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Genome-wide CRISPR–Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1

Nature Immunology volume 21, pages 178–185 (2020)Cite this article

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Abstract

Human leukocyte antigen (HLA)-independent, T cell–mediated targeting of cancer cells would allow immune destruction of malignancies in all individuals. Here, we use genome-wide CRISPR–Cas9 screening to establish that a T cell receptor (TCR) recognized and killed most human cancer types via the monomorphic MHC class I-related protein, MR1, while remaining inert to noncancerous cells. Unlike mucosal-associated invariant T cells, recognition of target cells by the TCR was independent of bacterial loading. Furthermore, concentration-dependent addition of vitamin B-related metabolite ligands of MR1 reduced TCR recognition of cancer cells, suggesting that recognition occurred via sensing of the cancer metabolome. An MR1-restricted T cell clone mediated in vivo regression of leukemia and conferred enhanced survival of NSG mice. TCR transfer to T cells of patients enabled killing of autologous and nonautologous melanoma. These findings offer opportunities for HLA-independent, pan-cancer, pan-population immunotherapies.

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Fig. 1: MC.7.G5 recognizes multiple cancer types through an HLA-independent mechanism.
Fig. 2: Whole-genome CRISPR–Cas9 library screening reveals MR1 as the candidate target of MC.7.G5.
Fig. 3: MR1 is the cancer cell-expressed target of MC.7.G5.
Fig. 4: MC.7.G5 does not recognize MR1 by known mechanisms.
Fig. 5: MC.7.G5 does not recognize healthy cells.
Fig. 6: MC.7.G5 remained inert to activated, stressed or infected healthy cells.
Fig. 7: MC.7.G5 mediates in vivo regression of leukemia and prolongs the survival of mice.
Fig. 8: Transfer of the MC.7.G5 T cell receptor redirects patient T cells to recognize autologous melanoma.

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Data availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

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References

  1. Vavassori, S. et al. Butyrophilin 3A1 binds phosphorylated antigens and stimulates human γδ T cells. Nat. Immunol. 14, 908–916 (2013).

    Article  CAS  Google Scholar 

  2. Kjer-Nielsen, L. et al. MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 491, 717–723 (2012).

    Article  CAS  Google Scholar 

  3. Corbett, A. J. et al. T-cell activation by transitory neo-antigens derived from distinct microbial pathways. Nature 509, 361–365 (2014).

    Article  CAS  Google Scholar 

  4. Gold, M. C. et al. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage. J. Exp. Med. 211, 1601–1610 (2014).

    Article  CAS  Google Scholar 

  5. Eckle, S. B. G. et al. Recognition of vitamin B precursors and byproducts by mucosal associated invariant T cells. J. Biol. Chem. 290, 30204–30211 (2015).

    Article  CAS  Google Scholar 

  6. Le Bourhis, L. et al. Antimicrobial activity of mucosal-associated invariant T cells. Nat. Immunol. 11, 701–708 (2010).

    Article  Google Scholar 

  7. Reantragoon, R. et al. Structural insight into MR1-mediated recognition of the mucosal associated invariant T cell receptor. J. Exp. Med. 209, 761–774 (2012).

    Article  CAS  Google Scholar 

  8. Lepore, M. et al. Parallel T-cell cloning and deep sequencing of human MAIT cells reveal stable oligoclonal TCRβ repertoire. Nat. Commun. 5, 3866 (2014).

    Article  Google Scholar 

  9. Lepore, M. et al. Functionally diverse human T cells recognize non-microbial antigens presented by MR1. eLife 6, 1–22 (2017).

    Google Scholar 

  10. Gherardin, N. A. et al. Diversity of T cells restricted by the MHC class I-related molecule MR1 facilitates differential antigen recognition. Immunity 44, 32–45 (2016).

