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 R., Dolton, Garry, Miles, John J., Burrows, Scott R., Gold, Marielle C., Lewinsohn, David M., and Sewell, Andrew K. (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.

[img]
Preview
PDF (Published Version) - Published Version
Available under License Creative Commons Attribution.

Download (2MB) | Preview
View at Publisher Website: http://dx.doi.org/10.4049/jimmunol.15014...
 
5
58


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 ID: 44943
Item Type: Article (Research - C1)
ISSN: 1550-6606
Additional Information:

© 2016 The Authors. This is an open-access article distributed under the terms of the CC-BY 3.0 Unported license.

https://creativecommons.org/licenses/by/3.0/

Funders: US Department of Veterans Affairs (DVA), National Institutes of Health (NIH), Wellcome Trust (WT), Biotechnology and Biological Sciences Research Council (BBSRC), Medical Research Council (MRC), Health and Care Research Wales (HCRW)
Projects and Grants: DVA Biomedical Laboratory Research and Development Merit Review Award I01 BX001231, DVA Biomedical Laboratory Research and Development Merit Review Award I01 BX000533, NIH Grant R01 AI078965, NIH Grant R01 AI048090, NIH Grant T32 AI078903-05, NIH Grant T32 HL83808-05, BBSRC Grant BB/H001085/1
Date Deposited: 02 Aug 2016 23:36
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1107 Immunology > 110702 Applied Immunology (incl Antibody Engineering, Xenotransplantation and T-cell Therapies) @ 100%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 100%
Downloads: Total: 58
Last 12 Months: 27
More Statistics

Actions (Repository Staff Only)

Item Control Page Item Control Page