Using solubel peptide-MHC tetramer, the trial of staining and cloning the melanoma antigen-specific cytotoxic lymphocytes

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The purpose of this project is to construct the soluble class I MHC-peptide complex ("tetramers") and to visualize the melanoma antigen-specific cytotoxic lymphocytes (CTL) using the produced tetramers.1. We have produced HLA-A2/gp100_<(209-217)>, HLA-A2/MART-1_<(27-35)>, and HLA-A24/EBV-P tetramers. The β2-microglobulin (β2m) and soluble HLA-A2 and A24 heavy chains linked at its carboxyl terminus to a BirA substrate peptide were expressed separately in Escherichia coli. The expressed HLA-A2 or A24-BirA substrate peptide and β2m subunits were refolded together in vitro in the presence of synthetic antigenic peptides, corresponding to the HLA-A2-restricted melanoma-immunogenic epitopes of gp100_<(209-217)> (ITDQVPFSV) and MART-1_<(27-35)> (AAGIGILTV), and HLA-A24 restricted viral-immunogenic epitope of EBV-P (TYGPVFMCL). Folded materials were then subjected to enzymatic biotinylation with BirA enzyme. The HLA-A2 or A24/peptide complexes were purified first on a get filtration column and … More subsequently on a Mono Q ion exchange column. Tetrameric complexes of the biotinylated HLA-A2 or A24/peptide were finally produced by mixing the purified biotinylated heterodimers with Streptavidine-phycoerythrin (PE) conjugates at a molar ratio of 4 : 1.2. The specific binding of the HLA-A2/gp100_<(209-217)> and HLA-A2/MART-1_<(27-35)> tetramers were assessed by staining the relevant peptide-specific tumor-infiltrating T-lymphocytes (TILs), TIL1520 and TIL1235 respectively, and analyzing them on flow cytometry. The HLA-A2/gp100_<(209-217)> tetramer specifically stained more than 90% of TIL1520 specific for the gp100_<(209-217)> peptide, but, did not stain TIL1370 specific for irrelevant gp100_<(154-162)> (G154 : KTWGQYWQV) peptide. In addition, using HLA-A24/EBV-P tetramer, we have analyzed EBV-P specific CTL that was generated by in vitro stimulation with the peptide from peripheral blood mononuclear cells (PBMCs) of HLA-A24 healthy donor and the CTL were found to stain with the tetramer.These results indicate that the produced peptide-MHC tetramers can be used to specifically bind to antigen-specific CTL restricted by both HLA-A2 and HLA-A24, and possibly to enrich specific CTL among a heterogeneous population. Less