Author response: TAPBPR mediates peptide dissociation from MHC class I using a leucine lever
2018-11-07

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Tapasin and TAPBPR are known to perform peptide editing on major histocompatibility complex class I (MHC I) molecules; however, the precise molecular mechanism(s) involved in this process remain largely enigmatic. Here, using immunopeptidomics in combination with novel cell-based assays that assess TAPBPR-mediated peptide exchange, we reveal a critical role for the K22-D35 loop of TAPBPR in mediating peptide exchange on MHC I. We identify a specific leucine within this loop that enables TAPBPR to facilitate peptide dissociation from MHC I. Moreover, we delineate the molecular features of the MHC I F pocket required for TAPBPR to promote peptide dissociation in a loop-dependent manner. These data reveal that chaperone-mediated peptide editing on MHC I can occur by different mechanisms dependent on the C-terminal residue that the MHC I accommodates in its F pocket and provide novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity. https://doi.org/10.7554/eLife.40126.001 Introduction Major histocompatibility complex class I (MHC I) molecules play a critical role in immmunosurveillance, particularly in the context of viral infections and cancer, by presenting antigenic peptides to CD8 +T cells. Prior to their cell surface export, MHC I molecules undergo peptide editing, a process that involves the exchange of low-affinity peptides for those of higher affinity. In addition to ensuring that only stable peptide:MHC I complexes are released to the plasma membrane, peptide editing ultimately controls the peptide repertoire that is displayed for immune detection. For over two decades, tapasin was the only known peptide editor for MHC I, facilitating peptide selection within the confines of the peptide loading complex (PLC) (Williams et al., 2002; Howarth et al., 2004; Chen and Bouvier, 2007; Wearsch and Cresswell, 2007). However, it is now well-recognised that the tapasin-related protein TAPBPR is a second independent peptide editor that performs peptide exchange outside the PLC (Boyle et al., 2013; Hermann et al., 2013; Hermann et al., 2015; Morozov et al., 2016). Furthermore, TAPBPR can work in cooperation with UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) to reglucosylate MHC I, causing recycling of MHC molecules to the PLC (Neerincx et al., 2017). Although TAPBPR usually functions as an intracellular peptide editor, we have recently made the fascinating discovery that when given access to surface expressed MHC I molecules, TAPBPR retains its function as a peptide exchange catalyst and can be utilised to display immunogenic peptides of choice directly onto the surface of cells (Ilca et al., 2018). We have therefore identified that manipulation of TAPBPR function may be utilised as a potential immunotherapeutic that facilitates the presentation of both neoantigens and viral-derived peptides, thereby overriding the endogenous cellular antigen processing pathway. Moreover, we have also developed two novel functional assays that enable detailed interrogation of TAPBPR-mediated peptide exchange on MHC I (Ilca et al., 2018). Precisely how tapasin and TAPBPR function at the molecular level remains largely enigmatic. The recently reported crystal structures of human TAPBPR in complex with mouse MHC I captured the endpoint of peptide editing, thereby suggesting that TAPBPR facilitates peptide exchange by widening the peptide binding groove of MHC I at the α2–1 region (Jiang et al., 2017; Thomas and Tampé, 2017). However, there remains an incomplete understanding of the step-by-step processes by which the two peptide editors, TAPBPR and tapasin, recognise peptide-loaded MHC I molecules and actively facilitate peptide dissociation to result in the conformations observed in the crystal structures. Indeed, McShan et al. have recently used NMR in an attempt to further delineate the dynamic process of peptide exchange on MHC I by TAPBPR (McShan et al., 2018). Intriguingly, the structure reported by Thomas and Tampe identified a loop of TAPBPR that was proposed to interact with the peptide binding groove of MHC I, where the C-terminus of bound peptide usually resides (Thomas and Tampé, 2017). While this study demonstrated the localisation of the loop, to date, there is no experimental evidence to support the notion that this loop mediates peptide exchange on