ERAAP and Tapasin Independently Edit the Amino and Carboxyl Termini of MHC Class I Peptides

This information is current as Takayuki Kanaseki, Kristin Camfield Lind, Hernando of September 24, 2021. Escobar, Niranjana Nagarajan, Eduardo Reyes-Vargas, Brant Rudd, Alan L. Rockwood, Luc Van Kaer, Noriyuki Sato, Julio C. Delgado and Nilabh Shastri J Immunol 2013; 191:1547-1555; Prepublished online 17

July 2013; Downloaded from doi: 10.4049/jimmunol.1301043 http://www.jimmunol.org/content/191/4/1547

Supplementary http://www.jimmunol.org/content/suppl/2013/07/17/jimmunol.130104 http://www.jimmunol.org/ Material 3.DC1 References This article cites 51 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/191/4/1547.full#ref-list-1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

ERAAP and Tapasin Independently Edit the Amino and Carboxyl Termini of MHC Class I Peptides

Takayuki Kanaseki,*,†,1 Kristin Camfield Lind,*,1 Hernando Escobar,‡ Niranjana Nagarajan,* Eduardo Reyes-Vargas,‡ Brant Rudd,‡ Alan L. Rockwood,‡ Luc Van Kaer,x Noriyuki Sato,† Julio C. Delgado,‡ and Nilabh Shastri*

Effective CD8+ T cell responses depend on presentation of a stable peptide repertoire by MHC class I (MHC I) molecules on the cell surface. The overall quality of peptide–MHC I complexes (pMHC I) is determined by poorly understood mechanisms that generate and load peptides with appropriate consensus motifs onto MHC I. In this article, we show that both tapasin (Tpn), a key component of the peptide loading complex, and the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP) are quintessential editors of distinct structural features of the peptide repertoire. We carried out reciprocal immuni- zation of wild-type mice with cells from Tpn- or ERAAP-deficient mice. Specificity analysis of T cell responses showed that Downloaded from absence of Tpn or ERAAP independently altered the peptide repertoire by causing loss as well as gain of new pMHC I. Changes in amino acid sequences of MHC-bound peptides revealed that ERAAP and Tpn, respectively, defined the characteristic amino and carboxy termini of canonical MHC I peptides. Thus, the optimal pMHC I repertoire is produced by two distinct peptide editing steps in the endoplasmic reticulum. The Journal of Immunology, 2013, 191: 1547–1555. http://www.jimmunol.org/ resentation of endogenous peptides by MHC class I (MHC To elicit robust CD8+ T cell responses, we selected peptides I; peptide–MHC I complexes [pMHC I]) on the cell entering the Ag presentation pathway to yield high-affinity pMHC P surface enables the immune system to detect and elimi- I that will persist on the cell surface. In addition to a characteristic nate infected or transformed cells. The peptides are generated length of 8–10 aa, the peptides presented by MHC I on the cell from intracellular proteins and loaded onto MHC I by the Ag surface are uniquely defined by the presence of conserved con- processing pathway (1, 2). The pathway begins in the cytoplasm sensus motifs. The set of peptides bound by a given MHC I mol- where antigenic precursors are fragmented to produce a pool of ecule shares conserved amino acids located at discrete positions, intermediate peptide fragments. The fragments are transported called anchor residues, that allow peptide binding to the MHC I into the endoplasmic reticulum (ER) where they are loaded onto (3). Amino acid substitutions at these anchor positions resulted in by guest on September 24, 2021 MHC I molecules. The resulting pMHC I are exported to the cell loss of stable interactions between peptides and MHC I that, in surface to serve as potential ligands for recognition by the CD8+ turn, inhibited CD8+ T cell responses. T cell Ag receptors. Because circulating CD8+ T cells make only The pool of peptides for MHC I presentation is produced from transient contacts with APCs, effective CD8+ T cell responses are endogenously synthesized proteins fragmented mainly by the multi- critically dependent on presentation of an optimally stable pMHC catalytic proteasome (4), as well as other proteases (5, 6). These Irepertoire. models suggest that cytoplasmic proteolysis is primarily respon- sible for generating the canonical C termini of antigenic peptides. The intermediate peptide fragments are transported into the ER by the TAP (7). Upon entering the ER, the peptides encounter the *Division of Immunology and Pathogenesis, Department of Molecular and Cell peptide loading complex (PLC) that facilitates loading of optimal Biology, University of California, Berkeley, Berkeley, CA 94720; †Department of Pathology, Sapporo Medical University, Sapporo, Hokkaido 060-8556, Japan; peptide onto MHC I (8, 9). The PLC consists of TAP, the chaper- ‡ARUP Institute for Clinical and Experimental Pathology, Department of Pathology, x ones tapasin (Tpn) and calreticulin, the thiol oxidoreductase ERp57, University of Utah School of Medicine, Salt Lake City, UT 84108; and Department b -microglobulin, and the MHC I H chain. Among these compo- of Pathology, Microbiology and Immunology, Vanderbilt University School of Med- 2 icine, Nashville, TN 37232 nents, Tpn is critical for the formation and function of the PLC 1T.K. and K.C.L. contributed equally to this work. (8–10). Tpn interacts directly with TAP, the MHC I H chain, Received for publication April 23, 2013. Accepted for publication June 4, 2013. and ERp57, thereby bringing the PLC components together and keeping the empty MHC I close to the source of incoming pep- This work was supported by the National Institutes of Health (N.S.). tides (9–15). Consistent with its central function in the PLC, Address correspondence and reprint requests to Dr. Nilabh Shastri, Division of Immunology, LSA 421, Department of Molecular and Cell Biology, University surface expression of MHC I molecules is profoundly diminished of California, Berkeley, Berkeley, CA 94720-3200. E-mail address: nshastri@ in Tpn-deficient mice (16, 17) and in several MHC I molecules berkeley.edu in human cells (9, 18). Furthermore, the loss of Tpn results in The online version of this article contains supplemental material. presentation of suboptimal pMHC I (9, 11, 17, 19–22). Thus, Abbreviations used in this article: BfA, brefeldin A; ER, endoplasmic reticulum; Tpn is the key mediator of peptide loading in the PLC. Never- ERAAP, endoplasmic reticulum aminopeptidase associated with Ag processing; ERAAP2/2TAP2/2, ERAAP and TAP double-deficient; ERAAP2/2Tpn2/2, theless, the molecular features of the peptide cargo affected by ERAAP and Tpn double-deficient; KbDb dko, Kb and Db double-deficient; MHC I, Tpn remain unknown. MHC class I; PLC, peptide loading complex; pMHC I, peptide–MHC I complex; The ER aminopeptidase associated with Ag processing (ERAAP) Tpn, tapasin; WT, wild-type. has emerged as yet another editor of the pMHC I repertoire in the Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 ER (23, 24). The loss of ERAAP caused profound changes in the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301043 1548 GENERATING CANONICAL PEPTIDES FOR MHC I pMHC I repertoire relative to wild-type (WT) mice (25–29). Anal- Measurement of intracellular IFN-g production by CTLs + ysis of CD8 T cell responses elicited in WT mice by ERAAP- The CD8+ T cell responses of the immunized mice were measured by deficient cells showed that classical as well as nonclassical MHC I assessment of intracellular IFN-g production. Spleen cell APCs were presented a distinct, highly immunogenic peptide repertoire (26, treated with 200 ng/ml LPS (Sigma) overnight, and CD4+ and CD8+ cells 27, 30, 31). Furthermore, examination of the sequence of pre- were depleted using magnetic beads (Dynal Biotech, Invitrogen) before + sented peptides in ERAAP-deficient cells by mass spectrometry incubation with restimulated CD8 T cells. A total of 1 ml/ml brefeldin A (BfA; GolgiPlug [BD Biosciences]) was added after 1 h, and total incu- revealed that the peptides were longer, often because of extra bation of CD8+ T cells and APCs was 5 h. Cells were stained for surface N-terminal residues (30). How ERAAP edits peptides presented markers followed by intracellular staining for IFN-g. Cells were analyzed by MHC I and whether editing occurs within the PLC is not known. by flow cytometry using an LSR II (BD) and with FlowJo Software Because Tpn physically brings together PLC components, cells (TreeStar). All plots were first gated on live cells based on forward/side scatter, followed by gating on CD8+ and IFN-g+. For blocking of particular without Tpn lack a functional PLC. We reasoned that the peptide MHC I molecules, APCs were first incubated for 30 min on ice with Ab editing events in the PLC might be evident in cells lacking Tpn supernatant before addition of CD8+ T cells as described earlier. For or ERAAP. analysis of pMHC I surface stability, APCs were incubated for 2 or 4 h at 37˚C in medium containing 8 mg/ml BfA (Sigma) followed by addition of In this study, we analyzed the peptide editing functions of Tpn + and ERAAP required for generating the optimal pMHC I repertoire. CD8 T cells and staining as described earlier. We examined peptide editing events in cells lacking Tpn or In vivo cytotoxicity assay ERAAP. By immunological, biochemical, and molecular analyses, WT female mice, depleted of NK cells at least 36 h before every im- we find that ERAAP and Tpn independently edited the N and C 2 2 munization, were primed i.p. with 2 3 107 male WT, ERAAP / ,or termini of the peptide repertoire presented by MHC I on the cell Tpn2/2 splenocytes. Mice were challenged on day 7 with a 1:1:1 mix of Downloaded from surface. labeled ERAAP2/2,Tpn2/2, and WT female APCs. Target cell labels were ERAAP2/2 labeled with 20 mM CellTracker Blue CMAC (7-amino- Materials and Methods 4-chloromethylcoumarin; Invitrogen), Tpn2/2 targets labeled with 0.125 Mice mM CFSE (low dose), and WT targets labeled with 1.25 mM CFSE (high dose; Invitrogen). Twenty hours after challenge with labeled cells, host ERAAP-deficient (25), Tpn-deficient (16), TAP1-deficient (32), and Kb mice were sacrificed and splenocytes analyzed by flow cytometry to deter- b b b and D double-deficient (K D dko) mice (33) have been described else- mine percentage of cells remaining. Plots were gated on live cells before http://www.jimmunol.org/ where. C57BL/6J mice were purchased from The Jackson Laboratory. analysis of target populations. Percent specific lysis was calculated as ERAAP- and Tpn-deficient mice were crossed to generate ERAAP and follows: 100 3 (1 2 [ratio of target output]/[ratio of target input]), where Tpn double-deficient (ERAAP2/2Tpn2/2) mice. Use of all mice was done input is determined before injection of targets and output represents targets with the approval of the Animal Care and Use Committee of the University recovered after challenge. To calculate ratios, (% population of interest)/ 2 2 2 2 of California at Berkeley. (%ERAAP / +%Tpn / +%WT). Antibodies Large-scale peptide sequencing by tandem mass spectrometry The following Abs used for flow cytometry analysis were purchased from Peptide sequencing using immunoaffinity purification of pMHC complexes b b BD Biosciences: anti–H-2K (AF6-88.5), anti–H-2A (25-9-17), anti-CD8a from detergent-solubilized spleen lysates was performed as reported pre-

