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Differences in the Expression of Human Class I MHC Alleles and Their Associated Peptides in the Presence of Proteasome Inhibitors

This information is current as Chance John Luckey, Jarrod A. Marto, Megan Partridge, Ed of September 26, 2021. Hall, Forest M. White, John D. Lippolis, Jeffrey Shabanowitz, Donald F. Hunt and Victor H. Engelhard J Immunol 2001; 167:1212-1221; ; doi: 10.4049/jimmunol.167.3.1212

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Differences in the Expression of Human Class I MHC Alleles and Their Associated Peptides in the Presence of Proteasome Inhibitors1

Chance John Luckey,2* Jarrod A. Marto,2† Megan Partridge,* Ed Hall,‡ Forest M. White,† John D. Lippolis,* Jeffrey Shabanowitz,† Donald F. Hunt,†§ and Victor H. Engelhard3*

We have studied the contributions of proteasome inhibitor-sensitive and -insensitive proteases to the generation of class I MHC- associated peptides. The cell surface expression of 13 different human class I MHC alleles was inhibited by as much as 90% or as little as 40% when cells were incubated with saturating concentrations of three different proteasome inhibitors. Inhibitor- resistant class I MHC expression was not due to TAP-independent expression or preexisting internal stores of peptides. Further-

more, it did not correlate with the amount or specificity of residual proteasome activity as determined in in vitro proteolysis assays Downloaded from and was not augmented by simultaneous incubation with multiple inhibitors. Mass spectrometry was used to directly characterize the peptides expressed in the presence and absence of proteasome inhibitors. The number of peptide species detected correlated with the levels of class I detected by flow cytometry. Thus, for many alleles, a significant proportion of associated peptide species continue to be generated in the presence of saturating levels of proteasome inhibitors. Comparison of the peptide-binding motifs of inhibitor-sensitive and -resistant class I alleles further suggested that inhibitor-resistant proteolytic activities display a wide diversity of cleavage specificities, including a trypsin-like activity. Sequence analysis demonstrated that inhibitor-resistant peptides http://www.jimmunol.org/ contain diverse carboxyl termini and are derived from protein substrates dispersed throughout the cell. The possible contributions of inhibitor-resistant proteasome activities and nonproteasomal proteases residing in the cytosol to the peptide profiles associated with many class I MHC alleles are discussed. The Journal of Immunology, 2001, 167: 1212–1221.

he recognition of antigenic peptides in association with duction of known epitopes (8–10). Finally, treatment of cells with class I MHC molecules at the cell surface provides a various inhibitors of proteasome activity leads to a significant de- ϩ mechanism by which CD8 T lymphocytes gain infor- crease in presentation of several peptide epitopes as well as re- T b mation about the proteins being made within cells. In most cells, duced surface expression of H2-K and HLA-A*0201 (2). These by guest on September 26, 2021 class I-associated peptides are derived from endogenously ex- inhibitors also diminish the ability of several human class I mol- pressed intracellular proteins and are between 8 and 11 aa long (1). ecules to form stable dimers (11, 12). The enzyme complex most often implicated in the generation of Despite this breadth of evidence, other studies have suggested class I- associated peptides is the proteasome (2). Proteasome in- that proteasome involvement in class I- restricted peptide produc- volvement in class I peptide production is based on several lines of tion is more limited (13). In particular, it has been suggested that evidence. First, the expression of the catalytically active protea- ubiquitination is not involved in the production of at least some ␤ some subunits LMP2 and LMP7, as well as the proteasome class I-associated peptides (14). More directly, cell surface expres- regulator PA28, have been shown to enhance the production of sion of two murine class I alleles is only partially blocked by class I-associated peptides in vivo and in vitro (3–5). Second, ubiq- proteasome inhibitors (15, 16). Furthermore, the ability of two uitination, which targets proteins for proteasome-mediated degra- human class I molecules to form stable dimers, a property thought dation, is required for generation of class I- associated epitopes to depend upon peptide availability for binding, was not blocked from some proteins (6, 7). Third, incubation of whole proteins or when proteasome activity was inhibited (17). A few specific TAP- synthetic polypeptides with proteasomes in vitro results in the pro- dependent epitopes have also been shown to be either insensitive to proteasome inhibition (15, 18) or destroyed by proteasome ac- *Department of Microbiology and Carter Immunology Center, University of Virginia tivity (19). Finally, cells grown for extended periods in proteasome Health Sciences Center, Charlottesville, VA 22908; and Departments of †Chemistry and ‡Information Technology and Communication, Research Computing Support inhibitors continue to express stable murine class I dimers (20). Group, University of Virginia, Charlottesville, VA 22901; and §Department of Collectively these studies strongly suggest that both proteasomes Pathology, University of Virginia, Charlottesville, VA 22904 and nonproteasomal proteases can independently generate class Received for publication November 13, 2000. Accepted for publication May I-associated peptides. 18, 2001. Neither the relative contributions of different proteolytic path- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ways to class I expression, nor the sequence and protein source of with 18 U.S.C. Section 1734 solely to indicate this fact. the peptides they produce, are well understood. Here, we have 1 This work was supported by U.S. Public Health Service Grants AI 20963 and AI combined proteasome inhibitors, flow cytometry, and mass spec- 21393 (to V.H.E.), and AI33993 (to D.F.H.). C.J.L. was supported by Medical Sci- trometry to address these issues. Cells treated with acid to remove entist Training Program Grant GM 07267. surface class I peptide complexes were allowed to re-express 2 C.J.L. and J.A.M. contributed equally to this work. newly formed complexes in the presence or absence of proteasome 3 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, VA inhibitors. We then used flow cytometry to measure total class I 22908. E-mail address:[email protected]. surface re-expression and mass spectrometry to analyze the peptides