MHC I. In contrast, Jiang et al. failed to capture the loop in proximity to the peptide-binding groove (Jiang et al., 2017), further questioning the relevance and importance of this loop in TAPBPR-mediated peptide exchange. Given the discordance between the data reported for the captured structures and the lack of functional evidence to support any role for this loop, it is vital to reconcile these discrepancies to understand whether the TAPBPR loop is involved in peptide exchange. Here, we investigate the functional importance of the K22-D35 loop using two newly developed assays in combination with immunopeptidomic analysis. Our data demonstrates that this loop is critical for peptide dissociation from MHC I. Furthermore, we highlight key molecular features governing TAPBPR:MHC I interaction and provide insight into the mechanism(s) of peptide selection on MHC I molecules. Results The TAPBPR K22-D35 loop lies at the interface with the MHC I peptide binding groove Prior to the recent determination of the TAPBPR-MHC I crystal structures (Jiang et al., 2017; Thomas and Tampé, 2017), we docked our model of TAPBPR onto a previously determined structure of HLA-A2, using our mutagenesis data that identified critical regions in the TAPBPR-MHC I interface (Hermann et al., 2013). Our docking identified a region of TAPBPR that lies close to the peptide binding groove of MHC I, in the proximity of the F pocket (Figure 1a, dotted circle). This region contained a loop that differs between tapasin and TAPBPR. In tapasin, this loop appears to be rather short and is not sufficiently well ordered in the crystal structure to be visible (Dong et al., 2009), while in TAPBPR this loop is significantly longer (Figure 1b). The two crystal structures of the TAPBPR-MHC I complex support our prediction regarding the arrangement of TAPBPR relative to MHC I, including this loop region being very near the F pocket (Figure 1c). However, the position and orientation of the loop in the structures is poorly defined. In the structure from Jiang et al. (2017), this loop is not sufficiently well ordered to be modelled. In the structure from Thomas and Tampé (2017), the loop has been modelled; however, the electron density into which it was built is not well defined: several side chains and even several of the backbone atoms do not fit the electron density well (Figure 1d). It is likely that alternative orientations of the loop would also satisfy the crystallographic data presented. Therefore, it is critically important to verify whether there is any functional role of this loop in peptide editing. Figure 1 Download asset Open asset TAPBPR loop interactions with MHC I. (a) Model of TAPBPR (pink) docked onto MHC I (blue) and β2m (cyan) based on interaction studies (Hermann et al., 2013). (b) Top panel, illustration of the proximity of the TAPBPR loop region to the peptide binding groove (viewed from the top of complex shown in panel a). Lower panel, schematic diagrams of the amino acid composition of the TAPBPR and tapasin loops compared to the length and orientation of a peptide. (c) Overlay of two recent X-ray structures of TAPBPR in complex with MHC I (Jiang et al., 2017; Thomas and Tampé, 2017) (PDB ID 5WER and 5OPI) oriented and coloured to illustrate the similarity to our TAPBPR:MHC I complex (panel a). The position of the TAPBPR loop is circled (black dashed line). (d) The electron density map (2Fo-Fc, green mesh) and the built model (maroon sticks, residues D23-E34) are shown for the loop region of TAPBPR (PDB ID 5OPI). Two views of the loop and density are shown rotated by 90 degrees. (e) Expression of TAPBPR loop variants in IFNγ treated HeLaM-TAPBPRKO and their interaction with MHC I and UGT1. Western blotting for calnexin is included as a loading control. Representative of three independent experiments. https://doi.org/10.7554/eLife.40126.002 TAPBPR loop mutants are stably expressed and bind MHC I and UGT1 To test whether the K22-D35 loop of TAPBPR was a functionally important region for mediating peptide selection, we first replaced all the residues in the loop with either glycine, alanine or serine, to produce a TAPBPR variant with a potentially functionless loop (TAPBPRØloop) (Table 1). More subtle mutations of this loop were designed based on the work by Springer and colleagues, who demonstrated that dipeptides carrying long hydrophobic residues are able to bind to the peptide binding groove of recombinant MHC I and enhance