    Article  CAS  Google Scholar 

  11. Keller, A. N. et al. Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells. Nat. Immunol. 18, 402–411 (2017).

    Article  CAS  Google Scholar 

  12. Reantragoon, R. et al. Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells. J. Exp. Med. 210, 2305–2320 (2013).

    Article  CAS  Google Scholar 

  13. McWilliam, H. E. G. et al. The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1. Nat. Immunol. 17, 531–537 (2016).

    Article  CAS  Google Scholar 

  14. Lamichhane, R. & Ussher, J. E. Expression and trafficking of MHC related protein 1 (MR1). J. Immunol. 38, 42–49 (2017).

    Google Scholar 

  15. Young, M. H. et al. MAIT cell recognition of MR1 on bacterially infected and uninfected cells. PLoS ONE 8, e53789 (2013).

    Article  CAS  Google Scholar 

  16. Gentles, A. J. et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat. Med. 21, 938–945 (2015).

    Article  CAS  Google Scholar 

  17. Gherardin, N. A. et al. Enumeration, functional responses and cytotoxic capacity of MAIT cells in newly diagnosed and relapsed multiple myeloma. Sci. Rep. 8, 4159 (2018).

    Article  Google Scholar 

  18. Parra-Cuadrado, J. F. et al. A study on the polymorphism of human MHC class I-related MR1 gene and identification of an MR1-like pseudogene. Tissue Antigens 56, 170–172 (2000).

    Article  CAS  Google Scholar 

  19. Seshadri, C. et al. A polymorphism in human MR1 is associated with mRNA expression and susceptibility to tuberculosis. Genes Immun. 18, 8–14 (2017).

    Article  CAS  Google Scholar 

  20. Lion, J. et al. MR1B, a natural spliced isoform of the MHC-related 1 protein, is expressed as homodimers at the cell surface and activates MAIT cells. Eur. J. Immunol. 43, 1363–1373 (2013).

    Article  CAS  Google Scholar 

  21. Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    Article  CAS  Google Scholar 

  22. Patel, S. J. et al. Identification of essential genes for cancer immunotherapy. Nature 548, 537–542 (2017).

    Article  CAS  Google Scholar 

  23. Reith, W., LeibundGut-Landmann, S. & Waldburger, J.-M. Regulation of MHC class II gene expression by the class II transactivator. Nat. Rev. Immunol. 5, 793–806 (2005).

    Article  CAS  Google Scholar 

  24. Laugel, B. et al. Engineering of isogenic cells deficient for MR1 with a CRISPR/Cas9 lentiviral system: tools to study microbial antigen processing and presentation to human MR1-restricted T cells. J. Immunol. 197, 971–982 (2016).

    Article  CAS  Google Scholar 

  25. Eckle, S. B. G. et al. A molecular basis underpinning the T cell receptor heterogeneity of mucosal-associated invariant T cells. J. Exp. Med. 211, 1585–1600 (2014).

    Article  CAS  Google Scholar 

  26. Alía, M., Ramos, S., Mateos, R., Bravo, L. & Goya, L. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J. Biochem. Mol. Toxicol. 19, 119–128 (2005).

    Article  Google Scholar 

  27. Irvine, D. J., Purbhoo, M. A., Krogsgaard, M. & Davis, M. M. Direct observation of ligand recognition by T cells. Nature 419, 845–849 (2002).

    Article  CAS  Google Scholar 

  28. Hulin-Curtis, S. L. et al. Histone deacetylase inhibitor trichostatin A sensitises cisplatin-resistant ovarian cancer cells to oncolytic adenovirus. Oncotarget 9, 26328–26341 (2018).

    Article  Google Scholar 

  29. Wooldridge, L. et al. MHC class I molecules with superenhanced CD8 binding properties bypass the requirement for cognate TCR recognition and nonspecifically activate CTLs. J. Immunol. 184, 3357–3366 (2010).

