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.

Data availability

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. Kim Lee Swee

    Department of Cancer Immunology and Immune Modulation, Boehringer Ingelheim, Biberach an der Riss, Germany
    Competing interests
    Kim Lee 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
  • Kim Lee Swee

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

Reviewing Editor

  1. Brian Altman

Version history

  1. Preprint posted: August 27, 2022 (view preprint)
  2. Received: October 19, 2022
  3. Accepted: September 20, 2023
  4. Accepted Manuscript published: September 21, 2023 (version 1)
  5. Accepted Manuscript updated: September 22, 2023 (version 2)
  6. Version of Record published: October 19, 2023 (version 3)

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.

Metrics

  • 1,083
    views
  • 188
    downloads
  • 0
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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. Kim Lee 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

Share this article

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

Further reading

    1. Cancer Biology
    2. Cell Biology
    Camille Dantzer, Justine Vaché ... Violaine Moreau
    Research Article

    Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

    1. Cancer Biology
    Fang Huang, Zhenwei Dai ... Yang Wang
    Research Article

    Aberrant alternative splicing is well-known to be closely associated with tumorigenesis of various cancers. However, the intricate mechanisms underlying breast cancer metastasis driven by deregulated splicing events remain largely unexplored. Here, we unveiled that RBM7 is decreased in lymph node and distant organ metastases of breast cancer as compared to primary lesions and low expression of RBM7 is correlated with the reduced disease-free survival of breast cancer patients. Breast cancer cells with RBM7 depletion exhibited an increased potential for lung metastasis compared to scramble control cells. The absence of RBM7 stimulated breast cancer cell migration, invasion, and angiogenesis. Mechanistically, RBM7 controlled the splicing switch of MFGE8, favoring the production of the predominant isoform of MFGE8, MFGE8-L. This resulted in the attenuation of STAT1 phosphorylation and alterations in cell adhesion molecules. MFGE8-L exerted an inhibitory effect on the migratory and invasive capability of breast cancer cells, while the truncated isoform MFGE8-S, which lack the second F5/8 type C domain had the opposite effect. In addition, RBM7 negatively regulates the NF-κB cascade and an NF-κB inhibitor could obstruct the increase in HUVEC tube formation caused by RBM7 silencing. Clinically, we noticed a positive correlation between RBM7 expression and MFGE8 exon7 inclusion in breast cancer tissues, providing new mechanistic insights for molecular-targeted therapy in combating breast cancer.