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MHC Multimer − Peptide HLA Micropolymorphisms Strongly Affect HLA Micropolymorphisms Strongly Affect Peptide−MHC Multimer−Based Monitoring of Antigen-Specific CD8 + T Cell Responses This information is current as Marit M. van Buuren, Feline E. Dijkgraaf, Carsten of September 28, 2021. Linnemann, Mireille Toebes, Cynthia X. L. Chang, Juk Yee Mok, Melanie Nguyen, Wim J. E. van Esch, Pia Kvistborg, Gijsbert M. Grotenbreg and Ton N. M. Schumacher J Immunol 2014; 192:641-648; Prepublished online 16 December 2013; Downloaded from doi: 10.4049/jimmunol.1301770 http://www.jimmunol.org/content/192/2/641 Supplementary http://www.jimmunol.org/content/suppl/2013/12/16/jimmunol.130177 http://www.jimmunol.org/ Material 0.DC1 References This article cites 40 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/192/2/641.full#ref-list-1 Why The JI? Submit online. by guest on September 28, 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology HLA Micropolymorphisms Strongly Affect Peptide–MHC Multimer–Based Monitoring of Antigen-Specific CD8+ T Cell Responses Marit M. van Buuren,* Feline E. Dijkgraaf,* Carsten Linnemann,* Mireille Toebes,* Cynthia X. L. Chang,† Juk Yee Mok,‡ Melanie Nguyen,‡ Wim J. E. van Esch,‡ Pia Kvistborg,* Gijsbert M. Grotenbreg,† and Ton N. M. Schumacher* Peptide–MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01– Downloaded from restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA- A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06–restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong http://www.jimmunol.org/ effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group. The Journal of Immunology, 2014, 192: 641–648. n 1996, Altman et al. (1) described how multimers of pep- Traditionally, MHC-based immunomonitoring projects have tide–MHCs (pMHCs) coupled to fluorochromes can be used focused on analyses of T cell reactivity toward a small number of I to monitor Ag-specific CD8+ T cells by flow cytometry. epitopes, restricted by a few HLA alleles that are present at high Since this first description, pMHC multimer–based immunomo- frequency within the Caucasian population. To illustrate this bias, nitoring has become a very widely used technique to understand .65% of melanoma-associated Ags described in literature are by guest on September 28, 2021 spontaneous and therapy-induced T cell reactivity in different restricted by the HLA-A*02 allele (6), even though this allele is fields, as illustrated by the .3000 citations to the original work only one of the many HLA-A alleles that are present. Notably, in (1). In addition to the use of pMHC multimers for the quantifi- non-Caucasian populations, the HLA-A*02:01 subtype (by far the cation of Ag-specific T cell responses (2), these tools have also most frequent subtype in Caucasian populations) is present in only been used for the isolation of Ag-specific T cells for both research a minor fraction of HLA-A*02+ individuals (7). purposes and adoptive cell therapy (3) and to describe the com- The variation between the subtypes of specific HLA alleles (tra- position of the naive T cell compartment (4, 5). ditionally referred to as four-digit subtypes) maps to a small number of sequence differences within the a1ora2 domains of the MHC H *Division of Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, chain, which together form the peptide-binding groove. Depending The Netherlands; †Department of Microbiology, Immunology Programme, National on their location, these sequence differences may influence T cell University of Singapore Graduate School for Integrative Sciences and Engineering, Singapore 117456; and ‡Division of Reagents, Sanquin Blood Supply, 1066 CX recognition by modifying the peptide binding properties of the MHC Amsterdam, The Netherlands (8) or altering the TCR exposed surface, or they may be without Received for publication July 3, 2013. Accepted for publication November 13, 2013. effect. This work was supported by European Union FP7 Grant SPHINX, Center for Trans- In this study, we have assessed the extent to which the ability to lational Molecular Medicine