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Size, Affinity, and Immunogenicity Peptide-Binding Repertoires Of HLA Class I Alleles Are Associated with Peptide-Binding Repertoires of Different Size, Affinity, and Immunogenicity This information is current as Sinu Paul, Daniela Weiskopf, Michael A. Angelo, John of September 29, 2021. Sidney, Bjoern Peters and Alessandro Sette J Immunol 2013; 191:5831-5839; Prepublished online 4 November 2013; doi: 10.4049/jimmunol.1302101 http://www.jimmunol.org/content/191/12/5831 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/11/05/jimmunol.130210 Material 1.DC1 http://www.jimmunol.org/ References This article cites 51 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/191/12/5831.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • 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 HLA Class I Alleles Are Associated with Peptide-Binding Repertoires of Different Size, Affinity, and Immunogenicity Sinu Paul,1 Daniela Weiskopf,1 Michael A. Angelo, John Sidney, Bjoern Peters, and Alessandro Sette Prediction of HLA binding affinity is widely used to identify candidate T cell epitopes, and an affinity of 500 nM is routinely used as a threshold for peptide selection. However, the fraction (percentage) of peptides predicted to bind with affinities of 500 nM varies by allele. For example, of a large collection of ∼30,000 dengue virus–derived peptides only 0.3% were predicted to bind HLA A*0101, wheras nearly 5% were predicted for A*0201. This striking difference could not be ascribed to variation in accuracy of the algorithms used, as predicted values closely correlated with affinity measured in vitro with purified HLA molecules. These data raised the question whether different alleles would also vary in terms of epitope repertoire size, defined as the number of associated epitopes or, alternatively, whether alleles vary drastically in terms of the affinity threshold associated with immuno- Downloaded from genicity. To address this issue, strains of HLA transgenic mice with wide (A*0201), intermediate (B*0702), or narrow (A*0101) repertoires were immunized with peptides of varying binding affinity and relative percentile ranking. The results show that absolute binding capacity is a better predictor of immunogenicity, and analysis of epitopes from the Immune Epitope Database revealed that predictive efficacy is increased using allele-specific affinity thresholds. Finally, we investigated the genetic and structural basis of the phenomenon. Although no stringent correlate was defined, on average HLA B alleles are associated with significantly narrower repertoires than are HLA A alleles. The Journal of Immunology, 2013, 191: 5831–5839. http://www.jimmunol.org/ olecular structures recognized by immune system re- resources that allow prediction of the binding affinity of peptides ceptors are called epitopes (1). Epitopes that bind, and to MHC class I and II molecules are provided at a number of are presented in the context of, class I and class II publically accessible websites, including the Immune Epitope M + + MHC molecules are typically recognized by CD8 and CD4 Database (IEDB) (20, 21), BIMAS (22), SYFPEITHI (23), NetMHC T cells, respectively. Binding of a peptide to the MHC molecule is (24), ProPred (25), ProPred1 (26), ABCpred (27), MULTIPRED one of the most selective steps in the classical MHC class I (28), and Rankpep (29). In general, MHC class I binding pre- pathway of Ag processing (2–4). The affinity with which an epi- diction tools scan a protein’s amino acid sequence to determine by guest on September 29, 2021 tope binds to the MHC molecule plays an important role in de- each subsequence’s ability to bind a specific MHC class I mol- termining its immunogenicity (5), and high-affinity MHC/epitope ecule. Whereas most MHC class I epitopes are 9 and 10 aa in interactions tend to be associated with higher immune responsive- length (20, 21, 23), it is known that shorter or longer peptides ness. However, whereas MHC binding is necessary for recognition can also be antigenic targets of class I responses. However, the by T cells, it is in itself not sufficient to define immunogenicity. availability of predictive tools for noncanonical sizes (i.e., other Indeed, recognition appears to be influenced by several other fac- than 9- and 10-mers) is more limited, and their performance is tors, such as abundance of proteins, Ag processing, immunodo- generally less robust, likely due to the fact that limited data are minance, and the presence of a suitable T cell repertoire (2–4, available to train and improve the corresponding algorithms. 