displayed under these conditions. Our results provide insight into both on 1 ϫ 106 cells as described above. The majority of cells were washed the relative contribution as well as the specificity of proteasome in- twice in PBS at 4°C, snap frozen in liquid nitrogen, and stored at Ϫ80°C hibitor-insensitive proteases that generate class I-associated peptides. for later use. Frozen cell pellets were then resuspended at a concentration of 1 ϫ 108 cells/ml in 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1- propane sulfonate, 20 mM Tris-HCl pH 8.0, 100 ␮M iodoacetamide, 5 Materials and Methods ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin A, 5 mM EDTA, Inhibitors 0.04% sodium azide, and 1 mM PMSF (lysis buffer; all from Boehringer Mannheim, Indianapolis, IN). After rocking for1hat4°C, lysates were 4 Lactacystin (LAC) (Calbiochem, La Jolla, CA) is a Streptomyces metab- clarified by centrifugation at 100,000 ϫ g for 1 h. Lysates were precleared olite that irreversibly inhibits proteasomes via covalent binding to the cat- for4hat4°Cwith recombinant protein A-Sepharose beads (Pharmacia, alytic sites of the ␤ subunits (21, 22). N-acetyl-L-leucinyl-L-leucinal-L-nor- Piscataway, NJ). Class I-specific Ab-saturated recombinant protein leucinal (LLnL; Calbiochem), also known as calpain Inhibitor I, reversibly A-Sepharose beads were then added to lysates for 12 h at 4°C. Beads were inhibits proteasomes as well as several other classes of proteases (23). spun down and lysate was removed to a separate tube. Beads saturated with Carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (z-L3VS) was a gener- Abs of other class I specificities could then be added sequentially to purify ous gift from Dr. H. Ploegh (Harvard University, Cambridge, MA) and has additional alleles. Beads were washed twice in lysis buffer, twice in 50 mM been shown to specifically inhibit proteasomes by binding to all of the Tris-HCl/1 M NaCl (pH 8.0), twice in 50 mM Tris-HCl/250 mM NaCl (pH known active sites and blocking all known in vitro measures of the pro- 8.0), and three times in 50 mM Tris-HCl (pH 8.0). Beads were then trans- teasome function (24). Brefeldin A (BFA; Sigma, St. Louis, MO) inhibits ferred in 50 mM Tris-HCl to the top insert of an Ultra-free-MC 5000 egress of all proteins through the secretory pathway at the level of the NMWL filter from Millipore (Bedford, MA) and excess buffer was spun cis-Golgi, including newly generated class I peptide complexes (25). through the filter. Filter insert was then moved into a Teflon tube (Savillex, ␤ Minnetonka, MN). Peptides were eluted from class I/ 2-microglobulin/Ab Cell lines and washed through the filter with 10% acetic acid. Downloaded from All tumor lines were of human origin and were maintained in RPMI 1640 Mass spectrometric data acquisition medium supplemented with 5% FCS containing SerXtend (Irvine Scien- tific, Santa Ana, CA) and 2 mM glutamine (cell medium) in a humidified Mass spectrometric data were acquired on a home-built Fourier transform 5% CO2 atmosphere at 37°C. The B lymphoblastoid cell line 721 and the ion cyclotron resonance mass spectrometer (FTMS) (34) equipped with a TAP mutant line 721.174 have been described previously (26, 27). Stable nano-HPLC microelectrospray ionization source. Nano-HPLC columns transfectants of the class I-A and class I-B locus-negative cell line C1R were constructed of 50-␮m inside diameter fused silica and packed with with human HLA-A or -B molecules were maintained in cell medium ϳ8cmof5-␮m diameter reverse-phase beads. An integrated micro-ESI ␮ ␮ containing either 300 g/ml G418 or 300 g/ml Hygromycin. Various emitter tip (ϳ1-␮m diameter) was located a few millimeters from the col- http://www.jimmunol.org/ C1R transfectants were generous gifts from Dr. P. Cresswell (Yale Uni- umn bed. Typically, 0.3% of a given class I peptide extract (ϳ3 ϫ 106 cell versity, New Haven, CT), Dr. A. McMichael (John Radcliffe Hospital, equivalents) was loaded onto a column and eluted directly into the mass Oxford, U.K.), Dr. R. Colbert (Children’s Hospital Medical Center, Cin- spectrometer with a linear 30-min gradient of 0–70% acetonitrile in 0.1% cinnati, OH), or were produced by this laboratory (28). acetic acid. Full scan mass spectra, over a mass-to-charge (m/z) range 300 Յ m/z Յ 2500, were acquired at a rate of 1 scan/s. After acquisition, Acid treatment and flow cytometry data were saved in the NetCDF format and imported into Matlab v5.3 Cells (1–2 ϫ 106) were centrifuged and the resulting pellet was resus- technical computing software (The Mathworks, Natick, MA) using the pended gently in 50 ␮l of 300 mM glycine (pH 2.5)/1% (w/v) BSA (acid NetCDF Toolbox (publicly available from Dr. Charles R. Denham, U.S. wash) and incubated for 4 min at 37°C. The suspension was neutralized by Geological Survey, Woods Hole, MA 02543). m/z and scanline coordinate dilution with 100 ␮l of cell medium containing 0.5 N NaOH and 0.2 M data were superimposed on a rectangular grid. Line/peak intensity data by guest on September 26, 2021 HEPES and centrifuged. Cells were resuspended into 200 ␮l of cell me- artifacts were displayed and removed by setting the intensity values for the dium in the presence or absence of 10 ␮g/ml BFA or various concentra- corresponding grid coordinates to zero. The filtered data were then saved in the CDF format using the Matlab NetCDF Toolbox and imported into tions of LAC, LLnL, or z-L3VS and incubated for5hat37°C to allow class I re-expression. In some experiments, cells were incubated for2hat Finnigan Xcalibur (Finnigan, San Jose, CA) for further analysis and 37°C in the presence or absence of inhibitors before acid treatment and display. further incubation with inhibitors. Cells were subjected to flow cytometry Tandem mass spectrometry (MS/MS) data acquisition analysis as described earlier (19). Live cells were gated, 10,000 events were counted, and the mean fluorescence intensity was recorded. MS/MS data were acquired on a Finnigan LCQ quadrupole ion trap mass spectrometer (Finnigan) equipped with a nano-HPLC micro-ESI source as Antibodies described above. Typically, 4% of a given class I peptide extract (ϳ4 ϫ 107 cell equivalents) was loaded onto a column and eluted directly into the Both PA2.1 and BB7.2 recognize HLA-A*0201 molecules (29, 30). 4D12 mass spectrometer with a linear 120-min gradient of 0–70% acetonitrile in recognizes HLA-B5 molecules (31). SFR8-B6 recognizes HLA-B mole- 0.1% acetic acid. Data-dependent spectral acquisition was performed as cules that express the Bw6 public Ag. HLA-B8 contains Bw6 while follows. A full scan mass spectrum was acquired over 280 Յ m/z Յ 2000. HLA-B5 does not (32). B123.2 recognizes all HLA-B and HLA-C mole- The instrument control computer then selected the top five most abundant cules. W6/32 recognizes all human mature peptide containing HLA-A, ion species, which were subjected to MS/MS analysis over the next five HLA-B, and HLA-C class I molecules (33). Biotinylated Ab to HLA-A1 scans. After acquiring MS/MS data on a particular ion species, its corre- was obtained from One Lambda (Canoga Park, CA). FITC-labeled goat sponding m/z value was ignored for a period equal to the observed chro- anti-mouse IgG and streptavidin were used as secondary staining reagents matographic peak width (ϳ1.5 min for the data shown herein). This data and were obtained from Cappel (Durham, NC). acquisition procedure minimized redundancy and allowed MS/MS analysis Immunoaffinity purification of peptides associated with class I on peptide species spanning a wide abundance dynamic range. A typical ϳ molecules chromatographic run contained 2000 total MS/MS scans, of which 35– 50% contained features characteristic of peptide dissociation spectra. After Cells (5 ϫ 108–1 ϫ 109) were pretreated with 250 ␮M LLnL for 2 h, acid acquisition, data were searched using SEQUEST; an algorithm that treated with 50 ␮l of acid wash per 1 ϫ 106 as described above, and then matches uninterpreted MS/MS spectra to theoretical peptides generated incubated with 250 ␮M LLnL for another 5 h. Alternatively, 1 ϫ 108–5 ϫ from user-specified databases (35). All data herein were searched against 108 cells were acid treated and then incubated in cell medium for 5 h. both human-only and nonredundant protein databases compiled at the Na- Greater numbers of cells were used for the inhibited samples to compensate tional Center Biotechnology Information (National Institutes of Health, for lower class I expression levels at the cell surface at the end of the Bethesda, MD), and all reported peptide sequences were verified by man- experiment. Class I surface expression was determined by flow cytometry ual interpretation. In vitro proteasome activity assays 4 Abbreviations used in this paper: LAC, lactacystin; LLnL, N-acetyl-L-leucinyl-L- The 20S proteasomes were purified from 721 cells as previously described leucinal-L-norleucinal; z-L VS, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone; 3 (36). Proteasomes were incubated for2hat24°C in 50 mM Tris-HCl and BFA, brefeldin A; FTMS, Fourier transform ion cyclotron resonance mass spectrom- ␮ ␮ eter; m/z, mass-to-charge; MS/MS, tandem mass spectrometry; ChT-L, chymotrypsin- 5 mM MgCl2 in the presence and absence of 100 M LAC, 250 M LLnL, ␮ like; T-L, trypsin-like; PGPH, peptidyl glutamyl peptide hydrolyzing; ER, endoplas- or 100 M z-L3VS. The fluorosubstrates z-Leu-Leu-Glu-AMC, Suc-Leu- mic reticulum; LC/FTMS, liquid chromatography interfaced directly to a FTMS. Leu-Val-Tyr-AMC, or Boc-Leu-Arg-Arg-AMC (Calbiochem) were then 1214 PROTEASOME INHIBITOR-INSENSITIVE CLASS I MHC PEPTIDES added to give a final concentration of 100 ␮M along with additional inhibitor necessary to maintain original inhibitor concentration. Fluorescence was measured every 20 min for 8–12 h using a Cytofluor II Multiwell Plate Reader (Bio-Rad, Hercules, CA) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. For each substrate, control samples from which 20S proteasomes were omitted were used to establish the background. End points chosen for all experiments were within the linear por- tion of the activity profile for samples incubated in the absence of inhibitors.

