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.
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.
Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom; LaugelBF@cardiff.ac.uk SewellAK@cardiff.ac.uk.
Bruno
B
Laugel
Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom;
Angharad
A
Lloyd
Department of Pulmonary and Critical Care Medicine, Oregon Health and Science University, Portland, OR 97239;
Erin W
0000-0002-9709-7624
EW
Meermeier
Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom;
Michael D
MD
Crowther
Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3AX, United Kingdom; and.
Thomas R
TR
Connor
Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom;
Garry
G
Dolton
QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4006, Australia.
John J
JJ
Miles
QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4006, Australia.
Scott R
SR
Burrows
Department of Pulmonary and Critical Care Medicine, Oregon Health and Science University, Portland, OR 97239;
Marielle C
0000-0003-2505-3291
MC
Gold
Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3AX, United Kingdom; and.
David M
DM
Lewinsohn
Division of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom; LaugelBF@cardiff.ac.uk SewellAK@cardiff.ac.uk.
Andrew K
0000-0003-3194-3135
AK
Sewell
D017951:Antigen Presentation / Q000276:immunology*; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D005434:Flow Cytometry; D000072669:Gene Editing / Q000379:methods*; D005822:Genetic Vectors; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D006801:Humans; D016086:Lentivirus; D008213:Lymphocyte Activation / Q000276:immunology*; D015778:Minor Histocompatibility Antigens / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D016176:T-Lymphocyte Subsets / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
D015395:Histocompatibility Antigens Class I; C094840:MR1 protein, human; D015778:Minor Histocompatibility Antigens