(53-6.7), anti-CD4 (RM4-5), and anti–IFN-g (XMG1.2). Abs CD16/32 (Fc viously (35). In brief, freshly isolated spleen cells from 25 C57BL/6 and by guest on September 24, 2021 b block, clone 93) and anti–H-2D (28-14-8) were purchased from eBio- 50 Tpn-deficient mice were lysed and pMHC I was immuno-purified using sciences. For in vivo depletions, purified anti-NK1.1 Ab (PK136) from Bio- mAbs Y3 (anti–H-2Kb) and B22.249 (anti–H-2Db). Samples were subject Xcell was used. To block presentation by MHC I to T cell lines, we used to fast protein liquid chromatography-HPLC fractionation, and sequence b b the following culture supernatants: anti–H-2K (5F1.5), anti–H-2D (B22.249), identification by nanochip electrospray ionization-quadrupole time of b or anti–H-2A (M5/114). In the immunoaffinity purification of pMHC I for flight mass spectrometer. Peptides were identified with high confidence b b mass spectrometry, anti–H-2K (Y3) and anti–H-2D (B22.249) were used. using an initial search with Spectrum Mill algorithm followed by expert manual spectral validation (36). To analyze the amino acid conservation Cell lines and DNA constructs of groups of more than seven peptides, we used WebLogo program (http:// ERAAP and TAP double-deficient (ERAAP2/2TAP2/2) fibroblasts were weblogo.berkeley.edu/). previously reported, and immortalized ERAAP2/2Tpn2/2 fibroblast cell lines were generated in the same manner as previously described (25). The use and generation of b-galactosidase (lacZ)–inducible T cell hybridomas Results B3Z, 30NXZ, 1AZ, 11P9Z, LPAZ, 27.5Z, and BEKo8Z have been de- ERAAP and Tpn differentially influence pMHC I repertoire scribed elsewhere (25, 31). Activation of T cell hybridomas was deter- mined by measurement of cleavage of the lacZ substrate chlorophenol red- ERAAP and Tpn are both ER-resident editors of the peptide rep- b-D-galactopyranoside (Roche). Splenocytes from indicated mice treated ertoire presented by MHC I. To assess the relative contributions of with 200 ng/ml LPS (Sigma) for 14–16 h were used as APCs for T cell ERAAP and Tpn to the overall peptide repertoire, we measured hybridomas. The ES-X9[SHL8] construct containing ER-localization se- pMHC I surface expression by flow cytometry (Fig. 1A–C). Spleen quence followed by N terminally extended antigenic SIINFEHL (SHL8) 2/2 2/2 2/2 2/2 peptide was previously described (34). cells from WT, Tpn , ERAAP , ERAAP Tpn ,TAP- deficient (TAP2/2), or KbDb dko strains were stained for Db and Peptide extraction and reverse-phase HPLC analysis Kb MHC I (Fig. 1A, 1B), as well as the MHC II molecule Ab as a The preparation, fractionation, and detection of the peptide extracts has negative control (Fig. 1C). Compared with WT C57BL/6 mice, loss been described previously (34). In brief, the peptides were eluted from the of ERAAP diminished MHC expression by ∼20%, whereas loss of cells by 10% formic acid, fractionated by reverse-phase HPLC, treated Tpn with or without ERAAP expression diminished MHC expres- with trypsin, and assayed as described previously. Synthetic peptides were ∼ prepared by D. King (University of California at Berkeley). sion by 90% (16, 17, 34). However, Tpn-deficient cells expressed relatively more pMHC I compared with cells lacking TAP1, the Immunizations and CTL lines peptide transporter, or cells completely lacking Kb,aswellasDb To generate T cell lines, we immunized female Tpn-deficient or WT mice MHC I. Thus, ERAAP and especially Tpn were required for main- i.p. with 2 3 107 spleen cells from male WT or Tpn-deficient mice, re- taining normal level of pMHC I expression on the cell surface. spectively. Ten days after immunization, spleen cells were restimulated We next assessed the influence of ERAAP and Tpn on the in vitro. Cultures contained 20 U/ml (first two restimulations) or 50 U/ml (subsequent restimulations) hIL-2 (BD Biosciences) and irradiated spleen generation of specific peptides bound to MHC I. We measured the cells from female mice of the same genotype used for immunization. presentation of a panel of endogenously processed peptides on 2 2 Additional restimulations were done every 7–10 d. the surface of spleen cells from WT B6, Tpn-deficient (Tpn / ), The Journal of Immunology 1549 Downloaded from

FIGURE 1. Tpn and ERAAP differentially affect surface pMHC I expression. (A–C) Surface (A) H-2Db,(B) H-2Kb, and (C) H-2Ab expression on spleen cells derived from WT, ERAAP2/2, Tpn2/2, ERAAP2/2Tpn2/2,TAP2/2, and KbDb dko mice. Bar graphs summarize the mean fluorescence intensity. FACS plots are gated on live cells. Top panels show representative data; bottom panels show data from three independent experiments. or ERAAP-deficient (ERAAP2/2) mice (Fig. 2). The absence of certain pMHC I were absent in Tpn-deficient mice, specific CD8+

ERAAP affected pMHC I presentation differentially (23, 25, 31), T cells would not be tolerized to them and would respond to the http://www.jimmunol.org/ ranging from no detectable change in the pKb ligand recognized novel pMHC I expressed by WT cells. We immunized Tpn2/2 by the 27.5Z hybridoma to an almost complete loss of the pMHC I mice with WT spleen cells expressing the normally diverse pMHC ligands recognized by the LPAZ and 11p9Z hybridomas. In con- I repertoire. After 10 d, splenocytes from recipient mice were trast, presentation of pMHC I ligands recognized by 1AZ, 30NXZ, restimulated for a week with WT spleen cells. The cultures were and BEko8Z hybridomas was markedly enhanced on surface of then analyzed for presence of CD8+ T cells that produced IFN-g ERAAP-deficient cells. In contrast, loss of Tpn was generally when stimulated with spleen cell APCs of the indicated genotype. deleterious for all the pMHC I ligands tested. Taken together, The Tpn2/2 anti-WT CD8+ T cells responded strongly to WT APCs these observations show that normal expression of pMHC I was but not to self APCs (Fig. 3A, 3B), showing that Tpn-deficient mice by guest on September 24, 2021 influenced by ERAAP and even more so by Tpn. perceived normal pMHC I as foreign in WT cells. We infer that Tpn deficiency caused the loss of many pMHC I normally present in Absence of Tpn causes selective loss of pMHC I ligands WT cells. Typical of peptides presented by MHC I, these pMHC I To further define the specific changes that occurred in the pMHC I required TAP for their presentation (Fig. 3B). repertoire because of loss of Tpn, we took advantage of the immune To establish the ligand specificity of the responding CD8+ Tcells, systems’ ability to detect differences between self and nonself. If we used spleen cells from KbDb dko mice as APCs. The Tpn2/2

FIGURE 2. Tpn and ERAAP differentially affect presentation of endogenous pMHC I by spleen cells. Splenocytes from indicated male mice were stimulated with 200 ng/ml LPS overnight before incubation with CD8+ T cell hybridomas 27.5Z, LPAZ, 11P9Z, 1AZ, 30NXZ, or BEKo8Z. Hybridomas specifically recognize peptides encoded by the indicated genes and presented on H-2Db, H-2Kb, or Qa-1b. Hybridoma response is determined by assessing conversion of the colorimetric substrate chlorophenol red-b-D-galactopyranoside to chlorophenol red measured at absorbance at 595 nm (A595). Data are representative of three independent experiments. 1550 GENERATING CANONICAL PEPTIDES FOR MHC I anti-WT T cells did not respond to KbDb dko spleen cells, indicating access to incoming peptides and empty MHC I. To directly assess that Tpn-dependent peptides were presented by Kb and Db MHC I in the role of PLC in determining ERAAP function, we analyzed WT cells, rather than nonclassical MHC I molecules (Fig. 3B). peptide processing in cells lacking the key PLC components TAP or Tpn (Fig. 5A, 5B). We transfected ERAAP2/2TAP2/2 or Tpn-deficient cells also express novel immunogenic pMHC I 2 2 2 2 ERAAP / Tpn / fibroblasts with N-terminally extended SHL8 To determine whether loss of Tpn editing also resulted in presenta- precursors in the presence or absence of WT ERAAP. The pep- 2 2 tion of novel pMHC I, we immunized WT mice with Tpn / cells. tides extracted from transfected cells were fractionated by HPLC The recipient T cells were restimulated in vitro and analyzed for to separate the untrimmed precursor from the trimmed peptide 2 2 2 2 responses to WT or Tpn / APCs. WT anti-Tpn / T cells produced products. The HPLC fractions were assayed for presence of an- 2 2 IFN-g in response to Tpn / , but not to self WT APCs, indicating tigenic peptides containing SHL8 with or without the N-terminal 2 2 presence of novel pMHC I in Tpn / cells (Fig. 4A, 4B). extension using the SHL8/Kb-specific B3Z hybridoma as de- Tpn expression appears to affect TAP stability and could thus scribed earlier (34). In ERAAP2/2TAP2/2 cells without ERAAP influence peptide transport into the ER (37). Therefore, it was pos- (vector alone), the N-terminally extended precursor peptide was sible that the peptides presented in the absence of Tpn could be the predominant peptide species (Fig. 5A, upper panel). Upon independent of TAP transport (38, 39). However, when we used coexpression of ERAAP, the precursor peptide was no longer de- 2 2 2 2 TAP / cells as APCs, WT anti-Tpn / T cells did not produce tected, and two peaks corresponding to the precisely cleaved IFN-g (Fig. 4B), indicating that peptide transport is required for SHL8 octapeptide and the KSHL8 nonapeptides were found (Fig. presentation of these pMHC I, and that this presentation is not 5A, lower panel). The SHL8 and KSHL8 peptides, respectively, a consequence of TAP deficiency. Furthermore, although blocking represent the final products presented by the Kb and Db MHC I Downloaded from the Ab MHC II molecule or the Kb MHC I molecule did not inhibit present in these cells. Likewise, the same precursor and processed IFN-g production in any of the five lines tested, blocking with the peptides were detected in presence or absence of ERAAP in Tpn- anti-Db Ab inhibited T cell responses more effectively than block- deficient cells (Fig. 5B). Thus, in the ER, ERAAP could trim ing with anti-Kb (Fig. 4C). The possible contribution of CD8+ antigenic precursors to their final products in the absence of TAP T cells restricted by other nonclassical MHC I to the overall CD8+ or Tpn. The results show directly that expression of either TAP or