Results Proteasome inhibitors differentially affect the expression of HLA-A1, HLA-B51, HLA-A*0201, and HLA-B8 To examine the involvement of different proteolytic activities in the generation of peptides presented by class I MHC molecules, cells were treated with acid to remove surface class I peptide complexes, and then allowed to re-express newly synthesized complexes in the presence or absence of proteasome inhibitors for 5 h (19). Immediately after treatment of the B-LCL 721 with acid, the expression of HLA-A*0201 was nearly undetectable as quantitated Downloaded from by ﬂow cytometry (Fig. 1A). In the absence of inhibitors, cells re-expressed 30% of the pretreatment level of HLA-A*0201 in 5 h. BFA, which blocks egress of newly generated class I peptide complexes at the level of the cis-Golgi (25), inhibited re-expression almost entirely. The other class I alleles on 721 (HLA-A1, HLA-

B8, and HLA-B51) behaved similarly (data not shown). Treatment http://www.jimmunol.org/ of cells with acid therefore makes it possible to selectively measure the surface expression of newly generated class I peptide complexes. Three different inhibitors were used, either alone or in combination, to block proteasome activity in 721 cells. LLnL, also known as calpain inhibitor I, reversibly inhibits proteasomes as well as several other classes of proteases (23). LAC and z-L3VS specifically inhibit proteasomes by covalent binding to all known FIGURE 1. Acid treatment and re-expression of HLA-A*0201, -B51, catalytic sites (21, 22, 24). Each of these inhibitors strongly -A1, and -B8 on 721 cells in the presence of proteasome inhibitors. Cells by guest on September 26, 2021 blocked the re-expression of two class I alleles on 721 cells. were treated as indicated below, then fixed, stained with the allele-specific HLA-A1 re-expression after 5 h was only 5–15% of that observed Abs BB7.2 (A*0201), 4D12 (B51), SFR8-B6 (B8), and a commercially in the absence of inhibitor, whereas that of HLA-B51 was 15–25% obtained Ab specific for HLA-A1, and analyzed by flow cytometry as (Fig. 1B). In contrast, re-expression of HLA-A*0201 under the described in Materials and Methods. B and C, 100% re-expression corre- same conditions was 50–60% of that observed in the absence of sponds to cells allowed to re-express the specific allele in the absence of any inhibitor for 5 h. Background staining with second Ab only was sub- inhibitor, whereas that of HLA-B8 was 40–50%. The concentra- tracted from all values before percent re-expression was calculated. The tions of each inhibitor used in the above experiments had been results are representative of three, four, and three experiments, respec- determined to be saturating for inhibition of class I re-expression tively. A, 721 cells were acid treated and incubated in cell medium or cell (data not shown). In addition, we measured the effect of inhibitors medium containing 10 ␮g/ml BFA for 5 h. A separate aliquot of 721 cells on the cleavage of model fluorosubstrates by proteasomes purified was acid treated and stained immediately (0 h). Secondary Ab alone stain- from 721 cells and which classically measure the chymotrypsin- ing is shown as a gray histogram. B, 721 cells were acid treated and then like (ChT-L), trypsin-like (T-L), and peptidyl glutamyl peptide- incubated for 5 h in the absence or presence of 10 ␮g/ml BFA, 100 ␮M ␮ ␮ ␮ ϩ ␮ hydrolyzing (PGPH) cleavage activity of proteasomes. Using the LAC, 250 M LLnL, 50 M z-L3VS (L3VS), 100 M LAC 250 M ␮ ϩ ␮ same high concentrations and preincubation conditions (see be- LLnL (LAC/LLnL), 100 M LAC 50 M z-L3VS (LAC/L3VS), or 250 ␮M LLnL ϩ 50 ␮M z-L VS (LLnL/L3VS). C, 721 cells were incubated low) as were used in vivo, each inhibitor blocked 89–99% of all 3 with 100 ␮M LAC (PreLAC) or 250 ␮M LLnL (PreLLnL) for 2 h, fol- three cleavage activities of proteasomes, and each activity was lowed by acid treatment and continued incubation with the same inhibitor inhibited by a minimum of 95% by at least one of the inhibitors for 5 h. Alternately, 721 cells were incubated in cell medium for 2 h (Table I). In particular, LAC gave essentially complete inhibition followed by acid treatment and incubation for5hintheabsence or pres- of both the ChT-L and T-L activities. Also, T-L was the major ence of 10 ␮g/ml BFA, 100 ␮M LAC, or 250 ␮M LLnL. activity that persisted in the presence of z-L3VS (10% residual activity) while PGPH was the major activity that persisted in the presence of either LLnL or LAC (10 and 11%, respectively). It is noteworthy that these differences in residual activities were not Re-expression of class I molecules in the presence of protea- reflected in significantly different levels of re-expression in vivo some inhibitors could be explained by a preexisting pool of class (Fig. 1). In addition, using combinations of the three inhibitors at I-binding peptides or peptide-MHC complexes. This pool would these saturating concentrations did not inhibit re-expression fur- have been generated by proteasome activity before inhibition but ther. Collectively, these results demonstrate that a significant frac- not yet expressed on the cell surface at the time of acid treatment. tion of the surface expression of these class I molecules was de- To examine this possibility, we pretreated cells with proteasome pendent on proteolytic activities that were insensitive to inhibitors inhibitors for 2 h before treatment with acid. Pretreatment with of the proteasome. either LAC or LLnL decreased the re-expression observed by an The Journal of Immunology 1215

