Figures and data
Peptide used for template development
Thermal stability of MHC-I monomers with template peptide.
Thermal stability of HLA-A*03:01, HLA-A*11:01, HLA-B*07:02, HLA-C*07:01 and HLA-C*07:02 (chromatograms) complexed with template peptide at 50°C, assessed using analytical size exclusion HPLC. Peptide–MHC I monomers were incubated with indicated peptides at 50°C for 10 minutes. Peptides used are listed in Table 1. One of at least three representative experiments is shown.
Optimal exchange conditions for template-peptide MHC-I monomers.
Thermal stability of the template peptide=MHC-I complex to determine optimal exchange temperature with one high-affinity peptide for all alleles tested. Primary data of temperature-based peptide exchange analyzed by gel filtration chromatography at indicated temperatures. Peptides used are depicted in Table 1. One of at least three representative experiments is shown.
Temperature exchange tetramer binding to clonal T cell lines.
Clonal CD8+ T cell lines stained with PE-conjugated temperature exchange multimers that either contained a relevant (pink) or irrelevant peptide (grey). The specificities of the T cell lines are depicted above each figure, as well as HLA-type and first three amino acids of the peptide. Y-axis shows the individually scaled counts and x-axis shows the mean fluorescent intensity of PE.
Comparison of temperature exchange multimers to conventional multimers on clonal T cell lines.
Clonal CD8+ T cell lines stained with 13.3 ng/μl of PE-conjugated temperature exchange tetramers (pink) or with conventional tetramers (blue). The specificities of the T cell lines are depicted with the HLA-type and first three amino acids of the peptide for each line. Y-axis shows the individually scaled counts and x-axis shows mean fluorescent intensity of PE.
Comparison of temperature exchange tetramers to conventional tetramers on PBMCs.
PBMCs of healthy individuals were incubated with peptide exchange (pink) or conventional (blue) tetramers. Donor PBMCs were CMV- or EBV-positive based on serology and were incubated with the relevant tetramer based on donor HLA-type. Tetramers were conjugated to APC (y-axis) as well as PE (x-axis). Gate shows the tetramer-binding CD8+ T cells and the percentage of total CD8+ T cells is depicted in the top right corner.
Clonal CD8+ T cell line and exchange peptide details
