Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling

  1. Doug K Tischer
  2. Orion David Weiner  Is a corresponding author
  1. University of California, San Francisco, United States

Abstract

T cells are thought to discriminate self from foreign peptides by converting small differences in ligand binding half-life into large changes in cell signaling. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the mechanical stability of the receptor-ligand interaction. Here we develop an optogenetic approach to specifically tune the binding half-life of a chimeric antigen receptor without changing other binding parameters and provide direct evidence of kinetic proofreading in T cell signaling. This half-life discrimination is executed in the proximal signaling pathway, downstream of ZAP70 recruitment and upstream of diacylglycerol accumulation. Our methods represent a general tool for temporal and spatial control of T cell signaling and extend the reach of optogenetics to probe pathways where the individual molecular kinetics, rather than the ensemble average, gates downstream signaling.

Data availability

All single cell measurements generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 4, Figure 4-figure supplement 1 and Figure 4-figure supplement 3.

Article and author information

Author details

  1. Doug K Tischer

    Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Orion David Weiner

    Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States
    For correspondence
    orion.weiner@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1778-6543

Funding

Genentech Foundation (Graduate Student Fellowship)

  • Doug K Tischer
  • Orion David Weiner

National Institutes of Health (GM109899)

  • Orion David Weiner

Novo Nordisk

  • Orion David Weiner

National Science Foundation (DBI-1548297)

  • Orion David Weiner

National Institutes of Health (GM118167)

  • Orion David Weiner

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Arup K Chakraborty, Massachusetts Institute of Technology, United States

Publication history

  1. Received: October 2, 2018
  2. Accepted: April 3, 2019
  3. Accepted Manuscript published: April 5, 2019 (version 1)
  4. Version of Record published: April 29, 2019 (version 2)

Copyright

© 2019, Tischer & Weiner

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Doug K Tischer
  2. Orion David Weiner
(2019)
Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling
eLife 8:e42498.
https://doi.org/10.7554/eLife.42498

Further reading

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    The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.

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    The interplay among different cells in a tissue is essential for maintaining homeostasis. Although disease states have been traditionally attributed to individual cell types, increasing evidence and new therapeutic options have demonstrated the primary role of multicellular functions to understand health and disease, opening new avenues to understand pathogenesis and develop new treatment strategies. We recently described the cellular composition and dynamics of the human oral mucosa; however, the spatial arrangement of cells is needed to better understand a morphologically complex tissue. Here, we link single-cell RNA sequencing, spatial transcriptomics, and high-resolution multiplex fluorescence in situ hybridisation to characterise human oral mucosa in health and oral chronic inflammatory disease. We deconvolved expression for resolution enhancement of spatial transcriptomic data and defined highly specialised epithelial and stromal compartments describing location-specific immune programs. Furthermore, we spatially mapped a rare pathogenic fibroblast population localised in a highly immunogenic region, responsible for lymphocyte recruitment through CXCL8 and CXCL10 and with a possible role in pathological angiogenesis through ALOX5AP. Collectively, our study provides a comprehensive reference for the study of oral chronic disease pathogenesis.