Table I. Effect of proteasome inhibitors on the in vitro activity of expressed on 721 cells in the presence of LAC (405 fluorescent purified proteasomesa units) was substantially greater than the level expressed on 721.174, even in the absence of LAC (104 fluorescent units). The % Inhibition lower level of expressed HLA-A*0201 on 721.174 cells is not Proteasome Activity ␤ Substrate Measured LAC LLnL z-L3VS likely to reflect differences in the translation of class I or 2-microglobulin molecules because restoration of TAP normalizes ex- b-LRR-amc T-L 99.1 96.7 90.3 pression of HLA-A*0201 in 721.174 cells (27). Therefore ϳ75% s-LLVY-amc ChT-L 99.9 97.2 98.5 of the HLA-A*0201 that is re-expressed on 721 cells in the z-LLQ-amc PGPH 89.3 90.1 95.9 presence of proteasome inhibitors is dependent on TAP func- a Purified 20S proteasome preparations were preincubated with each inhibitor for tion. Collectively, these results suggest that the proteasome 2 h. Fluorosubstrates were then added and the fluorescence was measured every 20 min for 8–12 h. Concentrations of inhibitors throughout preincubation and assay were inhibitor-insensitive proteolytic activity responsible for HLA-B8 ␮ ␮ ␮ 100 M LAC, 250 M LLnL, and 100 M z-L3VS. Values represent the change in and HLA-A*0201 re-expression resides in the cytosol. fluorescence obtained at the end of the assay as compared to that measured in the absence of inhibitor. All measured end points were taken from the linear portions of Proteasome inhibitor-insensitive re-expression of 13 different the response curve and are the mean of two experiments. human class I MHC molecules Our results with 721 cells suggested that class I alleles differ in additional 10% (Fig. 1C). Longer pretreatment times did not fur- their ability to bind peptides generated by proteasome inhibitor- ther decrease re-expression (data not shown). This is consistent insensitive activities. Most of the positions at which residues occur

with the hypothesis that there was a small internal pool of peptides that are relevant for class I binding are not at either the amino or Downloaded from that were either preexistent or made shortly after inhibitor addition carboxyl termini and are therefore unlikely to be affected by pro- and had not yet reached the surface at the time of acid treatment. tease speciﬁcity. However, previous studies have suggested that On the other hand, after complete exhaustion of this pool and in the there is limited trimming of the carboxyl termini of class I- asso- presence of saturating levels of proteasome inhibitors, nearly half ciated peptides, and they therefore are the result of initial endo- of the HLA-A*0201 and HLA-B8 molecules were still re-ex- protease cleavage (41–46). Since the carboxyl-terminal residue is

pressed on this cell line. Therefore, the high level re-expression of an important component of every known class I- binding motif, we http://www.jimmunol.org/ HLA-A*0201 and HLA-B8 in the presence of proteasome inhib- hypothesized that the carboxyl-terminal binding motifs of class I itors cannot be accounted for by a preexisting pool of class I-bind- molecules would correlate with their surface expression in the ing peptides or peptide-MHC complexes. presence of proteasome inhibitors. We therefore examined the cell surface expression of a set of human class I alleles with different TAP-independent expression of HLA-A*0201 and HLA-B8 does carboxyl-terminal binding motifs, which were expressed after not account for their proteasome inhibitor-insensitive expression transfection in the B-LCL C1R. The cell surface expression of most class I MHC molecules, in- C1R expresses only a low level of endogenous HLA-Cw04 and cluding HLA-B8 (37), is almost completely dependent on the func- does not express endogenous HLA-A or -B class I products (47). tion of TAP, which transports peptides generated in the cytosol We used the pan class I- specific mAb w6/32 to detect HLA-Cw04 by guest on September 26, 2021 into the endoplasmic reticulum (ER) lumen. HLA-A*0201 binds to in untransfected C1R cells as well as all transfected products. As peptides produced by this pathway, but also those produced in a measured with this Ab, surface expression levels of all transfected TAP-independent manner. We and others have suggested that this class I MHC molecules were similar and at least 10- to 20-fold pathway consists of peptides generated entirely within the ER lu- higher than that of endogenous HLA-Cw04 (data not shown). LAC men by as yet poorly described proteases (38, 39). Thus, the re- and LLnL both inhibited the re-expression of the HLA-Cw04 by expression of HLA-A*0201 on 721 cells treated with proteasome 70% (Fig. 2). This percentage inhibition was as great as that ob- inhibitors might be due to this alternate pathway. We therefore served with any other C1R transfectant, and combined with a much examined HLA-A*0201 re-expression in the B-LCL 721.174, a lower level of initial expression, indicates that the endogenous C mutant cell line derived from 721 that lacks a functional TAP locus accounts for Ͻ5% of the w6/32 staining observed with any transporter (26, 40). The constitutive surface expression of HLA- of the other C1R transfectants. In keeping with our earlier results, A*0201 on 721.174 is ϳ17% of that on 721 (Table II). Both cells the cell surface expression of all 13 HLA-A, -B, and -C alleles was re-expressed ϳ30% of their pretreatment levels of HLA-A*0201 at least partially insensitive to inhibitors of proteasome activity, after they were acid treated and incubated for5hintheabsence of but the extent of this insensitivity varied among alleles (Fig. 2). As inhibitors. Significantly, the level of HLA-A*0201 that was re- observed with 721, preincubation of the transfectants with inhibitors for longer than 2 h did not decrease re-expression further (data not shown). Similar recovery patterns were also observed Table II. Effect of LAC on the TAP-dependent and -independent when either C1R transfectants or melanoma cell lines were stained a expression of HLA-A*0201 with mAbs specific for HLA-B*2702, HLA-B*2704, HLA- B*2705, HLA-B8, and HLA-A*0201, indicating that recovery was HLA-A*0201 Expression not cell type or Ab dependent (data not shown). Incubation Conditions 721 (TAPϩ) 721.174 (TAPϪ) To test the hypothesis that re-expression was correlated with the carboxyl-terminal binding residue, we used the binding motif in- Medium 2120 349 formation available on the SYFPEITHI web site (48). Interest- Acid wash ϩ medium for 5 h 617 (29.1)b 104 (29.8) Acid wash ϩ LAC for 5 h 405 57 ingly, all alleles that bind peptides with basic carboxyl termini (HLA-B*2705, HLA-A68, and HLA-A3.1) demonstrated rela- a TAP normal 721 and TAP-deficient 721.174 cells were acid treated and allowed tively high levels of re-expression in the presence of proteasome to re-express HLA-A*0201 in the absence or presence of 100 ␮M LAC for 5 h. HLA-A*0201 levels were measured by flow cytometry after staining with PA2.1. inhibitors. This is consistent with the idea that peptides with basic Mean fluorescent intensity values are shown. This experiment is representative of four carboxyl termini are preferentially generated by proteasome inhib- experiments. b Values in parentheses represent percentage of normal expression in medium itor-insensitive proteolytic activity. Further support for this idea only. was evident when the re-expression of structurally related alleles 1216 PROTEASOME INHIBITOR-INSENSITIVE CLASS I MHC PEPTIDES