T cell response is presently unclear. Alternatively, some ligands Tpn, and therefore an intact PLC, is not required for N-terminal http://www.jimmunol.org/ may represent novel pMHC I conformations that are not recognized trimming of antigenic precursors by ERAAP. by conventional anti-MHC I Abs. Together, these results show that Tpn- and ERAAP-deficient cells express unique, loss of Tpn not only caused a profound loss of pMHC I, it also nonoverlapping pMHC I allowed generation of new and immunologically distinct pMHC I. In Tpn2/2 mice, many Kb and Db are less stable on the cell To further assess the relationship between peptide editing by surface than in their WT counterparts (16). To test the stability of ERAAP and Tpn, we examined the potential overlap between the immunogenic pMHC I in Tpn2/2 cells, we used WT or Tpn2/2 novel peptides generated in the absence of Tpn or ERAAP. T cell splenocytes as APCs after treatment with BfA, an inhibitor of lines generated in WT mice by immunization with Tpn or ERAAP-

ER-Golgi transport, for either 2 or 4 h (Fig. 4D). Although BfA deficient cells were tested for responses to various APCs. The by guest on September 24, 2021 2 2 treatment of WT APCs did not affect expression of pMHC I Tpn / anti-WT CD8+ T cell lines responded to both WT and 2 2 recognized by the Tpn2/2 anti-WT T cells, treatment of Tpn2/2 ERAAP / APCs equally well, suggesting that both cells pre- 2 2 APCs caused a dramatic loss of WT–anti-Tpn2/2–stimulating li- sented the unique pMHC I that were lost in Tpn / mice (Fig. 2 2 gands. Thus, even though pMHC I expressed by Tpn2/2 cells were 6A). In contrast, the WT anti-Tpn / lines recognized only Tpn- 2 2 markedly less stable than those expressed by WT cells, they were deficient cells but did not recognize either WT or ERAAP / cells 2 2 2 2 nevertheless highly immunogenic. (Fig. 6B). Likewise, WT anti-ERAAP / lines recognized ERAAP / APCs but did not respond to either WT or Tpn2/2 APCs (Fig. 6C). PLC components TAP and Tpn are not required for peptide The lack of cross-reactivity between T cells specific for Tpn2/2 or trimming by ERAAP ERAAP2/2 APCs showed that ERAAP and Tpn have distinct The loss and gain of novel pMHC I in either Tpn or ERAAP- and nonoverlapping roles in editing peptides for presentation deficient cells suggested the function of these two editors may on MHC I. be linked. For example, peptide trimming could be more effective if To further rigorously establish the distinction between the im- ERAAP interacted with the PLC that could provide ERAAP with munologically distinct Tpn or ERAAP-dependent ligands, we

FIGURE 3. Tpn-deficient mice elicit CD8+ T cell response to pMHC I expressed by WT cells. (A) Intracellular IFN-g+ produced by Tpn2/2 anti-WT CD8+ T cell lines in response to WT or Tpn2/2 APCs. (B) Tpn2/2 anti-WT CD8+ T cell IFN-g+ response against WT, Tpn2/2,TAP2/2,orKbDb dko APCs. Each point represents an individual mouse. Data are from one of two independent experiments (A) or pooled from two independent experiments (B). (A and B) Numbers indicate percent IFN-g+ cells in the CD8+ T cell gate. The Journal of Immunology 1551 Downloaded from http://www.jimmunol.org/

FIGURE 4. Tpn-deficient cells elicit CD8+ T cell responses in WT mice. (A)IFN-g+ response of WT anti-Tpn2/2 CD8+ T cell lines against WT or Tpn2/2 APCs. Numbers indicate percent IFN-g+ cells of total CD8+ T cells. (B) WT anti-Tpn2/2 CD8+ T cell responses against WT, Tpn2/2,orTAP2/2 APCs. (A and B) Numbers indicate percent IFN-g+ cells in the CD8+ T cell gate. (C) WT anti-Tpn2/2 CD8+ T cell responses against WT APCs previously treated with blocking Abs to MHC I Kb (a-Kb)orDb (a-Db), or to MHC II Ab (a-Ab). Percentages of CD8+IFN-g+ cells were normalized to no Ab control (No Ab). (D) IFN-g+ production by WT anti-Tpn2/2 (left) and Tpn2/2 anti-WT (right) CD8+ T cell lines against splenocyte APCs treated with BfA for 2 or 4 h before coculture with T cells. Percent of CD8+ IFN-g+ was normalized to untreated (2). Data are representative of three independent experiments (A, B) or are pooled from two independent experiments (C, D).

2 2 2 2 assessed the ability of WT mice to eliminate ERAAP / or Tpn / lower MHC I expression (17, 40). To distinguish the three pop- by guest on September 24, 2021 target cells in vivo (Fig. 7). We primed WT mice with splenocytes ulations of donor cells recovered from host animals in vivo, we from ERAAP2/2, Tpn2/2, or WT mice as a negative control. labeled each target cell population with a different fluorescent dye Seven days later, the mice were challenged with a cell mixture as shown schematically (Fig. 7A). After 20 h, spleens from host containing an equal number of WT, ERAAP2/2, and Tpn2/2 WT mice were analyzed for the presence of each labeled cell spleen cells as targets (Fig. 7A, 7B). We depleted NK cells in host population (Fig. 7B, output). A decrease in the percentage of cells mice before immunization and challenge to obviate the possible recovered relative to the WT (self) targets indicates elimination of effect of these cells in targeting Tpn2/2 or ERAAP2/2 cells with individual populations by the immune system of WT hosts.

FIGURE 5. Peptide trimming by ERAAP does not require Tpn or TAP. (A and B) The ES-X9[SHL8] construct was cotransfected with ERAAP cDNA or with empty vector into (A) ERAAP2/2TAP2/2 or (B) ERAAP2/2Tpn2/2 fibroblasts. Cell lysates were fractionated by RP-HPLC and trypsinized to release SHL8 peptides before detection with B3Z hybridoma in the presence of L cells expressing H-2Kb. Synthetic SHL8 and KSHL8 peptide run under identical conditions verified the HPLC fraction numbers. Data are representative of three independent experiments. 1552 GENERATING CANONICAL PEPTIDES FOR MHC I

FIGURE 6. Immunogenic pMHC I expressed by Tpn or ERAAP-deficient cells do not overlap. T cell responses against indicated splenocyte APCs. (A) Tpn2/2 anti-WT T cell response, (B) WT anti-Tpn2/2 T cell response, and (C) WT anti-ERAAP2/2 T cell response against spleen cell APCs derived from WT, Tpn2/2, or ERAAP2/2 mice. Immunizations to induce specific CD8+ T cells and intracellular cytokine staining to detect IFN-g production are described earlier. The p values were calculated by Mann–Whitney U test. **p , 0.01, ***p , 0.001. Data are pooled from two (C) or three (A, B) independent experiments.

Mice primed with ERAAP2/2 cells efficiently eliminated munogenic to WT T cells and are distinct from the unedited peptides ERAAP2/2 targets but did not influence the recovery of Tpn- presented by ERAAP-deficient cells. Our previous analysis of the 2/2 deficient or self-WT cells (Fig. 7C). In contrast, WT mice primed unedited peptides in ERAAP spleen cells had found that the Downloaded from with Tpn2/2 splenocytes eliminated Tpn2/2 targets, but not novel peptides were longer in length and varied at their N termini ERAAP2/2 or WT targets. Finally, there was no specific loss of any (30). Whether specific structural changes also occurred in peptides of these target cells in mice primed with self-WT cells. Furthermore, produced in absence of Tpn is not known. To define the Tpn- the requirement for prior immunization for the in vivo elimination dependent changes in the peptide repertoire, we isolated Kb and of ERAAP2/2 or Tpn2/2 targets suggests that these responses are Db pMHCIfromWTaswellasTpn2/2 splenocytes, eluted the

mediated by the adaptive immune system. The in vitro and bound peptides, and determined their amino acid sequences by http://www.jimmunol.org/ in vivo assessment of WT anti-Tpn2/2 and WT-anti ERAAP2/2 tandem mass spectrometry. From WT cells, we identified 210 and T cell lines demonstrates that the unedited peptide repertoires in 163 peptides bound to Db and Kb, respectively. In contrast, the cells deficient in ERAAP versus Tpn were distinct without any lower pMHC I expression in Tpn-deficient cells allowed recovery detectable overlap. of fewer peptides; 63 and 22 peptides bound to Db and Kb,re- spectively. We did not find any obvious differences in the intra- Tpn-deficient cells present peptides lacking canonical cellular localization of the source proteins for these peptides (data consensus motif not shown). Many peptides in Tpn2/2-deficient splenocytes were The earlier findings showed that loss of Tpn results in the presen- also found in WT mice (Fig. 8A, Supplemental Fig. 1A, Sup- tation of a novel set of peptides on the cell surface that are im- plemental Tables I, II). Remarkably, comparison of the unique by guest on September 24, 2021

