Construction Of A New Kind Of PR1-HLA-A~*0201-SCT Tetramer And Preliminary Identification

Major histocompatibility complex (MHC) class I/peptide tetrameric technology (the tetramer), which allow the rapid detection and isolation of antigen-specific T cells by fluorescence activated cell sorting, has become an essential tool for T cell analysis and have a broad range of applications in cancer research and clinical trials. However, it need express and purify MHC class I molecules andβ2-microglobulin (β2m) seperately, then refold them with antigenic peptides in vitro. This multi-step procedure and the very low efficiency in refolding course make the production of the MHC class I tetramer such a time-consuming and expensive work. Moreover, the stability of this reagent greatly relies on the affinity between antigenic peptides and MHC class I molecules. For low affinity peptides, it is very difficult to form stable MHC class I tetramer even the peptide can prime strong T cell response in vivo. These defects restrict the wide application of MHC class I tetramer severely and many efforts have been made to improve the practicality of MHC class I tetramer.The really important progress has not been made until Hansen TH and his staff expressed major histocompatibility complex class I molecules as single polypeptides wherein the antigenic peptide,β2m, and heavy chain are attached by flexible linkers. These molecules were named single chain trimers (SCTs). The covalently linked antigenic peptides greatly decrease the dependency of high affinity antigenic peptides and improve the stability of SCTs, make it an excellent probe for T cell analysis.Chronic myeloid leukemia (CML) is a clonal bone marrow stem cell disorder. PR1 (VLQELNVTV), which is an HLA-A*0201 restricted antigenic peptide derived from myeloid leukemia associated antigen protease 3, can prime specific CTLs responses against myeloid leukemia cells. The objective of our study is to produce a PR1-SCT tetramer reagent and utilize it in the analysis of PR1-specific CTLs. We hope it will be beneficial to adoptive cell immunotherapy of CML.We have successfully constructed PR1-SCT by ordering the components in the format PR1-(G4S)3-β2m-(G4S)4-HLA-A*0201 and cloned it into pQE31 vector. After expression in E.coli M15 [pREP4] strain, we harvested PR1-SCT in the format of inclusion bodies. The inclusion bodies were purified by Bio-Scale Mini Profinity IMAC Cartridges using Profinia Protein Purification System (Bio-Rad, USA), then refolded the purified inclusion bodies by dilution method in a refolding buffer containing 400 mmol/L L-Arginine to recover its native comfomation. We introduced 2 mol/L urea into the refolding buffer and the refolding efficiency increased greatly.Refolded PR1-SCT was biotinylated using a BirA enzyme and purified by Sephacryl-S100 column to remove free biotin. We analyzed the efficiency of biotinylation reation by streptavidin-shift assay, and confirm the recovery of native conformation of PR1-SCT utilizing HLA-Ⅰconformation-specific W6/32 monoclonal antibody in streptavidn-capture ELISA. Finally, biotinylated PR1-SCT was used as monomer to form tetramer with PE-labeled streptavidin by the ratio of 4:1.