FIGURE 2. Proteasome inhibitor effects on the re-expression of several class I alleles expressed in C1R. C1R or transfectants were preincubated, acid treated, and inhibited as described in Materials and Methods and the legend to Fig. 1. Cw04 is the untransfected line while A and B allele names represent allele stably transfected into C1R. These results are representative of three independent experiments. Downloaded from http://www.jimmunol.org/ was compared. For example, HLA-A69 consists of the ␣1 domain out 250 ␮M LLnL. These peptide mixtures were analyzed by liq- of HLA-A68 linked to the ␣2 domain of HLA-A*0201. Conse- uid chromatography interfaced directly to a LC/FTMS (34). In quently, its binding speciﬁcity at the P2 anchor is that of HLA- addition to temporal separation of peptide species, this method A68, but it prefers peptides with hydrophobic carboxyl termini like provides a 1000-fold improvement in mass-resolving power and a HLA-A*0201 (49, 50). Although the re-expression of HLA-A68 10- to 100-fold improvement in mass accuracy compared with pre- was ϳ65% in the presence of proteasome inhibitors, that of HLA- viously used triple quadrupole mass analyzers (9, 38, 53). This

A69 was only 30%. In addition, HLA-B*2705 binds peptides with enables us to unambiguously distinguish peptides differing by as by guest on September 26, 2021 basic, aromatic, or aliphatic carboxyl termini, whereas HLA- little as 0.01–0.1 Da compared with ϳ1.5 Da afforded by triple B*2704 prefers peptides with hydrophobic C termini (51, 52). quadrupole instrumentation. When combined with software allow- Again, re-expression of HLA-B*2704 was only about half that of ing two-dimensional displays of the data, this method allowed us HLA-B*2705 in the presence of proteasome inhibitors. These data to visualize 2500 different peptide species in a single display. imply that class I-associated peptides with basic carboxyl termini We first analyzed peptides associated with HLA-A1, the class I represent a significant component of the peptides generated by MHC allele that was the most sensitive to proteasome inhibitors of proteasome inhibitor-insensitive proteolytic activities. all those tested above. By comparing m/z vs retention time plots of Despite the above evidence, HLA-B8 and HLA-B*2702 show peptide extracts from equivalent numbers of inhibitor-treated and relatively high re-expression in the presence of proteasome inhib- untreated cells, we observed that the vast majority of individual itors although their published motifs do not include basic carboxyl- peptides normally presented by HLA-A1 were decreased in abun- terminal residues. Interestingly, within the group of alleles that do dance by at least 90% in the sample from cells treated with LLnL not bind to peptides with basic carboxyl termini, there is a sug- (Fig. 3, A and B). This demonstrates that proteasomes are respon- gestion that re-expression in the presence of proteasome inhibitors sible for the generation of most of the peptides associated with correlates with the ability to bind to a larger range of carboxyl- HLA-A1. In addition, the remaining species that were visible in terminal residues. This is most evident when the motifs of HLA- the sample from proteasome inhibitor-treated cells were frequently B*2702 and HLA-B*2704 are compared, as well as those of HLA- distinct from those in control cells. Also, when the intensity values B51 and HLA-B35. Collectively, these data suggest that some of the sample from inhibitor-treated cells were normalized for the proteases active in the presence of proteasome inhibitors generate level of HLA-A1 as determined from flow cytometry, the peptide class I- associated peptides with carboxyl termini that do not in- profile was clearly different from that of untreated cells (Fig. 3C). clude basic residues. The persistence of a small but distinct subset of peptides at ele- vated levels in the presence of proteasome inhibitors indicates that Class I-associated peptides expressed in the presence of proteolytic activities with specificities that are distinct from the proteasome inhibitors are a complex subset of those present uninhibited proteasome are responsible for their generation. under normal conditions We next looked at the impact of proteasome inhibitors on the The results above demonstrate that class I- associated peptides can distribution of peptides associated with HLA-B*2705 and HLA- be generated by at least one cytosolic protease activity that is re- Cw04. As shown above, HLA-Cw04 is expressed constitutively at sistant to proteasome inhibitors. However, they provide only sug- only 5% of the level of HLA-B*2705 and is re-expressed poorly in gestive information about the nature of the peptides presented. We the presence of LLnL, while HLA-B*2705 re-expression is largely therefore extracted the peptides associated with class I molecules unaffected by proteasome inhibitors. Therefore, the vast majority re-expressed on acid-treated cells that were incubated with or with- of peptides in these extracts should have been associated with The Journal of Immunology 1217 Downloaded from http://www.jimmunol.org/ FIGURE 3. HLA-A1-associated peptide profiles from cells incubated in the presence or absence of LLnL. 721 cells were either acid treated and FIGURE 4. HLA-B*2705-associated peptide profiles from cells incu- incubated in cell medium for5h(A) or preincubated with cell medium plus bated in the presence or absence of LLnL. C1R cells transfected with ␮ 250 M LLnL for 2 h, acid treated, and incubated with cell medium plus HLA-B*2705 were either acid treated and incubated in cell medium for 5 h ␮ 250 M LLnL for5h(B and C). HLA-A1 molecules were affinity purified (A) or preincubated with cell medium plus 250 ␮M LLnL for 2 h, acid using w6/32 Ab after removal of the other class I MHC molecules with the treated, and incubated with cell medium plus 250 ␮M LLnL for5h(B). HLA-A*0201-specific Ab BB7.2 and the pan HLA-B- and -C-specific Ab Class I MHC molecules were affinity purified using w6/32 Ab, and the B.123.2. HLA-A1-associated peptides were isolated and analyzed by LC/ isolated peptides were analyzed by LC/FTMS as described in Materials