FIGURE 7. Different ligands are used for rejection of Tpn- or ERAAP-deficient cells by WT mice in vivo. T cells from WT mice primed 7 d earlier with male WT, Tpn2/2, or ERAAP2/2 splenocytes were assessed for their ability to specifically eliminate WT, Tpn2/2, or ERAAP2/2 female targets in vivo. Targets were given distinct labels so they could be compared in the same host mouse: WT = CFSE (high dose); Tpn2/2 = CFSE (low dose); ERAAP2/2 = CMAC. (A) Schematic indicating the populations that represent ERAAP2/2, Tpn2/2, and WT. (B) Representative FACS plots of labeled targets before (input) and after (output) challenge. Input refers to proportions of each labeled cell type before challenge, whereas output refers to the labeled populations identified 20 h posttransfer. (C) Summary of in vivo killing assay from (B). Negative loss (gain) is plotted as zero. Data are representative of two inde- pendent experiments (B) or are pooled from two independent experiments (C). The Journal of Immunology 1553 peptides in Tpn2/2 cells with their WT counterparts revealed Discussion 2 2 significant differences. First, peptides in Tpn / cells were mark- We show in this article that generation of the optimal pMHC I edly longer than those in WT cells (Fig. 8B, Supplemental Fig. 1B). repertoire required independent editing by Tpn and ERAAP. The Second, the canonical asparagine (N) residue at the p5 position of absence of Tpn profoundly altered the pMHC I repertoire, causing Db bound peptides was consistently absent in peptides produced in loss of many pMHC I normally expressed in WT cells, as well as absence of Tpn (Fig. 8C, 8D). A loss of the conserved phenylala- gain of other novel pMHC I. Interestingly, the new pMHC I in nine or tyrosine residues (F/Y) at the p5 anchor position was less Tpn2/2 cells were immunologically distinct from those found in obvious in peptides bound to Kb (Supplemental Fig. 1C, 1D). cells lacking the N-terminal peptide editor, ERAAP. The CD8+ The most striking difference in the amino acid sequences was T cells in WT mice elicited by either ERAAP2/2 or Tpn2/2 cells found in the C-terminal (PV) position of Kb and Db peptides eluted were highly specific and did not cross-react. Amino acid se- 2 2 from Tpn / samples. Typically, the C-terminal position is oc- quences of unedited peptides revealed that differences between cupied by an aliphatic amino acid: Met (M), Ile (I), Leu (L), or Val the peptides in ERAAP versus Tpn-deficient cells were in their N- (V), as seen in peptides found in WT cells (Fig. 8C, 8D, Sup- and C-terminal anchor residues, respectively. Thus, Tpn defines 2/2 plemental Fig. 1C, 1D). However, in the Tpn samples, a higher the C terminus, whereas ERAAP shapes the N terminus of ca- frequency of abnormal amino acids was identified at PV. These nonical MHC I peptides. included Lys (K), Ser (S), Asn (N), Pro (P), and Ala (A) for Db The MHC I molecules present an extraordinarily diverse set of b (Fig. 8C, 8D), and Thr (T) and Pro (P) for K (Supplemental Fig. peptides to allow effective immune surveillance of virtually all 1C, 1D). Thus, the ability to choose the appropriate C-terminal endogenous proteins (3). Nevertheless, the canonical peptides amino acid, a key determinant of pMHC I stability, was lost in the presented by a given MHC I molecule share certain key features: Downloaded from absence of Tpn. a length of 8–10 aa, and the presence of conserved residues at the We verified the MHC I binding characteristics of a few repre- C terminus and at an internal p2 or p5 position (41). These con- sentative peptides by assessing their ability to stabilize Db or Kb served amino acids are called anchor residues because their on the surface of TAP-deficient RMA/s cells. Each peptide presence determines the stability of the pMHC I (42). Editing of bound to the respective MHC I, although the binding was lower the available peptide repertoire is crucial to ensure that only the b b and the decay was faster than the canonical K and D binding stable pMHC I reach the surface that are capable of triggering http://www.jimmunol.org/ peptides (Supplemental Fig. 2A–D). From Tpn-deficient mice, CD8+ T cell responses. To generate the optimal pMHC I repertoire even the peptides that contained both anchor residues, such as thus requires the editing mechanisms to determine the appropriate FSPLNPVRV (Db) and SLNRFIPL (Kb), were suboptimal binders C termini, the length, as well as the internal conserved residues. of MHC I. Thus, in contrast with the striking influence of ERAAP The extent to which these choices are determined by the shape of on the N termini, Tpn primarily influenced the C-termini peptides the peptide binding groove of the MHC I itself versus other key presented by MHC I. players in the pathway has remained unclear. by guest on September 24, 2021

FIGURE 8. MHC I in WT and Tpn-deficient cells present unique peptides. The H-2Db MHC I were immunoprecipitated from WT and Tpn-deficient spleen cells. Eluted peptides were sequenced by mass spectrometry and manually validated. (A) Numbers of distinct or shared peptides found in pMHC I expressed by WT or Tpn-deficient cells. (B–D) Analysis of the unique peptides from WT and Tpn-deficient cells. (B) The average lengths of peptides recovered from WT or Tpn-deficient cells. The indicated p value was calculated by two-tailed t test. (C) Conservation of p5 and C-terminal anchor residues (PV). Plots represent frequency of Asn (N) at P4–6 or the frequency of indicated amino acids at PV of H-2Db peptides. (D) Logo representation of H-2Db peptides eluted from WT or Tpn-deficient cells. Peptides are grouped according to their lengths, and the numbers of peptides in each group are indicated. The height of each bar is proportional to the degree of amino acid conservation, and the height of each letter composing the column is proportional to its frequency at the given position. Amino acids are colored as follows: hydrophobic (black), aromatic (purple), acidic (red), basic (blue), neutral (green), and the others (orange). 1554 GENERATING CANONICAL PEPTIDES FOR MHC I

The MHC I molecules are loaded with their peptide cargo in the Previous studies have assumed that cytosolic enzymes, such as ER within the PLC (8). The crucial role of the PLC in peptide the proteasome, generated peptides with canonical C-terminal loading has been demonstrated by the severe loss of peptide- residues (4, 45). However, our data showed that Tpn is the ulti- loaded MHC in cells without Tpn (16, 17). Tpn is the key element mate arbitrator for ensuring that peptides with appropriate C- that holds the PLC together and retains the MHC I molecules in terminal residue are presented. Consistent with a role for Tpn- the ER until loaded with appropriate peptides (43). When various mediated editing at the C termini of peptides, T134K mutation antigenic peptides were assessed for binding MHC I in the ER and in the a2 helix of MHC I was found to affect Tpn–MHC I subsequent display on the cell surface, presentation efficiency was interactions in human cells (46). Although the molecular mecha- determined by peptide affinity in presence of Tpn, but not other nism by which Tpn edits peptides is still unclear, putative models PLC components (20). Likewise, through in vitro reconstitution for peptide optimization via Tpn include accelerated dissociation in microsomes, Tpn mediated the binding of high-affinity peptide of unfavorable peptides (40, 44, 47) or maintenance of the MHC I to MHC I (44). Taken together with the severe reduction of in a peptide-receptive conformation until bound by a high-affinity pMHC I expression on the surface when Tpn was lost, these obser- peptide (48). Notably, Tpn–MHC interactions occurred with the vations suggest that most peptides require editing in the PLC. Which C-terminal end of the MHC I peptide binding groove. structural aspects of the canonical MHC I–bound peptides deter- In conclusion, Tpn and ERAAP edit distinct aspects of the mined the appropriate affinity threshold remained unclear. peptides loaded onto MHC I molecules in the ER and explain the We first used an immunological approach to characterize the canonical features of the MHC I peptide cargo. Because the changes changes in the peptide repertoire caused by loss of Tpn. We elicited in pMHC I ligands due to failure of these editing steps are non-

CD8+ T cell responses in WT mice immunized with spleen cells overlapping, it should be interesting to determine why Tpn or Downloaded from from Tpn-deficient mice and vice versa. Because the immune re- ERAAP are differentially targeted for immune evasion by viruses sponse has evolved to distinguish nonself from self, the CD8+ (49–51) and in cancer (40, 52). T cell responses are exquisitely specific for nonself pMHC I. Importantly, the T cell can reveal changes in the pMHC I reper- toire caused by alterations in Tpn expression that are not readily Acknowledgments + We thank David King for peptide synthesis and Hector Nolla for assistance detected by any other assay. The specificity of the CD8 T cell http://www.jimmunol.org/ responses showed that the absence of Tpn, like that of ERAAP with flow cytometry. described earlier (26), caused the loss of many WT pMHC I, as well as gain of other immunogenic pMHC I. As in ERAAP- Disclosures deficient cells, the new pMHC I expressed by Tpn-deficient cells The authors have no financial conflicts of interest. were also less stable, suggesting that the presented peptides were structurally distinct from their WT counterparts. Interestingly, different structural changes in the pMHC I rep- References ertoirewerecausedbythelossofTpnratherthanbylossofERAAP. 1. Shastri, N., S. Cardinaud, S. R. Schwab, T. Serwold, and J. Kunisawa. 2005. All the peptides that fit: the beginning, the middle, and the end of the MHC class I by guest on September 24, 2021 We compared the effects of ERAAP versus Tpn deficiency to de- antigen-processing pathway. Immunol. Rev. 207: 31–41. termine whether there were similarities in their editing functions. To 2. Cresswell, P. 2005. Antigen processing and presentation. Immunol. Rev. 207: 5–7. examine a broad set of endogenous pMHC I, we compared the 3. Rammensee, H.-G., K. Falk, and O. Ro¨tzschke. 1993. 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Avg. length (AA) c. Conservation of P5 Conservation of P a. Peptides (#) b. WT 75 WT 200 10 **p<0.01 100 Tpn-/- -/- 80 Tpn 150 50 Shared 9 60 100 151 40 Length 8 25 50 20

9 Prevalence (%) Phe or Tyr at P4-P6 (%) P4-P6 at Tyr or Phe

Number of peptides of Number 0 7 0 0 -/- -/- -/- WT Tpn WT Tpn WT Tpn MI L V F A T P Amino Acid

Length: 8 9

4 n=124 d. 4 n=26 3 3

2 bits 2 WT bits 1 I A I Y I I 1 I Y I 0

S 1 2 3 4 5 6 7 I 8 0 2 4 5 6 7 9 1 VAT SV F E F N LV R SQVA NG F A I 3 EPR A FD Y E Q KR 8 LM N N L V Y M C T V S T T P V N L T EKH V L F V C

