Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System.

Autor:	Yousefi OS; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, University of Freiburg, 79104 Freiburg, Germany.; Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany., Hörner M; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, University of Freiburg, 79104 Freiburg, Germany.; Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany., Wess M; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, University of Freiburg, 79104 Freiburg, Germany.; Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany., Idstein V; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, University of Freiburg, 79104 Freiburg, Germany.; Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany., Weber W; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, University of Freiburg, 79104 Freiburg, Germany.; Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany., Schamel WWA; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, University of Freiburg, 79104 Freiburg, Germany.; Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.; Center for Chronic Immunodeficiency (CCI), Medical Center Freiburg and Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany.
Jazyk:	angličtina
Zdroj:	Bio-protocol [Bio Protoc] 2020 Mar 05; Vol. 10 (5), pp. e3540. Date of Electronic Publication: 2020 Mar 05 (Print Publication: 2020).
DOI:	10.21769/BioProtoc.3540
Abstrakt:	T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic TCR, generated by fusing the PhyB interacting factor (PIF) to the TCRβ chain. Switching PhyB to the OFF conformation disrupts this interaction. Sufficiently long binding of PhyB tetramers to the PIF-TCR led to T cell activation as measured by calcium influx. Here, we describe protocols for how to generate the tetrameric ligand for our opto-ligand-TCR system, how to measure ligand-TCR binding by flow cytometry and how to quantify T cell activation via calcium influx. Competing Interests: Competing interestsOpto Biolabs provided us with the pxONE device. (©Copyright Yousefi et al.)
Databáze:	MEDLINE
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