FTMS as described in Materials and Methods. B, The data from the LLnL- and Methods. Equal numbers of cell equivalents of peptides were analyzed by guest on September 26, 2021 treated sample are normalized to represent the amount of HLA-A1-asso- and are shown in A and B. The boxed areas indicate a region in which the ciated peptides recovered from a number of cells equal to that displayed in peptide proﬁles were disparate, while the circled areas indicate two regions A. C, The data from the LLnL-treated sample are normalized to represent meant to serve as examples of similar peptide proﬁles. an equivalent amount of HLA-A1 molecules as recovered from the cells displayed in A.

of peptide species that are associated with HLA-B*2705 under normal conditions. HLA-B*2705. The m/z vs retention time plots of peptides expressed in the presence of LLnL was similar in complexity to that Class I-associated peptides expressed in the presence of of peptides expressed on untreated cells (Fig. 4). Thus, the re- proteasome inhibitors are derived from proteins expressed in a expression of HLA-B*2705 molecules in cells treated with LLnL variety of cellular locations and are produced from proteases was not due to the presentation of a small number of highly abun- with a broad spectrum of speciﬁcities dant peptides. Proteasome inhibition resulted in an observable de- To gain information about the proteasome inhibitor-resistant path- crease in only a subset of early eluting peptides (boxes in Fig. 4). ways of class I epitope generation in the cytosol, we characterized Unlike what was seen with HLA-A1, the majority of the peptides a subset of peptides re-expressed in the presence of proteasome that eluted later in the gradient seemed unaffected (two areas are inhibitors using a combination of online microcapillary HPLC and circled as examples in Fig. 4). collision-activated dissociation on a quadrupole ion trap mass To determine more accurately the relative abundances of indi- spectrometer. Forty-six peptides were sequenced from the mixture vidual peptide species in the two samples, we analyzed all of the associated with the HLA-B and -C alleles on 721 cells in the pres- masses visible in a representative 1-s interval of the two m/z vs ence of LLnL (Table III), while 36 were sequenced from the mix- retention time plots (indicated by the arrows in Fig. 4). In this time ture associated with HLA-B*2705 and HLA-Cw04 on C1R/ slice, 20 of 21 observed peptides changed in abundance by no B*2705 cells in the presence of LLnL (Table IV). more than a factor of 5 (data not shown). In contrast to what was All of the sequenced peptides were derived from endogenously observed with HLA-A1, these were equally distributed between expressed proteins. Forty-four of the 82 peptides were from known groups that increased or decreased in abundance. The abundance proteins with nuclear, cytosolic, mitochondrial, or peroxisomal lo- of the remaining peptide increased 9-fold upon proteasome inhi- cations and are therefore translated in the cytosol on free polyri- bition. These results demonstrate that activities sensitive to pro- bosomes. Based on the TAP dependence of the presenting class I teasome inhibitors are required for the generation of at most a molecules and the cytosolic localization of these proteins, gener- small subset of peptides associated with HLA-B*2705, and that ation of these peptides most likely occurs in the cytosol. Of the 22 proteasome inhibitor-resistant activities can generate the majority peptides derived from known membrane or secreted proteins, only 1218 PROTEASOME INHIBITOR-INSENSITIVE CLASS I MHC PEPTIDES

Table III. Sequences of peptides generated in the presence of proteasome inhibitors associated with HLA-B and -C locus products on 721 cellsa

Motif Sequence Source Protein Protein Localization Epitope Localization

Cw04 KYFDEHYEY CDK 2 Cytosol Cytosol PYLDLLLQI X-linked ubiquitin hydrolase Cytosol Cytosol B51 DAYVLPKLY Ribosomal protein S26 Cytosol Cytosol YAFNMKATV HSC-70 Cytosol Cytosol DALRSILTI tRNA ligase Cytosol Cytosol DANPYDSVKKI Diubiquitin Cytosol Cytosol DALDVANKIGII 60S Ribosomal protein Cytosol Cytosol NAYVNINRI Kalirin Cytosol Cytosol DAVVKHVL Cotamer ␣ subunit Cytosol Cytosol IPMIIHQL Importin ␣-chain Cytosol Cytosol DAEMTTRMV MECL-1 Cytosol Cytosol DALLIIPKV T complex protein 1, ␨2 subunit Cytosol Cytosol DGYEQAARV T complex protein 1, ␧ subunit Cytosol Cytosol DALLQMITI EF-2 Nuclear Nuclear DAENAMRYI sRNP Cap binding protein Nuclear Nuclear MPMNVADLI E1F-4A Nuclear Nuclear TPVRLPSI IRF-1 Nuclear Nuclear

VPYEPPEV p53 Nuclear Nuclear Downloaded from FAYVQIKTI Cytochrome P450 Mitochondrial Mitochondrial IPLPLGTVTI Naϩ/Kϩ-ATPase Membrane Transmembrane DAYALNHTL MHC class I Membrane Luminal/extracellular DPYEVSYRI BTG1 protein ? ? DAFKIWVI Hypothetical protein ? ? IPYQDLPHL Lysophospholipase homologue ? ? DAPAHHLF Hypothetical protein ? ? http://www.jimmunol.org/ B8 DAREIVNNV DNA topoisomerase Cytosol Cytosol LAAARLAAA Protein disulﬁde-isomerase Membrane Signal sequence DALLKFSHI Testis-enhanced gene transcript Membrane Luminal/extracellular DALKEKVI MET adenosyltransferase ? ? B51, B8, DIHHKVLSL Ras-GAP SH3-binding protein Cytosol Cytosol or Cw04 FLKIKPVSL DEC-205 Membrane Signal sequence AVILRALSL HLA-DP ␣-chain Membrane Signal sequence MMKLIINSL GP96 Membrane Luminal/extracellular ATKARLSSL Myocillan Membrane Luminal/extracellular DLHEKDFSL Poly(ADP-ribosyl) transferase ? ? by guest on September 26, 2021 B51 or YPFFRGVTI D123 protein Cytosol Cytosol Cw04 YFAERVTSL Cysteine-rich protein 1 Nuclear Nuclear LPSLRILYM Cytochrome c oxidase Membrane Transmembrane Uncertain YLPAGQSVL Prohibin Cytosol Cytosol LLIENVASL Glutathione peroxidase Cytosol Cytosol IQFPANLQL TFIID 105-kDa subunit Nuclear Nuclear LLALVGLLSL CD18 (LFA1) Membrane Signal sequence PLQPLTVTV Hypothetical protein ? ? FLQVCDWLY Hypothetical protein ? ? VRNNVIIVM Hypothetical protein ? ?

a 721 cells were preincubated with 250 ␮M LLnL for 2 h, acid washed, and incubated with 250 ␮M LLnL for 5 h. HLA-B and -C molecules were affinity purified using B1.23.2 and peptides were isolated as described in Materials and Methods. Peptides were then analyzed by LCQ mass spectrometry as described in Materials and Methods. All reported sequences were manually confirmed. ? indicates that the information cannot be determined from the known protein sequence.