4 n=7

3

-/- 2 Tpn bits 1

0 1 2 3 4 M 5 6 7 8 N P FP C

Supplementary Figure 1. Tapasin-deficient cells present unique H-2Kb peptides, which are longer and lack canonical anchor residues. Kb pMHC I were immunoprecipitated from B6 (WT) and Tpn-/- spleen cells. Eluted peptides were sequenced by mass spectrometry and manually validated. (a) Numbers of peptides specific to WT or Tpn-/- cells or found in both data sets (shared). (b) The average lengths of peptides specific to WT or Tpn-/-!"$" H-2Kb $'$+3$;>'"$?@Db peptides. (d) Logo representation of H-2Kb peptides specific to WT or Tpn-/- cells. Peptides are grouped according to their lengths, and the numbers of included peptides are indicated. The height of each column is proportional to the degree of amino acid conservation and the height of each letter composing the column is proportional to its frequency at the given position. Amino acids are colored as follows: hydrophobic (black), aromatic (purple), acidic (red), basic (blue), neutral (green), and the others (orange). Supplementary Figure 2

a. FSPLNPVRV FVHPGAATVPTM SNLYNHPQLI WAPENAPLKS FAPVAPKFTPV 4000 4000 4000 4000 4000

3000 3000 3000 3000 3000

2000 2000 2000 2000 2000

expression (MFI) expression 1000 1000 1000 1000 1000 b

0 0 0 0 0

H-2D -4 -3 -2 -1 0 1 2 3 4 -4 -3 -2 -1 0 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10-4 10-3 10-2 10-1 100 101 102 103 104 10-4 10-3 10-2 10-1 100 101 102 103 104 10-4 10-3 10-2 10-1 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10 Peptide Concentration (nM) Peptide Concentration (nM) Peptide Concentration (nM) Peptide Concentration (nM) Peptide Concentration (nM) b. FSPLNPVRV FVHPGAATVPTM SNLYNHPQLI WAPENAPLKS FAPVAPKFTPV 100 100 100 100 100

75 75 75 75 75

50 50 50 50 50 expression b

25 25 25 25 25 (relative to 0hr) to (relative H-2D 0 0 0 0 0 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 Recovery Time (hours) Recovery Time (hours) Rec overy Time (hours) Recovery Time (hours) Recovery Time (hours)

SLNRFIPL ISPYFINT VGQQFGAVGV c. 1000 1000 1000 -/- 750 750 750 Tpn Peptide

500 500 500 LYL8

expression (MFI) expression 250 250 250 b WI9 0 0 0 H-2K 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 Peptide Concentration (nM) Peptide Concentration (nM) Peptide Concentration (nM)

d. SLNRFIPL ISPYFINT VGQQFGAVGV 100 100 100

75 75 75

50 50 50 expression 25 25 25 b

3

(relative to 0hr) to (relative 0 0 0 H-2K 0 1 2 3 0 1 2 3 0 1 2 Recovery Time (hours) Recovery Time (hours) Recovery Time (hours)

Supplementary Figure 2. Peptides from tapasin-deficient cells form less stable pMHC I on the surface and decay faster. (a). Stabilization of H-2Db MHC I with Tpn-/- peptides. RMA/s cells, incubated overnight to stabilize empty pMHC I on the surface, were incubated with indicated Tpn-/- peptides (top of each graph) or control LTFNYRNL (LYL8, Kb-binding) or WMHHNMDLI (WI9, Db-binding) peptides for 1 hour at 22°C followed by incubation at 37°C for 3 hours prior to analysis by flow cytometry. (b) Decay of H-2Db surface UXZ[\@)Z"[^[X""_` ^"x?@{b. MFI for each sample is normalized to 0hr recovery time. (c) Stability of Kb peptides performed as in (a). (d) Decay of Kb peptides as in (b). Data are representative of three independent experiments (a and c) or contain pooled data from at least three independent experiments (b and d). .                

Data Peptide Accession Set Sequence Length Number Source Protein Name Nuclear factor NF-kappa-B p100 Tpn-/- AALQNLEQL 9 Q9WTK5 subunit C-1-tetrahydrofolate synthase, Tpn-/- AGILNGKLV 9 Q922D8 cytoplasmic

Tpn-/- FSHPNVLPV 9 O55222 Integrin-linked protein kinase Heterogeneous nuclear Tpn-/- FSPLNPVRV 9 O35737 ribonucleoprotein H KN motif and ankyrin repeat Tpn-/- GGPENTLVF 9 Q8BX02 domain-containing protein 2

Tpn-/- GINVNAAPF 9 Q60865 Caprin-1

Tpn-/- NSGINVMQV 9 O89090 Transcription factor Sp1

Tpn-/- SSLLSPMSV 9 P80315 T-complex protein 1 subunit delta

Tpn-/- YSSENISNF 9 O70400 PDZ and LIM domain protein 1 SAM domain and HD domain- Tpn-/- YVFPGASHN 9 Q60710 containing protein 1 Probable JmjC domain-containing Tpn-/- SLLGGHPRL 9 Q69ZK6 histone demethylation protein 2C

Tpn-/- LHLGVTPSVL 10 O89023 Tripeptidyl-peptidase 1

Tpn-/- SNLYNHPQLI 10 Q6NSQ7 Protein LTV1 homolog

Tpn-/- VGPLGSTIPM 10 Q9DCP9 DAZ-associated protein 2 Pre-mRNA-processing factor 40 Tpn-/- VMSGMMMSHM 10 Q9R1C7 homolog A V-type proton ATPase subunit B, Tpn-/- VSGVNGPLVI 10 P62814 brain isoform

Tpn-/- WAPENAPLKS 10 P18760 Cofilin-1 F-actin-capping protein subunit Tpn-/- AQYNLDQFTPV 11 P47754 alpha-2

Tpn-/- FAPVAPKFTPV 11 Q62523 Zyxin

Tpn-/- FAPVNVTTEVK 11 P10126 Elongation factor 1-alpha 1 Trafficking protein particle Tpn-/- LLTSGVEFESL 11 Q3U0M1 complex subunit 9 Glyceraldehyde-3-phosphate Tpn-/- SAPSADAPMFV 11 P16858 dehydrogenase

Tpn-/- SLGVGYRTQPM 11 Q8C2Q3 RNA-binding protein 14 Histone-lysine N-methyltransferase Tpn-/- FALQNATGDGKF 12 Q6P2L6 NSD3

Tpn-/- FVHPGAATVPTM 12 Q9DCP9 DAZ-associated protein 2

Tpn-/- SQYGWSGNMERI 12 P08113 Endoplasmin

B6 IRMMLEII 8 Q8CHY3 Dymeclin FH1/FH2 domain-containing protein B6 AALLNFDEF 9 Q6P9Q4 1 U3 small nucleolar RNA-interacting B6 AALLNTDLV 9 Q91WM3 protein 2 Tripartite motif-containing B6 AALQNAVAF 9 Q80VI1 protein 56

B6 AALQNLVKI 9 P70168 Importin subunit beta-1

B6 AALVNLDSL 9 Q8CHU3 Epsin-2 Dolichyl-P-Man:Man(7)GlcNAc(2)-PP- dolichyl-alpha-1,6- B6 AAMLNYTHI 9 Q8VDB2 mannosyltransferase tRNA (uracil-5-)-methyltransferase B6 AAPFDTVHI 9 Q8BNV1 homolog A Histone deacetylase complex B6 AAPINTQGL 9 Q8BIH0 subunit SAP130

B6 AAPISPWTM 9 Q3U133 Zinc finger protein 746 Methyltransferase-like protein B6 AAPRSFIFL 9 Q3UIK4 KIAA1627 Dual specificity protein B6 AAPSNLPYL 9 Q8BFV3 phosphatase 4

B6 AAVLNPRFL 9 Q7TPS5 Uncharacterized protein KIAA0528 Heat shock protein 75 kDa, B6 AGTRNIYYL 9 Q9CQN1 mitochondrial

B6 AKIQMLDAL 9 Q9D757 Death-associated protein-like 1 Anaphase-promoting complex subunit B6 AMLTNLESL 9 Q8BZQ7 2

B6 AMNVNDLFL 9 P61021 Ras-related protein Rab-5B

B6 AMNVNEIFM 9 P35278 Ras-related protein Rab-5C

B6 AMYVHAYTL 9 O70435 Proteasome subunit alpha type-3 Histone-lysine N- methyltransferase, H3 lysine-36 B6 ASMINVEYL 9 O88491 and H4 lysine-20 specific Ankyrin repeat domain-containing B6 ASVLNVNHI 9 Q99NH0 protein 17

B6 ASVSNPLFL 9 P70428 Exostosin-2 Signal recognition particle 54 kDa B6 ATIINEEVL 9 P14576 protein

B6 ATVSNGPFL 9 Q69ZF8 Male-specific lethal 2 homolog

B6 EAVKNLEWI 9 Q02788 Collagen alpha-2(VI) chain

B6 ERAQLAAQL 9 P70436 Homeobox protein DLX-4

B6 FAHTNIESL 9 P27773 Protein disulfide-isomerase A3 Phosphatidylinositol-5-phosphate B6 FAIDDHDYL 9 Q91XU3 4-kinase type-2 gamma

B6 FAIQNPTLI 9 Q9D2H6 Transcription factor Sp2 Macrophage colony-stimulating B6 FAPKNIYSI 9 P09581 factor 1 receptor

B6 FAPYNKPSL 9 Q8R3P2 Protein deltex-2 Eukaryotic translation initiation B6 FGPINSVAF 9 Q9QZD9 factor 3 subunit I Uncharacterized protein CXorf21 B6 FIMTNVDQI 9 Q9D3J9 homolog

B6 FSFVNSEFL 9 P68404 Protein kinase C beta type B6 FSLQNQLRL 9 Q9JJ28 Protein flightless-1 homolog ETS-related transcription factor B6 FSPFNPTSL 9 Q9Z2U4 Elf-4 Uncharacterized protein C1orf103 B6 FSVTNPHTM 9 Q8CDD9 homolog

B6 FSYLNPQEL 9 Q8C2S5 F-box/LRR-repeat protein 5 Glycerol-3-phosphate dehydrogenase B6 GALKNIVAV 9 Q3ULJ0 1-like protein

B6 GAPLNLVDL 9 Q9QWT9 Kinesin-like protein KIFC1 CMP-N-acetylneuraminate-beta- galactosamide-alpha-2,3- B6 GAVKNLTYF 9 Q91Y74 sialyltransferase Interferon-induced very large B6 GMIENGPFL 9 Q80SU7 GTPase 1

B6 GQVINLDQL 9 Q3UVG3 Protein FAM91A1

B6 GSVVNSVAL 9 P59326 YTH domain family protein 1 MAP kinase-activating death domain B6 IAPTNAEVL 9 Q80U28 protein Mitochondrial import inner B6 IAVANAQEL 9 P62075 membrane translocase subunit Tim13