peptide dissociation (Saini et al., 2015). Thus, we explored whether a leucine residue at position 30 of the mature TAPBPR protein, the only long hydrophobic residue within the entire loop, was involved in peptide exchange on MHC I. We created two TAPBPR variants in order to test this. First, we replaced the leucine with glycine in the TAPBPRWT molecule (TAPBPRL30G). Second, we reintroduced leucine 30 into TAPBPR with the dysfunctional loop (TAPBPRØG30L) (Table 1). Upon transduction into TAPBPR-deficient HeLaM cells (HeLaM-TAPBPRKO), steady state expression of all the TAPBPR loop mutants was similar to TAPBPRWT and all variants interacted equally well with both MHC I and UGT1 (Figure 1e), suggesting the overall protein stability and structure of TAPBPR was not significantly affected by the changes to the loop. Table 1 Panel of TAPBPR loop mutants. Residues altered are highlighted in red. https://doi.org/10.7554/eLife.40126.003 TAPBPR variantLoop sequenceWTKDGAHRGALASSEDØloopAAGGSGGGGSGGAAL30GKDGAHRGAGASSEDØG30LAAGGSGGGLGGGAA The K22-D35 loop is essential for mediating peptide exchange on HLA-A*68:02 Recently, we have developed two novel assays which can be used to measure TAPBPR-mediated peptide exchange on MHC I molecules. The first assay established takes advantage of the small proportion of TAPBPR that escapes to the cell surface upon its over-expression in cell lines (Ilca et al., 2018). We have demonstrated that plasma membrane expressed wild-type TAPBPR efficiently mediates peptide exchange on cell surface HLA-A*68:02 molecules found on HeLaM cells (Ilca et al., 2018). We have also previously shown that TAPBPR-mediated peptide exchange in this assay occurs directly on the cell surface, given that it works on cells incubated at 4°C, which inhibits membrane trafficking (Ilca et al., 2018). To initially explore the functional importance of the K22-D35 loop, we tested whether plasma membrane expressed TAPBPRØloop was capable of mediating peptide exchange on HLA-A*68:02 to a similar extent as TAPBPRWT. When exploring the ability of TAPBPR to promote peptide association, we found plasma membrane expressed TAPBPRØloop enhanced the binding of an exogenous fluorescent peptide specific for HLA-A*68:02 (YVVPFVAK*V) onto cells to a similar extent as TAPBPRWT (Figure 2a). This may be due, in part, to TAPBPR enhancing the trafficking of associated, peptide-receptive MHC I with it through the secretory pathway. However, when we tested the ability of TAPBPR to mediate peptide dissociation (i.e. removal of the bound fluorescent peptide in the presence of an unlabelled competitor peptide) (Figure 2b), in contrast to the efficient exchange observed with TAPBPRWT, very little, if any, dissociation of fluorescent peptide was observed with TAPBPRØloop (Figure 2c–2e). This suggests that the K22-D35 loop of TAPBPR is essential for mediating efficient peptide dissociation from HLA-A*68:02. Figure 2 with 1 supplement see all Download asset Open asset The TAPBPR K22-D35 loop is critical for peptide exchange. (a) Typical peptide binding when cells gated for expressing high levels of surface TAPBPR were incubated with 10 nM YVVPFVAK*V peptide for 15 min at 37°C on IFNγ treated HeLaM-TAPBPRKO cells -/+expression of TAPBPRWT, TAPBPRØloop, TAPBPRL30G or TAPBPRØG30L. (b) Schematic representation of the experimental workflow used to compare the efficiency of peptide exchange by plasma membrane bound TAPBPRWT with the plasma membrane bound TAPBPR loop mutants. (c) Histograms show the level of dissociation of YVVPFVAK*V (YVV*) in the absence (blue line) and presence of 100 nM non-labelled competitor peptide YVVPFVAKV (YVV)(orange line) or EGVSKQSNG (ETVΔ2/9), a peptide in which the anchors which permit HLA-A*68:02 binding are mutated to produce a non-binding derivative (black line). Similar patterns of dissociation were found on cells incubated at 4°C demonstrating that the peptide exchange occurs directly on the cell surface (see Figure 2—figure supplement 1). (d-e) Graphs show the percentage of fluorescent peptide YVVPFVAK*V (YVV*) remaining in the presence of (d) 100 nM or (e) increasing concentrations of the non-labelled competitor peptide YVVPFVAKV as a percentage of the bound YVVPFVAK*V observed in the absence of competitor peptide from four independent experiments. Error bars show -/+SD.