6 were from signal sequences, while 16 were derived from luminal addition, there was no obvious constraint on the occurrence of or transmembrane regions. Both TAP-independent (38) and -de- particular aliphatic residues at this position in the presence of pendent pathways (53–55) for the presentation of these peptides LLnL (Table III). Twenty-eight of 36 peptides sequenced from the have been described. Regardless of the exact mechanism, these mixture associated with HLA-B*2705 and HLA-Cw04 contained results demonstrate that proteasome inhibitor-resistant proteases the Arg element of the HLA-B*2705 motif at P2 while the re- responsible for class I epitope generation degrade a variety of pro- maining 8 contained a canonical HLA-Cw04 motif residue at this teins localized throughout the cell as well as proteins synthesized position (Table IV) (56). Unlike all other alleles from which se- in either the cytosol or the ER. quences were obtained, HLA-B*2705 binds peptides containing Thirty-eight of 46 peptides sequenced from the mixture associ- aromatic, hydrophobic, or basic C-terminal amino acids (51, 57). ated with the HLA -B and -C alleles on 721 cells conformed to the Of 28 HLA-B*2705-associated peptide sequences, 8 contained a established binding motifs for at least one of HLA-B51, HLA-B8, lysine or arginine at the carboxyl terminus, while 9 others ended in or HLA-Cw01 molecules (56) (Table III). The remaining eight aromatic residues and 9 ended in nonaromatic hydrophobic resi- peptides fit none of the published motifs well and may either be dues (Table IV). Thus, the peptides that persisted upon proteasome derived from an unidentified C locus product or bind to one of the inhibition demonstrated the complete range of C termini known to known B or C alleles despite the lack of a canonical binding motif. bind to HLA-B*2705. These results demonstrate that the proteases Regardless, all of the peptides sequenced contained either an aro- that generate class I- associated peptides in proteasome inhibitor- matic or large hydrophobic residue at the carboxyl terminus. In treated cells collectively have a broad spectrum of specificities. The Journal of Immunology 1219

Table IV. Sequences of peptides generated in the presence of proteasome inhibitors associated with HLA-B*2705 and -Cw04 in C1R cellsa

Sequence Source Protein Protein Localization Epitope Localization

HLA-B*2705 NRIVYLYTK 60S Ribosomal protein L34 Cytosol Cytosol VRMNVLADALK Ribosomal protein S15 Cytosol Cytosol GRIGVITNR 40S Ribosomal protein S4 Cytosol Cytosol IRGAIILAK Ribosomal protein S2 Cytosol Cytosol GRVGDVYIPR SR p46 splicing factor Nuclear Nuclear IRNDEELNK Histone H2A1 Nuclear Nuclear VRLLLPGELAK Histone H2B Nuclear Nuclear GRFSGLLGR IL-16 precursor Secreted Luminal/extracellular TRYQGVNLY Poly(A)-binding protein 1 Cytosol Cytosol QRNVNIFKF LDH-Ab Cytosol Cytosol GRFNGQFKTY 40S Ribosomal protein S21 Cytosol Cytosol GRSTGEAFVQF HNRNP 2H9 Nuclear Nuclear IRAAPPPLF Cathepsin A Lysosomal Signal sequence QRNLYIAGF B cell receptor-associated protein 31 Membrane Transmembrane GRWPGSSLYY Lamin B receptor Membrane Luminal/extracellular GRWPGSSLY Lamin B receptor Membrane Luminal/extracellular

ARLTDYVAF COP9 ? ? Downloaded from LRFQSSAVMAL Histone H3 Nuclear Nuclear ␤ SRSVALAVLAL 2-Microglobulin Membrane/secreted Signal sequence GRTFIQPNM GPAT Secreted Luminal/extracellular MRMATPLLM Invariant chain Membrane Luminal/extracellular ARFGLIQSM Hypothetical protein ? ? NRFAGFGIGL TB1 ? ? ARFGLIQSM Hypothetical protein ? ? http://www.jimmunol.org/ QRVNVQPEL RAB GGTase ? ? LRFQSSAVMALQ Histone H3 Nuclear Nuclear SRLSPPAGLFTS Stat5A Nuclear Nuclear HLA-Cw04 motif VYDLSIRGF Translin Nuclear Nuclear HPPPPPPPP C/EBP␤ Nuclear Nuclear PSPPPPPPP EBNA 2A Nuclear Nuclear VYDIAAKF Alkylglycerone-P synthase Peroxisomal Luminal AYGISKTGVSI Flight 3 Membrane Luminal/extracellular APEPSTVQILHSPAVE CD-22 Membrane Luminal/extracellular by guest on September 26, 2021 TFDDIVHSF Fatty acid synthase ? ? LFDDIDHNM Meningioma-Ag 5 ? ?

a Experimental details were as described in the footnote to Table III, except that the cells used were C1R-B*2705, and the Ab used for afﬁnity puriﬁcation was w6/32. b LDH, lactate dehydrogenase; EBNA, EBV-encoded nuclear Ag; HNRNP, heterogeneous nuclear ribonucleoprotein; GPAT, glutamine phosphoribosylpyrophosphate aminotransferase.

Discussion One possible explanation for our results was that proteasome Several studies using inhibitors of proteasome activity have either inhibitor-resistant class I peptide expression resulted from large supported or questioned the general involvement of proteasomes in internal pools of peptides. Although preincubation of cells with the generation of class I MHC-associated peptides. In this study, proteasome inhibitors demonstrated the existence of an internal we used 3 different inhibitors to examine the contribution of pro- store of peptides that could associate with class I MHC molecules, teasomes to the generation of peptides associated with 13 different class I re-expression occurred even after depletion of these stores. human class I MHC alleles. In the presence of these proteasome The minimal size of this pool, combined with the overall inhibition inhibitors, 9 of 13 human class I alleles continued to be expressed of class I MHC re-expression, demonstrates that there is not a large at Ͼ30% of control levels. This re-expression could not be ac- reservoir of peptides in the cells that are destined for class I MHC counted for by the use of peptides generated directly in the ER in binding. This is an important consideration given the role of class a TAP-independent manner. In addition, the allelic variation did I peptide presentation in controlling intracellular pathogens. Upon not correlate with differences in their afﬁnities for TAP (presum- initial infection, peptides generated from newly translated proteins ably via tapasin) (58). We used a combination of Fourier transform should be immediately available for class I binding and surface mass spectrometry and two-dimensional data displays to examine expression. the complexity of peptides associated with two alleles. In the pres- Another possible explanation for the continued expression of ence of proteasome inhibitors, expression of HLA-A1 was sub- some class I MHC molecules in the presence of proteasome in- stantially reduced, as was the expression of the vast majority of its hibitors is that these compounds do not completely block all ac- associated peptides. In contrast, expression of HLA-B*2705 was tivities of the proteasome. Several previous studies have demon- largely insensitive to proteasome inhibition and a diverse array of strated that the ChT-L activity of mammalian proteasomes is peptides continued to be presented. Thus, the peptides expressed in rapidly inactivated by relatively low concentrations of LLnL (23), the presence of proteasome inhibitors represents a signiﬁcant frac- LAC (21, 22, 59), or z-L3VS (59) and that inhibition by the latter tion of the peptides associated with some class I MHC alleles two compounds is irreversible (21, 59). Although the T-L and under normal conditions. PGPH activities of the proteasome are less sensitive than the 1220 PROTEASOME INHIBITOR-INSENSITIVE CLASS I MHC PEPTIDES

