Rubin, Bent</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An alternative method for T-cell receptor repertoire analysis: Clustering of human V-beta subfamilies selected in responses to staphylococcal enterotoxins B and E</atitle><jtitle>Molecular immunology</jtitle><addtitle>Mol Immunol</addtitle><date>1993-07</date><risdate>1993</risdate><volume>30</volume><issue>10</issue><spage>877</spage><epage>886</epage><pages>877-886</pages><issn>0161-5890</issn><eissn>1872-9142</eissn><coden>MOIMD5</coden><abstract>We have designed a convenient procedure for the analysis of Vβ repertoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucleotidyl transferase and submitted to one-side specificity PCR amplification with a constant region oligonucleotide primer. The amplified material is then analysed by reverse spot-test hybridization: after 32P-labelling, the amplification product is put to hybridize on a membrane where specially designed Vβ subfamily-specific probes are immobilized. The radioactivity fixed on each probe can then be easily quantified and the signal obtained is directly proportional to the initial amount of homologous RNA. We applied this technique to the study of Vβ gene selection following T-cell stimulation by staphylococcal enterotoxins B and E. We show that with these toxins two almost non-overlapping sets of T-cells are recruited and that this selection is likely to be dependent on specific amino acid residues shaping the fourth complementarity determining region of the TCR-β chain. These residues constitute two tandemly-conserved tripeptide sequences (Asp 39Pro 40Gly 41)-(Val 61)Ser 70Arg 71) and (Arg 66Phe 67Ser 68)-(Asp 88Ser 89Ala 90) in the SEB- and the SEE-responsive Vβ gene clusters respectively.</abstract><cop>Oxford</cop><pub>Elsevier Ltd</pub><pmid>8341282</pmid><doi>10.1016/0161-5890(93)90011-Y</doi><tpages>10</tpages></addata></record>
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source MEDLINE; ScienceDirect Journals (5 years ago - present)
subjects Amino Acid Sequence
Antigen-Antibody Reactions
Base Sequence
Biological and medical sciences
Cells, Cultured
Enterotoxins - immunology
Fundamental and applied biological sciences. Psychology
Genes. Genome
Humans
Immunoblotting
Immunoglobulin Variable Region - chemistry
Immunoglobulin Variable Region - genetics
Immunoglobulin Variable Region - immunology
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Multigene Family
Nucleic Acid Hybridization
Oligodeoxyribonucleotides - chemistry
Oligonucleotide Probes
Polymerase Chain Reaction
Receptors, Antigen, T-Cell, alpha-beta - chemistry
Receptors, Antigen, T-Cell, alpha-beta - immunology
Sequence Alignment
Sequence Homology, Amino Acid
Staphylococcus
Staphylococcus aureus - immunology
T-Lymphocytes - immunology
title An alternative method for T-cell receptor repertoire analysis: Clustering of human V-beta subfamilies selected in responses to staphylococcal enterotoxins B and E
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