****p≤0.0001, ***p≤0.001 using unpaired two-tailed t-tests. https://doi.org/10.7554/eLife.40126.004 L30 is a critical residue of TAPBPR for peptide exchange on HLA-A*68:02 Next, we tested the ability of both plasma membrane bound TAPBPRL30G and TAPBPRØG30L to promote peptide exchange in our assay. As observed above with TAPBPRØloop, plasma membrane expressed TAPBPRL30G and TAPBPRØG30L were able to promote the association of exogenous fluorescent peptide onto cells (Figure 2a). However, when we explored the ability of TAPBPRL30G to mediate fluorescent peptide dissociation from HLA-A*68:02, we found that it was incapable of mediating efficient peptide dissociation in the presence of unlabelled competitor (Figure 2c–2e). Strikingly, alteration of this single residue had the same effect on the function of TAPBPR as mutating the entire loop. The crucial role of L30 in mediating peptide dissociation was further supported with our observation that TAPBPRØG30L, in which the leucine residue alone is restored into the dysfunctional TAPBPRØloop molecule, transformed it into a functioning peptide exchange catalyst on HLA-A*68:02, albeit at reduced capability compared to TAPBPRWT (Figure 2c–2e). While TAPBPRWT efficiently promoted rapid peptide exchange at very low concentrations of competitor peptide, both TAPBPRØloop and TAPBPRL30G were extremely inefficient at mediating peptide exchange on HLA-A*68:02 molecules, even in the presence of high concentrations of competitor peptide (Figure 2e). These results suggest that L30 is a critical residue within the loop of TAPBPR for mediating peptide dissociation from HLA-A*68:02. Soluble TAPBPR lacking L30 cannot facilitate peptide dissociation from HLA-A*68:02 We have also recently shown that soluble TAPBPR, consisting only of the lumenal domains of the wild-type molecule (i.e. lacking its transmembrane region and cytoplasmic tail) can also efficiently promote peptide exchange on three MHC I molecules: HLA-A*68:02, HLA-A2 and H-2Kb (Ilca et al., 2018). Therefore, we can also use soluble TAPBPR as a second means to assay the ability of TAPBPR mutants to mediate efficient peptide exchange on MHC I. Using this approach, the majority of MHC I molecules that TAPBPR will have access to will be folded with bound peptides of relativity high affinity expressed on the surface of cells. The lumenal domains of the TAPBPR variants with C-terminal His-tags were purified using Ni-affinity chromatography from the culture supernatants of transfected 293T cells (Figure 3a). Differential scanning fluorimetry revealed all TAPBPR loop variants had a similar melting temperature as TAPBPRWT (Figure 3b), indicating the alterations made to the loop had not significantly affected protein folding and stability. Comparison of the ability of the soluble TAPBPR variants to mediate peptide exchange on HLA-A*68:02 molecules on HeLaM cells revealed that TAPBPRWT was most efficient, followed by TAPBPRØG30L, which exhibited ~33% peptide exchange activity relative to TAPBPRWT (in the presence of 10 nM ETVSK*QSNV) (Figure 3c and d). However, both TAPBPRL30G and TAPBPRØloop were unable to efficiently mediating peptide exchange, displaying only ~3% of the exchange activity of TAPBPRWT (in the presence of 10 nM ETVSK*QSNV) (Figure 3c and d). As previously shown (Ilca et al., 2018), soluble TAPBPRTN5, a mutant which cannot bind to MHC I, did not mediate any peptide exchange (Figure 3c and d). This hierarchy (WT>ØG30L>L30G>TN5) was maintained over a wide range of exogenous TAPBPR concentrations (Figure 3e). Furthermore, the same hierarchical order of peptide exchange efficiency for the variants was observed using another HLA-A*68:02-binding peptide, YVVPFVAK*V (Figure 3c & d). Figure 3 Download asset Open asset Soluble TAPBPR loop variants exhibit reduced ability to mediate peptide exchange on surface HLA-A*68:02 molecules. (a) Expression and purity of soluble forms of WT, L30G, ØG30L, and Øloop TAPBPR variants after their purification from the culture supernatant using Ni-affinity. (b) Differential scanning fluorimetry demonstrates the three TAPBPR loop mutants have equivalent thermal denaturation profiles as TAPBPRWT. (c) Histograms of the typical fluorescent peptide binding to IFNγ-treated HeLaM cells incubated -/+100 nM exogenous soluble TAPBPR variant for 15 min at 37°C, followed by incubation with 10 nM ETVSK*QSNV or YVVPFVAK*V for an additional 15 min. TAPBPRTN5, in which isoleucine at position 261 is mutated to lysine, to produce a TAPBPR variant which cannot bind to MHC I, is included as a control (d) Bar graphs show the reproducibility of results in (c). (e) Dose response curves of fluorescent peptide binding to IFN-γ treated HeLaM cells incubated with increasing concentrations of the soluble TAPBPR variants prior to the addition of 10 nM ETVSK*QSNV. Error bars represent MFI -/+SD from four independent experiments. ****p≤0.0001 using unpaired two-tailed t-tests. https://doi.org/10.7554/eLife.40126.006 The K22-D35 loop is essential for soluble TAPBPR to bind peptide-loaded MHC I When we determined the ability of the soluble TAPBPR variants to bind to HLA-A*68:02, TAPBPRWT was the most efficient binder, followed by TAPBPRØG30L (Figure 4a). However, TAPBPRL30G and TAPBPRØloop were unable to make productive interactions with surface expressed HLA-A*68:02 (Figure 4a). As expected, TAPBPRTN5, which has a disrupted binding site for MHC I, was unable to bind to cells (Hermann et al., 2013) (Figure 4a). As this inability of soluble TAPBPRØloop and TAPBPRL30G to interact with MHC I contradicted our finding with their membrane-bound counterparts (Figure 1e), we determined whether these soluble TAPBPR variants could bind to the total cellular MHC I from cell lysates in pull-down experiments. All soluble TAPBPR variants (with the exception of TAPBPRTN5) were capable of binding significant amounts of MHC I, although less MHC I was detected bound to TAPBPRL30G and TAPBPRØloop (Figure 4b). A major difference between surface MHC I on intact cells (Figure 4a) and total cellular MHC I in detergent (Figure 4b) is the availability of peptide-receptive molecules. Therefore, we speculated that TAPBPR molecules with alterations to the loop are still able to bind to peptide-receptive MHC I but are unable to physically make MHC I peptide-deficient due to their inability to dissociate peptide. Consistent with this, incubation of cells at 26°C, which increases the expression of peptide-receptive MHC I on the plasma membrane (Ljunggren et al., 1990; Schumacher et al., 1990; Garstka et al., 2015), resulted in the TAPBPR loop variants, but not TAPBPRWT, exhibiting increased binding to cells (Compare Figure 4c with Figure 4a summarised in Figure 4d). While no significant change was observed in the ability of exogenous TAPBPRWT to bind to cells incubated at the lower temperature, a ~ 7 fold increase in binding of TAPBPRL30G and TAPBPRØloop was observed at 26°C compared to 37°C, and the binding of TAPBPRØG30L increased by ~2.5 fold (Figure 4d). When given access to surface expressed peptide-receptive MHC I upon incubation at 26°C, all soluble TAPBPR variants exhibited a corresponding increase in the ability to mediate peptide exchange (compare Figure 3c (37°C) with Figure 4e (26°C), summarised in Figure 4f). These findings suggest that TAPBPR variants lacking L30 are indeed able to bind to peptide-receptive MHC I but are unable to physically make MHC I peptide-deficient due to their inability to efficiently facilitate peptide dissociation. Consistent with this, when cells cultured at 26°C were incubated with an HLA-A*68:02-binding peptide prior to testing soluble TAPBPR binding, the peptide significantly reduced the ability of TAPBPRØloop to bind to cells, while the binding of TAPBPRWT was unaffected (Figure 4—figure supplement 1). Figure 4 with 1 supplement see all Download asset Open asset Residues K22-D35 are essential for soluble TAPBPR to bind peptide-loaded MHC I. (a and c) Histograms of soluble TAPBPR loop variant binding to HeLaM-HLA-ABCKO cells expressing HLA-A*68:02 incubated with 100 nM TAPBPR at (a) 37°C or (c) 26°C for 30 min. TAPBPRTN5, a TAPBPR variant which cannot bind to MHC I, is included as a negative control. (b) TAPBPR pull-downs on IFNγ-treated HeLaM-TAPBPRKO cells incubated with soluble TAPBPR loop mutants reveal all variants are capable of binding to MHC I, but do not bind to UGT1. TAPBPRTN5 is included as a non-MHC binding control. Data is representative of three independent experiments. (d) Bar graph comparing soluble TAPBPR variant binding to HeLaM-HLA-ABCKO+A*68:02 cells at 37°C with 26°C from three independent experiments. Error bars represent -/+SD. (e) Histograms show typical fluorescent peptide binding to IFNγ induced HeLaM cells treated -/+100 nM soluble TAPBPR variants for 15 min at 26°C, followed by incubation with 10 nM ETVSK*QSNV for 15 min at 26°C. (f) Bar graph compares ETVSK*QSNV peptide binding to HeLaM cells treated -/+ soluble TAPBPR variants at 37°C with 26°C from three independent experiments. Error bars represent -/+SD. n/s = not significant, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, using unpaired two-tailed t-tests. https://doi.org/10.7554/eLife.40126.007 Mutation of the TAPBPR K22-D35 loop alters the peptide repertoire presented on MHC I Having shown a role for the K22-D35 loop of TAPBPR in mediating efficient peptide dissociation from MHC I, we next determined whether the peptide repertoire presented by MHC I molecules was altered in cells upon mutation of the loop. When we compared the immunopeptidomes of IFNγ-treated HeLaM-TAPBPRKO cells expressing TAPBPRWT with cells expressing TAPBPRØloop, we found significant changes in the peptides presented on MHC I (Figure 5a). 