Post-Endoplasmic Reticulum Rescue of Unstable MHC Class I Requires Proprotein Convertase PC7

This information is current as Ralf M. Leonhardt, Dorothee Fiegl, Elke Rufer, Axel of September 30, 2021. Karger, Barbara Bettin and Michael R. Knittler J Immunol 2010; 184:2985-2998; Prepublished online 17 February 2010; doi: 10.4049/jimmunol.0900308

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Post-Endoplasmic Reticulum Rescue of Unstable MHC Class I Requires Proprotein Convertase PC7

Ralf M. Leonhardt,*,1 Dorothee Fiegl,†,1 Elke Rufer,†,1 Axel Karger,‡ Barbara Bettin,‡ and Michael R. Knittler†

The function of the peptide-loading complex (PLC) is to facilitate loading of MHC class I (MHC I) molecules with antigenic peptides in the endoplasmic reticulum and to drive the selection of these ligands toward a set of high-affinity binders. When the PLC fails to perform properly, as frequently observed in virus-infected or tumor cells, structurally unstable MHC I peptide complexes are generated, which are prone to disintegrate instead of presenting Ags to cytotoxic T cells. In this study we show that a second quality control checkpoint dependent on the serine protease proprotein convertase 7 (PC7) can rescue unstable MHC I, whereas the related convertase furin is completely dispensable. Cells with a malfunctioning PLC and silenced for PC7 have substantially reduced MHC I surface levels caused by high instability and significantly delayed surface accumulation of these molecules. Instead of acquiring Downloaded from stability along the secretory route, MHC I appears to get largely routed to lysosomes for degradation in these cells. Moreover, mass spectrometry analysis provides evidence that lack of PLC quality control and/or loss of PC7 expression alters the MHC I-presented peptide profile. Finally, using exogenously applied peptide precursors, we show that liberation of MHC I epitopes may directly require PC7. We demonstrate for the first time an important function for PC7 in MHC I-mediated Ag presentation. The Journal of Immunology, 2010, 184: 2985–2998. http://www.jimmunol.org/ ntigenic peptides are loaded onto MHC class I (MHC I) quality control function of the PLC (3). In the mutant PLCs of molecules in the endoplasmic reticulum (ER) within these cells, MHC I largely acquires suboptimal ligands, which fail A a large multisubunit complex known as the peptide- to confer normal stability. We note that this strongly resembles the loading complex (PLC) (1). One major function of this complex is effects of viral evasion targeting the function of the loading to optimize the peptide cargo transferred into the binding groove of complex (5–7). Following ER-export, however, initially labile MHC I in a sense that only stably binding, high-affinity ligands are MHC I molecules with low structural integrity were found to loaded (1). Consequently, cells that express a defective PLC, owing substantially improve over time, with their stability eventually to absence or mutation of its central constituents’ TAP (2, 3) or arriving at the cell surface as highly stable molecules (3). Similar tapasin (4), generate a large fraction of structurally fragile MHC I- observations have also been described for tapasin-deficient cells by guest on September 30, 2021 peptide complexes that display low thermal stability and a high (4), suggesting that post-ER stabilization of MHC I-peptide tendency to disintegrate during transport along the secretory route complexes is likely to become generally applied under circum- or at the cell surface. Upon loss of their antigenic ligand, empty stances in which unstable MHC I peptide complexes leave the ER MHC I molecules are not only useless for immunosurveillance by and enter the exocytic pathway. The underlying mechanism was cytotoxic T cells, but may become harmful when they in- shown to require a class of serine proteases, the so-called pro- discriminately pick up peptides from the extracellular milieu. protein convertases (pPCs) (3), and is likely to involve peptide We recently showed that reconstitution of the TAP-deficient cell exchange in late secretory compartments. Consistent with this line T2, with the truncated transporter 1-2DN, fully restores finding, others have found pPCs to generate MHC I-ligands in the peptide transport and supply to MHC I, but fails to restore the trans-Golgi network (TGN) of TAP-deficient cells (8, 9). The human genome encodes at least seven highly related members of the subtilisin/kexin-like pPC family, three of which *Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06519; †Friedrich-Loeffler-Institute, Federal Research Institute for Animal display a highly restricted expression pattern in testis (PC4) or Health, Institute of Immunology, Tuebingen; and ‡Friedrich-Loeffler-Institute, Fed- endocrine/neuroendocrine cells (PC1 and PC2) (10). The remaining eral Research Institute for Animal Health, Institute of Molecular Biology, Greifs- four members (furin, PACE4, PC5, and PC7) show a widespread wald-Insel Riems, Germany 1 tissue distribution and primarily reside in the TGN, in post-TGN R.M.L., D.F., and E.R. contributed equally to this work. vesicles or in the endocytic system (10), where they process a large Received for publication January 28, 2009. Accepted for publication January 13, number of polypeptides of cellular (11) and/or pathogenic (12) 2010. origin. In particular, viral fusion proteins frequently require This work was supported by the Friedrich-Loeffler-Institute and the Federal Ministry of Education and Research of the Federal Republic of Germany Grant 01 KI 0720. cleavage-activation by pPCs to become functional (13), so that Address correspondence and reprint requests to Dr. Michael R. Knittler, Friedrich- pPC-dependent processing might introduce important viral epito- Loeffler-Institute, Federal Research Institute for Animal Health, Institute of Immu- pes to the MHC I Ag presentation pathway. However, which pPCs nology, Paul-Ehrlich-Strasse 28, D-72076 Tuebingen, Germany. E-mail address: are involved in generating and processing MHC I ligands in the michael.knittler@fli.bund.de compartments of the late secretory route is unknown. Abbreviations used in this paper: BFA, brefeldin A; Endo H, endoglycosidase H; ER, endoplasmic reticulum; HDA, hexa-D-arginine; MFI, mean fluorescence intensity; Inthisstudywefoundthat,ofthefourbroadlyexpressedpPCs,only MHC I, MHC class I; MS, mass spectrometry; PLC, peptide-loading complex; PC7andfurinaretranscribedinT2cells.Toinvestigatewhetherfurin, pPC, proprotein convertase; shRNA, short hairpin RNA; siRNA, small interfering PC7, or both are involved in post-ER stabilization of MHC I com- RNA; TGN, trans-Golgi network; wt, wild type. plexes, we performed specific and constitutive silencing (RNA in- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 terference) of the two pPCs in the cell line T2(1-2DN), characterized www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900308 2986 FUNCTIONAL ROLE OF PC7 IN ANTIGEN PRESENTATION by a defective quality control in the PLC (3, 14). Our experiments Abs show that in the absence of PC7, unstable MHC I molecules are not Rabbit antisera 116/5 and D90 recognize the C terminus of rat TAP2 (18) or rat rescued in post-ER compartments during their intracellular transport TAP1 (19); 3B10.7 is a rat mAb binding the heavy chains of HLA-A and -B to the cell surface. Consistent with this finding, PC7-silenced T2(1- (20); and 4E is a b2m-dependent mouse mAb that recognizes an epitope 2DN) transfectants [T2(1-2DN/PC7↓)] are characterized by delayed, common to all assembled HLA-B molecules (21). Mon-152 is a mouse mAb reduced, and unstable surface expression of MHC I complexes. In raised against human furin and recognizes an epitope in the cysteine-rich re- gion (Alexis, Enzo Life Sciences, Loerrach, Germany). V-20 is an affinity contrast, no such effects on intracellular MHC I maturation and purified goat polyclonal antiserum raised against human PC7, recognizing an surface expression could be observed for T2(1-2DN) cells knocked internal epitope mapping between residues 680–730 (Santa Cruz Bio- down for furin [T2(1-2DN/FUR↓)]. Moreover, in T2(1-2DN/PC7↓) technology, Santa Cruz, CA). Anti-PC7 is a rabbit antiserum raised against cells a substantial amount of post-ER-MHC I routes to the lysosomal a synthetic peptide corresponding to aa 298–310 of the external catalytic do- main of human PC7 (Alexis). Anti-Lamp1 and anti-EAA1 are polyclonal pathway. In support of our findings, MALDI-mass spectrometry rabbit antisera raised against the C-termini of human LAMP1 and EAA1 (MS)-analysis provides experimental evidence that the MHC I- (Abcam, Cambridge, U.K.). The rabbit polyclonal antiserum to human tapasin presented peptide profile is altered in the absence of PLC quality (R.gp48N) was a gift from Dr. P. Cresswell. Anti-ER60 is a polyclonal rabbit control and/or lack of PC7 function. Furthermore, using exogenously antiserum binding ERp57/ER60 (22). In flow cytometric experiments, mAb applied peptide precursors, we show that liberation of MHC I epit- against human transferrin receptor (CD71, BD Biosciences, San Jose,CA) was used as control. FITC-, tetramethylrhodamine isothiocyanate (TRITC)- and opes may directly require PC7. Based on our findings, we suggest HRP-conjugated Abs were purchased from Dianova (Hamburg, Germany). that the post-ER stabilization of suboptimally loaded MHC I mole- cules occurs in the TGN and/or post-TGN vesicles via an exchange Immunofluorescence of bound peptide ligands for ones that depend on the proteolytic T2 cells were fixed at room temperature for 15 min in 3% para- Downloaded from activity of the pPC PC7 for their generation. formaldehyde, quenched with 10 mM glycine for 10 min, permeabilized with 0.1% saponin (Sigma-Aldrich) and incubated serially with the in- dicated primary and appropriate fluorescence-labeled secondary Abs. All Materials and Methods images were captured by using an Axiovert 200M/Apotome microscope Cell lines and cell culture (Zeiss, Goettingen, Germany). Colocalization of fluorophores was mea- sured utilizing the AxioVision colocalization software module (Zeiss). The software automatically analyzes the number of pixels by which two dif- T2 is a human TAP-deficient lymphoblastoid cell line expressing HLA-A2 http://www.jimmunol.org/ and HLA-B51 (15). Transfectants of T2 expressing rat TAPwt or TAP1- ferent fluorophores are colocalized. 2DN were cultured in IMDM (Invitrogen, Carlsbad, CA) supplemented with 1 mg/ml G418 (PAA) and/or 750 ng/ml puromycin (Sigma-Aldrich, Preparation of endomembranes from T2 transfectants St. Louis, MO) (16). The human hepatoma cell line HepG2 (17) was Five 3 107 cells were incubated in ice-cold 10 mM Tris pH 7.5 for 10 min. cultured in DMEM (Invitrogen) supplemented with 10% FCS, 1 mM so- Next, the cells were homogenized and the nuclei were removed by cen- dium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen) and non- trifugation at 1.600 3 g; 20 mM HEPES pH 7.5 / 25 mM CH3COOK / 5 essential amino acids (Invitrogen). All culture media contained 100 U/ml mM (CH3COO)2Mg4H2O / 1 mM DTT / Complete protease inhibitor- penicillin and 100 mg/ml streptomycin (Invitrogen). mixture (Roche, Basel, Switzerland) were added to the supernatant and centrifuged at 1.600 3 g. Afterward, the postnuclear supernatants were RNA interference subjected to ultracentrifugation at 100,000 3 g to isolate endomembranes. by guest on September 30, 2021 Vectors encoding short hairpin RNA (shRNA) targeting furin (59-GG- Pulse-chase analysis, immunoprecipitation, endoglycosidase H GCCTTCATGACAACTCATT-39) or PC7 (59-GCTATGACCTCAACTCT- digest, and Western blotting AA-39) were constructed on the basis of the plasmid pSUPER (Oligoengine, Seattle, WA) according to the manufacturer’s instructions. Constructs were Cells were starvedfor 2 h inmethionine/cysteine-free RPMI;500mCi Promix sequenced before cotransfection with pPUR (Promega, Madison, WI) into (Amersham) was added for 30 or 60 min. The chase was initiated by the G418-resistant T2(1-2DN) cells by electroporation using a Bio-Rad gene addition of a 10-fold excess of unlabeled methionine/cysteine. Equal num- pulser (Bio-Rad, Hercules, CA) at 270 Vand 500 mF. After coselection with bers of cells were lysed in PBS containing 1% Triton X-100 (Sigma- puromycin (750 ng/ml) and G418 (1 mg/ml) for 4–6 wk, stable transfectants Aldrich). Immunoprecipitations were performed from equivalent amounts of were subcloned and screened for the silencing of furin and PC7 by RT-PCR precleared lysates by using anti-MHC I Abs. For identification of immuno- and Western blot analysis. For small interfering RNA (siRNA)-mediated isolated MHC I, precipitated complexes were separated by SDS-PAGE. knockdown of PC7 annealed double-stranded HP (HiPerformance) validated Fluorographs were scanned by a Chromoscan 3 microdensitometer (Joyce- siRNA with high knockdown efficiency ($70%) were designed and pur- Loebl,Gateshead, U.K.) orquantifiedusing GelEval1.22 software (FrogDance chased from Qiagen (Valencia, CA). To knock down PC7 expression, 5 mg Software, Dundee, U.K.). Immunoprecipitation, endoglycosidase H (Endo oligo duplex RNA specific for PC7 (Hs PCSK7 2 HP siRNA, target sequence; H) digest, and Western blotting were performed as described earlier (16). 59-TCGGAGCATTGTGACCACTGA-39; Qiagen) were transfected into 2 3 106 T2(1-2DN) cells using the Lonza/amaxa Nucleofector system and the Flow cytometry T2 cell Nucleofector kit (Lonza, Cologne, Germany). As a negative control, Flow cytometry was performed as described previously (16). To acid-remove AllStars Negative Control siRNA (Qiagen) was used. After nucleofection, surface MHC I molecules, cells were treated with IMDM/50 mM Na-citrate T2(1-2DN) cells were maintained for 24 h and screened for the silencing of pH 3.0 for 3 min before neutralization with 150 mM NaH PO , pH 10.5. PC7 by Western blot analysis. 2 4 Treatments with hexa-D-arginine (HDA) (Calbiochem, San Diego, CA) at 100 mM were performed for 14 h at 37˚C in FCS-free IMDM after an acid- RT-PCR and quantitative PCR wash. Decay of surface-MHC I was assessed in standard brefeldin A (BFA) For RT-PCR, mRNA was isolated from T2 or HepG2 cells and reverse- assays, in which cells were treated for different times with 10 mg/ml of the transcribed into cDNA using the SuperScript first-strand synthesis Kit drug. MHC I surface levels were determined with Ab 4E. For thermosta- (Invitrogen) in combination with oligo(dT)-primer (Invitrogen). Standard bility assays with living cells, T2 and 721.220 transfectants were heat Taq-driven PCR (39 cycles/annealing at 65˚C) was performed using primer shocked at 45–60˚C for 10 min. To examine the stability of surface-HLA- pairs 59-GCAACGGAGGCCAACACAACG-39 /59-GCGTTGAGGAGGC- B51, cells were directly immunostained with anti-MHC I Ab 4E. To analyze CGAAACCG-39 (PC7), 59-CTACGGCACCTCCGTGCAGCC-39 /59-CTT- the stability of intracellular HLA-B51, cells were first fixed, permeabilized, GAATCCTGGGCCATTGCACG-39 (PC5), 59-CAAACAATTCCTACTG- and stained with Ab 4E. The specificity of intracellular MHC I staining was CATCGTGGGC-39 /59-CCGTTCACTTTCCAGTCGCTCGC-39 (PACE4), controlled with different human cell lines not expressing HLA-B5 (data not 59-CGAGGATGACGGCAAGACAGTGG-39 /59-CGGCGATTATAGGA- shown). To characterize endosomal recycling of MHC I, primaquine CAGGGTG AGC-39 (furin) and 59-ATGACAACTTTGGTATCGTGGAA- (Sigma-Aldrich) was applied for 4 h at a concentration of 250 mM. GG-39 /59-GAAATGAGCTTGACA AAGTGGTCGT-39 (GAPDH). Primer MS analysis of MHC I-associated peptides pairs were designed to cover all known isoforms. Standard SYBR-green- based real-time quantitative PCR was performed on a Cepheid SmartCycler HLA-B51–associated self-peptides were analyzed by MALDI-MS. For the (Cepheid, Sunnyvale, CA) in two triplicates using the same primer pairs. isolation of surface-MHC I molecules, cells were preincubated in the cold The Journal of Immunology 2987 for 2 h with 4E Ab. Afterward, unbound anti-HLA-B51 Abs were removed depending on pPCs (3). To investigate which members of the pPC by extensive washing, and cells were lysed in 1% n-octylglucoside for 30 family (10) are necessary to allow this post-ER rescue to occur, we min. Because T2 cells do not synthesize detectable amounts of im- first determined by RT-PCR the T2-specific expression profile of munoglobulins by themselves (data not shown), postnuclear lysates could be directly incubated for 2 h with protein A-Sepharose. The successful the four broadly expressed pPCs (furin, PC5, PC7, and PACE4). isolation of surface HLA-B51 was controlled by Western blot, and the As shown in Fig. 1A, transcripts for PC7 and furin were detected obtained MHC I signals were quantified by densitometric scanning of four in T2 cells, whereas PC5 and PACE4 were completely absent. In different blot exposures. Beads with immunoisolated MHC I molecules contrast, the human hepatoma cell line HepG2, which served as were transferred into an ultrafiltration tube with a 3-kDa cutoff (Millipore, Bedford, MA), washed with 10 volumes of 20 mM Tris, pH 7.8, 0.1% a positive control in this experiment, expressed all four pPCs. To Zwittergent 3-12 (Calbiochem, San Diego, CA) and 20 volumes of bi- confirm the expression of PC7 and furin in T2 cells also on the destilled water. Peptides were eluted by incubation in 0.1% trifluoroacetic protein level, we performed Western blot analysis, which demon- acid in bidistilled water for 30 min, followed by ultrafiltration and ly- strated the presence of both pPCs in a T2-derived endomembrane ophilization. MALDI-MS analysis of the eluted peptides was performed on fraction (Fig. 1B). Moreover, the cellular t1/2 of both pPCs in T2 a Bruker Ultraflex Instrument and spectra were processed by flexAnalysis ∼ 2.0 software (Bruker Daltonics, Bremen, Germany). Obtained data were cells was similar and was determined to lie in the range of 2h normalized to the relative amount of immunoisolated HLA-B51 molecules (Fig. 1C). As expected, furin was predominantly detected in com- determined by immunoblot. partments that also contained the TGN marker TGN46, whereas the cellular distribution of PC7 displayed only a minor overlap with that of furin and TGN46 in most T2 cells (Fig. 1D, compare first, Results second and third rows). In fact, comparative immunofluorescence

The expression profile of pPCs in human T2 cells is limited to analysis of intact and permeabilized cells demonstrated that in the Downloaded from PC7 and furin T2 cells PC7 mostly localizes to vesicles that line up immediately We previously observed that reconstitution of the human TAP- beneath the plasma membrane and apparently not on the plasma deficient cell line T2 with the truncated peptide transporter 1-2DN membrane itself (Fig. 1D, fourth row). Thus, consistent with the causes the formation of unstable MHC I molecules, which after work of Wouters et al. (23), PC7 and furin appear to be spatially their export from the ER undergo stabilization by a mechanism separated to a large extent in the cell. http://www.jimmunol.org/

FIGURE 1. Expression of pPCs in human T2 cells. A, pPC or GAPDH expression in T2 or HepG2 cells was analyzed by RT-PCR. B, Protein expression of furin and PC7 in T2 and HepG2 by guest on September 30, 2021 cells. Membrane lysates of T2 cells were analyzed by Western blot using furin- or PC7-specific anti- sera. C, Cycloheximide chase experiments. T2 cells were incubated in the presence of 100 mM cycloheximide, lysed at indicated time points, and analyzed by immunoblotting with anti-furin and anti-PC7 Abs. Signals obtained for pPCs were quantitated by densitometric scanning. One repre- sentative experiment of two independent experi- ments is shown. D, Intracellular localization of furin and PC7. T2 cells were stained for furin (Mon-152, TRITC, left) and PC7 (V-20, FITC, middle)(upper row). Furthermore, cells were stained for TGN46 (anti-TGN46, TRITC, left) and furin (Mon-152, FITC, middle)(second row)or PC7 (V-20, FITC, middle)(third row). Individual red and green channels are shown in grayscale. Overlaps (merge) between the respective im- munostainings are depicted as color images (right). In the bottom row T2 cells were stained with anti- PC7 antiserum recognizing the external catalytic domain before fixation (left) and after fixation and permeabilization (right). Depicted are overlays of FITC and phase-contrast images. In all color im- ages, nuclei are visualized by DAPI (blue). Scale bars indicate 10 mm. To quantify the degree of relative colocalization, we obtained image data from 20–30 cells as described in Materials and Methods. 2988 FUNCTIONAL ROLE OF PC7 IN ANTIGEN PRESENTATION

In summary, according to the above pPC expression profile, PC7 RT-PCR analysis showed that the knockdown of either pPC in the and furin are the only convertases in T2 cells that might potentially stable cell lines T2(1-2DN/FUR↓) and T2(1-2DN/PC7↓) was effi- be involved in post-ER stabilization of MHC I. cient and specific (Fig. 2A, upper panel). To quantify the silencing efficiency we performed quantitative PCR confirming 8–10-fold lower mRNA levels for furin and PC7, respectively (Fig. 2A, lower PC7 but not furin is required for normal MHC I surface levels panel). To further demonstrate specificity of pPC silencing, we in the context of a defective PLC performed Western blotting using detergent-solubilized endo- PC7 or furin (or both) may be responsible for the post-ER rescue of membrane fractions of the different transfectants (Fig. 2B). As unstable MHC I formed in the T2-derived transfectant T2(1-2DN) expected from the experiments in T2 cells (Fig. 1B), the translation that expresses a defective PLC. To distinguish between possible products of the two pPCs were readily detected in the original cell roles for PC7 and furin, we stably silenced either of the two pPCs line T2(1-2DN), whereas they were almost undetectable in their in the T2(1-2DN) cell line via specific shRNA expression. respective knockdown derivatives. Quantification of the obtained Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. PC7 but not furin is required for high MHC I surface levels in T2(1-2DN) cells. A, pPC or GAPDH expression in T2(1-2DN) or its stable knockdown derivatives T2(1-2DN/FUR↓) and T2(1-2DN/PC7↓) was analyzed by RT-PCR (upper panel) or qPCR (lower panel). B, Cell lysates and membrane extracts of the different T2 transfectants were analyzed by Western blot using the indicated Abs. C, Surface expression of HLA-B51 was assessed by flow cytometry using the Ab 4E (filled histograms, left panel). In additional experiments, mAb against transferrin receptor (CD71) was used as control (filled histograms, right panel). Background staining was analyzed by incubating with secondary Ab alone (unfilled histograms, left and right panels). One representative experiment of two independent experiments is shown. D, HDA sensitivity of MHC I surface expression in T2(1-2DN) cells silenced for furin or PC7. Cells were acid washed to remove assembled surface MHC I and incubated for 14 h in the presence of 100 mM pPC-inhibitor HDA. Subsequently, cells were immunostained with the mAb 4E to assess surface expression of HLA-B51 by flow cytometry (left panel). Surface MHC I staining in the presence of HDA is plotted as a percentage of reduction in MFI compared with the MFI of the control experiments (right panel). One representative result of three separate experiments is shown. The Journal of Immunology 2989 signals by densitometric scanning revealed a reduction for furin and was largely heat-stable in TAPwt transfectants (Fig. 3A, lane 1; for PC7 by .98% compared with the original cell line T2(1-2DN), quantification, see Fig. 3B), whereas in cells expressing truncated whereas the protein levels for TAP1, TAP2 (or its truncated variant TAP (1-2DN), $50% of the MHC I peptide complexes did not 2DN) and ER60 were not affected by the silencing vectors. survive the incubation at 37˚C (Fig. 3A, lane 2; for quantification, Next, to assess whether either of the two pPCs has a role in Ag see Fig. 3B). Thermal instability of HLA-B51 was also seen for the presentation, we determined the steady-state surface levels of the T2(1-2DN) transfectants silenced for furin or PC7 (Fig. 3A, lanes 3 MHC I allele HLA-B51 in the different T2 transfectants by flow and 4; for quantification, see Fig. 3B). To elucidate whether the cytometry (Fig. 2C, left panel). As expected from our previous observed heat sensitivity of MHC I was due to suboptimal peptide study, T2 cells transfected with wild type [wt; (TAPwt)] or trun- binding, lysates were preincubated with a high affinity HLA-B51 cated (1-2DN) TAP had similar MHC I surface levels, in the latter peptide ligand (QPRAPIRPI) before incubation at 37˚C. As can be case allowed for by a functional pPC-dependent post-ER stabili- seen from Fig. 3A (for quantification, see Fig. 3B), the pretreatment zation mechanism (3). As shown in Fig. 2C, additional furin with peptides lead to a significant stabilization of HLA-B51 in T2 knockdown in T2(1-2DN) did not alter MHC I expression, sug- (1-2DN), T2(1-2DN/FUR↓) and T2(1-2DN/PC7↓). Thus, all T2- gesting that furin is unlikely to be necessary for the proper opera- transfectants expressing the TAP variant 1-2DN have a comparable tion of this mechanism. In striking contrast, however, PC7 defect in the intracellular formation of stable MHC I peptide knockdown caused a substantial reduction in the HLA-B51 steady- complexes. state surface levels in the context of 1-2DN-expression, (Fig. 2C, We next compared the thermostability of intracellular to that of left panel). This reduction was specific, because transferrin receptor cell surface-HLA-B51 complexes in intact cells. Because assem- was expressed equally on all cell lines (Fig. 2C, right panel), in- bled MHC I molecules have a much higher heat resistance under Downloaded from dicating an essential role for PC7 in the post-ER rescue of unstable physiologic conditions than in detergent extracts (3, 25), trans- MHC I molecules. Moreover, we note that we also observed sub- fectants were heat-shocked for 10 min at 60˚C to disrupt unstable stantial downmodulation of HLA-B51 surface expression in T2(1- MHC-peptide complexes or were left untreated. To determine the 2DN) cells via transient transfection with either of three unrelated thermostability of intracellular HLA-B51, cells were first fixed, PC7-targeting siRNAs (data not shown), further underscoring the permeabilized, and immunostained with the conformation-sensi-

importance of and specific requirement for PC7 in the rescue of tive Ab 4E (Fig. 3C, left column), whereas cells were directly http://www.jimmunol.org/ unstable HLA-B51. immunostained (Fig. 3C, right column) for flow cytometry for the To further corroborate this finding, we pharmacologically analysis of surface-HLA-B51. inhibited pPCs in all above cell lines using the cell-permeable As expected from our previous studies (3) T2(TAPwt) is charac- peptide HDA, which efficiently blocks both furin and PC7 with an terized by a high thermostability of intracellular HLA-B51 at 60˚C, inhibition constant (Ki) of 106 and 1875 nM, respectively (24). In whereas MHC I complexes in T2(1-2DN), T2(1-2DN/FUR↓) and our previous studies we observed that HDA treatment specifically T2(1-2DN/PC7↓) disintegrated to a drastic extent under these impaired MHC I surface expression in T2(1-2DN), but not in T2 conditions (60–70% reduction of the intracellular immunostain- (TAPwt) cells, consistent with a requirement for post-ER stabili- ing). However, despite the abundant population of largely fragile zation of MHC I only in the former cell line (Fig. 2D) (3). In intracellular MHC I molecules, we found that T2(1-2DN) harbors by guest on September 30, 2021 keeping with our observation that furin is not required for this highly stable surface-HLA-B51, in fact similar in stability to that process to occur, the furin-silenced cell line T2(1-2DN/FUR↓) fully found for T2(TAPwt) (Fig. 3C, right column) (3). A comparably retained the sensitivity to the drug observed in the mother cell line. high thermostability of surface-HLA-B51 was also observed for the However, HDA did not further affect (the already at steady-state furin-silenced cell line T2(1-2DN/FUR↓). However, most in- low) MHC I surface levels on T2(1-2DN/PC7↓) cells, suggesting terestingly, surface MHC I in T2(1-2DN/PC7↓) cells readily dis- that it is indeed the target of the drug that is downregulated in this integrated in response to the heat shock, indicating that PC7 is cell line (Fig. 2D). We additionally note that, because HDA has no essential to allow the process of post-ER stabilization to occur. To effect on HLA-B51 levels in T2(TAPwt) cells (Fig. 2D) (3), PC7 is control the specificity of our observation for T2(1-2DN/PC7↓) unlikely to have a general effect on loading or trafficking of MHC I cells, we performed a second independent PC7 knockdown ex- in T2-derived cell lines, but is only specifically required in cells that periment with T2(1-2DN) cells in which we used siRNA oligonu- fail to generate stable MHC I in the ER. We provide both genetic cleotides targeting a sequence of the PC7 gene different from that of and pharmacological evidence that PC7 is required, whereas furin the PC7-shRNA construct (Fig. 3D; Materials and Methods). The is dispensable for efficient post-ER rescue of unstable MHC I. effective and specific knockdown of PC7 by siRNA was verified by Western blot experiments with lysates from T2(1-2DN) cells that Stability of surface MHC I is severely compromised in were transfected or not with PC7 or control siRNA (Fig. 3D, left T2(1-2DN) transfectants lacking PC7 panel). In agreement with the results shown in Fig. 3C, we observed Our previous studies (3) demonstrated that the MHC I molecules that PC7 siRNA silenced T2(1-2DN) cells are characterized by that formed in the cell line T2(1-2DN) were initially unstable, but thermolability of intracellular and surface-expressed HLA-B51, gained stability after export from the ER to finally accumulate at whereas in contrast the control siRNA transfectants possess the the cell surface as highly stable molecules. Hence, loss of surface same high thermostability of surface MHC I as the nontransfected MHC I in the PC7-silenced cells would likely be the consequence T2(1-2DN) cells (Fig. 3D, middle and right panels). of a deficient stabilization process normally operating along the Interestingly, improved surface-over-intracellular stability of secretory route. HLA-molecules was also observed in LCL 721.220 cells To address this directly, we performed thermostability experi- expressing tapasin-C95A (data not shown), suggesting that post-ER ments that make use of the established correlation between heat rescue of fragile MHC-peptide complexes occurs independently of resistance of MHC I and the affinity of its peptide cargo (3, 4). To this what particular molecular mechanism actually generates these end, lysates of the different T2-transfectants were incubated for 1 h unstable MHC I molecules. Consistent with the concept of PC7 at 4˚ or 37˚C before intact assembled MHC I molecules were im- being responsible for this phenomenon, we found PC7 to be muno-isolated with the HLA-B–specific conformation-sensitive expressed in LCL 721.220 cells by RT-PCR (data not shown). Im- mAb 4E (21). In accordance with our earlier findings (3), HLA-B51 portantly, we note that this finding is consistent with the results of 2990 FUNCTIONAL ROLE OF PC7 IN ANTIGEN PRESENTATION Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 3. Thermostability of MHC I molecules in T2(1-2DN) cells silenced for furin or PC7. A, Lysates of T2 transfectants were pretreated or not with 10 mM HLA-B51-binding peptide (QPRAPIRPI) and incubated for 1 h at 4˚ or 37˚C. Thermostability of HLA-B51 was analyzed by immunoprecipitation of assembled MHC I using the conformation-sensitive Ab 4E. Precipitates were analyzed in Western blots probed with the Ab 3B10.7. B, The levels of precipitated MHC I H chain were quantitated from the Western blot by densitometric scanning and peak integrals were plotted as bar graphs in arbitrary units. C, Thermostability of intracellular and cell surface MHC I. Transfectants were collected in PBS/0.1% NaN3 and heat shocked at 60˚C for 10 min. Assembled intracellular (left column) or surface (right column) HLA-B51 was immunostained with the anti-MHC I Ab 4E and analyzed by flow cytometry. Background staining was analyzed by incubating with secondary Ab alone (unfilled histograms). Data are representative of two independent experiments. D, Effect of siRNA mediated silencing of PC7 on the thermostability of intracellular and surface MHC I of T2(1-2DN) cells. To knock down PC7 ex- pression, siRNA specific for PC7 were transfected into T2(1-2DN). As a negative control, AllStars Negative Control siRNA (Qiagen) was used. To verify the knockdown of PC7 cell lysates of T2(1-2DN) and the siRNA transfectants were analyzed by Western blot using the indicated Abs (left panel). Cells from the same knockdown experiment were collected in PBS/0.1% NaN3 and heat shocked for 10 min. Assembled intracellular (middle panel, left column) or surface (middle panel, right column) HLA-B51 was immunostained with the anti-MHC I Ab 4E and analyzed by flow cytometry (middle panel). Data are representative of three independent experiments (see histogram, right panel). The Journal of Immunology 2991

Williams et al. (4), who demonstrated HLA-Bp2705 expressed at presence or absence of MHC I-binding peptides. Thermoresistant the cell surface of tapasin-deficient LCL 721.220 cells to be vastly MHC I was subsequently immuno-isolated with conformation- more stable than its respective intracellular fraction, whereas sensitive mAb 4E and incubated with Endo H. This experimental when tapasin was coexpressed, intracellular HLA-Bp2705 was setup allows us to gain insight into how the thermostability of the highly stable. Altogether, this strongly suggests that rescue of pulse-labeled post-ER MHC I population develops over time. In suboptimally loaded HLA-molecules cannot only occur in dif- accordance with our earlier observations (3), MHC I molecules in ferent types of cells and in the context of different alterations to T2(TAPwt) cells had obtained full thermostability at the time when the PLC; it also can involve different HLA-alleles (e.g., HLA- they were exported from the ER. In contrast, in the cell lines T2(1- Bp5101, HLA-Bp2705). 2DN) and T2(1-2DN/FUR↓), the ER-exported MHC I was initially Combined with our data on steady-state surface-MHC I levels unstable, but then steadily enhanced its thermostability during post- and HDA sensitivity, this finding strongly suggests that PC7 is ER transport to the cell surface (Fig. 4A–C, compare black versus the pPC required for the rescue of unstable MHC I molecules gray bars). In contrast, in the case of T2(1-2DN/PC7↓), post-ER generated in the context of a PLC devoid of a proper quality MHC I molecules do not display such intracellular improvement of control function. stability (Fig. 4D, compare black versus gray bars), but seem to have an accelerated protein turnover. In support of this, pulse-chase Newly synthesized MHC I is largely routed to lysosomes for experiments that were performed in the presence of the lysoso- ↓ degradation in T2(1-2DN/PC7 ) cells motropic reagent chloroquine and stabilizing peptides revealed that To analyze the maturation and stabilization of MHC I complexes in T2(TAPwt), T2(1-2DN), and T2(1-2DN/FUR↓) only a minor directly during their intracellular transport to the surface, we per- fraction of MHC I molecules enters the chloroquine-sensitive Downloaded from formed comparative pulse-chase experiments with the different T2 pathway, whereas in T2(1-2DN/PC7↓) a substantial amount of transfectants (Fig. 4). To this end, cell extracts from different time post-ER MHC I routes to lysosomal degradation (Fig. 4D, compare points after pulse-labeling were heat-treated at 37˚C for 1 h in the gray versus white bars). http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 4. Analysis of intracellular MHC I pro- cessing in T2(TAPwt) (A), T2(1-2DN) (B) and T2(1- 2DN) transfectants silenced for furin (C) or PC7 (D). To analyze maturation and thermostability of MHC I, T2 transfectants were pulse-labeled for 30 min and chased for the indicated times in the presence and absence of chloroquine. Subsequently, cells were lysed with or without 10 mM peptide (QPRAPIRPI) and incubated for 1 h at 37˚C. Immunoprecipitates recognized by the Ab 4E were digested with Endo H and separated by SDS-PAGE. Autoradiography was performed, and four different exposures were taken and scanned using a densitometric scanner to ensure linearity. Obtained signals of MHC I heavy chains (post-ER) were quantitated, and the bands with the highest intensity were set to 100%. 2992 FUNCTIONAL ROLE OF PC7 IN ANTIGEN PRESENTATION

The kinetics of HLA-B51 maturation in the respective T2 rival of newly synthesized proteins at the cell surface. The decay of derivatives provide evidence that a PC7-dependent process rescues the pre-existing pool of surface MHC I was then analyzed by flow unstable MHC I from lysosomal degradation and instead allows this cytometry. As can be seen in Fig. 5A, the survival of surface HLA- population to acquire optimal high affinity peptide ligands in a post- B51 is almost identical among T2(TAPwt), T2(1-2DN) and T2(1- ER compartment. 2DN/FUR↓). BFA treatment for 16 h reduced the mean fluores- cence intensity (MFI) of surface-stained MHC I in these three cell Surface HLA-B51 in T2(1-2DN/PC7↓) cells presents an altered lines by ∼40%. In contrast, in T2(1-2DN/PC7↓) cells the MFI was peptide repertoire reduced by .65%, showing that HLA-B51 indeed turned over ↓ Our experiments in Figs. 3 and 4 show that, in T2(1-2DN/PC7 ) more rapidly in this cell line. cells, intracellular and surface MHC I are characterized by re- Furthermore, we analyzed whether the recovery of HLA-B51 markable physical lability and increased susceptibility to lysosomal surface levels after a short acid treatment (to remove pre-existing degradation. Thus, a higher turnover rate of surface-HLA-B51 in extracellularly exposed MHC I) would be affected in T2(1-2DN/ T2(1-2DN/PC7↓) would be predicted. To test this hypothesis, the PC7↓). In these experiments, we observed similar kinetics for the different T2 transfectants were treated with BFA to inhibit the ar- reappearance of surface MHC I complexes in T2(TAPwt), T2(1- Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 5. Survival, recovery, and recycling of MHC I-peptide complexes on the surface of T2(1-2DN) cells silenced for furin or PC7. A, Survival of surface-MHC I. Transfectants were treated with BFA for 0, 2, 4, 8, and 16 h before MHC I surface levels were assessed by flow cytometry using the Ab 4E. Grading of the obtained histograms from light gray (0 h) to black (16 h) indicates the different incubation times (left panel). Results are presented as a percentage of reduction in MFI at different hours compared with the MFI at 0 h (right panel). B, Surface recovery of MHC I. Cells were acid washed to remove assembled surface MHC I and then incubated at 37˚C for 0, 1, 2, and 4 h. Levels of surface HLA-B51 were determined by flow cytometry using the mAb 4E. Grading of the obtained histograms from light gray (0 h) to dark gray (4 h) indicates the different incubation times. Black histograms represent MHC I surface staining of untreated control cells (left panel). Results are depicted as percentage of increase in MFI at different hours compared with the MFI of untreated transfectants (right panel). C, Localization of MHC I in endolysosomal compartments. T2(TAPwt), T2(1-2DN), T2(1-2DN/FUR↓), and T2(1-2DN/PC7↓) were fixed, permeabilized, and stained with Abs specific for HLA-B51, EEA1 (marker of early endosomes) or Lamp1 (marker of late endosomes/lysosomes). Depicted are overlays of immunofluorescence stainings of HLA-B51 (green)/EAA1 (red) (upper row) and HLA-B51 (green)/ Lamp1 (red) (lower row). Nuclei are visualized by DAPI (blue). Scale bars indicate 10 mm. To quantify the degree of relative colocalization, we obtained image data from 20–30 cells as described in Materials and Methods. D, Endocytic recycling of MHC I. Expression of surface HLA-B51 in the presence or in the absence of primaquine was analyzed by flow cytometry. Results are plotted as percentage of reduction of the MFI compared with the MFI of the control experiments. The depicted histogram summarizes the results of three independent experiments. The Journal of Immunology 2993

2DN), and T2(1-2DN/FUR↓). Strikingly, in the case of T2(1-2DN/ reduction of surface MHC I is much less pronounced (12%) for T2(1- PC7↓) cells, the recovery remained substantially below that of the 2DN/PC7↓) cells. We speculate this to reflect the higher degree of other T2 transfectants, demonstrating that PC7 is essential for disintegration of MHC I (i.e., the irreversible dissociation of MHC H a normal accumulation rate of MHC I at the plasma membrane in chain and b2-microglobulin) in T2(1-2DN/PC7↓) cells, because in these cells (Fig. 5B). In line with our data from Fig. 4, we interpret this study the PC7-dependent rescue mechanism does not act to this delay to reflect the loss of suboptimally loaded MHC I by ly- preserve suboptimally loaded HLA-B51. Our findings in Fig. 5C and sosomal degradation. To explore whether post-ER MHC I molecules 5D suggest that in T2(1-2DN/PC7↓) cells an increased amount of are enriched in endolysosomal compartments of T2(1-2DN/PC7↓) post-ER HLA-B51 is enriched in endolysosomal compartments, and cells, we analyzed colocalization of HLA-B51 with early endosomes these molecules enter the endosomal recycling pathway to the and late endosomes/lysosomes by immunofluorescence microscopy plasma membrane, to a smaller extent. (Fig. 5C). T2(TAPwt), T2(1-2DN), T2(1-2DN/FUR↓), and T2(1- Peptide exchange along the exocytic pathway is likely to underlie 2DN/PC7↓) were permeabilized and stained with Abs specific for the observed stabilization of MHC I in T2(1-2DN) cells. Thus, the HLA-B51, EEA1 (marker of early endosomes) or Lamp1 (marker of presented peptide repertoire would be predicted to differ signifi- late endosomes/lysosomes). In the case of T2(TAPwt), T2(1-2DN), cantly between T2(TAPwt) and T2(1-2DN) on the one hand and and T2(1-2DN/FUR↓), HLA-B51 showed an overlap with early between T2(1-2DN) and T2(1-2DN/PC7↓) on the other hand. To endosomes and late endosomes/lysosomes in the range of 3% address this issue directly, peptides were acid eluted from im- (EEA1) and #1% (Lamp1), respectively. In contrast, the values of munopurified surface-HLA-B51-complexes (Fig. 6A) and their colocalization of HLA-B51 with EAA1 and Lamp1 containing spectra were analyzed by MALDI-MS (Fig. 6B). Comparison of the Downloaded from compartments are up to 3-fold higher in T2(1-2DN/PC7↓), sug- set of isolated peptides was conducted on the basis of their mo- gesting an increased endolysosomal localization of post-ER MHC I lecular mass in the range of 800–1350 Da (Fig. 6B, I–IV) and in the absence of PC7 (Fig. 5C). To assess whether internalization controlled by a mock elution experiment (Fig. 6B, V). The spectra from the cell surface and retention or degradation in endosomes or show that the overall distribution of masses is comparable for T2 lysosomes leads to the enhanced loss of surface-expressed HLA-B51 (TAPwt), T2(1-2DN), T2(1-2DN/FUR↓), and T2(1-2DN/PC7↓) in T2(1-2DN/PC7↓) cells, we analyzed MHC I surface expression in (Fig. 6B, I-IV). However, in the spectra derived from T2(1-2DN) the presence or in the absence of primaquine (Fig. 5D), which in- and T2(1-2DN/FUR↓) cells (Fig. 6B, II and III), which are very http://www.jimmunol.org/ hibits recycling of endocytosed proteins including MHC I back to the much related, a collection of additional peptide peaks (signals: a–l) cell surface. As seen in Fig. 5D, T2(TAPwt), T2(1-2DN), and T2(1- in the range of 1032–1298 Da are present (Fig. 6B, II and III, in- 2DN/FUR↓) cells show up to 28% reduction of surface-exposed dicated by arrows), which are clearly missing or strongly reduced in HLA-B51 in the presence of primaquine, whereas the drug-mediated the profile of T2(TAPwt) cells (Fig. 6B, I). In turn, only few of the by guest on September 30, 2021

FIGURE 6. Profile of MHC I peptide complexes on the surface of T2(1-2DN) cells silenced for furin or PC7. MALDI-MS profiles of self peptides eluted from surface HLA-B51 immunopurified with 4E Ab from 2 3 107 T2(TAPwt) (I), T2(1-2DN) (II), T2(1- 2DN/FUR↓)(III) and T2(1-2DN/PC7↓)(IV) cells, respectively. As a control, 4E-conjugated sepharose beads were acid eluted (V). A, Successful isolation of surface HLA-B51 was controlled by Western blot analysis (left panel), and the obtained MHC I signals were quantified by densitometric scanning (right panel). Four different exposures were taken to ensure linearity. The obtained MHC I signals from T2 (TAPwt) were set to 100%. B, The mass spectra were visualized and analyzed using flexAnalysis software. Data were normalized to the relative amount of im- munoisolated HLA-B51 molecules. Spectra are rep- resentative of two independent peptide preparations. Differential peptide peaks (signals: a–z) are indicated by arrows (newly appearing signals), asterisks (re- duced/disappearing signals), and boxes (increased signals). 2994 FUNCTIONAL ROLE OF PC7 IN ANTIGEN PRESENTATION peaks (signals: u–w) of the T2(TAPwt) spectrum were undetectable independent of cytosol and peptide transporter (35–37). Most in- within the profiles of T2(1-2DN) and T2(1-2DN/FUR↓). We note terestingly, studies on TAP-negative APCs suggested that pro- that this partial overlap of the peptide spectra is expected, as the loss cessing of exogenous peptides for presentation by MHC I involves of quality control function in the PLC will not abolish the occa- post-Golgi peptide exchange (38). However, whether and to what sional binding of abundant high-affinity peptides in the ER. In fact, extent pPCs are involved in this pathway is not known. To analyze only the bias in loading toward such ligands would be predicted to the involvement of PC7 in the generation of MHC I peptide com- be impaired. However, the additional peaks in the spectra of T2(1- plexes, radioiodinated peptide substrates with or without pPC 2DN) and T2(1-2DN/FUR↓) strongly suggest that in this study cleavage site (RRRR-gB and RRAR-gB) were incubated with the a certain population of HLA-B51 molecules is stably loaded with different T2 transfectants for 4 h at 30˚C in FCS-free medium. For peptide ligands that apparently do not traverse, or are ignored by, the isolation of MHC I molecules, cells were extensively washed the MHC I pathway in T2(TAPwt). Hence, they are likely to rep- and lysed in n-octylglucoside. Lysates were incubated in the cold resent the mass-spectrometric signature of peptide-exchange–me- with 4E Ab coupled to CNBr-activated sepharose. Immuno-iso- diated post-ER stabilization of MHC I. lated complexes were eluted, and the amount of radioactive pep- Upon comparing the peptide peaks of the different T2(1-2DN) tides bound to immuno-isolated MHC I was determined by transfectants, we noted that about half of the peptide population that g-counting. As seen in Fig. 7, significant amounts of radiolabeled was specifically detected for T2(1-2DN), and T2(1-2DN/FUR↓) peptide bound to MHC I could be detected when the T2 trans- disappears from the T2(1-2DN/PC7↓) spectrum (signals: a–c and f– fectants were incubated with the pPC-cleavable peptide RRRR-gB j; Fig. 6B, IV), and that some of the prominent peaks of T2(1-2DN) and not with the uncleavable peptide RRAR-gB. This finding im- and T2(1-2DN/FUR↓) show reduced (Fig. 6B, indicated by aster- plies that pPC cleavage is essentially required for the HLA-B51- Downloaded from isks) or increased signal strength (signals: x–z; Fig. 6B, indicated presentation of the gB-peptide. In the case of T2(1-2DN) and T2(1- by boxes) in the profile of T2(1-2DN/PC7↓). We also noted that, in 2DN/FUR↓), the amount of radiolabeled MHC I-bound peptides the range of 954–1174 Da, a new collection of prominent peptide was ∼3–4-fold higher when compared with the situation of the T2 peaks were visible (signals: m–t) that appear in neither T2(TAPwt) transfectant expressing TAPwt. This is expected because the stable nor T2(1-2DN) or T2(1-2DN/FUR↓), suggesting that in T2(1-2DN/ MHC generated in T2(TAPwt) cells is less likely to exchange its

PC7↓) cells the entry of HLA-B51 into the lysosomal pathway (Fig. high affinity ligands. Strikingly, the amount of radiolabeled pep- http://www.jimmunol.org/ 4) might also contribute to the formation of stable MHC I peptide tides bound by MHC I in T2(1-2DN/PC7↓) reached only #25% of complexes that escape to certain extent lysosomal degradation that found for T2(1-2DN) and T2(1-2DN/FUR↓), demonstrating (Figs. 4, 5) and are transported back to the cell surface. that PC7 is essential for efficient loading of an epitope that requires Our findings provide evidence that the absence of PLC-mediated prior pPC-mediated liberation from a precursor. quality control of MHC I loading and/or lack of PC7 causes the presentation of an altered MHC I peptide repertoire on the cell surface.

PC7 mediates the liberation of epitopes from exogenous peptide by guest on September 30, 2021 precursors WedecidedtoanalyzethecontributionofPC7toMHCIAgprocessing by a cell biologic assay, in which we used a defined 15-mer peptide of EBV glycoprotein B (EBVgB, GP110), which is a well-known target substrate of pPCs (26, 27). The EBVgB-peptide gB427-441 (LRRRRR↓DAGNATTPV, sequence in single-letter code) contains the pPC-cleavage motif—(R-X-K/R-R↓) confirmed by ProP 1.0 analysis (28)—conserved among most herpes virus gBs (29) (posi- tions 3–6, indicated in bold letters and by a down arrow at the cleavage site) as well as a nonameric peptide (downstream the cleavage site, positions 7–15, indicated by italics) with sequence properties of a high-affinity HLA-B51 binding ligand (SYFPEITHI 1.0-score: 24) (30). For our studies, alanine at position 11 of gB427-441 was re- placed by tyrosine to enable radioiodination of the pPC-substrate (RRRR-gB, LRRRRR↓DAGNYTTPV, replacement indicated by underlined letter). After SYFPEITHI 1.0 (30) and ProP 1.0 (28) FIGURE 7. PC7-mediated processing of exogenous oligopeptides con- analysis of RRRR-gB, we obtained results as good as for the original taining a suitable MHC I epitope downstream of a pPC cleavage site. peptide. For the control experiments, we used a related gB-peptide Radio-iodinated peptide substrates with or without pPC cleavage site (200 (RRAR-gB) in which the basic pPC-cleavage motif was destroyed by mM RRRR-gB and RRAR-gB) were added to the different T2-trans- replacement of arginine at position 5 with alanine (LRRRARDAG- fectants (5 3 107 cells) for 10 min at 10˚C after removal of surface MHC I NYTTPV, replacements indicated by underlined letter). by 3 min incubation with 50 mM citrate (pH 3) and subsequent neutrali- The experimental setup of our analysis is based on two obser- zation of pH. This step was followed by 4 h incubation at 30˚C in FCS-free vations. First, exogenous applied peptide derivates often used as medium. Afterward, cells were extensively washed and lysed in 1% n- substrate-based inhibitors reach pPC-containing cell compartments octylglucoside for 30 min. Lysates were incubated in the cold with 4E Ab coupled to CNBr-activated sepharose for 1 h and immuno-isolated com- (31). Second, T2 cells and T2 transfectants are able to process and plexes were eluted with 0.5% SDS. The amount of radioactive peptides present exogenous peptide substrates from fluid phase by MHC I bound to immuno-isolated MHC I was determined by g-counting (upper (32, 33) through a cellular pathway that is apparently independent panel), and the obtained values were normalized for the quantity of im- of TAP, but requires vesicular transport between secretory com- muno-isolated HLA-B51 molecules analyzed by Western blot (lower partments (34), indicating that such Ag substrates could serve in T2 panel). The depicted histogram summarizes the results of three in- cell lines as a source for an alternate MHC I presentation pathway dependent experiments. The Journal of Immunology 2995

Our findings demonstrate that in a situation in which the PLC 43–45) (some of the mentioned studies describe loading of pPC- quality control function fails, PC7 and not furin is involved in the dependent epitopes in the TGN during secretion, whereas loading is increased processing of exogenous MHC I-precursor peptides that thought to occur via endosomal recycling in others). Unfortunately, contain a pPC cleavage site in combination with a suitable most of these reports did not fully clarify (or did not address) which downstream MHC I epitope. particular pPCs was acting in their experimental system, and all above studies focused at the generation of particular epitopes, but Discussion not on bulk-loading of naturally occurring intracellular peptides. In Stability of the interaction of peptides with MHC I is highly relevant this context, it should be mentioned that none of the commercially for their suitability as an effective T cell epitope and in turn a suc- available pharmacologic pPC inhibitors displays sufficient speci- cessful immune response. To achieve this stability is the major ficity to convincingly discriminate between furin and PC7 activity. function of the PLC (1), and the amount of suboptimally loaded and Also, vaccinia virus-driven overexpression of pPCs used in some of structurally fragile MHC I drastically increases when this quality these studies (46) has been shown to be prone to cause cleavage control checkpoint does not work properly. Defective PLCs, which artifacts, which is an issue that may be particularly critical when T lack their quality control function are observed in cells expressing cell activation is used as a readout because few cleavage events mutants of tapasin or TAP or do not express these central con- might be fully sufficient to generate enough MHC-peptide com- stituents of the complex at all (2–4). Because tumors of diverse plexes to drive profound CTL-mediated target cell lysis. Conse- tissue origin tend to radically downmodulate or even completely quently, silencing by RNA interference is probably the most specific lose expression of tapasin or TAP, poor quality of MHC I loading is approach to assess functional differences between individual pPCs a physiologic scenario with potentially important immunologic when working with human cells. Downloaded from consequences for the pathology of cancer. Furthermore, recent We also note that the peptide-exchange mechanism we propose studies have revealed different examples of viral interference with provides an elegant explanation of why the peptides generated by the recruitment of MHC I into the PLC (7, 39), degradation or in- PC7 can stabilize HLA-B51 in T2(1-2DN) cells, whereas the ligands hibition of PLC components, or suppression of the quality control available in the ER obviously cannot. Certainly, it is highly unlikely function of the complex (5, 6). The formation of unstable MHC I that the pPC-generated peptide pool has a generally higher affinity molecules was observed in many of these studies (5–7), and even for MHC I as the peptides generated by the proteasome. Rather, it is http://www.jimmunol.org/ tapasin-independent alleles displayed enhanced thermolability in plausible to assume that it is the acidic environment in the TGN that the context of expression of a viral TAP inhibitor (4). Thus, the way confers the high stringency on MHC I loading and allows only op- timal peptide ligands to bind stably (i.e., not to dissociate). in which cells deal with a malfunctioning PLC (i.e., whether they However, such a pPC-dependent second checkpoint would have succeed in rescuing unstable MHC I and ensure the surface pre- to introduce relevant epitopes into the MHC I-presented repertoire sentation of relevant Ags) is likely to be an important factor in the to become immunologically significant. In this context, it is im- outcome of pathologic processes in vivo. portant to note that many viral proteins are processed by pPCs (e.g., Given this, a second backup mechanism along the secretory route diverse fusion proteins including those from influenza A virus,

aiming to recover MHC I molecules that carry low-affinity peptide by guest on September 30, 2021 respiratory syncytial virus, Ebola virus, severe acute respiratory cargo prone to dissociate, and thus of predictably low immunoge- syndrome virus, human CMV, hepatitis B virus, HIV) (13, 26, 47, nicity, would be intuitively plausible. Our finding that the rescue of 48). It is obvious that under conditions of an acute infection these unstable HLA-B51 occurs in a post-ER compartment [also by viral proteins will become highly abundant in the secretory Leonhardt et al. (3)] strongly suggests the underlying mechanism to compartments, and it has been shown that MHC I-presented viral involve peptide-exchange. The reason is that acid-catalyzed disso- Ags can originate from pPC-processed viral polypeptides (49). ciation of peptides and their reassociation with MHC I has been Thus, the pPC-dependent rescue of MHC I complexes could force observed at pH conditions corresponding to the increasingly acidic the surface presentation of important viral epitopes with signifi- environment along the endocytic and exocytic pathways (40–42). cant immunologic consequences. Because severe viral in- Peptide exchange, in turn, would be predicted to occur preferentially terference with the assembly and function of the PLC has been at predestabilized MHC I peptide complexes, thus explaining why observed (5–7, 50), this pPC-dependent pathway might allow for the pPC-dependent mechanism is so much more effective in T2(1- a functional rescue of MHC I-mediated Ag presentation and 2DN) than in T2(TAPwt) cells. Furthermore, the existence of such consequently lead to an enhanced clearance of infected cells. ↓ a mechanism, as well as its breakdown in T2(1-2DN/PC7 ) cells, is Thus, we propose that this alternative PC7-dependent pathway of also directly supported by the qualitative and quantitative changes in MHC I loading might be part of a cellular counter strategy that the MALDI-mass spectra characteristic for the four analyzed T2 undermines viral efforts to block the Ag presentation machinery. transfectants (Fig. 6B). Finally, our finding (based on pharmacologic Similar considerations may also apply for anticancer immune and genetic evidence) that this pathway essentially requires PC7 responses, because some tumor-associated Ags, such as gp100, are activity, argues that in T2(1-2DN) cells an already fragile MHC I also cleaved by pPCs (51). This should be considerd when pPC population acquires pPC-dependent ligands upon further acid de- inhibitors are used in the context of cancer therapies as suggested by stabilization in the late secretory compartments in which PC7 re- several laboratories, based on findings that pPCs promote tumor sides (23). Interestingly, the capacity of MHC I to bind peptides at growth and metastasis (52). However, we note that PC7 could also acidic pH corresponding to the TGN has been shown to vary among protect tumors from NK cell-mediated attack by securing high MHC different alleles, some of which even display superior binding at I levels despite low TAP or tapasin function in malignant cells. acidic versus neutral pH (29). Routing of HLA-B51 to lysosomes in One striking point is the magnitude of PC7-mediated MHC I T2(1-2DN/PC7↓) cells, however, obviously fails to efficiently res- rescue that we observed. To our knowledge, the contribution of no cue the molecules, which can be likely attributed to the substantially single protease in the MHC I pathway other than the proteasome (53) lower pH in these compartments preferring irreversible dissociation and ERAAP (54) has been reported to measurably enhance overall of MHC I rather than promoting peptide exchange. In addition, we MHC I surface levels, although many accessory proteases such as note that our model is in line with several reports that demonstrate TPP2 (55), leucine aminopeptidase (56), puromycin-sensitive that pPC-dependent peptides can be presented by MHC I (3, 8, 9, aminopeptidase (57), thimet oligopeptidase (58), and bleomycin 2996 FUNCTIONAL ROLE OF PC7 IN ANTIGEN PRESENTATION hydrolase (59) have been analyzed in knockout systems. This thus, rescue of unstable MHC I-complexes necessarily requires enormous potency strongly suggests that PC7-mediated MHC I further trimming of pPC-generated precursors. In particular, be- loading will make a substantial contribution to the repertoire of Ags cause pPCs cleave after paired basic amino acids and HLA-B51 presented in cells harboring malfunctioning PLCs as a consequence strongly prefers hydrophobic C-terminal anchor residues (62), PC7- of pathologic processes also in vivo. generated peptides would at least require C-terminal trimming. One of the most intriguing findings of this study is that only PC7 is Interestingly, recent studies suggest that carboxypeptidases might required for post-ER rescue of unstable MHC I, whereas furin serve for this function (43, 44), and at least carboxypeptidase D is appears to be completely dispensable. This is supported by the ubiquitously expressed and localized to late secretory compart- striking differences in stability (Figs. 3C,3D,5A) and steady-state ments (63). Future studies will be required to identify these down- levels (Fig. 2C) of surface-MHC I observed between T2(1-2DN/ stream proteases acting in the post-ER rescue of unstable MHC I- FUR↓) and T2(1-2DN/PC7↓). In fact, that these levels completely complexes. lose sensitivity toward the pPC inhibitor HDA in cells lacking PC7 The MHC I-loading experiments in Fig. 7, in which we used (Fig. 2D) underscores the exclusive involvement of this particular exogenous oligopeptides of a natural pPC target (EBVgB) con- pPC, whereas a practically complete furin knockdown is totally taining a suitable MHC I epitope downstream the proprotein neutral in this sense. Of note, HDA has a broad inhibitory capacity cleavage site, bypassed the need for further C-terminal substrate against most pPCs (including PC7), blocks furin activity efficiently trimming (e.g., carboxypeptidase D) and thus clearly created an in vivo, and actually has a .10-fold higher potency toward furin ideal model substrate situation for PC7 and MHC I. Nevertheless, than toward PC7 (24). Further corroborating the selective re- our studies revealed that the observed increased MHC I loading of quirement for PC7, but not furin, more than half of the peptide processed ligands in T2 transfectants with defective PLC is es- Downloaded from peaks that are specifically detected in the T2(1-2DN)- and T2(1- sentially dependent on PC7 and not on furin. In accordance with our 2DN/FUR↓)-derived peptide spectra disappear from the respective other findings, this strongly supports the idea that PC7 is able to spectrum of T2(1-2DN/PC7↓) (Fig. 6B). However, given the highly produce stable MHC I ligands directly from appropriate precursor overlapping cleavage specificities between all pPCs (26), the re- substrates and could function as one of the main executers within stricted requirement for PC7 in the process is surprising. Because it this pathway. Although several studies described MHC I loading of

is unlikely that furin and PC7 cleave radically different subsets of exogenous peptide substrates in T2 cells (32–34), it is not clear http://www.jimmunol.org/ pPC-substrates, many of which they actually share, the functional where exogenous ligands are processed and gain access to MHC I. differences between these two pPCs in the context of Ag pre- One of the possible scenarios that have been discussed allows sentation probably have different reasons. We nevertheless note processing of exogenous substrates and peptide binding to MHC I that several polypeptides that get cleavage-activated by pPCs are in post-Golgi compartments (64). Therefore, it is tempting to proteases themselves (e.g., matrix metalloproteases or carbox- speculate that for exogenous oligopeptides that fit the substrate ypeptidases) (10), and thus high substrate selectivity even in requirements of pPCs, PC7 could play an important role in this a limited number of cases might drive dramatic changes in the supplemental pathway, which seems to work much more efficiently proteolytical environment that MHC I traverses on its way to the under conditions in which the PLCs are unable to generate stable plasma membrane. Indeed, toward some substrates profound dif- MHC I peptide complexes. by guest on September 30, 2021 ferences between furin- and PC7-activity have been described (60). Interestingly, PC7 is known to display a widespread distribution We cannot exclude that such subtle differences in cleavage speci- in different organs and cell types (12), including expression in ficity then cause a drastic change in the pattern of activated pro- professional APCs such as dendritic cells and macrophages (65; teases along the exocytic route and eventually allow or do not allow data not shown). The PC7-dependent rescue pathway may thus be for the efficient generation of alternative epitopes for unstable a ubiquitous backup module present in all cells, ready to handle MHC I. In this scenario, PC7 but not furin would have the function otherwise detrimental attacks of viral effectors against PLC-me- of a master switch, specifically activating downstream effector- diated MHC I loading. proteases for the rescue process to occur. In view of our findings shown in Fig. 5, it might also be possible that PC7 but not furin Acknowledgments promotes MHC I recycling back to the cell surface from early en- We thank Dr. Meyers for helpful comments on the manuscript and Drs. dosomes, preventing deeper entry into the endocytic route. Cresswell and Urade for supplying rabbit anti-tapasin (R.gp48N) and rabbit Alternatively, it could be a spatial separation of furin and PC7 that anti-ER60 antiserum. underlies the functional differences of these two pPCs in Ag pre- sentation. In accordance with the results of our immunofluorescence Disclosures experiments depicted in Fig. 1D, studies by Wouters et al. (23) The authors have no financial conflicts of interest. suggest that the mature active forms offurin and PC7 display distinct subcellular distributions, with furin predominantly residing in the TGN and PC7 being localized to TGN-derived secretory vesicles. References Most interestingly, secretory vesicles budding from the trans-cis- 1. Wright, C. A., P. Kozik, M. Zacharias, and S. Springer. 2004. Tapasin and other ternae of the Golgi have been shown to be more acidic than the trans- chaperones: models of the MHC class I loading complex. Biol. Chem. 385: 763– Golgi in islet cells, and these differences in pH correlate with the 778. 2. Tan, P., H. Kropshofer, O. Mandelboim, N. Bulbuc, G. J. Ha¨mmerling, and processing of a pPC substrate (61). Hence, it is possible that only the F. Momburg. 2002. Recruitment of MHC class I molecules by tapasin into the PC7-containing late secretory compartments provide the pH neces- transporter associated with antigen processing-associated complex is essential sary to catalyze efficient peptide exchange in HLA-B51, whereas for optimal peptide loading. J. Immunol. 168: 1950–1960. 3. Leonhardt, R. M., K. Keusekotten, C. Bekpen, and M. R. Knittler. 2005. Critical furin-containing compartments do not. Alternatively, PC7-containing role for the tapasin-docking site of TAP2 in the functional integrity of the MHC secretory vesicles may be the superior environment for generation class I-peptide-loading complex. J. Immunol. 174: 5104–5114. and/or survival of MHC I ligands, because of the presence or ab- 4. Williams, A. P., C. A. Peh, A. W. Purcell, J. McCluskey, and T. Elliott. 2002. sence of downstream proteases further processing the prodomains Optimization of the MHC class I peptide cargo is dependent on tapasin. Im- munity 16: 509–520. liberated by pPCs. This is an important issue, because MHC I-bound 5. Park, B., Y. Kim, J. Shin, S. Lee, K. Cho, K. Fru¨h, S. Lee, and K. Ahn. 2004. peptides are much shorter in length than most of these prodomains; Human cytomegalovirus inhibits tapasin-dependent peptide loading and The Journal of Immunology 2997

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