title: A robust and scalable TCR-based reporter cell assay to measure HIV-1 Nef-mediated T cell immune evasion.
abstract: HIV-1 evades cytotoxic T cell responses through Nef-mediated downregulation of HLA class I molecules from the infected cell surface. Methods to quantify the impact of Nef on T cell recognition typically employ patient-derived T cell clones; however, these assays are limited by the cost and effort required to isolate and maintain primary cell lines. The variable activity of different T cell clones and the limited number of cells generated by re-stimulation can also hinder assay reproducibility and scalability. Here, we describe a heterologous T cell receptor reporter assay and use it to study immune evasion by Nef. Induction of NFAT-driven luciferase following co-culture with peptide-pulsed or virus-infected target cells serves as a rapid, quantitative and antigen-specific measure of T cell recognition of its cognate peptide/HLA complex. We demonstrate that Nef-mediated downregulation of HLA on target cells correlates inversely with T cell receptor-dependent luminescent signal generated by effector cells. This method provides a robust, flexible and scalable platform that is suitable for studies to measure Nef function in the context of different viral peptide/HLA antigens, to assess the function of patient-derived Nef alleles, or to screen small molecule libraries to identify novel Nef inhibitors.
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada.
authors: Gursev
authors: G
authors: Anmole
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada.
authors: Xiaomei T
authors: XT
authors: Kuang
authors: International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto 860-0811, Japan.
authors: Mako
authors: M
authors: Toyoda
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada.
authors: Eric
authors: E
authors: Martin
authors: Faculty of Health Sciences, Simon Fraser University, Burnaby, BC V5A1S6, Canada.
authors: Aniqa
authors: A
authors: Shahid
authors: Faculty of Health Sciences, Simon Fraser University, Burnaby, BC V5A1S6, Canada.
authors: Anh Q
authors: AQ
authors: Le
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada.
authors: Tristan
authors: T
authors: Markle
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada; Faculty of Health Sciences, Simon Fraser University, Burnaby, BC V5A1S6, Canada.
authors: Bemuluyigza
authors: B
authors: Baraki
authors: Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors: R Brad
authors: RB
authors: Jones
authors: Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors: Mario A
authors: MA
authors: Ostrowski
authors: International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto 860-0811, Japan.
authors: Takamasa
authors: T
authors: Ueno
authors: Faculty of Health Sciences, Simon Fraser University, Burnaby, BC V5A1S6, Canada; BC Centre for Excellence in HIV/AIDS, St. Paul's Hospital, Vancouver, BC V6Z1Y6, Canada.
authors: Zabrina L
authors: ZL
authors: Brumme
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada; Faculty of Health Sciences, Simon Fraser University, Burnaby, BC V5A1S6, Canada; BC Centre for Excellence in HIV/AIDS, St. Paul's Hospital, Vancouver, BC V6Z1Y6, Canada. Electronic address: mark_brockman@sfu.ca.
authors: Mark A
authors: MA
authors: Brockman
mesh_terms: D018920:Coculture Techniques; D015536:Down-Regulation / Q000276:immunology; D015497:HIV-1 / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis; D006801:Humans; D057131:Immune Evasion / Q000276:immunology*; D007118:Immunoassay; D019169:Jurkat Cells; D050778:NFATC Transcription Factors / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000276:immunology; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000276:immunology*
chemical_list: D015395:Histocompatibility Antigens Class I; D050778:NFATC Transcription Factors; D011948:Receptors, Antigen, T-Cell; D054301:gag Gene Products, Human Immunodeficiency Virus; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1
keywords: HLA class I; Human immunodeficiency virus; Immune evasion; Nef; T cell receptor