The P5-type ATPase ATP13A1 modulates major histocompatibility complex I-related protein 1 (MR1)-mediated antigen presentation

The monomorphic antigen-presenting molecule major histocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included β2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression.

The monomorphic antigen-presenting molecule major histocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included β 2 -microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression.
The monomorphic major histocompatibility complex (MHC) class I-related protein 1 (MR1) presents smallmolecule metabolites to mucosal-associated invariant T (MAIT) cells (1)(2)(3). While the full spectrum of MR1 ligands is still being elucidated (2,(4)(5)(6)(7)(8)(9)(10), the best characterized MAITactivating MR1 ligands are derivatives of pyrimidine intermediates of riboflavin biosynthesis, a pathway specific to certain fungi and bacteria and, thus, intrinsically non-self for humans (1,11). Accordingly, MAIT cells are activated upon recognition of MR1 ligands derived from a variety of microbes capable of riboflavin synthesis such as Escherichia coli (E.coli), Salmonella typhimurium, and Mycobacterium tuberculosis (12)(13)(14)(15). Molecularly, MAIT cells are characterized by high expression of the C-type lectin CD161, the interleukin (IL)-18 receptor, CD26 (16)(17)(18), and the canonical Vα7.2-Jα33/12/ 20 T cell receptor (TCR) α chain in humans (19)(20)(21). Owing to their restricted TCR repertoire, their relative abundance, and their effector-memory phenotype, MAIT cells are counted as an innate-like lymphocyte subset (22). Their high numbers and poised phenotype allow them to rapidly carry out effector functions in response to their cognate microbial antigens and place them at the interface between the innate and the adaptive immune system (22,23). Furthermore, the MR1 antigen presentation pathway is an attractive target for both therapeutic and vaccination strategies as the monomorphic nature of the presenting molecule renders it independent of individual variations in human leukocyte antigen (HLA) expression (24)(25)(26)(27)(28). A better understanding of this nonclassical antigen presentation pathway is, thus, crucial for harnessing the protective potential of MAIT cells in such therapeutic approaches.
MR1 transcript is detectable in almost all human cell lines and tissues but endogenous surface expression of MR1 protein is difficult to detect in most human cells even upon exposure to microbial MR1 ligand (29,30). Nevertheless, such low MR1 surface levels are capable of inducing a potent interferon-γ (IFNγ) response in MAIT cells (29,30). Since endogenous MR1 is difficult to detect, most of the current knowledge has been obtained in overexpression systems (30)(31)(32)(33)(34)(35). In one widely used model of MR1 overexpression, the majority of cellular MR1 resides in the ER in a partially folded, ligandreceptive state in the absence of exogenous or microbial ligands (34,36). Upon ligand binding, the MR1 heavy chain-β 2microglobulin (β 2 m)-complex translocates to the cell surface (34,37). ER egress is thought to require neutralization of the positive charge on the lysine 43 (K43) residue within the MR1 ligand binding groove (34,37). In other studies, MR1 has been found to partially colocalize with LAMP1 in endolysosomal compartments (35,38), and the relative contributions of these two intracellular pools to antigen presentation under physiological conditions remain to be determined (39). Importantly, a growing body of evidence suggests that the loading and trafficking of MR1 in the context of intracellular bacterial infection differ from the presentation of exogenous ligand (30,35,(39)(40)(41)(42).
In light of the ubiquitous expression of MR1 transcript and the observation that activating MR1 ligands can be derived from both pathogenic and commensal microorganisms, tight regulation of this highly sensitive antigen presentation pathway is required (31,41). Using a previously described functional genetic screen utilizing the near-haploid human cell line HAP1 (43,44), we identified the P 5 -ATPase ATP13A1 as one of the proteins involved in the modulation of MR1 surface expression.
The difference in the number of positive and negative candidate genes identified reflects the fact that the screen setup was more powerful in detecting putative positive regulators than negative ones. The screen was carried out in the presence of Ac6FP, which results in the translocation of most MR1 molecules to the surface. Thus, a further increase in MR1 surface expression, as would be expected upon KO of a negative regulator, may be masked. Loss of a positive regulator, on the other hand, leads to reduced MR1 surface levels and was more easily detected in our system. Therefore, we focused our analysis on the putative positive regulators of MR1 surface expression.
One of the most prominent and functionally interesting putative positive regulators of MR1 surface expression was the P 5 -ATPase ATP13A1 ( Fig. 1 and Table S2). While information on mammalian ATP13A1 has been sparse, recent evidence points toward a dislocase function (73). The yeast homologue Spf1p (also known as Cod1p) localizes to the ER and is involved in ion homeostasis (69,74). Consequently, loss of spf1 causes hypoglycosylation of proteins and results in ER stress (69,(74)(75)(76)(77)(78). In the context of MR1 antigen presentation, ATP13A1 was a particular promising target because of its localization to the ER and its putative transporter function.

ATP13A1 modulates the size of the cellular MR1 pool
To investigate the role of ATP13A1 in MR1-mediated antigen presentation, we generated HAP1.MR1 ATP13A1 KO clones. The same HAP1.MR1 parent clone used in the original screen (clone D9) was transiently transfected with plasmids encoding the Cas9 protein, the fluorescent protein mRuby, and one of three different sgRNAs targeting different regions of the ATP13A1 gene (Fig. S4). Single mRuby + cells were sorted, and individual clones were screened for loss of ATP13A1 expression by Western blot (Fig. 2A). Strikingly, total cellular MR1 levels were reduced in all but one HAP1.MR1 ATP13A1 KO clones regardless of the sgRNA used (Figs. 2, A and B and S5). A notable exception was clone 3-15, which displayed reduced cellular MR1 levels despite expressing ATP13A1 (see below and Fig. S5). Sequencing revealed that a six base pair deletion in clone 3-15 left the reading frame of ATP13A1 intact but altered a highly conserved region within the amino acid sequence (79) (Fig. S6). The importance of this GxPF sequence is not only underscored by its conservation across yeast, murine, and human ATP13A1 homologues (75,79,80) (NCBI Reference Sequences NP_010883.3, NP_573487.2, and NP_065143.2, respectively), but also by the observation that a construct lacking part of this motif was unable to rescue the spf1 KO phenotype in yeast (75). Alterations to this region appear to have resulted in the production of a non-or dysfunctional protein, which could explain the "KO-like" phenotype of clone 3-15 (Fig. S5). This makes clone 3-15 a very interesting control as it suggests that loss of ATP13A1 function and not merely expression of the protein is responsible for the MR1 phenotype.
When ATP13A1 expression was restored in a subset of clones comprising one KO clone for each sgRNA and clone 3-15, total MR1 levels were rescued, confirming that this phenotype is indeed attributable to loss of ATP13A1 and that the mutation found in clone 3-15 is not dominant negative (Fig. 2, C and D). In a T cell stimulation assay, the three HAP1.MR1 ATP13A1 KO clones elicited reduced MAIT cell activation compared with the parental clone when pulsed with the MR1 ligand 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU; added as 5-amino-ribityl uracil (5-A-RU) + methylglyoxal (MG)) ( Fig. 2E). Importantly, ATP13A1 overexpression increased the antigen presentation capacity of all three HAP1.MR1 ATP13A1 KO clones, and this trend was statistically significant at some antigen concentrations (Figs. 2E and S7). Although statistical significance varied across experimental replicates, the trends reported here were consistent across sgRNAs used for KO, MAIT cell donors, and assays, together strengthening the general conclusion that the MR1 phenotype can be at least partially rescued by reconstitution of ATP13A1 expression.  Table S2.
ATP13A1 modulates MR1-mediated antigen presentation  Fig. S5. B, quantification of data shown in A. Each symbol represents a clone and median is shown for the groups. Red symbols represent ATP13A1 KO clones, black symbols represent ATP13A1 sufficient clones. Color coding as shown in A. Clone 3-13 was not included as the shape of the band precluded reliable quantification. Data are representative of two experiments. Statistical significance of differences was analyzed using a twotailed Mann-Whitney test. C, ATP13A1 expression was reconstituted in a subset of clones using a lentiviral expression system and MR1 expression in Next, we tested MR1 cell surface expression in one clone per sgRNA that was deficient for ATP13A1 ("ATP13A1 KO clones") and one clone per sgRNA that was mRuby + upon sorting but still expressed the protein ("ATP13A1-sufficient clones"). Details on editing of the clones used for the individual experiments can be found in Table S3 and Fig. S6. Consistent with the results from the gene trap screen, surface levels of folded MR1 in HAP1.MR1 clones deficient for ATP13A1 were lower than those in their ATP13A1-sufficient counterparts, independently of the sgRNA used to knock out ATP13A1 (Fig. 2F). This difference was statistically significant in the presence of Ac6FP and as with the total protein content, the reduced MR1 surface expression could be reversed by overexpression of ATP13A1 (Fig. 2F). Importantly, surface expression of classical MHC class I molecules was not similarly impacted by loss of ATP13A1, suggesting that the phenotype is specific to MR1 (Fig. 2G). Indeed, MHC class I surface levels were elevated in the ATP13A1 KO clones, possibly because of increased availability of β 2 m or components of the PLC, which contribute to the stabilization of MR1 in the ER (36).
Additionally, β 2 m was not differentially expressed in the KO compared with the sufficient clones at the transcript ( Fig. 2H) or protein level (Fig. S8), indicating that the difference in MR1 surface expression was likely not due to a lack of its smaller subunit. This conclusion is corroborated by the rescue of both total and surface levels of MR1 in the reconstituted HAP1.MR1 ATP13A1 KO clones (Fig. 2, C and F), which demonstrates that β 2 m is not a limiting factor for MR1 expression in these cells. However, MR1 transcript levels were statistically significantly reduced in the ATP13A1 KO clones (Fig. 2I). Thus, we cannot exclude an effect of ATP13A1 on MR1 mRNA stability in the HAP1 system.
To confirm that the MR1 phenotype was not specific to the HAP1.MR1 parent clone used in the screen, we used sgRNA2 to knock out ATP13A1 in two other HAP1.MR1 parent clones (D3 and F2, compare Fig. S1). These newly derived clones replicated the loss of total MR1 by Western blot and largely also the MR1 surface phenotype except for one of the three tested F2 ATP13A1 KO clones, which had likely become polyploid (Figs. S9 and S10). Overall, these data indicate that the effect of ATP13A1 is not related to the number or site of insertions of the lentiviral MR1 expression cassette.

