One-pot, mix-and-read peptide-MHC tetramers

Christian Valdemar Vinge Leisner; Nina Loeth; Kasper Lamberth; Sune Justesen; Christina Sylvester-Hvid; Esben G Schmidt; Mogens Claesson; Soren Buus; Anette Stryhn

doi:10.1371/journal.pone.0001678

One-pot, mix-and-read peptide-MHC tetramers

Christian Valdemar Vinge Leisner, Nina Loeth, Kasper Lamberth, Sune Justesen, Christina Sylvester-Hvid, Esben G Schmidt, Mogens Claesson, Soren Buus, Anette Stryhn

Department of Immunology and Microbiology

74 Citations (Scopus)

Abstract

BACKGROUND: Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL's. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient "one-pot, mix-and-read" strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.

Original language	English
Journal	PLoS ONE
Volume	3
Issue number	2
Pages (from-to)	e1678
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0001678
Publication status	Published - 2008

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10.1371/journal.pone.0001678

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title = "One-pot, mix-and-read peptide-MHC tetramers",

abstract = "BACKGROUND: Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL's. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient {"}one-pot, mix-and-read{"} strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.",

author = "Leisner, {Christian Valdemar Vinge} and Nina Loeth and Kasper Lamberth and Sune Justesen and Christina Sylvester-Hvid and Schmidt, {Esben G} and Mogens Claesson and Soren Buus and Anette Stryhn",

note = "Keywords: Biotinylation; Epitope Mapping; Histocompatibility Antigens Class I; Humans; Immunologic Techniques; Major Histocompatibility Complex; Methods; Peptides; Streptavidin; T-Lymphocytes, Cytotoxic",

year = "2008",

doi = "10.1371/journal.pone.0001678",

language = "English",

volume = "3",

pages = "e1678",

journal = "PLoS ONE",

issn = "1932-6203",

publisher = "Public Library of Science",

number = "2",

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TY - JOUR

T1 - One-pot, mix-and-read peptide-MHC tetramers

AU - Leisner, Christian Valdemar Vinge

AU - Loeth, Nina

AU - Lamberth, Kasper

AU - Justesen, Sune

AU - Sylvester-Hvid, Christina

AU - Schmidt, Esben G

AU - Claesson, Mogens

AU - Buus, Soren

AU - Stryhn, Anette

N1 - Keywords: Biotinylation; Epitope Mapping; Histocompatibility Antigens Class I; Humans; Immunologic Techniques; Major Histocompatibility Complex; Methods; Peptides; Streptavidin; T-Lymphocytes, Cytotoxic

PY - 2008

Y1 - 2008

N2 - BACKGROUND: Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL's. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient "one-pot, mix-and-read" strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.

AB - BACKGROUND: Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL's. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient "one-pot, mix-and-read" strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.

U2 - 10.1371/journal.pone.0001678

DO - 10.1371/journal.pone.0001678

M3 - Journal article

C2 - 18301755

SN - 1932-6203

VL - 3

SP - e1678

JO - PLoS ONE

JF - PLoS ONE

IS - 2

ER -

One-pot, mix-and-read peptide-MHC tetramers

Abstract

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