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

Laugel, Bruno, Lloyd, Angharad, Meermeier, Erin W., Crowther, Michael D., Connor, Thomas ORCID: https://orcid.org/0000-0003-2394-6504, Dolton, Garry Michael, Miles, John James, Burrows, Scott R., Gold, Marielle C., Lewinsohn, David M. and Sewell, Andrew K. ORCID: https://orcid.org/0000-0003-3194-3135 2016. 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. Journal of Immunology 197 (3) , pp. 971-982. 10.4049/jimmunol.1501402

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Abstract

The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1−/− clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1−/− clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences Medicine
Subjects:	Q Science > QR Microbiology > QR180 Immunology
Additional Information:	This is an open-access article distributed under the terms of the CC-BY 3.0 Unported license.
Publisher:	American Association of Immunologists
ISSN:	0022-1767
Funders:	Wellcome Trust, BBSRC
Date of First Compliant Deposit:	20 June 2016
Date of Acceptance:	18 May 2016
Last Modified:	05 Jul 2023 18:38
URI:	https://orca.cardiff.ac.uk/id/eprint/92013

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