ATP13A1-deficient HAP1.MR1 clones do not suffer from elevated ER stress
Having established that ATP13A1 KO clones display an MR1 phenotype attributable to ATP13A1 deficiency, we next investigated the mechanism by which ATP13A1 expression modulates MR1 protein levels. Since ATP13A1 has been implicated in the transport of cations (69,74), we hypothesized that it could indirectly influence MR1 protein folding, loading, or stability in the ER by modulating ion concentrations in this compartment. Consistent with this, a large body of literature has linked deficiency for Spf1p and ATP13A1 to ER stress in yeast and human cells, respectively (69,74,(76)(77)(78). We, thus, tested whether HAP1.MR1 clones deficient for ATP13A1 differentially upregulated components of the unfolded protein response (UPR). Although all tested clones responded with upregulation of the ER stress markers HERPUD1 (81) and ATF4 (82) when treated with the ER stress-inducing inhibitor thapsigargin (TG) (83), we found no evidence for differential UPR induction in the ATP13A1 KO clones (Fig. 3, A-D). The same pattern was evident when analyzing the splice variants of XBP1, a transcription factor that controls the expression of UPR proteins in response to ER stress and is itself activated by alternative splicing of its mRNA (84) (Fig. 3E). The data also indicate that the HAP1.MR1 ATP13A1 KO clones do not suffer from elevated ER stress at baseline, suggesting that the lower MR1 protein levels are not due to a general defect in protein folding.
Divalent cations such as calcium and manganese are important cofactors for many enzymes and disturbing their homeostasis leads to defects in glycoprotein processing (69,(74)(75)(76). Since the MR1 protein sequence features a known N-linked glycosylation site at Asn86 (32), we next determined whether MR1 in the HAP1.MR1 KO clones might be hypoglycosylated. To investigate the glycosylation state of MR1 in the HAP1.MR1 ATP13A1 KO clones, whole cell lysates were digested with Endoglycosidase H (EndoH). Since EndoH cannot cleave the highly complex polysaccharide residues generated during posttranslational modification in the Golgi the polyclonal transduced cell lines was analyzed by Western blot. Lysates of HAP1 KO clones and the parent clones were made prior to those of the reconstituted lines. D, quantification of data shown in C. Each symbol represents one clone or cell line and median is shown for the groups. Red symbols represent ATP13A1 KO clones, black symbols represent ATP13A1 sufficient clones. Color coding as shown in A. The starred square in the KO group represents clone 3-15. E, the HAP1.MR1 parent clone, the indicated KO clones, and the reconstituted cell lines were pulsed with 5-A-RU+50 μM MG and incubated with sorted human MAIT cells for at least 36 h. IFNγ in the supernatants was quantified by ELISA. Each symbol represents the mean of technical duplicates for one clone or cell line and median is shown for the groups. Statistical significance of differences was analyzed with a Two-way RM ANOVA with matching across columns (by sgRNA) and rows (by clone/cell line) and Sidak's multiple comparison test. The HAP1.MR1 parent clone D9 is shown for reference but was not included in statistical analyses. Data are representative of three experiments except for clone 3-1 and its reconstituted cell line although statistical significance varied across repeats (see Fig. S7). F and G, ATP13A1 sufficient (suff; solid lines), KO (KO; dashed lines), and reconstituted (rec; filled) cells were incubated with 5 μg/ml of Acetyl-6-formylpterin (Ac6FP, blue) or DMSO (black) for 5 h before staining for MR1 (F) and MHC class I (G) surface expression. Isotype/fluorescence minus one (FMO) controls are from one sufficient, one KO, and one reconstituted clone each. Representative histograms are shown on the left and pooled quantification from three experiments is shown on the right. Each symbol represents the mean of replicate experiments for one clone/ cell line and median is shown for each group. Geometric mean fluorescence intensity was determined for the most prominent peak and normalized to the DMSO-treated parent clone. Statistical significance of differences was calculated using a Two-way RM ANOVA with matching across columns (by sgRNA) and rows (by clone/cell line) and Sidak's multiple comparison test. H and I, MR1 and B2M transcript levels were compared in three ATP13A1 sufficient clones and three ATP13A1 KO clones. Each symbol represents the mean of three experiments for one clone (two experiments for clone 3-2) and mean and SD are shown for each group. Statistical significance of differences was analyzed using a paired t test. For HAP1 clones shown in E-I see Table S3. KO clones are highlighted in red and the ATP13A1 mutant clone 3-15 is highlighted in bold and starred. ns = p > 0.05. IFNγ, interferon-γ; KO, knockout; LoD, limit of detection; MAIT, mucosal-associated invariant T cell; MHC, major histocompatibility complex. A-E, HAP1.MR1-derived clones were treated with 0.1 μM thapsigargin (TG, +) or DMSO (−) for 3 h. Protein lysates were separated by SDS-PAGE and immuno-blotted for HERPUD1 (green) and loading control GAPDH (red) (A and C) or ATF4 (green) and GAPDH (red) (B and D). Each symbol represents one clone and median is shown for the groups. Statistical significance of differences was calculated using a two-tailed Wilcoxon matched-pairs signed rank test. Relative signal intensities below 0 were set to 0. E, in the same experiment, cDNA of indicated HAP1.MR1 clones was analyzed for the XBP1 splice variant on a 2.5% agarose gel. Data in A-E are representative of two independent experiments except for the HAP1 WT, which was not included in the repeat. F, cell lysates of the indicated HAP1 clones were digested with endoglycosylase H (EndoH, +) or not (−). Digests were separated by SDS-PAGE and immuno-blotted for MR1 (green) and loading control ACTB (red). MR1-deficient HAP1 wild-type (WT) lysates were included to identify unspecific binding. Data in F are representative of four similar independent experiments including one that used the same lysates as the Western blot in Fig Table S3). ATP13A1 KO clones are highlighted in red and the ATP13A1 mutant clone 3-15 is highlighted in bold and starred. noRT, no reverse transcriptase control. apparatus, sensitivity to EndoH digestion is a proxy of ER residency (85,86). It has previously been shown that in many cell lines MR1 primarily resides in a pre-Golgi compartment at steady state as reflected by its sensitivity to digestion with EndoH (32,34,70). Accordingly, the cellular MR1 pool in the HAP1.MR1 parent clone D9 was also sensitive to digestion with EndoH (Fig. 3F). Interestingly, the much smaller pool of MR1 molecules in the HAP1.MR1 ATP13A1 KO clones was equally sensitive to the endoglycosidase, indicating that these molecules are glycosylated normally within the ER. This makes it seem unlikely that a general dysfunction of the ER glycosylation machinery is the cause for the reduced MR1 protein levels at baseline. Importantly, protein levels of classical MHC class I molecules were unaffected in the HAP1.MR1 ATP13A1 KO clones (Fig. S11), further corroborating this conclusion. Of note, the antibody used to detect MHC class I by Western blot does not react with HLA-A and HLA-G alleles (87). Hence, potential allele-specific effects on MHC class I molecules would not be detected in this assay and can, thus, not be excluded.
ATP13A1 specifically affects MR1-mediated antigen presentation but not classical peptide presentation HAP1 cell cultures are prone to becoming polyploid over time, due to a competitive growth disadvantage of haploid cells (88). Consistent with this, the functional data in Figure 2 are representative of two experiments, but one HAP1.MR1 ATP13A1 KO clone (3-1) lost its MR1 phenotype in a third repeat, which correlated with morphological changes indicative of a loss of the haploid state (Fig. S7). Therefore, we next validated a role for ATP13A1 in the monocytic cell line THP-1, which is frequently used as a model system for MR1mediated antigen presentation (2,30,89,90). We generated ATP13A1 KO clones from a THP-1 parent clone derived by limiting dilution (Fig. 4A) and confirmed disruption of ATP13A1 at the genomic level by Next-Generation Sequencing. This allowed us to investigate whether ATP13A1 deficiency impacted endogenous MR1. While MR1 mRNA levels were unaffected ( Fig. 4B), MR1 surface expression was reduced in five THP-1 ATP13A1 KO clones compared with ATP13A1sufficient controls (Fig. 4C). This trend did not reach statistical significance in one repeat due to one sample having high isotype staining (Fig. S12). Importantly, endogenous MR1 surface levels were lower in ATP13A1 KO clones not only at baseline but also in the presence of the MR1-stabilizing ligand Ac6FP (Fig. 4C). Indeed, the fold-change of MR1 geometric mean fluorescence intensity (GeoMean) induced by Ac6FP was smaller in these clones (Fig. 4E, top panel and Fig. S12C, top panel), suggesting that the observed differences in MR1 surface expression are not simply due to the smaller pool of MR1 molecules available. While surface expression of classical MHC class I molecules was more heterogeneous than MR1 in ATP13A1 KO clones, there was no statistically significant difference compared with ATP13A1-sufficent clones (Figs. 4D and S12B). As expected, MHC class I surface levels were unaffected by incubation with Ac6FP (Fig. 4E, bottom panel and Fig. S12C, bottom panel).
When ATP13A1 expression was reconstituted in the THP-1 ATP13A1 KO clone 2, MR1 surface expression was restored (Fig. S13). Total MR1 protein expression could not be investigated in these clones as endogenous MR1 levels are undetectable by Western blot (see e.g., Fig. S13B).
Consistent with reduced levels of MR1 at the cell surface, the THP-1 ATP13A1 KO clones presented the MR1 ligand 5-OP-RU (added as 5-A-RU + MG) to sorted human MAIT cells less efficiently than their ATP13A1 sufficient counterparts (Fig. 5A). This trend was observed across multiple experiments using different MAIT cell donors and 5-A-RU concentrations, although it did not always reach statistical significance (Fig. S14A). Since the antigen presentation pathways for exogenous ligand and intracellular bacteria differ mechanistically (31,39,42), we also tested the effect of ATP13A1 deficiency on MAIT cell activation in the context of bacterial infection. As with exogenous synthetic ligand, THP-1 ATP13A1 KO clones infected with E.coli showed a trend of eliciting a lower IFNγ response from sorted human MAIT cells as compared with ATP13A1 sufficient clones (Figs. 5B and S14B). Importantly, this functional defect appeared to be specific to MR1-mediated antigen presentation since the response of a peptide-specific T cell line to peptide-pulsed ATP13A1 KO clones was comparable to that elicited by ATP13A1 sufficient clones (Figs. 5C and S14C). Similarly, presentation of the CD1d ligand α-Galactosylceramide (αGalCer) to sorted human invariant natural killer T (iNKT) cells was not significantly affected by loss of ATP13A1 (Figs. 5D and S14D). Of note, ATP13A1 was not a hit in a recent whole-genome siRNA screen for modulators of antigen presentation by CD1d (91).
Lastly, when ATP13A1 expression was restored in ATP13A1 KO clone 2, its capacity to present 5-OP-RU was partially rescued (Figs. 5E and S14E). Overexpression of ATP13A1 in the THP-1 parent clone, however, did not increase antigen presentation. Thus, it appears plausible that the transporter operates within a tightly controlled regulatory network optimized to prevent aberrant MAIT cell activation.

