Peptide used for template development.

Thermal stability of MHC-I monomers with template peptide.

Thermal stability of HLA-A03:01, HLA-A11:01, HLA-B07:02, HLA-C07:01, and HLA-C*07:02 complexed with their template peptides was assessed at 50°C 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. The black line represents primary data without thermal exchange, the green line shows thermal exchange without peptide (resulting in complex instability), and the pink line represents thermal exchange in the presence of peptide (yielding a stable complex).

Optimal exchange conditions for template-peptide MHC-I monomers.

A) 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. The black line represents primary data without thermal exchange, the green line shows thermal exchange without peptide (resulting in complex instability), and the pink line represents thermal exchange in the presence of peptide (yielding a stable complex). The thermal exchange temperatures corresponding to each histogram are indicated above the panels and are 23, 30, 30, and 30°C, respectively. Peptides used are depicted in Table 1. One of at least three representative experiments is shown. B) The exchange efficiency was calculated from the area under the curve measured by HPLC and normalized to binding of the optimal peptide and timing as indicated in each histogram. Mean values ± SD from three independent experiments are shown. C) LILRB1 over-expression (OE) in K562. Grey: negative control, Purple line: K562-WT plus anti-LILRB1-Fitc, Purple filled: K562-LILRB1OE plus anti-LILRB1-Fitc (N=3). D) 4 different conventional tetramer staining on K562-LILRB1OE. Open blackline: negative control. (N-6) E) thermal exchange using a high affinity peptide or no peptide, followed by staining of the K562-LILRB1OE and flow cytometric analysis (N=4).

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.

Thermal stability of template peptide with MHC-I monomers.

Thermal stability of the template peptide MHC-I complex at 50°C using analytical size exclusion HPLC for HLA-A*03:01, HLA-A*11:01, HLA-B*07:02 and HLA-C*07:02 (chromatograms). Input, no peptides and plus high-affinity peptide. One of at least three representative experiments is shown.

Thermal stability optimization of the template peptide MHC-I monomers.

Thermal stability of template peptide MHC-I complex to determine optimal temperature with one high-affinity peptide for all alleles. Primary data of temperature based peptide exchange analyzed by gel filtration chromatography at indicated temperatures. Timing is 1 hours, unless other indicated. The exchange peptides were used are depicted in Table 1. One of at least three representative experiments is shown.

Dilution of temperature exchange multimers on T cell lines.

Clonal CD8+ T cell lines stained with PE-conjugated temperature exchange multimers that either contained an irrelevant (negative control; grey) or a relevant peptide (pink) in different concentrations. The different concentrations are depicted in three different shades of pink (1:6, :1:8 and 1:4). The specificity of the T cell clones are depicted above each figure as the HLA-restriction and first three amino acids of the peptide. Y-axis shows the individually scaled counts and x-axis shows mean fluorescent intensity of PE.

Comparison of temperature exchange (TE) multimers to conventional multimers at different concentrations.

Clonal CD8+ T cell lines were unstained (negative control; grey) or stained with PE-conjugated temperature exchange multimers (pink) or with conventional multimers (blue). The specificity of the T cell lines are depicted on the left side as the HLA-restriction and first three amino acids of the peptide. The concentration of the multimers is depicted above. Y-axis shows the individually scaled counts and x-axis shows mean fluorescent intensity of PE.

Comparison of temperature exchange multimers to conventional multimers on irrelevant PBMCs.

PBMCs of healthy individuals were incubated with peptide exchange (pink) or conventional (blue) multimers. The peptide-HLA combinations of the multimers and from which virus the peptide is derived, are depicted above each figure. Each figure is an overlay of two donors that were CMV- or EBV-positive based on serology and had an irrelevant HLA-type for the multimers. Multimers were conjugated to APC (y-axis) as well as PE (x-axis). Gate shows the multimer-binding CD8+ T cells and the frequency of total CD8+ T cells is depicted in the top right corner.

Flow cytometry gating strategy example.

Representative example of gating strategy used for flow cytometry. A) Gating strategy used for CD8+ T cell clones. All events were gated on lymphocytes and single cells. B) Gating strategy used for PBMCs. All events were gated on lymphocytes, single cells, live cells, CD14-CD19-CD16-, CD8+CD4- and multimer+.

Clonal CD8+ T cell line and exchange peptide details.

Donors used for detection of virus-specific CD8+ T cells using temperature-based sensitive multimers.