Quantum dot/pMHC multimers vs. phycoerythrin/pMHC tetramers for identification of HLA-A*0201-restricted pHBV core antigen18-27-specific T cells.

Abstract

Detection of human leukocyte antigens-A2-restricted p-hepatitis B virus (HBV) core antigen-specific cytotoxic T lymphocytes (CTLs) is important in the study of HBV immunopathogenesis and vaccine design. Currently, major histocompatibility complex (MHC) class I/peptide-(p) MHCI tetramers are considered the optimal tools to detect antigen-specific CTLs. However, the MHC-tetramer technique also has certain drawbacks and is under continuous development. The quantum dot (QD) bioconjugates nanotechnology with its unique inorganic-biological properties has been developing fast. However, QD/pMHC multimers have seldom been used for the identification of the C18–27 epitope, which is important in HBV infection. QD/pMHC multimers were synthesized by metal-affinity coordination and an avidin-biotin system. In the present study they were characterized by transmission electron microscopy, dynamic light scattering and fluorescence spectrophotometry. C18–27-specific CTLs were obtained by ex vivo expansion of CD8+ T cells. Cultured CTLs were tested for the secretion level of interferon (IFN)-γ by ELISA and for cytotoxicity by lactate dehydrogenase release assay. Then, the performance of phycoerythrin (PE)/pMHC tetramers and QD/pMHC multimers were compared by flow cytometry. The synthesized QD/pMHC multimers dispersed well and their emission spectrum exhibited only slight differences compared with original QDs. C18–27-specific CTLs not only secreted IFN-γ but also effectively targeted T2 cells pulsed with peptide C18–27. The frequencies of C18–27-specific CTLs determined by QD/pMHC multimers were higher compared with PE/pMHC tetramers. The present results suggested that QD/pMHC multimers may be able to characterize greater numbers of C18–27-specific CTLs with increased sensitivity compared to conventional strategies.