ATP13A1 deficiency affects MR1 levels at or immediately after protein synthesis
Since MR1 transcript levels did not significantly differ between ATP13A1 KO and sufficient THP-1 clones (Fig. 4B), and we found no evidence for a differential global induction of the unfolded protein response in the HAP1.MR1 ATP13A1 KO clones (Fig. 3), we hypothesized that MR1 may be specifically targeted for ER-associated degradation (ERAD). To test this hypothesis, we inhibited proteasomal degradation with the reversible peptide aldehyde inhibitor MG-132 (92,93). In parallel, we inhibited ERAD at a different step with the irreversible p97/VCP inhibitor NMS-873 (94). p97/ VCP aids in the extraction of ubiquitinated target proteins from the ER lumen into the cytosol and, thus, encourages misfolded proteins toward proteolysis (94,95). Even though proteasomal degradation was successfully inhibited in this experiment as shown by the stabilization of HERPUD1 (Fig. 6A), this inhibition did not lead to an increase in full-ATP13A1 modulates MR1-mediated antigen presentation length MR1 protein levels (Fig. 6B). This suggests that MR1 is not differentially degraded by ERAD upon ATP13A1 KO under homeostatic conditions. Similar results were obtained in the HAP1.MR1 ATP13A1 KO clones (Fig. S15). The low levels of HERPUD1 at baseline further confirmed that THP-1 ATP13A1 KO cells, like HAP1.MR1 ATP13A1 KO cells (Fig. 3, A-E), do not suffer from elevated ER stress at baseline. Like their HAP1.MR1 counterparts, THP-1 cells also Figure 4. MR1 surface expression is reduced in THP-1 ATP13A1 KO clones. A THP-1 WT clone was transiently transfected with a CRISPR/Cas9 plasmid encoding sgRNA 2 targeting the first exon of ATP13A1 and cells were sorted into single wells. A, clones were screened by Western blot. Membranes were probed with anti-ATP13A1 (green) and anti-β actin (ACTB; red) antibodies. Molecular weight of marker bands is shown in kDa to the left of the blot. Data are representative of at least three experiments for each clone except for WT THP-1, which was not always included. B, MR1 transcript levels were compared in five ATP13A1 sufficient clones and five ATP13A1 KO clones. Each symbol represents the mean of technical replicates for one clone, and mean and standard deviation are shown for each group. Statistical significance was calculated using a two-tailed t test with Welch's correction. Data are representative of two experiments. C and D, those ten clones were incubated with Acetyl-6-formylpterin (Ac6FP, blue) or DMSO (black) for 5 h before staining for MR1 (C) and MHC class I (D) surface expression. Histograms for two representative clones are shown on the left and cumulative data from all ten clones are shown on the right with median. The same parent sample is shown in both histograms for comparison. E, the same data as in C and D shown as fold-change of Geometric Mean Fluorescence Intensity (GeoMean) with Ac6FP compared with DMSO control. Results in C-E are supported by another experiment each although the repeat for C differed in statistical significance (see Fig. S12). Statistical significance was calculated using multiple t-tests and corrected for multiple comparisons with the Holm-Sidak method in C and D and the Mann-Whitney test in E. ATP13A1 KO clones are highlighted in red. ns = p > 0.05. L/D, live/dead stain; MHC, major histocompatibility complex. ATP13A1 modulates MR1-mediated antigen presentation responded to thapsigargin with upregulation of HERPUD1, showing that their ER stress response is intact but not triggered by loss of ATP13A1 alone (Fig. 6A).
To further characterize the effect of ATP13A1 on the kinetics of MR1 expression, we performed pulse-chase experiments after metabolic labeling of nascent proteins with radioactive amino acids. In order to be able to immunoprecipitate nascent MR1 molecules regardless of their folding state, we overexpressed human influenza haemagglutinin (HA)-tagged MR1 in one THP-1 ATP13A1 KO clone and one THP-1 ATP13A1-sufficient clone as well as the THP-1 parent clone (Fig. S13). Confirming the phenotype seen in the HAP1.MR1 ATP13A1 KO clones, both total and cell surface protein levels of recombinantly overexpressed MR1-HA were lower in the absence of ATP13A1 (Fig. S13, B, C and E). MR1 transcript levels were less than twofold different (Fig. S13D).
Immunoprecipitation of a radiolabeled cohort of proteins revealed that the rate of MR1 protein degradation within the first 4 min after translation was not increased in the ATP13A1 KO cells compared with the parent or the ATP13A1 sufficient cells (Fig. 6C). However, a trend toward lower MR1 total protein levels was evident even at the time of labeling, although this did not reach statistical significance (Fig. 6E). These differences were small but consistent between experimental repeats and suggest that ATP13A1 modulates either the rate of MR1 protein synthesis or the stability of the nascent MR1 polypeptide immediately after its cotranslational translocation into the ER. Importantly, the abundance and kinetics of folded MHC class I molecules were not affected in ATP13A1 KO cells (Fig. 6D). The increase in MHC class I signal over the course of the experiment can be explained by ongoing postpulse folding, since the antibody used is specific for the folded conformation (96, 97).  Fig. S14). Statistical significance of differences between indicated groups was analyzed using multiple unpaired t tests and correcting for multiple comparisons with the Holm-Sidak method. ns = p > 0.05. IFNγ, interferon-γ; iNKT, invariant natural killer T cell; KO, knockout; MAIT, mucosal-associated invariant T cell. ATP13A1 modulates MR1-mediated antigen presentation To exclude the possibility that the reduced MR1-HA expression in the ATP13A1 KO background was caused by differences in transduction efficiency, we transduced the clones with an MR1-Emerald construct featuring an internal ribosomal entry site (IRES) (98). In these cells, MR1 and Emerald are cotranscribed but not fused on the protein level. Thus, expression of the green fluorescent protein can be used as an indicator of transduction efficiency without affecting MR1 protein function. Although overall transduction efficiency was indeed slightly lower in the THP-1 ATP13A1 KO clone 2 (Fig. 7, A and B), MR1 surface levels were reduced in these cells compared with the THP-1 parent and the ATP13A1-sufficient clone 15 across a range of transduction levels (Fig. 7, C and E). Importantly, MHC class I surface expression was not affected (Fig. 7, D and F). In conclusion, in the absence of ATP13A1, we observed reduced expression of endogenous MR1 as well as of three exogenously expressed constructs, corroborating the conclusion that MR1 protein levels are stabilized by this P 5 -type ATPase.

