Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation.

Autor:	Zhao X; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore., Hamidinia M; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore., Choo JAL; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore., Too CT; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore; Immunology Programme, Life Sciences Institute, National University of Singapore., Ho ZZ; Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School., Ren EC; Singapore Immunology Network, A*STAR., Bertoletti A; Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School., MacAry PA; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore; Immunology Programme, Life Sciences Institute, National University of Singapore; NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore., Gould KG; Department of Immunology, Wright-Fleming Institute, Imperial College London., Brzostek J; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore; micjmb@nus.edu.sg., Gascoigne NRJ; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore; Immunology Programme, Life Sciences Institute, National University of Singapore; NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore; micnrjg@nus.edu.sg.
Jazyk:	angličtina
Zdroj:	Journal of visualized experiments : JoVE [J Vis Exp] 2019 Feb 28 (144). Date of Electronic Publication: 2019 Feb 28.
DOI:	10.3791/59126
Abstrakt:	Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu