Author response: TAPBPR alters MHC class I peptide presentation by functioning as a peptide exchange catalyst
2015-09-21

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Our understanding of the antigen presentation pathway has recently been enhanced with the identification that the tapasin-related protein TAPBPR is a second major histocompatibility complex (MHC) class I-specific chaperone. We sought to determine whether, like tapasin, TAPBPR can also influence MHC class I peptide selection by functioning as a peptide exchange catalyst. We show that TAPBPR can catalyse the dissociation of peptides from peptide-MHC I complexes, enhance the loading of peptide-receptive MHC I molecules, and discriminate between peptides based on affinity in vitro. In cells, the depletion of TAPBPR increased the diversity of peptides presented on MHC I molecules, suggesting that TAPBPR is involved in restricting peptide presentation. Our results suggest TAPBPR binds to MHC I in a peptide-receptive state and, like tapasin, works to enhance peptide optimisation. It is now clear there are two MHC class I specific peptide editors, tapasin and TAPBPR, intimately involved in controlling peptide presentation to the immune system. https://doi.org/10.7554/eLife.09617.001 eLife digest Our immune system protects us from infections and destroys cells that are turning cancerous. A group of proteins called MHC class I molecules are essential for this protection. These molecules let the immune system know what is going on inside our cells by displaying chopped up fragments of proteins (or peptides) on the surface of the cell. If these peptides are from infectious or disease-causing agents the immune system is triggered into action and can recognise and kill the cell. There is still much to discover regarding how MHC molecules choose which peptides to display. A very complex pathway within our cells controls this displaying of peptides to the immune system. Recently, a protein called TAPBPR was identified as a new player in MHC class I biology, but its role was unclear. Hermann, van Hateren et al. now reveal that TAPBPR plays a central role in restricting which peptides are loaded onto and presented by MHC class I molecules. The results suggest that TAPBPR acts as a quality control checkpoint, closely monitoring and ensuring that the loaded peptide is stable within the MHC class I molecule. The discovery of TAPBPR's role in peptide selection increases our understanding of how peptides are chosen and stabilized, and sets the stage for learning more about how cells decide which peptides to reveal to the immune system. https://doi.org/10.7554/eLife.09617.002 Introduction Major histocompatibility complex (MHC) class I molecules convey a selection of the peptidome of a cell to the immune system. As well as being of crucial importance for the detection of intracellular pathogens, particularly viruses, it is now apparent that peptide presentation by MHC class I is extremely relevant in the recognition of tumours (Rötzschke et al., 1990; Duan et al., 2014; Gubin et al., 2014; Snyder et al., 2014; Yadav et al., 2014). Despite this, the molecular mechanisms controlling peptide selection for immune recognition are still poorly understood. Currently, the loading of peptide onto MHC class I is known to be orchestrated by the co-factor tapasin and is thought to occur predominantly within the peptide loading complex (PLC) in the endoplasmic reticulum (Sadasivan et al., 1996; Li et al., 1997; Ortmann et al., 1997; Lehner et al., 1998; Tan et al., 2002). Tapasin enhances the rate and the extent of MHC class I peptide loading and improves the discrimination that occurs between peptides to ensure MHC class I molecules are loaded with high-affinity peptides, thus prolonging cell surface expression of MHC class I molecules (Williams et al., 2002; Howarth et al., 2004; Chen and Bouvier, 2007; Wearsch and Cresswell, 2007; van Hateren et al., 2013). MHC class I allomorphs differ in their dependence on tapasin for efficient peptide loading (Greenwood et al., 1994; Peh et al., 1998; Williams et al., 2002). Furthermore, the ability to select and assemble with an optimal peptide cargo in the absence of tapasin is inversely correlated to the enhancement observed in the presence of tapasin (Williams et al., 2002; van Hateren et al., 2013; Rizvi et al., 2014). It has recently become apparent that, in addition to tapasin, there is a second MHC class I-specific chaperone, the tapasin-related protein TAPBPR (Boyle et al., 2013). In contrast to tapasin, TAPBPR is not an integral component of the peptide loading complex and cannot compensate for the loss of tapasin (Boyle et al., 2013; Hermann et al., 2013). Currently, the precise function of TAPBPR in the MHC class I pathway is unknown (Hermann et al., 2015). A functional role in MHC class I antigen presentation is supported by the finding that the presence of TAPBPR slows the anterograde trafficking of MHC class I and prolongs the association of MHC class I with the PLC. This raises the possibility that TAPBPR serves as an additional quality control checkpoint in the MHC class I antigen presentation pathway (Boyle et al., 2013). Although tapasin and TAPBPR only share 22% identity (Teng et al., 2002), both bind MHC class I, and their orientation on MHC class I is similar (Dong et al., 2009; Hermann et al., 2013). This shared orientation raises the possibility that there is some common functionality between the two molecules, particularly in regard to the ability to influence the peptide repertoire presented on MHC class I. Here, we sought to determine whether, like tapasin, TAPBPR has a peptide editing function. Results Expression and purification of TAPBPR In order to test whether TAPBPR has peptide editing functionality we chose an approach analogous to that previously established for tapasin, namely a cell free in vitro assay that allows monitoring of peptide binding to MHC class I in real time via fluorescence anisotropy (Chen and Bouvier, 2007). Due to the intrinsically low affinity between tapasin and MHC class I molecules in vitro, soluble tapasin function on MHC class I is only measurable when the two molecules are brought into close proximity via a Jun/Fos leucine zipper (Chen and Bouvier, 2007) or when tapasin is conjugated to ERp57 (Wearsch and Cresswell, 2007). Since TAPBPR is not integrated into the PLC and can be co-immunoprecipitated with MHC I (Boyle et al., 2013, Hermann et al., 2013), it is likely that TAPBPR has a higher affinity for MHC class I than tapasin. We therefore tested whether TAPBPR functioned in vitro without the need for an artificial intermolecular tether. To this end, the luminal domains of TAPBPR were cloned into the pHLsec expression vector and transiently transfected into HEK293F cells. This resulted in the efficient production of a secreted form of TAPBPR with a C-terminal His-tag, allowing for purification from the culture supernatant using Ni-affinity and size exclusion chromatography. TAPBPR protein eluted as a single major peak of high purity as verified by Coomassie staining following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1A). TAPBPRTN5 in which the isoleucine at position 261 was mutated to lysine, which inhibits the interaction of TAPBPR with MHC class I (Hermann et al., 2013), was produced in the same manner and was also efficiently expressed and produced a major single protein peak following size-exclusion chromatography (Figure 1B). Differential scanning fluorimetry revealed melting temperatures for TAPBPR and TAPBPRTN5 to be between 51.5°C and 52.5°C indicating that the single point mutation did not adversely affect TAPBPR folding (Figure 1C). These mammalian-expressed, purified TAPBPR proteins were used in subsequent fluorescence anisotropy experiments to characterise the equilibrium and kinetic parameters between MHC class I and different peptide ligands. Figure 1 Download asset Open asset Expression of TAPBPR. Size exclusion chromatograms of (A) TAPBPR and (B) TAPBPRTN5 purified from cell culture supernatants. The protein peaks were analysed by SDS-PAGE followed by Coomassie staining. (C) Differential scanning fluorimetry of TAPBPR and TAPBPRTN5 demonstrate equivalent thermal denaturation profiles. The data is representative of two independent experiments. https://doi.org/10.7554/eLife.09617.003 TAPBPR enhances peptide dissociation from HLA-A2 in vitro To investigate if TAPBPR is able to directly edit peptides on MHC class I molecules in a manner similar to that attributed to tapasin, we adapted the in vitro peptide exchange assay previously developed for measuring tapasin function (Chen and Bouvier, 2007). Using fluorescent polarisation we tested whether TAPBPR enhanced peptide dissociation from HLA-A2, an allomorph that co-immunoprecipitates readily with TAPBPR (Hermann et al., 2013). HLA-A*02:01 molecules were loaded with the fluorescent peptide FLPSDC*FPSV (C* indicates the position of the fluorophore) before excess unlabelled FLPSDCFPSV peptide was added in the presence or absence of TAPBPR. Dissociation of FLPSDC*FPSV was only apparent in the first six hours in the presence of competitor peptide and TAPBPR (Figure 2A). Dissociation of FLPSDC*FPSV was also observed when a vast excess of unlabelled NLVPMVATV was used in the presence of TAPBPR (Figure 2B). To ensure the enhancement of peptide dissociation was a consequence of direct interaction between TAPBPR and HLA-A2, we repeated the peptide dissociation assays using TAPBPRTN5 (I261K) which inhibits the ability of TAPBPR to bind to HLA-A2 (Hermann et al., 2013). No enhancement in the dissociation of FLPSDC*FPSV was observed in the presence of TAPBPRTN5 (red line Figure 2B). Thus the ability of TAPBPR to enhance peptide dissociation is a direct consequence of TAPBPR binding to HLA-A2. Figure 2 with 1 supplement see all Download asset Open asset TAPBPR functions as a peptide loading catalyst and peptide editor for HLA-A2. (A,B) Dissociation, (C) association and (D) peptide competition of fluorescent peptide FLPSDC*FPSV on HLA-A*02:01 in the absence or presence of TAPBPR or TAPBPRTN5 as measured by fluorescence polarisation. (A) 0.15 μM HLA-A*02:01fos molecules were mixed with 1.2 μM human β2m and loaded with 0.1 μM FLPSDC*FPSV and (B) 0.5 μM HLA-A*02:01 molecules were loaded with 0.125 μM FLPSDC*FPSV. The complexes were then split and incubated with 1000 fold molar excess of (A) FLPSDCFPSV or (B) NLVPMVATV with either A) buffer (No protein) or supplemented with 0.75 μM TAPBPR, or (B) buffer (No protein) or supplemented with 0.25 μM TAPBPR or TAPBPRTN5. Note, the slight difference in dissociation rate observed in A & B is likely to be related to the concentration of TAPBPR used in each experiment and not to the sequence of the competing peptide used. The data shown in Figure 2A,B is representative of 13 independent experiments, which all produced similar results. (C) 0.5 μM HLA-A*02:01 molecules were made peptide receptive and then the binding of 0.125 μM FLPSDC*FPSV was followed in the presence or absence of 0.05 μM TAPBPR or TAPBPRTN5. One representative association experiment of 13 experiments is shown. (D) 0.5 μM HLA-A*02:01 molecules were made peptide-receptive and incubated with 0.125 μM high affinity peptide FLPSDC*FPSV and various concentrations of the lower affinity competing peptide NLVPMVATV (0–20 μM) in presence or absence of 0.25 μM TAPBPR or TAPBPRTN5. One of two experiments is shown. (E–G) Comparison of the dissociation of seven peptides from HLA-A*02:01fos in the presence or absence of TAPBPR or tapasin-Jun as performed in Figure 2—figure supplement 1. The results of three (E) and four (F,G) independent experiments were combined and are shown. Data from each experiment was processed in GraphPad Prism using one-phase exponential decay non-linear regression. The half-lives that were calculated for the dissociation of the indicated peptide in the presence of either (E) Competitor only, (F) Competitor and Tapasin-Jun or (G) Competitor and TAPBPR were plotted as a percentage of the half-life calculated for the dissociation of KLWEAESK*L in the equivalent condition. Error bars show the standard deviation of the relative half-lives measured in the different experiments. While there were slight modifications of experimental conditions between replicate experiments, results consistent with the presented results were observed. https://doi.org/10.7554/eLife.09617.004 TAPBPR enhances peptide association with HLA-A2 in vitro Next we examined the effect that TAPBPR has on the kinetics of peptide association with HLA-A2. To test this, HLA-A*02:01 molecules refolded with a UV conditional ligand were made peptide-receptive by photolysis and the binding of the fluorescent peptide FLPSDC*FPSV was monitored by fluorescence polarisation in the absence or presence of TAPBPR. We found that TAPBPR catalysed binding of FLPSDC*FPSV to peptide-receptive HLA-A*02:01 (Figure 2C). In contrast, no enhancement in the association of FLPSDC*FPSV with HLA-A2 was observed in the presence of TAPBPRTN5 (Figure 2C), further demonstrating that a direct association between TAPBPR and HLA-A2 is required for TAPBPR to influence HLA-A2 peptide binding characteristics. TAPBPR enhances the selection of high affinity peptide by HLA-A2 in vitro To ascertain whether TAPBPR is able to discriminate between peptides, competition assays were performed in which peptide-receptive HLA-A*02:01 molecules were incubated with a mix of two peptides; 0.125 μM of the high affinity labelled peptide FLPSDC*FPSV, and a variable concentration of the peptide NLVPMVATV, which binds HLA-A*02:01 with lower affinity than FLPSDCFPSV. In the presence of TAPBPR, NLVPMVATV became a poorer competitor (Figure 2D). TAPBPRTN5 had no effect on peptide competition (Figure 2D). These results suggest that, like tapasin (Chen and Bouvier, 2007), TAPBPR can enhance the selection of high affinity peptides for binding to MHC I in vitro. Comparison of TAPBPR and tapasin-jun mediated peptide dissociation from HLA-A2 in vitro Next we compared the ability of TAPBPR or tapasin-jun to enhance dissociation of FLPSDC*FPSV and a panel of six additional peptides bound to HLA-A2. The dissociation rates of all peptides were enhanced in the presence of TAPBPR and in the presence of tapasin-jun as compared to competing peptide alone (Figure 2—figure supplement 1). However, peptide specific differences were apparent in the ability of tapasin-jun and TAPBPR to enhance dissociation. As different concentrations of active tapasin-jun and TAPBPR proteins were present in these assays, it was not possible to directly compare the dissociation half-lives of specific peptides in the presence of tapasin-jun with TAPBPR. Therefore we compared the hierarchies of dissociation relative to one peptide (KLWEAESK*L) over four independent experiments. This revealed reproducible differences in the hierarchies of peptide dissociation between tapasin-jun and TAPBPR on HLA-A2 (Figure 2E–G). Interestingly the half-life hierarchy for tapasin was similar to the hierarchy observed in the absence of either cofactor (competitor only) (Figure 2E,F), with the exception of the dissociation of GLDDIKDLK*V whose dissociation was relatively much slower in the presence of tapasin-jun compared to the other peptides than was observed in the presence of competitor only. This suggests that tapasin enhancement of peptide dissociation generally parallels peptide-MHC complex stability, as previously reported (Howarth et al., 2004). However, the half-life of peptides in the presence of TAPBPR does not follow this trend (Figure 2G). Specifically we observed that in comparison to the other peptides, TAPBPR enhanced dissociation of KLVK*EVIAV to a much smaller extent than was apparent in the presence of tapasin-jun or absence of either cofactor (competitor only) (Figure 2E–G). The same was also true of FLPSDC*FPSV (Figure 2E–G). Taken together our results suggest firstly that both tapasin and TAPBPR enhance peptide dissociation, and secondly that there are subtle differences in regard to their peptide specificity. TAPBPR interacts with HLA-B as well as HLA-A molecules We previously reported that TAPBPR has preference for HLA-A68 over HLA-B15 expressed in HeLa cells (Boyle et al., 2013). We wondered if TAPBPR generally exhibited preference for HLA-A allomorphs or whether TAPBPR might interact with HLA-B allomorphs. To explore this, we expressed a small panel of individual HLA allomorphs in the MHC class I negative cell line 721.221, and determined their interaction with endogenous TAPBPR, in a system in which competition between MHC class I allomorphs is absent. Immunoprecipitation of TAPBPR, followed by western blotting for MHC class I, confirmed the strong association between TAPBPR and the HLA-A allomorphs HLA-A2 and HLA-A68 (Figure 3). However, an association between TAPBPR and HLA-B allomorphs was also detected, although this was weaker (HLA-B8 and -B15), or below the limits of detection (HLA-B40), compared to that for HLA-A2 (Figure 3). Therefore, although there is clearly a strong association with those HLA-A allomorphs tested, TAPBPR interacts with a broader range of HLA molecules than originally appreciated (Boyle et al., 2013). Figure 3 Download asset Open asset TAPBPR associates with HLA-A and HLA-B molecules. TAPBPR was isolated by immunoprecipitation (using R014) from the MHC class I negative cell line 721.221 and 721.221 stably transduced with HLA-A2, A68, B8, B15 or B40. Western blot analysis was performed for TAPBPR and the MHC class I heavy chain (using HCA2 and HC10) on lysates (labelled lys) and TAPBPR immunoprecipitates as indicated. The data is representative of three independent experiments. https://doi.org/10.7554/eLife.09617.006 TAPBPR function on HLA-B8 in vitro Given the interactions detected between TAPBPR and the studied HLA-B allomorphs, we asked whether TAPBPR was involved in peptide selection on HLA-B*08:01, an allomorph which interacts weakly with TAPBPR (Figure 3) and which is well-characterised in regard to tapasin function (Greenwood et al., 1994; Lehner et al., 1998; Peh et al., 1998; Chen and Bouvier, 2007; Wearsch and Cresswell, 2007). We first asked whether TAPBPR enhanced peptide dissociation from HLA-B*08:01fos molecules. We found that dissociation of EIYK*RWIIL was enhanced in the presence of TAPBPR (Figure 4A and Figure 4—figure supplement 1A). No enhancement of the dissociation of EIYK*RWIIL was observed in the presence of TAPBPR using HLA-B8:01-T134K in which the T134 residue had been mutated to lysine, which abrogates binding of TAPBPR to MHC I (Hermann et al., 2013) (Figure 4B and Figure 4—figure supplement 1B), demonstrating that the enhanced peptide dissociation was dependent on a direct interaction between TAPBPR and HLA-B8. We also tested the ability of TAPBPR to enhance dissociation of two additional peptides, ELRSRK*WAI and FLRGRK*YGL from HLA-B*08:01. The dissociation of ELRSRK*WAI was not altered in the presence of TAPBPR (Figure 4C and Figure 4—figure supplement 1C) and only a very slight change in dissociation of FLRGRK*YGL was observed in the presence of TAPBPR (Figure 4D and Figure 4—figure supplement 1D). Therefore, our results suggest that TAPBPR can enhance peptide dissociation from HLA-B8 but that this effect is peptide specific. Tapasin has also been reported to exhibit peptide specificity in regard to peptide dissociation (Chen and Bouvier, 2007). Like TAPBPR, tapasin-jun accelerated dissociation of fluorescein isothiocyanate (FITC) labelled versions of both EIYK*RWIIL and FLRGRK*YGL from HLA-B*08:01fos, while the dissociation of ELRSRK*WAI was insensitive to tapasin-jun (Chen and Bouvier, 2007). Figure 4 with 1 supplement see all Download asset Open asset TAPBPR can function as a peptide loading catalyst and peptide editor for HLA-B8. (A–D) Dissociation, (E–G) association and (H) peptide competition of fluorescent peptides on HLA-B*08:01 in the absence or presence of TAPBPR. 2 μM HLA-B*08:01fos or HLA-B*08:01fos T134K molecules were mixed with 20 μM human β2m and made peptide receptive then loaded with 1 μM (A,B) EIYK*RWIIL (C) ELRSRK*WAI or (D) FLRGRK*YGL. Dissociation was subsequently followed after the addition of a 250 molar excess of (A,B) EIYKRWIIL (C) ELRSRKWAI, or (D) FLRGRKYGL in the absence or presence of 0.75 μM TAPBPR. A total of eight dissociation experiments have been conducted for wild-type HLA B*08:01fos, and the T134K mutant was included in six of these experiments. (E–G) 0.6 μM HLA B*08:01fos molecules were mixed with 6 μM human β2m and made peptide-receptive, then the binding of 1 μM of (E) EIYK*RWIIL, (F) ELRSRK*WAI or (G) FLRGRK*YGL was followed in the absence or presence of 0.175 μM TAPBPR. One of three experiments is shown. (H) 0.6 μM HLA-B*08:01fos molecules were mixed with 0.6 μM human β2m and were made peptide-receptive, then incubated with 0.1 μM high affinity peptide ELRSRK*WAI and various concentrations of the lower affinity competing peptide EIYKRWIIL (0–25 μM) in presence or absence of 0.3 μM TAPBPR. One of six experiments is shown. Fluorescence polarisations measurements were taken after being left at room temperature overnight. While there were slight modifications of experimental conditions between replicate experiments, results consistent with the presented results were observed. https://doi.org/10.7554/eLife.09617.007 As with HLA-A2, we observed that TAPBPR could enhance peptide binding to peptide-receptive HLA-B*08:01fos molecules. However, this was only observed with EIYK*RWIIL (Figure 4E) and the binding of ELRSRK*WAI or FLRGRK*YGL to peptide receptive HLA-B*08:01fos was not altered in the presence of TAPBPR (Figure 4F,G). This contrasts with the results previously published by Chen and Bouvier, who observed that tapasin-jun enhanced binding of FITC labelled versions of ELRSRK*WAI and FLRGRK*YGL to peptide-receptive HLA-B*08:01fos molecules (Chen and Bouvier, 2007). Taken together the data indicate that TAPBPR can enhance the loading of peptide onto HLA-B*08:01, and that this is peptide specific. Furthermore, there are differences in peptide specificity between tapasin and TAPBPR with respect to HLA-B*08:01 peptide-loading. Finally we showed that, like tapasin (Chen and Bouvier, 2007), TAPBPR increased peptide exchange on HLA-B*08:01 since the low affinity peptide EIYKRWIIL became a poorer competitor against the high affinity labelled peptide ELRSRK*WAI in the presence of TAPBPR (Figure 4H). TAPBPR expression alters the peptide repertoire presented by MHC class I on cells Given that TAPBPR functions as a peptide exchange catalyst for MHC class I in vitro, we next determined if TAPBPR expression had any effect on the peptide repertoire expressed by MHC class I molecules in cells. To do this, we created a HeLa cell line in which TAPBPR was knocked out using the clustered regularly interspaced short palindromic repeats (CRISPR) system. We found that upon treatment with IFN-γ, TAPBPR expression was induced in HeLa, but not in the HeLa-TAPBPR KO line (Figure 5A). Tapasin expression was not affected by the sgRNA targeted to TAPBPR (Figure 5A). In the absence of TAPBPR, cell surface expression of MHC class I in IFN-γ treated HeLa did not significantly change as determined by W6/32 staining and flow cytometry (Figure 5B). When the amino acid sequences of the peptides eluted from MHC class I molecules from IFN-γ treated HeLa and HeLa-TAPBPR KO cells were compared, the most noticeable difference was a change in the diversity of peptides isolated (Figure 5C). In IFN-γ treated HeLa cells, we isolated 1607 different peptides on MHC class I, compared with 2074 in IFN-γ treated HeLa-TAPBPR KO cells (Figure 5C). 1398 peptides were shared between the two cell lines, but 209 peptides were unique to HeLa and 676 were unique to HeLa-TAPBPR KO (Figure 5C). When the peptides were subdivided into those predicted to bind to HLA-A*68:02 or HLA-B*15:03 based on anchor sequences, an increased repertoire was observed for both allomorphs in the absence of TAPBPR (Figure 5C). with these a similar of increased peptide diversity was also observed in IFN-γ treated cells upon TAPBPR depletion using to TAPBPR (Figure supplement 1). Figure with 2 see all Download asset Open asset TAPBPR expression alters the peptide repertoire presented by MHC class I on cells. (A) The class I complex was from IFN-γ treated HeLa and HeLa-TAPBPR KO cells. Western blot analysis was performed for TAPBPR tapasin MHC class I and on lysates and TAPBPR immunoprecipitates as indicated. The data is representative of three independent experiments. (B) analysis of MHC class I detected with W6/32 on IFN-γ treated HeLa and HeLa-TAPBPR KO cells with an control on both cell is included as a class I complexes were isolated by affinity chromatography using W6/32 from (C) IFN-γ treated HeLa and HeLa-TAPBPR KO cells or (D) HeLa and HeLa peptides were analysed using show the total of peptides and also the of peptides as HLA-A*68:02 and HLA-B*15:03 based on their peptide using the The of peptides shared between the cell and the of peptides unique to each cell line is shown. The data was from analysis performed on one independent repeats have been performed in two different cell cells shown in Figure supplement and cells shown in Figure in which a similar of increased peptide diversity in TAPBPR cells was observed. of and C-terminal anchor show of peptide on HLA-A*68:02 or show anchor at and combined for shared peptides found in both IFN-γ treated HeLa and HeLa TAPBPR KO cells expression in the presence of and peptides unique to HeLa TAPBPR KO cells (red in the presence of shared peptides found in both HeLa and HeLa TAPBPR expression in the presence of and peptides unique to HeLa in the presence of Figure 6 Download asset Open asset to the peptide repertoire expressed on MHC class I molecules in IFN-γ treated cells upon TAPBPR depletion or tapasin analysis of (A) HLA-A2 expression with and (D) HLA-B expression with 4E) on the cell surface of TAPBPR stably and tapasin cells treated with and without IFN-γ for The data is representative of three independent experiments. class I complexes were isolated using affinity chromatography using (B) and (E) from IFN-γ treated TAPBPR stably and tapasin cells. peptide were analysed using show the total of The data was from analysis performed on one independent repeats the peptide repertoire expressed on MHC class I conditions of TAPBPR and TAPBPR in two other cell have been performed cells shown in Figure and cell shown in Figure supplement in which a similar of increased peptide diversity upon TAPBPR depletion was observed. show the of peptides on (C) HLA-A2 and (F) eluted from IFN-γ treated TAPBPR stably and tapasin cells. To these we compared the amino acid sequences of peptides eluted from MHC class I molecules isolated from HeLa and HeLa cells TAPBPR by the most difference was a change in the diversity of peptides isolated (Figure We found the total of different peptides isolated from MHC class I was in cells TAPBPR, from MHC class I peptides from HeLa cells to MHC class I peptides isolated from HeLa cells TAPBPR. peptides were shared between the two cell (Figure when these peptides were further into those predicted to bind to HLA-A*68:02 and based on their an increased repertoire was observed for both allomorphs in the absence of TAPBPR (Figure Taken these results suggest that TAPBPR the peptide repertoire presented on MHC class I molecules. We compared the anchor found at the and C-terminal position of the shared