6–10). Previous studies indicated 500 nM as an MHC affinity Several different computational approaches toward prediction threshold associated with potential immunogenicity for HLA class algorithms are available, including those based on artificial neural I–restricted T cells (5). networks (30), the average relative binding method (31), the sta- Computational prediction of MHC class I binding capacity has bilized matrix method (SMM) (32, 33), scoring matrices derived been used in epitope identification and vaccine discovery stud- from positional scanning of combinatorial peptide libraries (34), ies for many years (11–19). Various bioinformatics tools and the NetMHCpan method (35), hidden Markov models (28), and position-specific scoring matrices (29). The output of the different Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La methods is typically given either in units of predicted affinity (IC50 Jolla, CA 92037 nanomolar) or as a percentile score reflecting the relative affinity 1S.P. and D.W. contributed equally to this work. of a selected peptide compared with a universe of random se- Received for publication August 8, 2013. Accepted for publication October 2, 2013. quences. The efficacy of different methodologies for predicting This work was supported by National Institutes of Health Contracts high-affinity MHC binding peptides has been addressed in several HHSN272200900042C and HHSN272201200010C (to A.S.). studies by our group, from both the bioinformatics (21, 36) and Address correspondence and reprint requests to Dr. Alessandro Sette, Division of T cell epitope identification perspectives (4, 13, 37–40). Vaccine Discovery, La Jolla Institute for Allergy and Immunology, 9420 Athena However, a key question to be addressed is whether predicted Circle, La Jolla, CA 92037. E-mail address: [email protected] binding affinity or percentile rank is the best predictor of potentially The online version of this article contains supplemental material. immunogenic peptides, especially across a diverse set of different Abbreviations used in this article: DENV, dengue virus; IEDB, Immune Epitope HLA class I molecules. In this study, we set out to address this Database; SFC, spot-forming cell; SMM, stabilized matrix method. question by investigating the MHC binding and associated T cell Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 epitope repertoires of a panel of different HLA class I molecules www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302101 5832 DIFFERENT PEPTIDE-BINDING REPERTOIRES OF HLA CLASS I ALLELES using bioinformatics analyses, experimental testing of in vitro CA) as previously described (41). All mouse experiments were performed binding with purified HLA molecules, and in vivo immunoge- following Institutional Animal Care and Use Committee–approved animal nicity testing of selected peptides in HLA transgenic mice. The protocols. Mice between 8 and 12 wk of age were immunized s.c. with a pool of 10 individual peptides (10 mg peptide) in 100 ml PBS emulsified results unexpectedly revealed that different HLA class I molecules in CFA. Two weeks after immunization the mice were sacrificed, and are associated with distinctively different repertoires of peptide splenic CD8+ T cells were isolated. binders and associated epitope affinities. IFN-g ELISPOT assay Materials and Methods CD8+ T cells were isolated by magnetic bead positive selection (Miltenyi Biotec, Bergisch Gladbach, Germany). A total of 2 3 105 CD8+ T cells Peptide sets were stimulated with 1 3 105 naive splenocytes as APCs and 10 mg/ml A set of dengue virus (DENV) sequences recently used for epitope iden- individual peptides in 96-well flat-bottom plates (Immobilon-P; Millipore, tification studies in endemic areas (41) was used in the initial analysis Bedford, MA) coated with anti–IFN-g mAb (clone AN18; Mabtech, presented in this study. Essentially, full-length DENV polyprotein se- Stockholm, Sweden). Each peptide was tested in triplicate. Following quences for each serotype were retrieved from the National Center for a 20-h incubation at 37˚C, the wells were washed with PBS/0.05% Biotechnology Information Protein database using the query “txid11053 Tween 20 and then incubated with biotinylated IFN-g mAb (clone AND polyprotein AND 3000:5000[slen]” with the corresponding National R4-6A2; Mabtech) for 2 h. The spots were developed using Vectastain Center for Biotechnology Information taxonomy ID being substituted for ABC peroxidase (Vector Laboratories, Burlingame, CA) and 3-amino- each serotype. To avoid geographical bias, the number of unique isolates 9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO) and counted by (varying by at least 1 aa from all other isolates) from any one country was computer-assisted image analysis (KS-ELISPOT reader; Zeiss, Munich, limited to 10.
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