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
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eLife assessment
This important research uses complementary CRISPR screening strategies to reveal novel pathways that prevent T cells from killing tumor cells. The evidence presented to support the claims is solid, although some additional assays defining the features of these novel pathways and their clinical relevance are still required. Overall, this work will be of broad interest to immunologists, cancer biologists, and those interested in cell adhesion and cell-cell communication.
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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 2 C, 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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eLife assessment
This important research uses complementary CRISPR screening strategies to reveal novel pathways that prevent T cells from killing tumor cells. The evidence presented to support the claims is solid, although some additional assays defining the features of these novel pathways and their clinical relevance are still required. Overall, this work will be of broad interest to immunologists, cancer biologists, and those interested in cell adhesion and cell-cell communication.
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Reviewer #1 (Public Review):
This paper accomplishes the authors' goal of using two complementary CRISPR approaches to identify novel determinants of CTL killing in vitro. Through these screens, the authors identify two new genes, ILKAP and ICAM1, that both modulate CTL killing across different cancer cell types. The dissection of how different ICAM1 proteins (membrane-bound and secreted) was also performed in a rigorous fashion. The use of multiple unrelated cancer cell lines greatly increases the strength of the findings and potential future applicability. Major weaknesses of the manuscript first include how ILKAP is connected to the control of ICAM1, which is unclear from the data presented in the paper. Secondly, while the authors use many different mutational variants of ICAM1 to dissect its function, the specific role of each of …
Reviewer #1 (Public Review):
This paper accomplishes the authors' goal of using two complementary CRISPR approaches to identify novel determinants of CTL killing in vitro. Through these screens, the authors identify two new genes, ILKAP and ICAM1, that both modulate CTL killing across different cancer cell types. The dissection of how different ICAM1 proteins (membrane-bound and secreted) was also performed in a rigorous fashion. The use of multiple unrelated cancer cell lines greatly increases the strength of the findings and potential future applicability. Major weaknesses of the manuscript first include how ILKAP is connected to the control of ICAM1, which is unclear from the data presented in the paper. Secondly, while the authors use many different mutational variants of ICAM1 to dissect its function, the specific role of each of these mutations is not well described. A rigorous examination of secreted ICAM1 on CTL killing is not presented, and since membrane-bound and secreted ICAM1 have opposing functions on CTL killing, the clinical relevance of modulating ICAM1 is unclear. Finally, the authors do not consider how ICAM1 may affect antigen-presenting cells and other myeloid cells in the tumor which are critical intermediaries in the antitumor immune response. Overall, once these points of weakness are addressed, this work is expected to have a high impact in the field, as it presents new targets outside the PD-L1 / PD-1 axis that may aid in CTL killing of tumors across multiple cancer types.
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Reviewer #2 (Public Review):
Mattes et al. used a CRISPR screen to determine tumor-intrinsic factors modulating the efficacy of T cell-mediated cell lysis. For this purpose, colon carcinoma cell lines were modified with gain-of-function (CRISPRa) and loss-of-function (CRISPRi) sgRNA libraries. Modified colon cancer cells were subsequently exposed to antigen-specific tumor cell lysis by CD8+ cytotoxic T cells and surviving colon cancer cells were analyzed for over- and underrepresented genes. The screen replicated findings from previous studies showing the importance of IFNy, TNFa, and autophagy pathways for T cell-mediated lysis of cancer cells. In addition, the authors identified two genes involved in cell adhesion that modulate T cell-mediate cell lysis: ILKAP and ICAM1. Subsequently, the authors validate their findings in in-vitro …
Reviewer #2 (Public Review):
Mattes et al. used a CRISPR screen to determine tumor-intrinsic factors modulating the efficacy of T cell-mediated cell lysis. For this purpose, colon carcinoma cell lines were modified with gain-of-function (CRISPRa) and loss-of-function (CRISPRi) sgRNA libraries. Modified colon cancer cells were subsequently exposed to antigen-specific tumor cell lysis by CD8+ cytotoxic T cells and surviving colon cancer cells were analyzed for over- and underrepresented genes. The screen replicated findings from previous studies showing the importance of IFNy, TNFa, and autophagy pathways for T cell-mediated lysis of cancer cells. In addition, the authors identified two genes involved in cell adhesion that modulate T cell-mediate cell lysis: ILKAP and ICAM1. Subsequently, the authors validate their findings in in-vitro experiments and show that a soluble form of ICAM1 is negatively affecting tumor cell lysis. Finally, they analyze publicly available gene expression data from cancer patient cohorts and show that high ICAM1 expression, in combination with high expression of genes associated with soluble ICAM1 generation, has a negative impact on patient survival. Beyond these findings, the CRISPR screening dataset from this study serves as a comprehensive resource for other researchers in the onco-immunology space.
The conclusions of this paper are well supported by data, but some aspects of the role of soluble ICAM1 in T cell-mediated tumor cell lysis and the limitations of the employed experimental system should be clarified and extended.
1. For their screening, the authors use an in-vitro model of antigen-specific tumor cell lysis based on expanded CMV-specific CD8+ T cells and CMV peptide-pulsed colon cancer cells. While this model allows for the efficient induction of cellular cytotoxicity by expanded primary T cells, it has one critical caveat: pulsing colon cancer cells with CMV peptide adds the antigen artificially to the MHC complex on the cell surface. Cell-intrinsic factors of antigen processing and presentation are not required for tumor cell recognition in this system. However, antigen processing and presentation pathways represent important targets of tumor evasion in cancer patients. Factors affecting these processes won't be detected in this study. To consider antigen processing and presentation as well, the authors could, for instance, have used an additional model with T cells containing a transgenic TCR specific for an antigen typically expressed on colon cancer cells (or another cancer cell line).
2. The authors demonstrate the negative impact of soluble ICAM1 on T cell-mediated cytotoxicity in their co-culture assay. However, they lack to provide evidence on how this is facilitated. One option, as the authors speculate in their discussion and cartoon, could be that soluble ICAM1 occupies LFA1 on T cells thereby preventing the binding of T cells to ICAM1 on the surface of tumor cells. To demonstrate that this is indeed the case, the authors could, for instance, have used microscopy and measured T cell and tumor cell interaction duration and frequency under conditions with and without soluble ICAM1 present.
3. Regarding the analysis of clinical relevance, the authors show that patients with high levels of ICAM1 expression in combination with high levels of protease expression have poor survival. The rationale behind this is that the proteases cleave ICAM1 off the membrane leading to high levels of soluble ICAM1 that then negatively affects T cell-mediate tumor cell lysis. To demonstrate that indeed the combination of both factors, ICAM1 expression, and protease expression, is responsible for poor survival, the authors should also have analyzed the impact of each of these factors alone on patient survival. If their hypothesis is true, the combination of high ICAM1 and protease expression should have a worse impact on survival than each factor alone.
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