    Article  CAS  Google Scholar 

  30. Lissina, A. et al. Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers. J. Immunol. Methods 340, 11–24 (2009).

    Article  CAS  Google Scholar 

  31. Tungatt, K. et al. Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs. J. Immunol. 194, 463–474 (2014).

    Article  Google Scholar 

  32. Betts, M. R. et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J. Immunol. Methods 281, 65–78 (2003).

    Article  CAS  Google Scholar 

  33. Haney, D. et al. Isolation of viable antigen-specific CD8+ T cells based on membrane-bound tumor necrosis factor (TNF)-α expression. J. Immunol. Methods 369, 33–41 (2011).

    Article  CAS  Google Scholar 

  34. Ryan, M. D., King, A. M. Q. & Thomas, G. P. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J. Gen. Virol. 72, 2727–2732 (1991).

    Article  CAS  Google Scholar 

  35. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    Article  CAS  Google Scholar 

  36. Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

    Article  Google Scholar 

  37. Maciocia, P. M. et al. Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies. Nat. Med. 23, 1416–1423 (2017).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank F. Zhang for deposition of the GeCKO v.2 library at the Addgene plasmid repository (Addgene plasmid no. 1000000048); D. Trono for the deposition of pRRL.sin.cppt.pgk-gfp.wpre (Addgene plasmid no. 12252), envelope plasmid pMD2.G (Addgene plasmid no. 12259), and packaging plasmids pMDLg/pRRE (Addgene plasmid no. 12251) and pRSV-Rev (Addgene plasmid no. 12253); and J. Riley, University of Pennsylvania, who kindly provided the pELNS vector. A.K.S. is a Welcome Senior Investigator (WT100327MA), M.D.C. was funded by the Welsh Assembly Government via a Health and Care Research Wales PhD studentship. M.L. is funded by a Consolidator Award via the Wellcome Institutional Strategic Support Fund to the Cardiff University College of Biomedical and Life Sciences. S.A.E.G. was funded by a Tenovus Cancer Care PhD studentship. J.M. was supported by Program Grant APP1113293 from the National Health and Medical Research Council Australia.

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  1. These authors contributed equally: Michael D. Crowther, Garry Dolton.

Authors and Affiliations

  1. Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK

    Michael D. Crowther, Garry Dolton, Mateusz Legut, Marine E. Caillaud, Angharad Lloyd, Meriem Attaf, Sarah A. E. Galloway, Cristina Rius, Barbara Szomolay, Ann Ager, Anna Fuller, Jamie Rossjohn & Andrew K. Sewell

  2. Division of Hematology, University of Utah School of Medicine, Salt Lake City, UT, USA

    Colin P. Farrell & John D. Phillips

  3. Systems Immunity Research Institute, Cardiff University, Cardiff, UK

    Barbara Szomolay, Ann Ager, Jamie Rossjohn & Andrew K. Sewell

  4. Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, UK

    Alan L. Parker

  5. Center for Cancer Immune Therapy, Herlev Hospital, Copenhagen University, Copenhagen, Denmark

    Marco Donia & Inge Marie Svane

  6. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia

    James McCluskey

  7. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia

    Jamie Rossjohn

  8. Infection and Immunity Program & Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    Jamie Rossjohn

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Contributions

A.K.S. and G.D. conceived project. M.D.C., G.D., M.E.C., S.A.E.G., M.A., A.L. and C.R. undertook the T cell experiments. M.D.C., M.L., C.P.F., B.S. and J.P. performed the genome-wide CRISPR experiments and/or analyses. A.F. generated lentiviral vectors and edited the manuscript. M.D. and I.M.S. supplied the patient PBMC and melanoma and renal carcinoma cell lines. A.A. provided advice on mouse experiments. A.L.P. provided expertise and ovarian cancer ascites. J.R. and J.M. provided the MR1 tetramer reagents. G.D. and A.K.S. supervised the work. M.D.C., G.D. and A.K.S. wrote and edited the manuscript.

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Correspondence to Andrew K. Sewell.

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Cardiff University has filed patents based on these findings.

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Crowther, M.D., Dolton, G., Legut, M. et al. Genome-wide CRISPR–Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1. Nat Immunol 21, 178–185 (2020). https://doi.org/10.1038/s41590-019-0578-8

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