Tapasin enhances MHC class I peptide presentation according to peptide half-life Mark Howarth*†‡, Anthony Williams†, Anne B. Tolstrup§¶, and Tim Elliott†ʈ *Medical Research Council Human Immunology Unit, Oxford University, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; †Cancer Sciences Division, Southampton University, Southampton General Hospital, Southampton SO16 6YD, United Kingdom; and §Inoxell, Kogle Alle´5, DK-2970 Hoersholm, Denmark Edited by Stanley G. Nathenson, Albert Einstein College of Medicine, Bronx, NY, and approved June 24, 2004 (received for review September 30, 2003) Understanding how peptides are selected for presentation by MHC Materials and Methods class I is crucial to vaccination strategies based on cytotoxic T General Reagents. Peptides were synthesized by using F-moc lymphocyte priming. We have studied this selection of the MHC chemistry (Peptide Protein Research, Eastleigh, U.K.) and were class I peptide repertoire in terms of the presentation of a series of Ͼ95% pure by HPLC and mass spectrometry. Serum-free media individual peptides with a wide range of binding to MHC class I. was AIM-V (Sigma). This series was expressed as minigenes, and the presentation of each peptide variant was determined with the same MHC class I Antibodies. 25-D1.16 (D1) recognizes H2-Kb-SIINFEKL (20), peptide-specific antibody. In wild-type cells, the hierarchy of pre- kindly provided by R. Germain (National Institutes of Health, sentation followed peptide half-life. This hierarchy broke down in Bethesda). Y3 recognizes a conformation-sensitive epitope of cells lacking tapasin but not in cells lacking calreticulin or in cells H2-Kb. 148.3 recognizes human TAP1, kindly provided by R. lacking transporter associated with antigen processing-associated Tampe (University of Marburg, Marburg, Germany). Anti- ERp57. We demonstrate a key role for tapasin in shaping the MHC body to the truncated nerve growth factor receptor (⌬NGFR) class I peptide repertoire, as enhancement of presentation in the was from the hybridoma 8737 (American Type Culture Col- presence of tapasin correlated with peptide half-life. lection). Monoclonal antibodies were purified from hybridoma supernatant on a protein A-Sepharose or protein G-Sepharose HC class I molecules may be the ultimate proteomic Fast Flow (Amersham Pharmacia) column as appropriate. Mmicroarray (1). On the surface of a single cell, MHC class Calreticulin expression was determined with the rabbit anti- I molecules provide a readout of the expression level of up to Calregulin antiserum C-17 (Santa Cruz Biotechnology). 10,000 proteins (2). This array is interpreted by cytotoxic T lymphocytes and natural killer cells, allowing them to monitor Cell Lines. Cells were grown in RPMI medium 1640 with 10% FCS ͞ ␮ ͞ the events inside the cell and detect infection and tumorigenesis. (Globepharm, Surrey, U.K.), 50 units ml penicillin, 50 g ml MHC class I molecules consist of a complex between heavy streptomycin, and 2 mM glutamine (R10) at 37°C with 5% CO2. chain, ␤2-microglobulin (␤2m), and a peptide of usually 8–11 LBL 721.220 (.220) is a human tapasin-deficient B lymphoblastoid b b residues (3). Folding of MHC class I heavy chain in the endo- cell line (21). .220 K and .220 K tapasin were kind gifts of J. plasmic reticulum (ER) is initially assisted by the chaperone McCluskey (University of Melbourne, Melbourne) (17). Fibroblast calnexin and ERp57, a thiol-dependent oxidoreductase that cell lines, from M. Michalak (University of Alberta, Edmonton, assists disulfide bond formation (4). When ␤2m associates with Canada), were generated from wild-type or calreticulin-knockout class I heavy chain, calnexin dissociates and class I becomes part mice, as described (22). RMA-S is a TAP-deficient mouse T cell of a peptide loading complex comprising transporter associated line. with antigen processing (TAP), tapasin, calreticulin, and ERp57 ⌬ (5–9). TAP transports peptides from the cytosol into the ER. Plasmids. The retroviral packaging plasmids CMVbipep- NGFR Tapasin helps the assembly of class I molecules with peptides and CMVbipep-neo have been described (23, 24). Minigene inserts were generated from primers by PCR for insertion at the SpeI and (10). Calreticulin is an ER chaperone for the folding of a wide ⌬ variety of glycoproteins. How the proteins in this complex MluI sites of CMVbipep- NGFR and encoded the octameric peptide preceded by methionine, which is cleaved by cytosolic cooperate in the loading of class I with high-affinity peptide is b b unclear. High-affinity peptides are of a length that allows both N-aminopeptidase activity. Wild-type K or T134K K ,akindgift their N and C termini to fit in the class I binding groove and have of K. Gould (Imperial College London), was subcloned from pKG Xho Hin an allele-specific binding motif (11, 12). into CMVbipep-neo at the I and cII restriction sites and introduced into cells with retroviral transduction. ⌬NGFR- IMMUNOLOGY An outstanding problem in the field of antigen presentation