Discussion
MAIT cells are one of the most recent additions to the family of innate-like, unconventional T cells (99). Unlike classical, MHC class I and II-restricted T cells, they are characterized by a limited TCR repertoire, reactivity to conserved, nonpeptidic antigens, and an effector-memory phenotype, which allows them to rapidly exert effector functions (15,22). MAIT cells have been implicated in the control of numerous infectious diseases (recently reviewed in (15)) and emerging data extend their activity to protective as well as pathogenic roles in sterile inflammation, autoimmunity, tissue repair, and cancer (100-106). Their unusually high abundance in both blood and tissues, combined with their restriction by a monomorphic antigen-presenting molecule, makes them a particularly attractive therapeutic target (24,25,28,105,106). However, to effectively leverage the potential of these cells in prophylactic and therapeutic applications, a better understanding of the MR1-MAIT cell axis is needed. In particular, the cellular and molecular mechanisms governing MR1 loading and trafficking are an area of ongoing research (10,31,36,39,41).
Here, we performed a gene trap screen and identified 199 putative modulators of MR1 surface expression in the presence of the stabilizing ligand Ac6FP. The hits included both proteins known to be involved in antigen presentation and protein trafficking and those not previously associated with this process. Intriguingly, classical MHC class I and components of the PLC (TAP1, TAP2, and tapasin) were putative negative regulators of MR1 surface expression in our screen. This differs from a recent CRISPR screen performed by McWilliam, Mak et al. (36), who found tapasin, calreticulin, and TAP among the positive regulators of MR1 surface expression. In contrast to earlier data (107), McWilliam, Mak, and colleagues show that MR1 surface expression is reduced in tapasin-deficient cell lines and primary cells. Furthermore, their study confirms previous reports of physical interaction of MR1 and tapasin by coimmunoprecipitation (32,36). Although shown to physically interact with MR1 in the ER (36), neither calnexin (CANX; log2MI = 0.687; fcpv = 0.442) nor calreticulin (CALR; log2MI = 2.375; fcpv = 0.124) nor TAPBPR (TAPBPL; log2MI = 1.159; fcpv = 1) was statistically significantly enriched in our screen for modulators of MR1 surface expression. This discrepancy could be due to technical differences such as using a gene-trap approach rather than the CRISPR/Cas9 system or reflect biological differences between the model systems used. The differing observations emphasize the need for further research in this area.
Due to its localization in the ER, its predicted transporter function, and its high enrichment in the MR1 low population in this study, we focused on the P 5 -ATPase ATP13A1. The data presented here support the conclusion that this transmembrane helix dislocase modulates the size of the intracellular pool of MR1 and consequently, MR1 surface expression and antigen presentation. ATP13A1 is the mammalian orthologue of the yeast ATPase Spf1p (also known as Cod1p) and is unique among the five known mammalian P 5 -ATPases (80). The only member of the P 5 A subgroup in humans, it differs from its P 5 B paralogues ATP13A2-5 in its intron/exon structure and is predicted to have resulted from an earlier gene duplication than the other isoforms (80,108). In addition, ATP13A1 features a stretch of 67 amino acids, which is not present in the other mammalian isoforms in either human or mouse but is in the yeast, Caenorhabditis elegans, and Drosophila melanogaster orthologues (79,80). Like all P-type ATPases, ATP13A1 features a central DKTGTLT motif containing the catalytic aspartate (77,79,80,109), and it also contains conserved nucleotide-binding domains (79,80). Topology predictions have identified at least ten putative transmembrane domains for mammalian ATP13A1 (80). Both ATP13A1 and ATP13A4 localize to the ER and have been reported to be involved in calcium homeostasis (69,74,110). However, direct calcium transport activity has not been shown for ATP13A1 and ATPase activity of purified Spf1p was not dependent on or enhanced in the presence of calcium in vitro (74). The concentration of manganese in microsomes isolated from Δspf1 cells was decreased compared with that in wildtype microsomes, though other divalent cations such as calcium and magnesium were not tested in this study (69).
In addition to their implication in ER ion homeostasis, P 5 A-ATPases are associated with a wide variety of cellular functions including ER stress and the UPR, protein processing and PVDF membranes, and visualized by autoradiography. Cycloheximide was included during the pulse in the control sample (CHX pulse). Molecular weight indications were inferred based on protein marker on the PVDF membranes. ATP13A1 KO clones are highlighted in red. Quantification in C and D shows pooled data from two independent experiments with median and range. E, quantification at t = 0 min for the two experiments shown in C and data from an experiment with steady-state analysis only. Each symbol represents one experiment and median is shown for each group. Statistical significance of the difference was calculated using a paired t test. F, quantification at t = 0 min for the two experiments shown in D. ns = p > 0.05. Red symbols represent ATP13A1 KO cells, black symbols represent ATP13A1 sufficient cells. MHC, major histocompatibility complex. turnover, membrane insertion and targeting of secretory proteins, intracellular sterol distribution and vesicular transport, and many more (111). Despite, or perhaps because of, these pleiotropic phenotypes, the substrate of this group of transporters remained enigmatic until very recently (111). In a landmark publication, McKenna et al. (73) demonstrated that human and yeast P 5 A-ATPases act as protein dislocases, removing mistargeted tail-anchored mitochondrial proteins wrongly inserted into the ER membrane. In this manuscript, we show that loss of ATP13A1 corresponded to a drastic decrease in total cellular MR1 levels in two different cell lines and examined possible mechanisms by which this transporter may impact the amount of MR1 available in the cell.

