HLA Micropolymorphisms Strongly Affect Peptide−MHC Multimer−Based Monitoring of Antigen-Specific CD8 + 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

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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 © 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 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. , 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 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- 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). Typing was per- scribed (11), using the PeliScreen HLA class I ELISA kit (Sanquin). In brief, formed according to the manufacturer’s protocol using SBT Excellerator after UV-mediated peptide exchange in the presence of indicated peptides, HLA-A, HLA-B, and HLA-C typing kits (GenDx). biotinylated HLA class I molecules were added to wells coated with SA. HLA Downloaded from class I complexes bound to the well were quantified using an HRP-conjugated T cell staining b + anti– 2-microglobulin Ab. Green-colored oxidation product was produced by For pMHC multimer staining of CD8 T cell clones, 1 ml PE-pMHC and 2 adding ABTS as a substrate for HRP. ml allophycocyanin–pMHC multimers were used to stain cells in a 15-min The following peptide sequences were submitted to the NetMHCpan incubation at 37˚C. Subsequently, cells were stained with anti–CD8-FITC server (http://www.cbs.dtu.dk/services/NetMHCpan/, version 2.4. Accessed: (BD Biosciences) and near-infrared (near-IR) Live/Dead stain (Invitrogen) June 14, 2012) (12): CMVNLV (NLVPMVATV), EBVGLC (GLCTLVAML), for 30 min on ice and washed twice with FACS buffer. Background signal EBVYVL (YVLDHLIVV), FLUGIL (GILGFVFTL), GnTVVLP (VLPDVFIRC),

was determined by staining with pMHC multimers loaded with a control http://www.jimmunol.org/ HA-2 (YIGEVLVSV), MAGE-A10 (GLYDGMEHL), MAGE-C2 YIG GLY ALK peptide, HIVSLY (SLYNTVATL). (ALKDVEERV), MAGE-C2KVL (KVLEFLAKL), Meloe-1TLN (TLNDECWPA), For pMHC combinatorial encoding, each pMHC complex was coupled to and MART-1ELA (ELAGIGILTV). Predicted IC50 values (in nanomolars) for a unique combination of two fluorochromes (fluorochrome concentrations binding to HLA-A*02:06 were obtained for each peptide. Peptides with IC50 as described in Ref. 16). The melanoma-associated epitope panel con- values of ,50, 50–500, and .500 nM were regarded as strong, weak, and sisting of 145 different peptides has been described previously (16). nonbinders, respectively. Detection of Ag-specific T cells Generation of pMHC complexes Ag-specific T cells were detected using an LSR II flow cytometer Indicated virus-derived, melanoma-associated, and UV-cleavable peptides were (Becton Dickinson) with FACSDiva software (Becton Dickinson). To synthesized in-house as described previously (13). Recombinant HLA-A*02 identify Ag-specific T cells when staining CD8+ T cell clones, the following

