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Efficient CRISPR/Cas9 Disruption of Autoimmune-Associated Genes Reveals Key Signaling Programs in Primary Human T Cells

Warren Anderson, Jerill Thorpe, S. Alice Long and David J. Rawlings
J Immunol November 13, 2019, ji1900848; DOI: https://doi.org/10.4049/jimmunol.1900848
Warren Anderson
*Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA 98101;
†Department of Pathology, University of Washington, Seattle, WA 98195;
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Jerill Thorpe
‡Benaroya Research Institute at Virginia Mason, Seattle, WA 98101;
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S. Alice Long
‡Benaroya Research Institute at Virginia Mason, Seattle, WA 98101;
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David J. Rawlings
*Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA 98101;
§Department of Pediatrics, University of Washington, Seattle, WA 98109; and
¶Department of Immunology, University of Washington, Seattle, WA 98109
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Key Points

  • CRISPR gene disruption in CD4+ T cells is enhanced by donor DNA template delivery.

  • Disruption of key signaling proteins in human CD4+ T cells mimics murine data.

  • Hyperactive signaling in human T cells can drive compensatory regulatory responses.

Abstract

Risk of autoimmunity is associated with multiple genetic variants. Genome-wide association studies have linked single-nucleotide polymorphisms in the phosphatases PTPN22 (rs2476601) and PTPN2 (rs1893217) to increased risk for multiple autoimmune diseases. Previous mouse studies of loss of function or risk variants in these genes revealed hyperactive T cell responses, whereas studies of human lymphocytes revealed contrasting phenotypes. To better understand this dichotomy, we established a robust gene editing platform to rapidly address the consequences of loss of function of candidate genes in primary human CD4+ T cells. Using CRISPR/Cas9, we obtained efficient gene disruption (>80%) of target genes encoding proteins involved in Ag and cytokine receptor signaling pathways including PTPN22 and PTPN2. Loss-of-function data in all genes studied correlated with previous data from mouse models. Further analyses of PTPN2 gene–disrupted T cells demonstrated dynamic effects, by which hyperactive IL-2R signaling promoted compensatory transcriptional events, eventually resulting in T cells that were hyporesponsive to IL-2. These results imply that altered phosphatase activity promotes evolving phenotypes based on Ag experience and/or other programming signals. This approach enables the discovery of molecular mechanisms modulating risk of autoimmunity that have been difficult to parse in traditional mouse models or cross-sectional human studies.

Footnotes

  • This work was supported by grants from the National Institutes of Health (DP3-DK111802 to D.J.R. and 5TL1TR002318-02 to W.A.). Additional support was provided by a JDRF Career Development Award (to S.A.L.), the Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children’s Research Institute, the Children’s Guild Association Endowed Chair in Pediatric Immunology (to D.J.R.), the Hansen Investigator in Pediatric Innovation Endowment (to D.J.R.), and the Benaroya Family Gift Fund (to D.J.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • The online version of this article contains supplemental material.

  • Received July 22, 2019.
  • Accepted October 10, 2019.
  • Copyright © 2019 by The American Association of Immunologists, Inc.
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The Journal of Immunology: 206 (3)
The Journal of Immunology
Vol. 206, Issue 3
1 Feb 2021
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Efficient CRISPR/Cas9 Disruption of Autoimmune-Associated Genes Reveals Key Signaling Programs in Primary Human T Cells
Warren Anderson, Jerill Thorpe, S. Alice Long, David J. Rawlings
The Journal of Immunology November 13, 2019, ji1900848; DOI: 10.4049/jimmunol.1900848

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Efficient CRISPR/Cas9 Disruption of Autoimmune-Associated Genes Reveals Key Signaling Programs in Primary Human T Cells
Warren Anderson, Jerill Thorpe, S. Alice Long, David J. Rawlings
The Journal of Immunology November 13, 2019, ji1900848; DOI: 10.4049/jimmunol.1900848
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Print ISSN 0022-1767        Online ISSN 1550-6606