ATP13A1 modulates MR1-mediated antigen presentation
Since spf1 mutants suffer severe ER stress (69, 74, 76-78), we hypothesized that the MR1 phenotype we observed in the ATP13A1-deficient cells may be caused by ER-associated degradation due to misfolding of either MR1 itself or a crucial chaperone. However, we did not find any evidence for elevated ER stress in ATP13A1-deficient HAP1.MR1 or THP-1 cells, and inhibition of proteasomal degradation did not rescue MR1 levels at steady state. This indicates that MR1 molecules do not accumulate in an unfolded state in the ER, which would necessitate clearance by the UPR and is in stark contrast to the yeast system (74,77,78,112). Of note, the metazoan ER stress response comprises three overlapping branches while the equivalent system in yeast cells relies on IRE1 sensing only (113). With less in-built redundancy, this simpler system may be less robust, providing a potential explanation as to why yeast cells appear to be more dependent on Spf1p for maintaining ER homeostasis. Alternatively, ER stress may be a downstream effect of Spf1p deficiency rather than a direct consequence of it, and mammalian cells may have more sophisticated mechanisms to counteract such secondary effects.
Early investigations identified a hypoglycosylation phenotype in spf1 mutants (69,75,76). Yet, the small population of MR1 molecules detectable in HAP1.MR1 ATP13A1 KO clones was still sensitive to endoglycosidase digestion, confirming that glycoprotein processing in the ER was not affected upon loss of the transporter. This observation is consistent with reports that EndoH-sensitive core glycosylation is intact in spf1 KO yeast strains, and only higher-order polysaccharide residues could not be processed in these mutants (76).
Since the reduction of total MR1 protein was not accompanied by a statistically significant difference on the mRNA level in THP-1 cells, we focused our efforts on posttranscriptional events. Pulse-chase experiments revealed that the difference in total MR1 protein levels was detectable within minutes of translation and affected MR1 molecules regardless of their folding state. This is consistent with a model where ATP13A1 function is important for the stabilization of nascent MR1 polypeptides immediately after translation and suggests a very early bifurcation into a cohort of MR1 molecules that successfully complete protein synthesis and initial folding and a cohort of molecules that is immediately degraded. Alternatively, the rate of MR1 protein synthesis may be affected in these cells. The observed trend that radioactive labeling of MR1 was less efficient in an ATP13A1 KO clone at early time points compared with THP-1 WT cells or an ATP13A1sufficient clone derived from the same parent supports this hypothesis. Intriguingly, protein translocation into the ER through Sec61 is impaired in an spf1 yeast mutant (114), providing a potential mechanistic basis for this model. However, such an effect on protein translocation would be expected to have a global effect, yet antigen presentation by classical MHC class I and CD1d was unaffected in our ATP13A1 KO cells. Pertinent to this, a recent siRNA screen to identify modulators of CD1d-mediated antigen presentation did not find ATP13A1 (91). A recent CRISPR screen for MR1 chaperones, however, also found sgRNAs targeting ATP13A1 to be enriched in cells with low MR1 surface expression (36), further supporting the role for this ATPase described here.
Although Spf1p has been shown to modulate ion concentrations in the ER, it has also been postulated that its substrates may be more diverse, potentially even including macromolecules such as aminophospholipids based on sequence similarities to P 4 -type ATPases (74,111). Since it is still unclear how soluble MR1 ligands access the ER where they encounter partially folded, ligand-receptive MR1 molecules (37,41) it is tempting to speculate that ATP13A1 may also transport vitamin metabolites, implying a role analogous to TAP, which transports antigenic peptides into the ER for loading onto MHC class I molecules (115). Pertinent to this, Sørensen and colleagues recently identified phosphatidylinositol 4-phosphate as a potent stimulator of Spf1p ATPase activity and hypothesized that the sterol flippase activity of P 5 A-ATPases may underlie the diverse phenotypes observed upon mutation of the transporters (114). Interestingly, two putative negative regulators of MR1 surface expression identified in our screen (VAC14; log2MI = 2.087; fcpv = 7.39 × 10 −6 ; and FIG4; log2MI = 1.879; fcpv = 1.05 × 10 −3 ) are components of a complex that regulates the interconversion of phosphatidylinositol-derived signaling lipids (52), further strengthening a potential link between MR1mediated antigen presentation and sterol homeostasis.
As mentioned above, recent work by McKenna et al. (73) further extended the list of P 5 A-ATPase substrates to short transmembrane helices of mislocalized proteins. In this study, the authors obtained the first structural information on Spf1p, which provides an explanation for the unusual substrate selectivity of P 5 A-ATPases: compared with other P-type transporters, Spf1p features an unusually large binding pocket with a lateral opening facing the lipid phase of the ER membrane. This topology is ideally suited for its interaction with short, helical transmembrane segments, which the ATPase removes from the lipid bilayer. In this way, tail-anchored mitochondrial proteins that are incorrectly inserted into the ER membrane can be removed and released into the cytosol for retargeting to the correct organelle. Using quantitative proteomics, the authors show that loss of ATP13A1 leads to decreased protein abundance of a variety of membraneanchored proteins (73), consistent with our findings on reduced MR1 protein levels in ATP13A1 KO cells. Unfortunately, the proteomics analysis does not include data on MR1 ATP13A1 modulates MR1-mediated antigen presentation (73). Of note, HLA-A protein levels were not affected (73), consistent with our results. Although misoriented N-terminal signal sequences can also be substrates for ATP13A1 (73) and MR1 features such a peptide at its N-terminus, ATP13A1 did not coimmunoprecipitate with MR1 in our hands (data not shown). Similarly, a recent coimmunoprecipitation screen provided no evidence that MR1 directly interacts with the transporter (36).
It remains a likely possibility that the defect in MR1 antigen presentation is an indirect rather than a direct consequence of loss of ATP13A1. Since inhibition of proteasomal degradation did not rescue the MR1 phenotype, it further remains to be determined whether MR1 molecules in the ATP13A1 KO cells are degraded by a different means or whether the rate of MR1 protein synthesis is reduced in these mutants. If protein synthesis was affected, further questions would arise as to the mechanism by which the ER transporter regulates translation and, most importantly, how this is specific to MR1.
In summary, we provide evidence that the P 5 -ATPase ATP13A1 is an important cellular factor impacting MR1 surface expression and antigen presentation through a role in MR1 biogenesis in the ER. Our findings suggest that the mechanism underlying this effect does not involve differential glycosylation or general ER stress but appears to act on nascent MR1 polypeptides upon or immediately after protein synthesis. Further investigations into the transcriptional and translational effects of ATP13A1 deficiency as well as the extent to which these effects can be generalized to in vivo models are needed to confirm our data and determine the molecular mechanism responsible for the MR1 phenotype.