Grant AMPVACS, Dutch Cancer Society Grant NKI correctly measure Ag-specific T cell responses against a given 2012-5463, and Singapore National Research Foundation Fellowship NRF2007NRF- epitope by pMHC multimer technology is influenced by such RF001-226. micropolymorphic differences between HLA subtypes. Our re- M.M.v.B. designed, performed, and interpreted experiments and wrote the paper; F.E.D. designed, performed, and interpreted experiments and wrote the paper; C.L., M.T., P.K., sults demonstrate that minor variations between HLA subtypes J.Y.M., M.N., and W.J.E.v.E. performed experiments and interpreted results; C.X.L.C. greatly affect the ability to detect Ag-specific CD8+ T cell responses provided essential reagents; G.M.G. provided essential reagents and experimental ad- vice; and T.N.M.S. supervised the project, designed and interpreted all experiments, and in both model systems and clinical samples. Furthermore, in wrote the paper. many cases, this lack of T cell detection reflects altered TCR Address correspondence and reprint requests to Prof. Ton N.M. Schumacher, Divi- interaction of the pMHC complex, rather than impaired MHC sion of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX binding of epitopes, and can therefore not be predicted by Amsterdam, The Netherlands. E-mail address: [email protected] pMHC binding assays. Collectively, these data indicate that the The online version of this article contains supplemental material. generation of pMHC-based monitoring technology for large Abbreviations used in this article: IR, infrared; MFI, mean fluorescence intensity; sets of HLA subtypes, as performed in this study for HLA- pMHC, peptide–MHC; SA, streptavidin; TIL, tumor-infiltrating lymphocyte. A*02, will be of importance for the development of personal- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 ized pMHC-based immunomonitoring. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301770 642 HLA MICROPOLYMORPHISMS STRONGLY AFFECT pMHC-BASED ANALYSIS To obtain bulk T cell populations (specific for MART-1ELA and GnTVVLP) Materials and Methods + HLA-A*02 subtypes of an HLA-A*02:06 melanoma patient, TILs were first expanded using a 14-d rapid expansion protocol (19). In brief, irradiated (40 Gy) PBMC Amino acid sequences of the HLA-A*02 alleles were obtained from the feeders of three different healthy donors were combined and taken in culture ImMunoGeneTics information system/HLA database (http://www.ebi.ac.uk/ with TILs (ratio of 20:1 for feeders/T cells) in the presence of anti-CD3 ipd/imgt/hla/align.html, version 3.8.0. Accessed: June 14, 2012) (9). The full- (OKT3; final concentration, 30 ng/ml) and IL-2 (final concentration, 3000 length protein sequences of HLA-A*02:02, HLA-A*02:05, HLA-A*02:06, IU/ml). Half of the medium was replaced after 5 d and cultures were split 1:1 HLA-A*02:07, HLA-A*02:11, HLA-A*02:71, and HLA-A*02:77 were on day 7 and when needed afterward. After 12–14 d, T cells were frozen. aligned to the reference sequence HLA-A*02:01. MART-1ELA–andGnTVVLP-specific T cell populations were isolated from expanded TILs by sorting of live single CD8+ T cells stained with PE/ Three-dimensional structures of HLA-A*02 subtypes allophycocyanin-labeled pMHC multimers. Obtained MART-1ELA and Graphic representations of the CMV -specific TCR (RA14) in complex GnTVVLP cell populations were further expanded using the above-described NLV rapid expansion protocol. with the CMVNLV-HLA-A*02:01 complex (10) (Brookhaven Protein Data Bank code 3GSN) were drawn using PyMOL software. TILs were stored in liquid nitrogen and were thawed 1 d prior to analysis. As a standard, cell numbers were determined by trypan blue staining, with pMHC ELISA and pMHC binding predictions an average recovery of 93%. HLA haplotyping A streptavidin (SA)-based sandwich ELISA was used to assess the ability of peptides to stabilize the HLA-A*02:01, HLA-A*02:02, HLA-A*02:05, HLA- Four-digit resolution HLA haplotyping was performed by the Leiden A*02:07, HLA-A*02:11, HLA-A*02:71, and HLA-A*02:77 subtypes, as de- University Medical Center (Leiden, The Netherlands).
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