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I Peptides Amino and Carboxyl Termini of MHC Class ERAAP And 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 Why The JI? Submit online. by guest on September 24, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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.
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