Experimental procedures
Cell culture THP-1 and HEK293T cells were purchased from ATCC. HAP1 WT 1524 was obtained from the Nijman lab (Nuffield Department of Medicine, University of Oxford). MAIT cells, MelanA-reactive CD8+ T cells, and iNKT cells were sorted from human peripheral blood mononucleocytes (PBMCs) isolated from leukocyte cones obtained from the NHS Blood and Transplant Unit as described below. This work was covered by HTA license number 12433. Cells were kept at 37 C with 5% CO 2 . In general, THP-1 cells and their derivatives were cultured in RPMI-1640 (Sigma-Aldrich or Gibco) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich or Gibco), 2 mM Glutamine (Sigma-Aldrich), 1 mM Sodium Pyruvate (Gibco), 1× Non-essential amino acids (Gibco), 100 U/ml penicillin +100 μg/ml streptomycin (Sigma-Aldrich), 10 mM HEPES (Gibco), and 50 μM β-mercaptoethanol (Gibco). HEK293T cells were cultured in DMEM (Sigma-Aldrich) supplemented in the same way. Sorted human T cells were cultured in IMDM (Gibco) supplemented with 5% pooled human serum (NHS Blood and Transplant Unit), approximately 5000 U/ml IL-2 (as described (116)), and supplements as above. HAP1 cells and their derivatives were cultured in IMDM (Gibco) supplemented with 10% FCS and 100 U/ml penicillin +100 μg/ml streptomycin only. Cells were subcultured every 2 to 4 days depending on confluency. Adherent cells were detached using either PBS + 1 mM EDTA (VWR) or Trypsin-EDTA (Gibco). Cells were stored in 10% DMSO (Sigma-Aldrich) in FCS in liquid nitrogen storage tanks for long-term storage.

PBMC isolation and MAIT/iNKT cell sorting
PBMCs were isolated from leukocyte cones obtained from the NHS Blood and Transplant Unit. Samples with suitable MAIT cell numbers as determined by flow cytometry were enriched for CD2 + cells by MACS sorting (Miltenyi) according the manufacturer's protocol. A representative enriched sample was >95% positive for CD2 as determined by flow cytometry (eBioscience, clone RPA-2.10). Cells were stored at 4 C o/n before surface staining for CD3 (eBioscience, clone SK7; not always included), CD161 (eBioscience, clone HP-3G10), and the Vα7.2 TCR (BioLegend, clone 3C10) to enrich for Vα7.2 + CD161 high MAIT cells on a SONY MA900, a FACSAria Fusion, or an FACSAria III sorter (both BD Biosciences) in the WIMM Flow Cytometry Facility. Sort purity was routinely >90%. Invariant natural killer T (iNKT) cells were sorted from PBMC with the antibody clone 6B11 (BioLegend) and maintained as described (117). MelanA-reactive CD8+ T cells were expanded with peptide-pulsed dendritic cells and subsequently sorted using tetramers as previously described (118).

Generation of overexpressing cell lines
To generate cell lines overexpressing human MR1, the pHR-SIN-MR1-IRES-Emerald lentiviral expression vector previously made in our lab (98) was digested with XhoI and NotI endonucleases, and the Emerald coding sequence was replaced with a 21 base pair adapter designed to include a BamHI restriction site to enable screening by PCR. The undigested plasmid was used for the IRES-Emerald transductions. The plasmid encoding the HA-tagged MR1 has been described previously (5). Briefly, the MR1 coding region was amplified from the pHR-SIN-MR1-IRES-Emerald vector (98) with a forward primer having a BglII overhang and a reverse primer introducing an HA sequence and a NotI restriction site. This was then cloned into the pHR-SIN-IRES-Emerald vector using the BamHI and NotI restriction sites upstream and downstream of the IRES-Emerald cassette, respectively. This vector was derived from the pHR-SIN-CSGW plasmid (119,120). Of note, the MR1 coding sequence used carries two silent mutations: A138G and C939T. All oligonucleotide sequences can be found in Table S4. The lentiviral expression vector for human ATP13A1 was purchased from VectorBuilder as a bacterial stock (pLV[Exp]-Bsd-{sffv}>hATP13A1[NM_020410.2]). Lentiviral particles were produced in HEK293T cells using packaging vectors encoding gag/pol (pCMVR8.91) and vsv-g (pMDG). Polyclonal MR1-overexpressing cell lines were flow sorted based on MR1 surface expression. ATP13A1 overexpressing cells were selected with Blasticidin S (Sigma-Aldrich).

Flow cytometry
Cells were incubated with Acetyl-6-formylpterin (Schirks Laboratories) as specified in the figure legends, harvested, and transferred to round-bottom 96-well plates for staining with Aqua live/dead stain (Life Technology or Biolegend), followed by Fc receptor blocking (normal immunoglobulin, octopharma or Fc Receptor Binding Inhibitor Antibody, eBioscience), and surface staining on ice. If applicable, cells were split into two wells and primary antibody mix containing either the target antibody or the isotype control antibody was added. When applicable cells were fixed in 2% PFA (Electron Microscopy Sciences) or ICS fixation buffer (eBioscience) before acquisition. Buffer containing 2 mM EDTA was used throughout the procedure for HAP1 cell lines. On occasion, Propidium Iodide (Sigma-Aldrich) was added to samples just before acquisition instead of staining with Aqua live/dead stain. For ploidy staining, cells were fixed in 70% ethanol and stained with 10 μg/ml Propidium Iodide (PI) in the presence of 0.5 mg/ml RNase A before analysis on a flow cytometer in the 561 to 610/20 or the 561 to 620/15 channel on linear scale. Single-color controls were either cells or OneComp eBeads Compensation Beads (Life Technologies). Cells were analyzed on an Attune NxT analyzer equipped with four lasers (Life Technologies), a CyAn ADP analyzer (Beckman Coulter), or an LSR Fortessa X50 fitted with five lasers (BD Biosciences) in the WIMM Flow Cytometry Facility, and data were analyzed and displayed using FlowJo v9 or v10 (FlowJo). Y-axes of histograms show modal counts and x-axes are in logarithmic or biexponential scale except for FSC and SSC, which are on a linear scale. Antibodies used were clone 26.5 for MR1 (BioLegend), clone MOPC-173 as IgG2A control (BioLegend), and clones W6/32 (BioLegend) and G46-2.6 (BD Biosciences) for MHC class I. Fluorophores are indicated on the plot axes.
For analysis of HAP1 CRISPR lines generated for bulk validation, cells were dissociated with 0.05% Trypsin-EDTA (Thermo Fisher Scientific), counted using a TC20 automated cell counter (Bio-Rad), and cell number across samples was equalized. Cells were incubated with primary antibody dilution for 1 h at 4 C. After two washes, cells were incubated with Alexa488-conjugated secondary antibody for 45 min at 4 C. Cells were fixed in ICS Fixation Buffer for 30 min at RT, washed twice, and incubated in 1× Permeabilization Buffer (BD Bioscience) for 10 min at RT. Cell pellet was then washed and incubated with 0.8 mg/ml RNase A (Applichem) for 1.5 h before two additional washing steps and incubation with 10 μg/ml PI. According to the manufacturer instruction, RNAse A and PI incubation steps were performed in 1× Permeabilization Buffer.