B6 IGPKNYEFL 9 Q8BHY8 Sorting nexin-14

B6 ILHTNLVYL 9 Q62280 Protein SSXT

B6 IQIANVTTL 9 Q9CU65 Zinc finger MYM-type protein 2 Peroxisomal carnitine O- B6 IQLMNTAHL 9 Q9DC50 octanoyltransferase

B6 IRPGATAVI 9 Q9WTR6 Cystine/glutamate transporter Interferon-inducible double stranded RNA-dependent protein B6 ISPENHISL 9 Q9WTX2 kinase activator A Delta-1-pyrroline-5-carboxylate B6 ISPFNFTAI 9 Q8CHT0 dehydrogenase, mitochondrial

B6 ISPVNPVAI 9 Q91WT8 RNA-binding protein 47

B6 KAIVNVIGM 9 Q99NB9 Splicing factor 3B subunit 1

B6 KGVFNVEVV 9 Q8R1A4 Dedicator of cytokinesis protein 7

B6 KSVENFVSL 9 Q8C3J5 Dedicator of cytokinesis protein 2

B6 KSVINTTLV 9 O35114 Lysosome membrane protein 2

B6 LAIRNDEEL 9 Q8CGP5 Histone H2A type 1-F

B6 LGLSNLTHL 9 Q9EQU3 Toll-like receptor 9 Conserved oligomeric Golgi complex B6 LLASHRTWI 9 Q921L5 subunit 2

B6 LQLLNTDYL 9 Q9R0L6 Pericentriolar material 1 protein

B6 LSHTNILVL 9 Q9EQU3 Toll-like receptor 9 Interferon-inducible double stranded RNA-dependent protein B6 LSLPNTDYI 9 Q9WTX2 kinase activator A

B6 LSNINSVFL 9 Q9Z0M6 CD97 antigen B6 MGILNTDTL 9 Q8C4V1 Rho GTPase-activating protein 24 Clathrin coat assembly protein B6 NGVINAAFM 9 Q61548 AP180

B6 NMPWNVDTL 9 Q61081 Hsp90 co-chaperone Cdc37

B6 QPPYNPTYM 9 Q9D7I0 Protein shisa-5

B6 QSLTNILHL 9 Q7TMR0 Lysosomal Pro-X carboxypeptidase Mothers against decapentaplegic B6 RALQLLDEV 9 P97471 homolog 4

B6 RALSNLESI 9 Q8R317 Ubiquilin-1 Procollagen-lysine,2-oxoglutarate B6 RSLLLLAPL 9 Q9R0E2 5-dioxygenase 1

B6 SAIINEDNL 9 Q8BG67 Protein EFR3 homolog A Guanine nucleotide exchange factor B6 SAISNLDYI 9 Q9R0C8 VAV3

B6 SALANYIHL 9 Q8BPS4 Integral membrane protein GPR180 DNA-dependent protein kinase B6 SALINLVEF 9 P97313 catalytic subunit

B6 SALLLKDVL 9 Q8BZ36 RAD50-interacting protein 1 PQ-loop repeat-containing protein B6 SAPINSVLL 9 Q8C4N4 2 Cartilage intermediate layer B6 SATINAEFV 9 Q66K08 protein 1

B6 SGIRNISFM 9 O35598 ADAM 10 Mediator of RNA polymerase II B6 SGLLNQQSL 9 Q9DB40 transcription subunit 27

B6 SGLQNFEAL 9 Q99KD5 Protein unc-45 homolog A

B6 SGVWNVTEL 9 P70257 Nuclear factor 1 X-type Echinoderm microtubule-associated B6 SHVTNVDFL 9 Q05BC3 protein-like 1 Signal transducer and activator of B6 SILVNQEVL 9 P42228 transcription 4 Microtubule-associated B6 SKLANIDYL 9 Q811L6 serine/threonine-protein kinase 4

B6 SLIILKQVM 9 Q9Z2U1 Proteasome subunit alpha type-5

B6 SLITNKVVM 9 P23492 Purine nucleoside phosphorylase

B6 SMAENSIPL 9 P58462 Forkhead box protein P1 Zinc finger CCCH domain-containing B6 SMGVNDIDI 9 Q3TIV5 protein 15

B6 SMVQNRVFL 9 Q8C3J5 Dedicator of cytokinesis protein 2

B6 SQISNTEFL 9 Q3TCJ1 Protein FAM175B

B6 SQVINPTAI 9 Q61191 Host cell factor

B6 SSLINGSFL 9 Q4JIM5 Tyrosine-protein kinase ABL2

B6 SSLMLESFF 9 Q91XQ0 Dynein heavy chain 8, axonemal Natural resistance-associated B6 SSLQNYAKI 9 P41251 macrophage protein 1 Peptidyl-prolyl cis-trans B6 SSPVNPVVF 9 Q9D868 isomerase H Glycosyltransferase-like domain- B6 SSVLNLTEL 9 Q8BW56 containing protein 1

B6 STIRLLTSL 9 P80318 T-complex protein 1 subunit gamma

B6 STLINSLFL 9 P42209 Septin-1

B6 STLVNSLFL 9 P28661 Septin-4 Tetratricopeptide repeat protein B6 SVLFNLTTM 9 Q8K2L8 15

B6 TALENLSTL 9 P19096 Fatty acid synthase

B6 TAPVNIAVI 9 Q99JF5 Diphosphomevalonate decarboxylase 1-phosphatidylinositol-4,5- bisphosphate phosphodiesterase B6 TQPLNHYFI 9 A3KGF7 beta-2

B6 TSLLNLTVI 9 Q8BM54 E3 ubiquitin-protein ligase MYLIP

B6 VAPYNTTQF 9 Q8R409 Protein HEXIM1

B6 VAVVNKVDI 9 O35604 Niemann-Pick C1 protein

B6 VAYMNPIAM 9 Q9CVI2 Protein FAM133B Ribosomal protein S6 kinase alpha- B6 VGIENFELL 9 Q8C050 5 Cytidine monophosphate-N- B6 VGLTNINVV 9 Q61419 acetylneuraminic acid hydroxylase

B6 VGVENVAEL 9 Q9ET01 Glycogen phosphorylase, liver form

B6 VGVNNPVFL 9 Q9DBN5 Peroxisomal Lon protease homolog 2

B6 VMLENYSHL 9 Q60585 Zinc finger protein 30

B6 VQNINIENL 9 P27005 Protein S100-A8

B6 VQPFNYVTL 9 Q9Z2G1 Protein fem-1 homolog A-A

B6 VSLLDIDHL 9 Q8BQM4 HEAT repeat-containing protein 3

B6 VSLNLRQVL 9 Q99N32 Beta-klotho Coiled-coil domain-containing B6 VSPLNVTAV 9 Q9CR27 protein 53 Lethal(2) giant larvae protein B6 VSVANVDLL 9 Q80Y17 homolog 1 DNA-directed RNA polymerase II B6 VVVENGELI 9 P08775 subunit RPB1

B6 WAVSNREML 9 Q80YQ8 Protein RMD5 homolog A 5'-AMP-activated protein kinase B6 WKVVNPYYL 9 Q5EG47 catalytic subunit alpha-1

B6 YALANAQQV 9 Q8VBU8 Protein BANP

B6 YAPDNVWFI 9 Q80V94 AP-4 complex subunit epsilon-1

B6 YAPINANAI 9 Q9R190 Metastasis-associated protein MTA2 Guanine nucleotide exchange factor B6 YQLLLKDFL 9 Q9CWR0 GEFT

B6 YQQMNPEAL 9 P54841 Transcription factor MafB Serine/threonine-protein kinase B6 YSIVNASVL 9 Q9ERE3 Sgk3 B6 SLPTNLIHL 9 Q6WKZ8 E3 ubiquitin-protein ligase UBR2

B6 YVVDNIDHL 9 Q9R1A8 E3 ubiquitin-protein ligase RFWD2

B6 ASLVNSPSYL 10 Q3U3D7 Transmembrane protein 131-like

B6 FALANHLIKV 10 Q9WVK4 EH domain-containing protein 1 Ubiquitin carboxyl-terminal B6 FAVVNHQGTL 10 Q5DU02 hydrolase 22 Uncharacterized protein C5orf37 B6 GALENAKAEI 10 Q9DBS8 homolog DNA (cytosine-5)-methyltransferase B6 LSLENGTHTL 10 P13864 1

B6 RSLDNGGYYI 10 P16277 Tyrosine-protein kinase BLK

B6 SAHQNYAEWL 10 Q99PL5 Ribosome-binding protein 1 Mitogen-activated protein kinase kinase kinase 7-interacting B6 SAIHNFYDNI 10 Q99K90 protein 2 Bromodomain adjacent to zinc B6 SALENGRYEL 10 O88379 finger domain protein 1A

B6 SAPVNGSVFI 10 Q91XF4 E3 ubiquitin-protein ligase RNF167 28S ribosomal protein S6, B6 SAVENILEHL 10 P58064 mitochondrial p53-associated parkin-like B6 SAVRNGLLLL 10 Q80TT8 cytoplasmic protein

B6 SAYLNMFEHI 10 Q99N16 Cytochrome P450 4F3 3-hydroxy-3-methylglutaryl- B6 SGPTNEDLYI 10 Q01237 coenzyme A reductase

B6 SMAGNIIPAI 10 Q9Z1F9 SUMO-activating enzyme subunit 2 Phosphotidylinositol phosphatase B6 SSAEVIITTL 10 P0C5E4 PTPRQ Mitochondrial import receptor B6 SSIRNFLIYV 10 B1AXP6 subunit TOM5 homolog

B6 TAIENSWIHL 10 Q9CQG2 Putative methyltransferase METT10D

B6 VGLPNESQAL 10 Q00175 Progesterone receptor

B6 VMVSNPATRM 10 Q61191 Host cell factor

B6 VSLLNPPETL 10 P51943 Cyclin-A2

B6 YAGSNFPEHI 10 P61161 Actin-related protein 2

B6 YALENFVENL 10 Q8VI75 Importin-4 G2/M phase-specific E3 ubiquitin- B6 YGPENTLPTL 10 Q5RJY2 protein ligase-like protein Rho-related GTP-binding protein B6 YSVANHNSFL 10 Q9D3G9 RhoH T-complex protein 11-like protein B6 AAAKNLSDMTL 11 Q8K1H7 2

