Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen specific tumor cell killing by cytotoxic T cells

Abstract

Cytotoxic CD8+ T lymphocytes (CTLs) are key players of adaptive anti-tumor immunity based on their ability to specifically recognize and destroy tumor cells. Many cancer immunotherapies rely on unleashing CTL function. However, tumors can evade killing through strategies which are not yet fully elucidated. To provide deeper insight into tumor evasion mechanisms in an antigen-dependent manner, we established a human co-culture system composed of tumor and primary immune cells. Using this system, we systematically investigated intrinsic regulators of tumor resistance by conducting a complementary CRISPR screen approach. By harnessing CRISPR activation (CRISPRa) and CRISPR knockout (KO) technology in parallel, we investigated gene gain-of-function as well as loss-of-function across genes with annotated function in a colon carcinoma cell line. CRISPRa and CRISPR KO screens uncovered 187 and 704 hits respectively, with 60 gene hits overlapping between both. These data confirmed the role of interferon‑γ (IFN-γ), tumor necrosis factor α (TNF-α) and autophagy pathways and uncovered novel genes implicated in tumor resistance to killing. Notably, we discovered that ILKAP encoding the integrin-linked kinase-associated serine/threonine phosphatase 2C, a gene previously unknown to play a role in antigen specific CTL-mediated killing, mediate tumor resistance independently from regulating antigen presentation, IFN-γ or TNF-α responsiveness. Moreover, our work describes the contrasting role of soluble and membrane-bound ICAM-1 in regulating tumor cell killing. The deficiency of membrane-bound ICAM-1 (mICAM-1) or the overexpression of soluble ICAM-1 (sICAM-1) induced resistance to CTL killing, whereas PD-L1 overexpression had no impact. These results highlight the essential role of ICAM-1 at the immunological synapse between tumor and CTL and the antagonist function of sICAM-1.

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All data generated or analyzed are included in the manuscript. Source data files are provided for figure 2 and figure 3

Article and author information

Author details

  1. Ann-Kathrin Herzfeldt

    Department of Cancer Immunology and Immune Modulation, Boehringer Ingelheim, Biberach an der Riss, Germany
    Competing interests
    Ann-Kathrin Herzfeldt, was an employee at this time of Boehringer Ingelheim Pharma GmbH Co. KG. The author has noother relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
  2. Marta Puig Gamez

    Department of Cancer Immunology and Immune Modulation, Boehringer Ingelheim, Biberach an der Riss, Germany
    Competing interests
    Marta Puig Gamez, was an employee at this time of Boehringer Ingelheim Pharma GmbH Co. KG. The author has noother relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
  3. Eva Martin

    Department of Drug Discovery Sciences, Boehringer Ingelheim, Biberach an der Riss, Germany
    Competing interests
    Eva Martin, was an employee at this time of Boehringer Ingelheim Pharma GmbH Co. KG. The author has noother relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
  4. Lukasz Miloslaw Boryn

    Ardigen SA, Kraków, Poland
    Competing interests
    Lukasz Miloslaw Boryn, was an Ardigen S.A. employee. The funder provided support in the form of salaries for the authors..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8091-1071
  5. Praveen Baskaran

    Department of Global Computational Biology and Digital Sciences, Boehringer Ingelheim, Biberach an der Riss, Germany
    Competing interests
    Praveen Baskaran, was an employee at this time of Boehringer Ingelheim Pharma GmbH Co. KG. The author has noother relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2275-3516
  6. Heinrich J Huber

    Drug Discovery Sciences, Boehringer Ingelheim, Biberach an der Riss, Germany
    Competing interests
    Heinrich J Huber, was an employee at this time of Boehringer Ingelheim Pharma GmbH Co. KG. The author has noother relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4454-2971
  7. Michael Schuler

    Dept of Drug Discovery Services, Boehringer Ingelheim, Biberach an der Riss, Germany
    Competing interests
    Michael Schuler, was an employee at this time of Boehringer Ingelheim Pharma GmbH Co. KG. The author has noother relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
  8. John E Park

    Department of Cancer Immunology and Immune Modulation, Boehringer Ingelheim, Biberach an der Riss, Germany
    For correspondence
    john.park@boehringer-ingelheim.com
    Competing interests
    John E Park, was an employee at this time of Boehringer Ingelheim Pharma GmbH Co. KG. The author has noother relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5674-6026
  9. Lee Kim Swee

    Department of Cancer Immunology and Immune Modulation, Boehringer Ingelheim, Biberach an der Riss, Germany
    Competing interests
    Lee Kim Swee, was an employee at this time of Boehringer Ingelheim Pharma GmbH Co. KG. The author has noother relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..

Funding

Boehringer Ingelheim (none)

  • Ann-Kathrin Herzfeldt
  • Marta Puig Gamez
  • Eva Martin
  • Lukasz Miloslaw Boryn
  • Praveen Baskaran
  • Heinrich J Huber
  • Michael Schuler
  • John E Park
  • Lee Kim Swee

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

Copyright

© 2023, Herzfeldt et al.

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. Ann-Kathrin Herzfeldt
  2. Marta Puig Gamez
  3. Eva Martin
  4. Lukasz Miloslaw Boryn
  5. Praveen Baskaran
  6. Heinrich J Huber
  7. Michael Schuler
  8. John E Park
  9. Lee Kim Swee
(2023)
Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen specific tumor cell killing by cytotoxic T cells
eLife 12:e84314.
https://doi.org/10.7554/eLife.84314

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https://doi.org/10.7554/eLife.84314

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