| Summary: | 碩士 === 國立清華大學 === 分子與細胞生物研究所 === 99 === The major histocompatibility complex (MHC) molecules present peptides derived from pathogen products or tumor antigens and play a critical role in initiating T cell response against infectious agents and tumor cells. The generation of MHC-peptide complexes requires a complicated and stepwise assembly procedure, finally resulting in the transport of MHC-peptide complexes to the surface of an antigen presenting cell, a process often affected by many cellular variables. The purpose of this study is to develop a platform where antigen-specific T cells can be efficiently stimulated, expanded, and isolated. In the past, the recombinant MHC class I complexes were constructed by combining heavy chain, 刍2m and the presented peptide. This approach was called 3-component approach (3C approach). In this study, we used the 2-component approach (2C approach) including the construction of a fusion protein where the peptide coding sequence is incorporated with the 刍2m subunit coding sequence by a flexible Gly/Ser linker, assembled in vitro with the extracellular domain of the MHC class I heavy chain. To this end, we have utilized sepharose beads as a solid phase loaded with recombinant 2C MHC-peptide complexes, together named as the artificial peptide presentosomes (APPs). The advantage of the APPs is three-fold. First, intrinsic intracellular variables complicating efficient “antigen uptake, processing and presentation” can be circumvented. Second, MHC-peptide complex antigens, or “T cell epitopes”, can be quantified and customized. Third, various co-stimulatory molecules can be loaded simultaneously to augment T cell response. We have successfully generated five 2C human MHC class I-peptide complexes, namely, HLA-A2/HA540-548-fused 刍2m, HLA-A2/M158-66-fused 刍2m, HLA-A2/HER2/neu369-377-fused 刍2m, HLA-A2/MAGE-C2336-344-fused 刍2m, and HLA-A11/LMP2340-349-fused 刍2m, representing antigens for stimulating the influenza H5N1 HA-specific, the influenza H5N1 M1-specific, the breast tumor oncogene HER2/neu-specific, the melanoma specific, and the EBV-transformed lymphoma-specific T cell response, respectively. The proper folding of the five complexes was confirmed by direct and sandwich ELISA revealing their high affinity with conformation-specific mAb CR11-351 or LGIII 147.4.1. HLA-A2/HA540-548-fused 刍2m and HLA-A2/M158-66-fused 刍2m loaded APPs have been tested and found to stimulate robust HLA-A2-restricted, antigen-specific T cell secretion of IFN-讪, an indicator of antigen-specific T cell activation. The APP platform may serve as a research tool to study T cell biology, a diagnostic tool to assess T cell function in diseases, and a therapeutic or vaccine vehicle providing highly immunogenic T cell epitopes.
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