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Measuring αβ T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging

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Optical Tweezers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2478))

Abstract

T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR–pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC–TCR interactions and have potential for adaptation to other receptor-ligand systems.

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Acknowledgments

We would like to acknowledge previous members of the Lang and Reinherz groups for developing and refining the protocols herein. Flow Cytometry experiments were performed in the VMC Flow Cytometry Shared Resource. The VMC Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404). This work is supported by grants from the NIH (R01 AI136301 and P01 AI143565).

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA

    Hannah M. Stephens, Debasis Banik & Matthew J. Lang

  2. Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, USA

    Kristine N. Brazin, Robert J. Mallis & Ellis L. Reinherz

  3. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Kristine N. Brazin & Ellis L. Reinherz

  4. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Kristine N. Brazin & Ellis L. Reinherz

  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Robert J. Mallis

  6. Department of Genetics, Stanford University, Stanford, CA, USA

    Yinnian Feng

  7. Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA

    Matthew J. Lang

Authors
  1. Hannah M. Stephens
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  2. Kristine N. Brazin
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  3. Robert J. Mallis
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  4. Yinnian Feng
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  5. Debasis Banik
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  6. Ellis L. Reinherz
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  7. Matthew J. Lang
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Corresponding author

Correspondence to Matthew J. Lang .

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Editors and Affiliations

  1. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA

    Arne Gennerich

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Stephens, H.M. et al. (2022). Measuring αβ T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 2478. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2229-2_26

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  • DOI: https://doi.org/10.1007/978-1-0716-2229-2_26

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2228-5

  • Online ISBN: 978-1-0716-2229-2

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