ChT-L activity, they can nonetheless be blocked by higher con- tively high levels in the presence of proteasome inhibitors. Our centrations of any of these three inhibitors (21–23, 59). Impor- data suggest that aside from a preference for basic carboxyl-ter- tantly, preincubation with inhibitors greatly increases their effec- minal residues, another determinant of the re-expression of class I tiveness (59, 60), and we adopted this strategy in the present work. MHC alleles in the presence of proteasome inhibitors is their abil- Of the three activities, PGPH is the most insensitive to LAC (21), ity to bind to a larger range of carboxyl-terminal residues. while T-L is the most insensitive to LLnL (23). In the present Although the overall complexity of HLA-B*2705-associated study, we found that all of these activities of puriﬁed proteasomes peptides was similar in the presence and absence of proteasome in vitro were blocked at least 95% by at least one of the inhibitors inhibitors, individual peptides in the mixture either decreased by at the same concentrations and conditions used in vivo. If residual up to 5-fold or increased by up to 9-fold. Because of the complex- proteasome activity accounted for the high level re-expression of ity of the mixture, these changes seem unlikely to be due to peptide some class I alleles, it would be expected that this would vary competition. Instead, these peptides may be either generated or depending on the inhibitor used because of the differences in ef- destroyed by different proteasome activities in addition to possibly fectiveness of the inhibitors against different proteasome activities. being generated by nonproteasomal proteases. We have previously

This was not observed. Alternatively, if re-expression were due shown that the M158–66 epitope from influenza A virus is pro- simply to a fraction of proteasomes that were not inhibited at all, duced by a proteasome inhibitor-resistant protease and destroyed then one would not expect to see allele-specific differences in the by proteasomes (19). The data in the present study suggest that the extent of re-expression. Furthermore, saturating concentrations of final level of presentation of many epitopes is the result of inter-

LAC, LLnL, or z-L3VS did not inhibit class I re-expression to a play between different proteolytic activities. greater extent in combination than individually, despite the fact In summary, we have demonstrated that proteasome inhibitor- Downloaded from that they differ in chemical structure, mode of inhibition, and pro- resistant pathways produce a signiﬁcant fraction of peptide species ﬁle of in vitro activity that persisted in the face of inhibition. Fi- associated with many class I alleles. These peptides are derived nally, the peptide sequences presented by class I MHC molecules from a wide range of cellular proteins and display heterogeneous re-expressed in the presence of inhibitors were not enriched for C termini. This suggests that they are generated either by a mul- those with C-terminal residues that would be generated by residual tifunctional protease or by multiple proteases with different cleav-

PGPH or T-L activity. Collectively, these results suggest that pro- age specificities. Further characterization of these proteolytic ac- http://www.jimmunol.org/ teolytic activities other than the proteasome may be responsible for tivities will provide insight into mechanisms for class I epitope the high re-expression of some human class I MHC alleles in the generation. presence of proteasome inhibitors. However, it is also possible that residual proteasome activities are responsible for our observations. Acknowledgments Our data suggest that the specificity for the anchor residue at the We thank Janet Gorman for her expert technical assistance in Ab produc- peptide carboxyl terminus is an important determinant of the ex- tion, Dr. Jacques Retief for his assistance in database management at the pression of class I alleles in the presence of proteasome inhibitors. University of Virginia, and C. A. Mosse, V. L. Crotzer, and Dr. We observed that class I MHC molecules with a preference for T. J. Bullock for helpful discussions. basic carboxyl-terminal anchors were generally re-expressed at by guest on September 26, 2021 higher levels. In addition, by comparing pairs of highly related References class I MHC alleles with differences in carboxyl-terminal binding 1. Pamer, E., and P. Cresswell. 1998. Mechanisms of MHC class I-restricted antigen preference, we found that those with a preference for basic resi- processing. Annu. Rev. Immunol. 16:323. 2. Rock, K. L., and A. L. Goldberg. 1999. Degradation of cell proteins and the dues were expressed at higher levels. Consistent with our results, generation of MHC class I-presented peptides. Annu. Rev. Immunol. 17:739. Benham et al. (17) also observed that two alleles with basic car- 3. Fehling, H. J., W. Swat, C. Laplace, R. Kuhn, K. Rajewsky, U. Muller, and boxyl-terminal binding motifs, HLA-A3.1 and HLA-A11, contin- H. von Boehmer. 1994. MHC class I expression in mice lacking the proteasome subunit LMP-7. Science 265:1234. ued to form peptide-dependent, SDS-stable dimers in the presence 4. Van Kaer, L., P. G. Ashton-Rickardt, M. Eichelberger, M. Gaczynska, of proteasome inhibitors. However, we found that HLA-A68, K. Nagashima, K. L. Rock, A. L. Goldberg, P. C. Doherty, and S. Tonegawa. which binds to basic carboxyl-terminal anchors, was also insensi- 1994. Altered peptidase and viral-specific T cell response in LMP2 mutant mice. Immunity 1:533. tive to proteasome inhibitors, whereas Benham et al. (17) found 5. Fruh, K., and Y. Yang. 1999. Antigen presentation by MHC class I and its the opposite result. One possible explanation for this discrepancy regulation by interferon ␥. Curr. Opin. Immunol. 11:76. is that peptide-associated HLA-A68 is more sensitive to SDS than 6. Michalek, M. T., E. P. Grant, and K. L. Rock. 1996. Chemical denaturation and modification of ovalbumin alters its dependence on ubiquitin conjugation for other class I molecules. Interestingly, an endoprotease that pre- class I antigen presentation. J. Immunol. 157:617. dominately cleaves after Lys or Arg has recently been identified in 7. Sijts, A. M., I. Pilip, and E. G. Pamer. 1997. The Listeria monocytogenes-secreted p60 protein is an n-end rule substrate in the cytosol of infected cells: implications the cytosol of mammalian cells (20, 61). We suggest that this pro- for major histocompatibility complex class I antigen processing of bacterial protease produces at least some of the peptides with basic carboxyl teins. J. Biol. Chem. 272:19261. termini that are associated with class I MHC molecules in the 8. Groettrup, M., A. Soza, U. Kuckelkorn, and P. M. Kloetzel. 1996. Peptide antigen production by the proteasome-complexity provides efficiency. Immunol. Today presence of proteasome inhibitors. 17:429. 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