One-Pot, Mix-and-Read Peptide-MHC Tetramers

Abstract

BackgroundCytotoxic 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 FindingsWe 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/SignificanceWe 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.

Differential Clade-Specific HLA-B*3501 Association with HIV-1 Disease Outcome Is Linked to Immunogenicity of a Single Gag Epitope

“…In contrast, although PY9-260E bound with stronger avidity than PY9-260D to HLA-B*3501, neither version of the 9-mer PY9 appeared to bind HLA-B*3501 with adequate stability to be immunogenic. It may be helpful in the future to confirm the identification of novel epitopes using peptide-MHC I tetramers, as now can be done readily (40,43).…”

HLA-B*57 Micropolymorphism Shapes HLA Allele-Specific Epitope Immunogenicity, Selection Pressure, and HIV Immune Control

“…Tetramers were generated as previously described (32). For staining of PE-or allophycocyanin (APC)-conjugated tetramer-specific cells, PBMCs were thawed in R20 medium; rested for 1 h at 37°C, 5% CO 2 ; stained with HLA-class I tetramer for 30 min at room temperature; washed and surface stained with CD8 AlexaFluor 750 (eBioscience), CD3 AlexaFluor 700 (BD), and a LIVE/DEAD violet cell stain kit (Invitrogen) for 30 min; washed in PBS; and fixed in 2% paraformaldehyde.…”

Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

“…Recombinant biotinylated MHC-I molecules were produced as previously described (18)(19)(20). Briefly, relevant HLA-encoding genes (a1a2a3, residues 1-275) were cloned into a pET28a expression vector already containing a biotin signal peptide and a histidine affinity tag.…”

“…Tetramers of HLA-C*03:04 were produced as previously described (20). Briefly, biotinylated recombinant HLA class I H chains were diluted into a reaction buffer containing 50 mM Tris-maleate (pH 6.6), 0.1% Lutrol F68 NF (BASF, a surfactant compatible with cellular use), and excess of b 2 m and peptide and incubated for 48 h at 18˚C.…”