B6 AAPQNFTPSMI 11 Q9Z1T1 AP-3 complex subunit beta-1 Sterol regulatory element-binding B6 AAVQNPALTAL 11 Q3U1N2 protein 2

B6 AQYGNILKHVM 11 Q8R4R6 Nucleoporin NUP53 Histone-arginine methyltransferase B6 ASPMSIPTNTM 11 Q9WVG6 CARM1 Leucine-rich repeat-containing B6 ATAINMKNEAL 11 Q91YK0 protein 49

B6 FSLENLRTMNT 11 Q80TG1 Uncharacterized protein KIAA1267

B6 PGSANETSSIL 11 Q80X90 Filamin-B

B6 SAIMNPASKVI 11 Q68FD5 Clathrin heavy chain 1

B6 SKFDVSGYPTI 11 P08003 Protein disulfide-isomerase A4 PX domain-containing protein B6 SSIQNFQKGTL 11 Q8BX57 kinase-like protein Ribosomal RNA processing protein 1 B6 SSPASTPLSPM 11 Q91YK2 homolog B

B6 YQFDKVGILTL 11 Q8BKT7 THO complex subunit 5 homolog Linker for activation of T-cells B6 VSYPLVTSFPPL 12 O54957 family member 1

B6 SVMENSKVLGEAM 13 P26039 Talin-1

B6 SVMENSKVLGESM 13 Q71LX4 Talin-2 shared AAGVGDMVM 9 P62830 60S ribosomal protein L23 shared AIVEHLVTL 9 Q9Z1E3 NF-kappa-B inhibitor alpha shared AMGVNLTSM 9 P17918 Proliferating cell nuclear antigen H-2 class I histocompatibility shared FAYEGRDYI 9 P01899 antigen, D-B alpha chain Cyclin-dependent kinase inhibitor shared FGPVNHEEL 9 P46414 1B shared FININSLRL 9 P16460 Argininosuccinate synthase Ribonucleoside-diphosphate shared FQIVNPHLL 9 P07742 reductase large subunit Heterogeneous nuclear shared FSPLNPMRV 9 P70333 ribonucleoprotein H2 shared FSPMGVDHM 9 P70372 ELAV-like protein 1 shared GGVVNMYHM 9 P28063 Proteasome subunit beta type-8 Heterogeneous nuclear shared GMGFGLERM 9 Q9D0E1 ribonucleoprotein M Malignant T cell amplified shared IGIENIHYL 9 Q9DB27 sequence 1 shared IQAENAEFM 9 P14901 Heme oxygenase 1 shared KALINADEL 9 P16546 Spectrin alpha chain, brain shared MAPQNLSTF 9 Q99KV1 DnaJ homolog subfamily B member 11 shared NGPQNIYNL 9 Q8K3C0 Ribonuclease kappa shared NSMVLFDHM 9 Q64511 DNA topoisomerase 2-beta Protein phosphatase 1 regulatory shared RAIENIDTL 9 Q3UM45 subunit 7 2-oxoglutarate dehydrogenase E1 shared SAPVAAEPF 9 Q60597 component, mitochondrial shared SAVVDKDFL 9 P55264 Adenosine kinase shared SSPSNMLPV 9 Q99KN9 Clathrin interactor 1 shared VGIENIHVM 9 Q8VE11 Myotubularin-related protein 6 shared VMVTNVTSL 9 P26039 Talin-1 Phosphoinositide 3-kinase adapter shared YGLKNLTAL 9 Q9EQ32 protein 1

shared FAPVNVTTEV 10 P10126 Elongation factor 1-alpha 1 Putative glycerophosphodiester shared FGIHNGVETL 10 Q8C0L9 phosphodiesterase 5 B-cell scaffold protein with shared NSPFNSKFPA 10 Q80VH0 ankyrin repeats

shared RQPSIELPSM 10 P19973 Lymphocyte-specific protein 1

shared SAPRNFVENF 10 Q91WG4 Elongator complex protein 2 ETS-related transcription factor shared SAPSAIALPV 10 Q9JHC9 Elf-2 2-oxoglutarate dehydrogenase E1 shared SAPVAAEPFL 10 Q60597 component, mitochondrial

shared SGPRNPFPPP 10 Q60843 Krueppel-like factor 2 F-actin-capping protein subunit shared AQYNMDQFTPV 11 P47753 alpha-1 Myosin regulatory light chain shared SLGKNPTDAYL 11 Q3THE2 MRLC2 Stromal membrane-associated shared AQPGASGMLTPM 12 Q7TN29 protein 2 Glyceraldehyde-3-phosphate shared SAPSADAPMFVM 12 P16858 dehydrogenase

shared FAPVNVTTEVKSV 13 P10126 Elongation factor 1-alpha 1                  

Data Peptide Accession Set Sequence Length Number Source Protein Name 60 kDa heat shock protein, Tpn-/- ISPYFINT 8 P63038 mitochondrial

Tpn-/- KAPAMFNI 8 P97351 40S ribosomal protein S3a Nuclear envelope pore membrane protein Tpn-/- SIFQFGKP 8 Q8K3Z9 POM 121

Tpn-/- SLNRFIPL 8 Q8VBV7 COP9 signalosome complex subunit 8

Tpn-/- VIPQFQTV 8 O89090 Transcription factor Sp1

Tpn-/- VNDIFERI 8 Q64475 Histone H2B type 1-B

Tpn-/- VADGFISRM 9 P24452 Macrophage-capping protein

Tpn-/- VGQQFGAVGV 10 Q8K2F8 Protein LSM14 homolog A

Tpn-/- VNRQFMAETQF 11 Q60865 Caprin-1 Cytoplasmic dynein 1 light B6 AALIYTSV 8 Q8R1Q8 intermediate chain 1 Developmentally-regulated GTP-binding B6 AAYEFTTL 8 P32233 protein 1

B6 AIFNFQSL 8 Q9CR64 Transmembrane protein 167A

B6 AIVEFATV 8 Q91WT4 DnaJ homolog subfamily C member 17

B6 ANYEFSQV 8 P56528 ADP-ribosyl cyclase 1 Tyrosine-protein phosphatase non- B6 AQYKFIYV 8 P29351 receptor type 6

B6 ASYLLAAL 8 P99027 60S acidic ribosomal protein P2

B6 ATYIFNGL 8 Q9QXK3 Coatomer subunit gamma-2

B6 AVFTFEQL 8 Q3UW53 Protein Niban

B6 AVIKFLEL 8 P43247 DNA mismatch repair protein Msh2 SH3 domain-binding glutamic acid-rich- B6 AVYAFLGL 8 Q9JJU8 like protein Loss of heterozygosity 11 chromosomal B6 AVYSFEAL 8 Q99KC8 region 2 gene A protein homolog

B6 AVYTFETL 8 Q8R1F1 Niban-like protein 1

B6 AWIKVEQL 8 Q922P9 Nuclear protein NP60

B6 EAYRFTGL 8 Q8K015 Centromere protein O Fragile X mental retardation syndrome- B6 EIVTFERL 8 Q61584 related protein 1

B6 FAYRFSNL 8 Q8BU03 Periodic tryptophan protein 2 homolog

B6 FNLVYENL 8 Q8R4V1 NFAT activation molecule 1

B6 FQQEFPSL 8 Q3TLH4 BAT2 domain-containing protein 1

B6 FVYIFQEV 8 Q99K51 Plastin-3 Putative alpha-1,2-glucosyltransferase B6 GVLRFVNL 8 Q3UGP8 ALG10-B

B6 HGYTFANL 8 Q9R1T2 SUMO-activating enzyme subunit 1

B6 HVYYFAHL 8 Q80SU7 Interferon-induced very large GTPase 1 CCR4-NOT transcription complex subunit B6 IAFIFNNL 8 Q6ZQ08 1

B6 IALRYVAL 8 Q9JIF7 Coatomer subunit beta

B6 IAVSFREL 8 Q8BJW5 Nucleolar protein 11 Suppressor of tumorigenicity protein B6 IAYAFFHL 8 Q8K4P7 7-like protein

B6 IIAAMPNL 8 O89090 Transcription factor Sp1 Peptide-N(4)-(N-acetyl-beta- B6 IIITFNDL 8 Q9JI78 glucosaminyl)asparagine amidase Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha B6 IIPMFSNL 8 Q76MZ3 isoform Eukaryotic translation initiation B6 IIQEFPAL 8 Q9QZ05 factor 2-alpha kinase 4

B6 IIVSFVNA 8 Q921M3 Splicing factor 3B subunit 3 BTB/POZ domain-containing protein B6 INFDFNTI 8 Q8CDD8 KCTD20 Glutathione-requiring prostaglandin D B6 IRYIFAYL 8 Q9JHF7 synthase F-actin-capping protein subunit alpha- B6 ISFKFDHL 8 P47753 1

B6 ISPLFAEL 8 Q99KH8 Serine/threonine-protein kinase 24

B6 ISYLYNKL 8 Q61471 Protein Tob1

B6 ISYQFSNL 8 Q99PQ1 Tripartite motif-containing protein 12 Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta B6 IVPLFTNL 8 Q7TNP2 isoform

B6 IWIRVASL 8 Q8BRG6 Kelch-like protein 24

B6 KSYLFQLL 8 P97377 Cell division protein kinase 2

B6 LIYKFLNV 8 Q6P5F9 Exportin-1

B6 LQYEFTHL 8 Q9JL26 Formin-like protein 1

B6 LQYEFTKL 8 A2APV2 Formin-like protein 2 Mucosa-associated lymphoid tissue lymphoma translocation protein 1 B6 LSYQYSGL 8 Q2TBA3 homolog

B6 LVYQFKEM 8 Q60775 ETS-related transcription factor Elf-1 Vacuolar protein sorting-associated B6 MSFQFAHL 8 Q80TY5 protein 13B RING finger and WD repeat domain- B6 NNYVYAGL 8 Q8CIK8 containing protein 3

B6 PNQLFINL 8 Q9JJ28 Protein flightless-1 homolog Eukaryotic translation initiation B6 QSIEFSRL 8 P23116 factor 3 subunit A

B6 RNFIFSRL 8 Q60848 Lymphocyte-specific helicase B6 RNYQFDFL 8 Q8K4Z5 Splicing factor 3 subunit 1