Antigen presentation assays
For functional assays, allophycocyanins were incubated with the indicated stimuli for 5 h at 37 C. Technical duplicates were split either before or after pulsing with the antigen such that the final number of allophycocyanins was 50,000 per well. For stimulation with E.coli, DH5α bacteria were grown in Luria Broth overnight, washed in PBS, and diluted to an OD 600 of 0.5, corresponding to approximately 4 × 10 8 /ml as calculated based on the online Agilent Biocalculator (https://www. chem.agilent.com/store/biocalculators/calcODBacterial.jsp). Stimulation with live bacteria was carried out in medium without antibiotics, and gentamycin (Lonza) was added to a final concentration of 50 μg/ml after 3 h. Peptide (ELAGI-GILTV, purchased from Sigma-Aldrich) pulsing was performed in serum-free medium. α-Galactosylceramide (αGalCer) was purchased from Enzo Life Sciences. 5-A-RU was synthesized as described previously (121) and combined with methyl-glyoxal (Sigma-Aldrich) immediately before addition to the culture. Stimuli were washed off before coincubation with either 15,000 or 20,000 T cells per well for at least 36 h. Interferon γ (IFNγ) in the culture supernatants was measured by ELISA using commercially available antibody pairs (BD Biosciences, clones NIB42 for the capture antibody and 4S.B3 for the biotinylated detection antibody). Antigen binding was visualized with avidin-peroxidase (Sigma-Aldrich) and developing solution containing phenylenediamine (Sigma-Aldrich). The reaction was stopped with 2 M H 2 SO 4 (Fluka) when the standard curve had fully developed. Color intensity was measured at 490 nm in an iMark plate reader (Bio-Rad) and quantified using a four-parameter standard curve. Supernatants were diluted as appropriate and added in a final volume of 25 μl/well. Standard curves of recombinant IFNγ (PeproTech) were in 50 μl/well.

Western blots
HAP1 cells were harvested for Western blots with Trypsin/ EDTA or PBS + 1 mM EDTA to preserve the surface proteome. Sometimes cells were lysed by adding lysis buffer directly to the dish. To make protein lysates, cells were washed in PBS, pelleted, and lysed at 4 C in either RIPA buffer (Sigma-Aldrich) or self-made lysis buffers (either 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP40 (IGEPAL CA-630) or 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 1 mM EGTA, 2.5 mM sodium pyrophosphate). All lysis buffers were supplemented with protease inhibitors (Roche). Cell lysates were cleared by centrifugation, and protein-containing supernatants were collected into fresh tubes and quantified with the Pierce BCA protein assay kit (Thermo Scientific) using a twofold dilution curve of BSA (Thermo Scientific) as a standard. HAP1 WT or D9 parent control lysates were not always prepared on the same day as the clone lysates and at times reused in different experiments as indicated in the figure legends. Samples were prepared in 1× Loading Buffer and 1× Reducing Agent (both Invitrogen) to contain equal amounts of protein, boiled, and loaded onto a gel according to their size. Generally, proteins with a molecular weight (MW) below 100 kDa were separated on 4 to 12% Bis/ Tris gels (Life Technologies) in MES SDS Buffer (Invitrogen), and proteins with a molecular weight above 100 kDa were separated on 3 to 8% Tris/Acetate gels (Life Technologies) in MOPS Buffer (Invitrogen). Appropriate MW markers were included. Gels were transferred onto a PVDF or nitrocellulose ATP13A1 modulates MR1-mediated antigen presentation membrane (both Bio-Rad) using the TransBlot Turbo Transfer system (Bio-Rad), and blocked in PBS + 0.1% Tween-20 + 5% bovine serum albumin (BSA) (Sigma-Aldrich or Fisher Scientific). If needed, membranes were cut to size for incubation with different primary antibodies. Membranes were washed in PBS + 0.1 or 0.5% Tween-20 (Acros Organics), incubated with LI-COR secondary antibodies with agitation, washed, and dried between tissues before imaging with the Odyssey Near-Infrared imaging system. For Endoglycosidase H (EndoH, NEB) digestions, equal amounts of protein from RIPA cell lysates were denatured in supplied Denaturation Buffer before splitting each sample in two. One half was incubated with the enzyme in the appropriate Glycobuffer and the other half with Glycobuffer only. Loading buffer and reducing agent were added to each reaction, and samples were analyzed by SDS-PAGE as above. Primary antibodies were clone 6C5 (mouse, Santa Cruz) or D16H11 (rabbit, Cell Signaling Technology) for GAPDH, C4 (Santa Cruz) for ACTB, polyclonal (Cat. No. 13260-1-AP; rabbit, Proteintech) for MR1, polyclonal (Cat. No. 16244-1-AP; rabbit, Proteintech) for ATP13A1, clone HC10 (mouse, purified from hybridoma supernatant by HIU lab management) for MHC class I, clone EPR9649 (rabbit, Abcam) for HERPUD1, clone D4B8 (rabbit, Cell Signaling Technology) for ATF4, and clone EP2978Y (rabbit, Abcam) for β 2 m. Suitable IRDye 680RD and 800CW secondary antibodies were purchased from LI-COR. For quantification of fluorescencebased Western blots, rectangles were drawn around the bands, and signal intensity was determined as median with the border set to right/left and width 3 in the LI-COR Image Studio software. Although we have no definite proof that the data are in the linear range, we assume that this is the case, based on the images.

qRT-PCR
RNA was extracted using either the QIAGEN RNeasy kit or the Ambion RNAqueous kit according to the manufacturer's instructions including the respective DNase digestion protocol. RNA was quantified by Nanodrop (Thermo Scientific), and equal amounts of RNA were reverse transcribed using Ambion or Takara Bio kits. Only cDNA transcribed with the same kit was directly compared in qRT-PCR reactions. HAP1 WT or D9 parent control RNA was not always extracted on the same day as the clone RNA. Pooled "no RT" and "no RNA" controls were routinely included and occasionally gave a signal but always at a much higher C T value than the samples. qRT-PCRs were run as technical duplicates or triplicates on a Quant-Studio7 qRT-PCR machine (Life Technologies), and expression was normalized to a housekeeping gene (HKG). Taqman probes used can be found in Table S4.
ER stress assay HAP1.MR1-derived clones were treated with 0.1 μM thapsigargin (Sigma-Aldrich) or solvent control DMSO for 3 h. Protein lysates were obtained and Western blots performed as above. In the same experiments, RNA was extracted and reverse transcribed as above. XBP1 cDNA was PCR amplified with previously published primers (122) (Table S4) and analyzed for the XBP1 splice variant on a 2.5% agarose gel.

