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

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.

Characterization of Human Mucosal‐associated Invariant T (MAIT) Cells

“…The use of exogenously added APCs allows one to "separate" Ag presentation and T cell activation, thereby avoiding MR1-Ag presentation by MAIT cells. This is required, for example, in order to formally test for MR1-Ag dependence of the observed stimulation using a panel of APC lines that either express wild-type levels of MR1, overexpress MR1 (see Support Protocols 5 and 6) (Huang et al, 2008;Lepore et al, 2017;Reantragoon et al, 2012;Wang et al, 2018;Young et al, 2013), or lack MR1 (see Support Protocol 7) (Laugel et al, 2016;Wang et al, 2018). MAIT cell activation can be measured based on expression of activationspecific surface markers (Table 1).…”

“…In response to both CD3/CD28 and PMA/ionomycin, MAIT cells secrete TNF, IFNγ, and granzyme B (Dias, Sobkowiak, Sandberg, & Leeansyah, 2016;Gherardin, Souter, et al, 2018;Kurioka et al, 2017;Slichter et al, 2016), but minimal IL-17A production is detected (Gherardin, Souter, et al, 2018;Slichter et al, 2016). Similarly, incubation in the presence of APCs that lack MR1 (Laugel et al, 2016;Wang et al, 2018) (see Support Protocol 7), as compared to cells proficient in or overexpressing MR1 (Reantragoon et al, 2012;Wang et al, 2018) (see Support Protocols 5 and 9), or in the absence of relevant Ag but in the presence of vehicle control, media, or irrelevant Ag, and/or in the presence of MR1 blocking antibody as compared to isotype control antibody (see Alternate Protocol 9), or in the presence of non-activating competitively inhibiting Ags (Ac-6-FP) in addition to stimulating Ag (see Alternate Protocol 10) serve as controls for the MR1-Ag specific reactivity in MAIT cell activation assays.…”

“…APC lines knocked out for MR1 represent the ideal tool for determining MR1-Ag dependence of MAIT cell activation (Laugel et al, 2016;Wang et al, 2018). Alternatively, MR1-blocking Abs (e.g., 26.5, Huang et al, 2005; or 8F2.F9, Chua et al, 2011) compared to isotype control Abs can be used in combination with APCs expressing wild-type levels of MR1 or overexpressing MR1, as previously shown by our teams Eckle et al, 2014;Gherardin et al, 2016;Keller et al, 2017;Kjer-Nielsen et al, 2012;Patel et al, 2013;Reantragoon et al, 2013;Reantragoon et al, 2012) and others (Chua et al, 2011;Gold et al, 2013;Huang et al, 2009;Kurioka et al, 2017;Lepore et al, 2017;Salio et al, 2017;Young et al, 2013).…”

“…This can be achieved with PMA/ionomycin or bead-immobilized CD3/CD28 specific mAbs (see Alternate Protocol 12). Similarly, suitable controls for MR1-Ag-specific reactivity in MAIT cell activation assays include incubation in the presence of APCs that lack MR1 (Laugel et al, 2016;Wang et al, 2018) (see Support Protocol 7) compared to cells proficient in MR1 or overexpressing MR1 (Reantragoon et al, 2012;Wang et al, 2018) (see Support Protocols 5 and 6); incubation in the absence of relevant Ag but in the presence of vehicle control, medium, or irrelevant Ag; incubation in the presence of MR1blocking antibody compared to isotype control antibody (see Alternate Protocol 13); and incubation in the presence of non-activating competitively inhibiting Ags (Ac-6-FP) in addition to stimulating Ag (see Alternate Protocol 10).…”

“…At the same time, there are some conflicting studies on MAIT cell frequencies and phenotypes in disease, warranting further investigation. Furthermore, some studies allude to the existence of heterogenous populations of MR1-restricted T cells that differ from MAIT cells, named atypical MR1-restricted T cells or MR1T cells Harriff et al, 2018;Koay et al, 2019;Lepore et al, 2017;Meermeier et al, 2016;reviewed in Godfrey et al, 2019). Although some of these T cells are identified using the MR1 tetramer, they can differ from MAIT cells in their MR1-Ag reactivity pattern, including their reactivity to MR1-6-FP tetramer, and can feature atypical MAIT TCRα chain usage, transcription factor profiles, and phenotypic markers.…”