is expressing cells were purified by using antibody-coated CEL- whether there is catalysis of class I peptide loading, by analogy Lection Pan Mouse IgG Dynabeads (Dynal, Great Neck, NY). to HLA-DM, which edits the peptides bound to MHC class II Neo-expressing cells were selected with 0.5 mg͞ml G418. pBMN- according to their binding stability (13). Bulk analysis of class I Zpuro encoding wild-type or C95A human tapasin was a kind gift does indicate impaired loading either in the absence of tapasin of P. Cresswell (Yale University, New Haven, CT). KpnI and (5, 14, 15) or with a mutant class I that fails to associate with TAP BamHI sites were added to wild-type and C95A tapasin by PCR (16). This is reflected in lower steady-state levels of class I, faster decay of class I from the cell surface, and increased export of peptide-receptive class I to the cell surface. It is unclear whether This paper was submitted directly (Track II) to the PNAS office. this represents class I molecules that are empty or just have a Abbreviations: ␤2m, ␤2-microglobulin; ER, endoplasmic reticulum; TAP, transporter asso- suboptimal peptide cargo. Attempts to compare the repertoire of ciated with antigen processing; ⌬NGFR, truncated nerve growth factor receptor; MFI, peptides presented with and without tapasin by peptide elution median fluorescence intensity. have failed to detect clear qualitative differences (17–19). Here, ‡Present address: Department of Chemistry, Massachusetts Institute of Technology, Cam- we describe a method to sample the repertoire of peptides bridge, MA 02139. presented at the cell surface and use this method to evaluate the ¶Present address: Symphogen A͞S, Elektrovej 375, 2800 Lyngby, Denmark. roles of tapasin, calreticulin, and ERp57 in the loading of class ʈTo whom correspondence should be addressed. E-mail: [email protected]. I with peptide. © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306294101 PNAS ͉ August 10, 2004 ͉ vol. 101 ͉ no. 32 ͉ 11737–11742 Downloaded by guest on September 26, 2021 with the primers 5Ј-GTAGGTACCATGAAGTCCCTGTCTCT- 0.1% Triton X-100 lysis buffer and boiled in 25 ␮l of SDS loading GCTC-3Ј and 5Ј-GTAGGATCCTCACTCTGCTTTCT- buffer, and supernatant was run on a 10% polyacrylamide gel. The TCTTTG-3Ј for subcloning into pREP4, a kind gift of F. Momburg gel was fixed, placed in Amplify (Amersham Pharmacia) for 30 min, (German Cancer Research Center, Heidelberg). pREP4 plasmids and dried before exposure on a PhosphorImager screen (Fuji). The encoding wild-type or C95A tapasin were introduced by using screen was imaged with a Fluor-S MultiImager. Lipofectamine 2000 (Invitrogen). pREP4 transfectants were se- lected and maintained in 0.3 mg͞ml Hygromycin B (Invitrogen). Results Inserts were confirmed by sequencing in both directions. Generating a Peptide Hierarchy to Sample the MHC Class I Peptide Repertoire. To sample the class I peptide repertoire, we generated Retroviral Transduction. The retroviral packaging cell line Phoe- a single detection system with which we could analyze presentation nix-Amphotropic (American Type Culture Collection) was of peptides with a wide range of binding affinities to class I. The use transfected on day 1 by using Effectene (Qiagen, Valencia, CA). of a single detection system avoided the obfuscation of phenotypic On day 2, cells were placed in fresh medium at 32°C. On day 3, differences between T cells responding to epitopes of different ϫ cell medium was centrifuged at 402 g for 5 min. Cells to be dominance (25). We chose flow cytometry with an MHC-peptide- transduced were resuspended in this supernatant with 5 ␮g͞ml ϫ specific antibody as our readout because of its quantitative signal polybrene (Sigma) and 25 mM Hepes and centrifuged at 1,118 over a wide range of presentation levels and its independence of g for2hat32°C. Thereafter, cells were incubated at 37°C and costimulation (20). A hierarchy based on the H2-Kb-binding pep- resuspended in fresh medium the next day. tide SIINFEKL (chicken ovalbumin residues 257–264) was gener- ated by varying anchor residues to produce peptides with a wide Flow Cytometry. ϫ 5 Suspension cells were plated overnight at 3 10 range of binding affinities. Peptides were expressed stably as cells per ml. Adherent cells were used at 50–70% confluency and minigenes in the cytosol, and peptide presentation was detected by trypsinized on ice. Cells were immediately transferred to 4°Cto flow cytometry with the antibody 25-D1.16 (D1) (20), which could minimize loss of fast-decaying peptides, washed in fluorescence- recognize all of the peptide variants. activated cell sorting (FACS) wash (PBS͞2% FCS͞0.02% NaN ), 3 Peptides that bind to and stabilize Kb generally have an and incubated in a saturating concentration of 1° antibody for 40 min. Cells were washed twice and resuspended in fluorescein- aromatic residue at P5 (Tyr or Phe) and a small hydrophobic labeled goat anti-mouse IgG (Sigma) or phycoerythrin-labeled goat residue at the C terminus (Leu, Met, Ile, or Val) (11, 26).