B6 RSLKFYSL 8 Q99KP6 Pre-mRNA-processing factor 19 Vacuolar protein sorting-associated B6 RSYDFEFM 8 P40336 protein 26A Ras-GEF domain-containing family B6 RTYIFTFL 8 Q8JZL7 member 1B Chromodomain-helicase-DNA-binding B6 RVLIFSQM 8 P40201 protein 1

B6 SAFEFNEL 8 Q9Z2C4 Myotubularin-related protein 1

B6 SAFSFRTL 8 Q8BMI0 F-box only protein 38 Trafficking protein particle complex B6 SALIYSNL 8 O55013 subunit 3 Serine/threonine kinase 11-interacting B6 SALRFLNL 8 Q3TAA7 protein Uncharacterized protein C1orf85 B6 SALVFTRL 8 Q9JHJ3 homolog V-type proton ATPase 16 kDa B6 SAMVFSAM 8 P63082 proteolipid subunit

B6 SAPEYVFL 8 Q7TNB8 Protein strawberry notch homolog 2 1-phosphatidylinositol-4,5- bisphosphate phosphodiesterase B6 SAPLYTNL 8 Q8K4S1 epsilon-1

B6 SGFVFTRL 8 Q6PCN7 Helicase-like transcription factor

B6 SIANFTNV 8 Q91V92 ATP-citrate synthase Probable ATP-dependent RNA helicase B6 SIIIFTNT 8 Q4FZF3 DDX49

B6 SILQYSNV 8 Q9WUE4 Tumor suppressor candidate 4

B6 SIVQFYYM 8 Q9R190 Metastasis-associated protein MTA2

B6 SNLKYILV 8 A2AWL7 MAX gene-associated protein Voltage-dependent T-type calcium B6 SNYIFTAI 8 O88427 channel subunit alpha-1H

B6 SNYVFVFL 8 Q9Z0S9 Prenylated Rab acceptor protein 1

B6 SQYVFTEM 8 Q5SYH2 Transmembrane protein 199

B6 SSFVFLNL 8 Q64310 Surfeit locus protein 4 3-hydroxy-3-methylglutaryl-coenzyme A B6 SSFVFSTV 8 Q01237 reductase Eukaryotic translation initiation B6 SSIVFAEL 8 Q9Z2R9 factor 2-alpha kinase 1 Acyl-CoA synthetase family member 2, B6 SSYDFTSI 8 Q8VCW8 mitochondrial ATP-binding cassette sub-family G B6 SSYFFGKL 8 Q99P81 member 3 Peroxisomal 3,2-trans-enoyl-CoA B6 SSYTFPKM 8 Q9WUR2 isomerase Interferon-induced guanylate-binding B6 STFIYNSI 8 Q01514 protein 1

B6 STFTFADL 8 Q9ERU9 E3 SUMO-protein ligase RanBP2 Centromere/kinetochore protein zw10 B6 STVEFTNL 8 O54692 homolog

B6 STYKFFEV 8 Q9CZM2 60S ribosomal protein L15 B6 SVIKFENL 8 Q9CPV7 Probable palmitoyltransferase ZDHHC6 EGF-like module-containing mucin-like B6 SVITYVGL 8 Q91ZE5 hormone receptor-like 4 U6 snRNA-associated Sm-like protein B6 SVVRYVQL 8 O35900 LSm2

B6 TAFQFLQL 8 P51945 Cyclin-G1

B6 TAFRFSEL 8 A3KGB4 TBC1 domain family member 8B

B6 TAFVFPRL 8 Q8BK62 Olfactomedin-like protein 3

B6 TAPEYVFL 8 Q689Z5 Protein strawberry notch homolog 1

B6 TAYAFHFL 8 Q9R0H0 Peroxisomal acyl-coenzyme A oxidase 1

B6 TAYEFAKL 8 Q9EQ06 Estradiol 17-beta-dehydrogenase 11

B6 TIYEYGAL 8 P46412 Glutathione peroxidase 3

B6 TIYRFLKL 8 Q8CEF1 Protein fem-1 homolog C Hypoxia-inducible factor 1-alpha B6 TNLVYPAL 8 Q8BLR9 inhibitor

B6 TNYIFDSL 8 Q9CXF4 TBC1 domain family member 15

B6 TNYKFFML 8 Q5Y5T1 Probable palmitoyltransferase ZDHHC20

B6 TQYTFSSI 8 Q08943 FACT complex subunit SSRP1

B6 TSLRFVFL 8 Q9CQV4 Protein FAM134C Uncharacterized protein C4orf15 B6 TSYRFLAL 8 Q8QZX2 homolog

B6 TTLIFQKL 8 P15307 C-Rel proto-oncogene protein

B6 VALKFSQI 8 Q62187 Transcription termination factor 1

B6 VALLFRQL 8 Q6ZPE2 Myotubularin-related protein 5

B6 VAPGYPLL 8 P21995 Embigin

B6 VAYKFPEL 8 Q6WKZ8 E3 ubiquitin-protein ligase UBR2 Ubiquitin carboxyl-terminal hydrolase B6 VAYTYDNL 8 Q6ZQ93 34 Acidic leucine-rich nuclear B6 VFRLLPQL 8 Q9EST5 phosphoprotein 32 family member B

B6 VFTEVANL 8 Q62141 Paired amphipathic helix protein Sin3b

B6 VGFRFPIL 8 Q9QUG9 RAS guanyl-releasing protein 2 General transcription factor IIH B6 VIAELVNV 8 Q8K2X8 subunit 5 Putative sodium-coupled neutral amino B6 VIISFNSL 8 Q5I012 acid transporter 10 Dephospho-CoA kinase domain-containing B6 VIQVFQQL 8 Q8BHC4 protein

B6 VIVRFLTV 8 P62245 40S ribosomal protein S15a

B6 VMYRVIQV 8 Q61069 Upstream stimulatory factor 1 NADH dehydrogenase [ubiquinone] 1 B6 VNVDYSKL 8 Q62425 alpha subcomplex subunit 4 Vacuolar protein sorting-associated B6 VSFTYRYL 8 Q920Q4 protein 16 homolog Vacuolar protein sorting-associated B6 VSIQFYHL 8 Q5H8C4 protein 13A

B6 VSYLFSHV 8 Q9D7G0 Ribose-phosphate pyrophosphokinase 1

B6 VSYVFTEV 8 Q8K2V6 Importin-11

B6 VTVDFSKL 8 Q6NVF4 DNA helicase B

B6 VVFFFTRL 8 P50615 Protein BTG3

B6 VVFQFQHI 8 P70658 C-X-C chemokine receptor type 4

B6 VVYIYHSL 8 Q8CD15 Myc-induced nuclear antigen

B6 AMYIFLHTV 9 Q9CQZ0 ORM1-like protein 2 Dolichyl-diphosphooligosaccharide-- protein glycosyltransferase subunit B6 AVLSFSTRL 9 P46978 STT3A Uncharacterized protein C6orf89 B6 ELFPVFTQL 9 Q99KU6 homolog DNA-directed RNA polymerase II subunit B6 IGPTYYQRL 9 Q8CFI7 RPB2 Calcium/calmodulin-dependent protein B6 KIFEFKETL 9 Q8BW96 kinase type 1D PAB-dependent poly(A)-specific B6 KNYDFAQVL 9 Q8BGF7 ribonuclease subunit 2

B6 QILSDFPKL 9 Q99ME9 Nucleolar GTP-binding protein 1

B6 QSLAFLKDI 9 Q6NVF4 DNA helicase B Alpha-N-acetyl-neuraminyl-2,3-beta- galactosyl-1,3-N-acetyl- galactosaminide alpha-2,6- B6 QVYTFTERM 9 Q9R2B6 sialyltransferase

B6 RNYEYLIRL 9 Q8R3L2 Transcription factor 25

B6 RVAEFTTNL 9 Q8VDD5 Myosin-9 26S proteasome non-ATPase regulatory B6 RVYEFLDKL 9 P14685 subunit 3

B6 SGYDFENRL 9 Q8VE09 Tetratricopeptide repeat protein 39C

B6 SIAAFIQRL 9 Q9Z1R2 Large proline-rich protein BAT3 Putative homeodomain transcription B6 SIINFIERL 9 Q9QZ09 factor 1

B6 TGYAYTQGL 9 Q9JHI9 Solute carrier family 40 member 1

B6 TILEFSQNM 9 Q6P5F9 Exportin-1

B6 TIQEFLERI 9 P57725 SAM domain-containing protein SAMSN-1

B6 VGFTFPNRL 9 Q9Z0E0 Neurochondrin

B6 VGYRFVTAI 9 Q80TM9 Nischarin

B6 VIYPFMQGL 9 Q91VV4 DENN domain-containing protein 2D Ras-GEF domain-containing family B6 VNFEKFWEL 9 Q8JZL7 member 1B cAMP-dependent protein kinase B6 VNFPFLVKL 9 P05132 catalytic subunit alpha

B6 VNLREYPSL 9 Q8BVK9 Sp110 nuclear body protein DNA-directed RNA polymerase II subunit B6 VNYRHLALL 9 P08775 RPB1 IQ motif and SEC7 domain-containing B6 VTYSFRQSF 9 Q8R0S2 protein 1 Uncharacterized protein C6orf89 B6 RELFPVFTQL 10 Q99KU6 homolog Developmentally-regulated GTP-binding shared ASYEFTTL 8 Q9QXB9 protein 2 Signal transducer and activator of shared ATLVFHNL 8 P42227 transcription 3

shared HIYEFPQL 8 Q8C4B4 Protein unc-119 homolog B Probable ATP-dependent RNA helicase shared INFDFPKL 8 P54823 DDX6

shared KILTFDQL 8 P35980 60S ribosomal protein L18 POU domain class 2-associating factor shared SGPQFVQL 8 Q64693 1 Endothelial PAS domain-containing shared SNYLFTKL 8 P97481 protein 1

shared SVYVYKVL 8 Q64475 Histone H2B type 1-B

shared TIIIFHSL 8 O08575 Eyes absent homolog 2 Phosphatidylinositol-binding clathrin shared VAFDFTKV 8 Q7M6Y3 assembly protein

shared VNFEFPEF 8 P62082 40S ribosomal protein S7 ATP-binding cassette sub-family G shared VNIEFKDL 8 Q64343 member 1

shared ASYEFVQRL 9 Q9JHU4 Cytoplasmic dynein 1 heavy chain 1