461 peptides were found exclusively in the TAPBPRWT-expressing cells and 550 peptides were found exclusively in the cells expressing the Øloop variant (Figure 5a). Label-free quantitation by mass spectrometry revealed that there were also significant changes in the abundance of some peptides between TAPBPRWT and TAPBPRØloop, with 193 peptides exhibiting increased abundance in cells expressing TAPBPRWT (Red circles, Figure 5b) and 222 peptides displaying increased abundance in TAPBPRØloop-expressing cells (Blue circles, Figure 5b). These findings demonstrate that there are significant changes in the peptide repertoire presented on MHC I upon mutation of the TAPBPR loop. While there were still large differences in the peptide repertoires between TAPBPRWT and TAPBPRØG30L-expressing cells, based on a presence/absence approach (Figure 5a), restoration of the leucine residue into the loop appeared to somewhat reduce some of the changes observed in peptide abundance (Figure 5b). Assignment of the identified peptides to the MHC I allotype found in HeLaM cells revealed a similar HLA distribution between the various loop mutants (Figure 5c and d). Figure 5 with 4 supplements see all Download asset Open asset Mutation of the K22-D35 loop of TAPBPR changes the peptide repertoire presented on cells. Peptides eluted from W6/32-reactive MHC I complex isolated from IFNγ treated HeLaM-TAPBPRKO expressing either TAPBPRWT, TAPBPRØloop or TAPBPRØG30L were analysed using LC-MS/MS. In dataset 1 (a–d), cells were frozen immediately post-trypsination while in dataset 2 cells were to in for 30 min after The of identified peptides are in Figure The of all for the two is shown in Figure supplement diagrams compare all the identified peptides using a presence/absence displaying peptides between two cells highlight the peptide which are expressed between two cell lines after an of The of these peptides is in Figure and = of significantly peptides, demonstrates the of significantly peptides in a specific cell compare to all peptides in the Bar graphs the MHC I molecules or that the identified peptides in and the significantly peptides identified in were to using the In (c) and peptides not are in of the peptide repertoire from a further mutant lacking L30 and from a can be found in Figure supplements 2 and 3 of the affinity of peptides upon mutation of the loop those in and (f) can be found in Figure supplement Figure data 1 of TAPBPR to the HLA molecules found in HeLaM cells. Soluble TAPBPRWT or soluble TAPBPRØloop were incubated with the single antigen HLA class I for min at 3 in bound TAPBPR was detected using the and a were analysed using the The the MFI of for the HLA incubated in the absence and presence of 1 of TAPBPRWT or The expression of MHC I using by for the specific of used is included as a control. Download Figure data 2 1 peptides eluted from W6/32-reactive MHC I complexes from IFNγ treated HeLaM-TAPBPRKO cells expressing TAPBPRWT. Download Figure data 3 1 peptides eluted from W6/32-reactive MHC I complexes from IFNγ treated HeLaM-TAPBPRKO cells expressing Download Figure data 4 1 peptides eluted from W6/32-reactive MHC I complexes from IFNγ treated HeLaM-TAPBPRKO cells expressing TAPBPRØG30L. Download Figure data 5 2 peptides eluted from W6/32-reactive MHC I complexes from IFNγ treated HeLaM-TAPBPRKO cells expressing TAPBPRWT. Download Figure data 2 peptides eluted W6/32-reactive MHC I complexes from IFNγ treated HeLaM-TAPBPRKO cells expressing Download Figure data 7 2 peptides eluted from W6/32-reactive MHC I complexes from IFNγ treated HeLaM-TAPBPRKO cells expressing TAPBPRØG30L. Download Figure data 1 of eluted peptides used to Download Figure data 2 of eluted peptides used to Download Figure data 10 Peptides eluted from W6/32-reactive MHC I complexes from IFNγ treated