Metabolic labeling and pulse-chase experiments
For metabolic labeling, 1.5 × 10 6 THP-1 cells were starved in the absence of methionine and cysteine for 1 h. Following starvation, cells were pulse labeled with 50 μCi of 35 S-methionine and -cysteine for 1 min (EasyTag EXPRESS35S Protein Labeling Mix, PerkinElmer) and immediately transferred into 10× volume of chase medium containing excess amounts of unlabeled cysteine (500 μg/ml), methionine (100 μg/ml), and 100 μM cycloheximide to prevent incorporation of radioactive amino acids. Cells were harvested at 0, 2, and 4 min and lysed in native lysis buffer (50 mM Tris⋅Cl, pH 7.5150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100) for 1 h. Hemagglutinintagged MR1 was immunoprecipitated using monoclonal anti-HA antiserum (clone 12CA5). HLA class I proteins were immunoprecipitated from the supernatant of the first immunoprecipitation with anti-HA, using W6/32 antibody. Quantification was performed in ImageJ by drawing rectangles around the bands, multiplying the mean gray value by the area, and subtracting the value of an equivalent area in the same lane to account for differences in loading across lanes.

Gene trap mutagenesis and sort
Gene trap mutagenesis was carried out as described before (44) with small changes. Briefly, HEK293T cells were cotransfected with a gene trap vector encoding for a strong adenoviral splice acceptor as well as blue fluorescent protein (pGT-en2-BFP344) and retroviral packaging plasmids Gag-pol, VSVg, and pAdv (43). Virus-containing supernatant was harvested and concentrated by ultracentrifugation twice a day for three consecutive days. Concentrates from each day were stored at 4 C o/n and then pooled for transfection. 2 × 10 7 cells of HAP1.MR1 clone D9 were seeded in a T175 flask on Day 0 and transduced with concentrated gene-trap retrovirus in the presence of 8 μg/ml of protamine sulfate (MP Biomedicals) for three consecutive rounds of transduction on Days 1 to 3 before expansion and freezing. Expanded transduced HAP1.MR1 D9 cells were thawed and approximately 3 × 10 9 cells were pulsed with 5 μg/ml Ac6FP for 5 h before cell surface staining with anti-MR1 primary antibody (clone 26.5) and goat anti-mouse secondary antibody coupled to Alexa488 (Life Technologies). Stained cells were fixed, permeabilized, and stained with Propidium Iodide before sorting on a LE-SH800 sorter (Sony) fitted with a 100 μm chip over 4 days gating on haploid cells based on PI staining. MR1 hi and MR1 low populations comprising approximately 1% of total, respectively, were sorted (Fig. S2).

Recovery of insertion sites and bioinformatics analysis
Gene trap insertion sites were recovered using linear amplification-mediated polymerase chain reaction (LAM-PCR) similarly to the protocol described by Blomen et al. (123) and Brockmann et al. (44). Briefly, sorted MR1 hi and MR1 low cells were decross-linked, and DNA was extracted from the two populations separately. Long terminal repeat (LTR)-proximal gene regions were amplified from 4 × 1 μg genomic DNA in four separate reactions per MR1 population using a double-biotinylated primer. Biotinylated singlestranded DNA (ssDNA) was isolated, and Illumina sequencing adapters were added. Libraries were PCR-purified and sequenced on a HiSeq2500 machine (Illumina) generating 50 base pair (bp) single end reads with the 5 0 sequencing primer. All primer sequences can be found in Table S4. Recovered sequences were analyzed with the pipeline described by Brockmann et al. (44). In short, reads were mapped onto the human reference genome hg19 by running the aligner Bowtie (124) twice, allowing for 0 and 1 bp mismatches to avoid omitting reads that do not map uniquely when tolerating 1 bp mismatch. Reads were mapped using hg19 protein-coding gene coordinates (Refseq) and inter-sectBED (125). A customized BED file was used for both gene mapping and determining the orientation of the integration relative to the gene (sense or antisense). Only sense insertions in nonoverlapping gene regions outside the 3 0 untranslated region (UTR) were considered to have the potential to disrupt gene function and taken into account for further analysis. The number of unique disruptive insertions for a gene in the MR1 hi or MR1 low population was normalized to the total number of insertions in the respective populations and compared with the number of normalized insertions for that gene in the opposite population using a two-sided Fisher's exact test. Significance of enrichment in either of the two tails was determined by applying the Benjamini-Hochberg false discovery rate correction to the calculated p values. In addition, enrichment in either tail was expressed as the Mutation Index (MI), calculated for each gene as shown below. If a gene was sequenced in only one of the populations, one insertion was added in order to enable calculation of the mutational index.
For in-depth validation of ATP13A1, three one-component CRISPR/Cas9 plasmids with a different single guide RNA (sgRNA) target sequence within ATP13A1 each were designed based on the Sabatini/Lander CRISPR pooled library (128) (Table S4 and Fig. S4). sgRNA sequences were tested for specificity using the UCSC-genome browser BLAT tool (https://genome.ucsc.edu/cgi-bin/hgBlat) and cloned into the pX458_Ruby backbone (Addgene #110164). HAP1.MR1 parent clones or a THP-1 WT clone obtained by limiting dilution was transfected with CRISPR plasmids and sorted based on mRuby expression after 2 to 3 days. Single cells were sorted into 96-well plates, and clones were picked for expansion approximately 2 weeks after sorting. For sequencing of CRISPR clones, DNA was extracted using the QIAGEN Blood and Tissue Kit in combination with Ambion Proteinase K. For near-haploid HAP1 clones, genomic DNA around the sgRNA target site was amplified, amplicons were sent for Sanger sequencing at the HIU Sequencing Facility, and sequences were analyzed with SnapGene Viewer (GSL Biotech) and SerialCloner (SerialBasics). For polyploid THP-1 ATP13A1 KO clones, regions around the sgRNA target sited were amplified from genomic DNA using primers that generated overhangs compatible with Illumina adapters (Table S4). PCR products were gel-purified (QIAGEN) and bar-coded using the Illumina index adapter series D5 and D7. Barcoded amplicons were analyzed on an Agilent 2200 TapeStation System, pooled in equimolar ratios, cleaned up with Agincourt Ampure XP beads (Beckman Coulter), and quantified on a Qubit3 Fluorometer. The library was sequenced paired-end (read distribution: 151 bp each side) on an Illumina Miseq machine using the Miseq Reagent Nano Kit V2 300-cycle (Illumina). Sequences were analyzed using the CRISPResso (130) web implementation.

Data analysis and plotting
Data were analyzed in Excel for Mac 2011 (Microsoft) and plotted using Prism (GraphPad Software) Version 7 and 8. Statistical analysis was performed using Prism (GraphPad Software) Version 7 and 8. Figures were assembled in Inkscape (inkscape.org).

Data availability
Data of repeat experiments are included in the Supporting Information whenever practical. Data not included are available upon request (mariolina.salio@imm.ox.ac.uk; kulicke@ ohsu.edu). Acknowledgments-We gratefully acknowledge Kevin Clark, Sally-Ann Clark, Paul Sopp, and Craig Waugh in the flow cytometry facility at the MRC WIMM for providing cell sorting services, members of the Brummelkamp laboratory (Netherlands Cancer Institute, Amsterdam, The Netherlands) for bioinformatics analysis of the gene trap screen, the MRC WIMM Genome Engineering Services for the design and cloning of the CRISPR constructs, Tim Rostron and John Frankland in the Sequencing facility at the MRC WIMM for providing sequencing services, and David Lewinsohn for critical reading of the manuscript. We dedicate this study to the memory of the late Professor V. C., FRS, an inspirational colleague, mentor, and pioneer in the field of iNKT/CD1/MAIT cell biology, who passed away in January 2020.  Conflict of interest-M. S. holds consultancies with Nucleome Therapeutics and Enarabio. S. M. N. is a founder, shareholder, board member, and employee of Scenic Biotech BV, a company that employs haploid genetics for target discovery and development. All other authors declare that they have no conflicts of interest with the contents of this article.