MHC Class I Multimer Preparation

For determining kinetics of mLama4- or mAlg8-specific T cell infiltration into tumours, H-2K^b tetramers conjugated to PE were prepared with mutant Lama4 or Alg8 peptides and produced by the NIH Tetramer Core Facility (Emory University). For screening H-2K^b or H-2D^b predicted epitopes, we generated peptide-MHC Class I complexes in house. The peptide-MHC class I complexes refolded with a UV-cleavable conditional ligand were prepared as described with modifications^{37 (link)}. Briefly, recombinant H-2K^b and H-2D^b heavy chains and human β2 microglobulin light chain were produced in Escherichia coli, isolated as inclusion bodies, and dissolved in 4M urea, 20 mM Tris pH 8.0. MHC Class I refolding reactions were performed by dialyzing a molar ratio of heavy chain: light chain: peptide of 1:1:8 against 10 mM potassium phosphate, pH 7.4 for 48 h. The UV-cleavable peptide SIINFEJL used to refold H-2K^b was purchased from Peptide 2.0. Refolded peptide-MHC class I complexes were captured by ion exchange (HiTrap Q HP, GE), biotinylated, and purified by gel filtration FPLC. UV-induced ligand exchange and combinatorial encoding of MHC Class I multimers was performed as described^{38 (link)}, except that the peptide-MHC multimers used for flow cytometry staining were prepared by the addition of titrated amounts of streptavidin-fluorochrome in a 10 μL format^{39 (link)}.