Generating Peptide-Loaded pMHC Tetramers

Ultraviolet (UV)-cleavable peptide-loaded biotinylated pMHC-CD8wt and pMHC-CD8mut monomers were obtained from the National Institutes of Health Tetramer Core Facility. HCV (KLVALGINAV), preproinsulin (PPI, ALWMRLLPL), and NY-ESO-1 (SLLMWITQV) peptides of 95% purity were synthesized (Genscript) and loaded onto both pMHC-CD8wt and pMHC-CD8mut (with D227K/T228A mutations that abrogate CD8 binding) monomers using UV light as previously described (17 (link), 23 (link)). Monomers were conjugated to allophycocyanin-labeled streptavidin (Biolegend) to create tetramers to identify HCV-specific T cells.

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Williams C.M., Schonnesen A.A., Zhang S.Q., Ma K.Y., He C., Yamamoto T., Eckhardt S.G., Klebanoff C.A, & Jiang N. (2017). Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells. Frontiers in Immunology, 8, 894.

Publication 2017

allophycocyanin-labeled streptavidin

Allophycocyanin Mutations Peptides Preproinsulin Streptavidin T cells Tetramers Ultraviolet light

Corresponding Organization : The University of Texas at Austin

Other organizations : National Cancer Institute, National Institutes of Health, Center for Cancer Research, University of Pennsylvania, Memorial Sloan Kettering Cancer Center, Parker Institute for Cancer Immunotherapy

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Variable analysis

independent variables

UV-cleavable peptide-loaded biotinylated pMHC-CD8wt and pMHC-CD8mut monomers

dependent variables

Identification of HCV-specific T cells

control variables

HCV (KLVALGINAV), preproinsulin (PPI, ALWMRLLPL), and NY-ESO-1 (SLLMWITQV) peptides of 95% purity
PMHC-CD8mut (with D227K/T228A mutations that abrogate CD8 binding)

controls

Positive control: Not explicitly mentioned.
Negative control: pMHC-CD8mut (with D227K/T228A mutations that abrogate CD8 binding)

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