b by guest on September 28, 2021 subtype-specific H chains and human 2-microglobulin L chain were produced gating strategy was used: 1) selection of live (IR dye–negative) single- in Escherichia coli and isolated from inclusion bodies. MHC class I refolding cell lymphocytes; 2) selection of anti–CD8- FITC+ cells; and 3) selection of reactions and purification by gel filtration HPLC were performed as described CD8+ T cells that were double-positive for PE and allophycocyanin pMHC previously (14). HLA-A*02 subtypes were refolded in the presence of the multimers. A total of 50,000 CD8+ T cells were recorded. Data shown are following UV-sensitive peptides: KILGFVF-J-V, KMDI-J-VPLL, LLDSD-J- representative of three independent experiments. ERL, LTA-J-FLIFL, and SVRD-J-LARL. MHC complexes loaded with the For combinatorial coding analyses, an 11-color instrument setting was used indicated peptides of interest were generatedbyUV-inducedligandexchange. (16). To identify Ag-specific T cells, the following gating strategy was used: In brief, pMHC complexes with UV-sensitive peptide (100 mg/ml) were sub- 1) selection of live (IR dye–negative) single-cell lymphocytes; 2) selection of jected to 366 nM UV light (Camag) for 1 h at 4˚C in the presence of rescue anti–CD8-FITC+ and “dump” (anti-CD4, -CD14, -CD16, -CD19; Invitrogen) peptide (200 mM) (13, 15). negative cells; 3) selection of CD8+ T cells that were positive in two and only two pMHC multimer channels; and 4) cells positive in only one as well as Generation of pMHC multimers cells positive in three or more pMHC multimer channels were gated out, as For each peptide, pMHC-multimers were generated using two different described previously (16). Predefined cut-off values for the definition of positive responses were fluorescent SA conjugates (Invitrogen) (16). T cell clones were stained with $ + $ + dual color–labeled pMHC multimers. Tumor-infiltrating lymphocytes (TILs) 0.005% of total CD8 cells and 10 events. Five hundred thousand CD8 were analyzed by combinatorial pMHC multimer stainings, in which pMHC T cells were recorded per sample. Identified Ag-specific T cell responses multimers loaded with 25 different peptides were used simultaneously in were in all cases confirmed in an independent stain using a different fluo- each staining reaction, with each pMHC combination encoded by a unique rochrome combination. two-color combination (17). pMHC multimer titrations + Generation of CD8 T cell clones pMHC multimers charged with either GnTVVLP (VLPDVFIRC), MART- 1ELA (ELAGIGILTV), gp100KTW (KTWGQYWQV), or tyrosinaseYMD PBMC samples were obtained from healthy individuals or from patients (YMDGTMSQV) peptides, coupled to SA-PE and SA-allophycocyanin, with stage IV melanoma in accordance with local guidelines, and following were used to stain expanded TILs of an HLA-A*02:06+ melanoma patient informed consent. at the indicated concentrations. A 13 pMHC multimer staining condition Cell sorting was performed on a FACSAria I (BD Biosciences), MoFlo corresponds to an MHC concentration of 6 mg/ml. Anti–CD8-FITC (BD Legacy (Beckman Coulter), or MoFlo Astrios (Beckman Coulter). Ag-specific Biosciences) and near-IR Live/Dead stain (Invitrogen) were used to gate T cell clones specific for the viral Ags CMVNLV (NLVPMVATV), EBVGLC on CD8+ and living cells. (GLCTLVAML), EBVYVL (YVLDHLIVV), and FLUGIL (GILGFVFTL) were All FCS files are available for scientific research purposes upon request. obtained by pMHC multimer–assisted single cell sorting from PBMCs of healthy donors. Ag-specific T cell clones for the melanoma-associated Results Ags MAGE-A10 (GLYDGMEHL), MAGE-C2 (ALKDVEERV), GLY ALK Peptide exchange technology for a series of HLA-A*02 MAGE-C2KVL (KVLEFLAKL), GnTVVLP (VLPDVFIRC) and Meloe-1TLN (TLNDECWPA) were obtained by pMHC multimer–assisted single cell sorting subtypes + from TILs and PBMCs of melanoma patients (18). The HA-2YIG–specific CD8 To address to what extent pMHC-based immunomonitoring is T cell clone was a gift of M.H. Heemskerk (Leiden University Medical Center, Leiden, The Netherlands). Sorted single CD8+ T cells were expanded as de- influenced by micropolymorphic variation within the HLA class scribed previously (18). a1anda2 domains, we selected a set of eight different HLA-A*02 The Journal of Immunology 643 subtypes (HLA-A*02:01, HLA-A*02:02, HLA-A*02:05, HLA- ence, the other seven HLA-A*02 subtypes that were chosen differ A*02:06, HLA-A*02:07, HLA-A*02:11, HLA-A*02:71, HLA- from HLA-A*02:01 in one to four residues within the a1 and/or a2 A*02:77). Of this set of alleles, HLA-A*02:01 is the most frequently domains (Fig. 1A). The location of these polymorphic residues sug- occurring subtype within the Western European and North American gests that, in at least in some cases, these alterations are unlikely to be population and therefore the commonly used HLA-A*02 allele in directly “seen” by Ag-specific TCRs (Fig. 1B). As an example, the immunomonitoring studies. When taking HLA-A*02:01 as a refer- HLA-A*02:06 sequence differs from HLA-A*02:01 in only one Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Sequence similarity of HLA-A*02 subtypes. (A) The amino acid sequences of the a1anda2 domains of seven HLA-A*02 subtypes were aligned to the reference sequence of HLA-A*02:01 (HLA-A*02:02, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:11, HLA-A*02:71, and HLA-A*02:77, respectively). The a1 domain covers residues 1–90 and the a2 domain covers residues 91–181; red arrows indicate those positions in which differences from the

HLA-A*02:01 subtype are present. (B) Representations of the CMVNLV-specific TCR (RA14) in complex with the CMVNLV-HLA-A*02:01 complex (10) (Brookhaven Protein Data Bank code 3GSN). Shown are TCRa (magenta), TCRb (light pink), MHC class I H chain a1(cyan),a2 (aquamarine) a3 (slate) b2- microglobulin (b2m; lime green), and CMVNLV peptide (yellow; ribbon structure. The position of the different polymorphic residues within the subtypes is highlighted in red. The subtype panels are a zoom in of the HLA-A*02:01 a1anda2 domains bound to CMVNLV peptide (90˚ rotation around the x-axis of boxed area). In each subtype figure the side chains of the HLA-A*02:01 allele are shown that are altered within the indicated subtype. 644 HLA MICROPOLYMORPHISMS STRONGLY AFFECT pMHC-BASED ANALYSIS residue, a conservative phenylalanine to tyrosine substitution at po- to detect CD8+ T cell responses against a series of Ags, HLA- sition 9 (F9Y) in the H chain. This residueformsakeycomponentof A*02:01–restricted T cell clones specific for 10 different viral and peptide-binding pocket B that accommodates an “anchor residue” at melanoma Ags were generated (CMVNLV,EBVGLC,EBVYVL, position 2 in HLA-A*02–binding peptides (20–22). Despite the ad- FLUGIL,) or expanded (GnTVVLP,MAGE-A10GLY,MAGE-C2ALK, ditional OH group present in the HLA-A*02:06 H chain, the B MAGE-C2KVL, and Meloe-1TLN (18), and HA2YIG). For all these pocket of HLA-A*02:01 and HLA-A*02:06 share the same T cell clones, HLA-A*02:01 was the only A*02 subtype present. preference for small aliphatic side chains at this position (22, 23). Thus, no other alleles were expressed that could also form the re- Therefore, based on their similar peptide-binding preferences, this striction element of the epitopes concerned. Subsequently, pMHC alteration forms an example of a micropolymorphism that could be multimers of all eight HLA-A*02 subtypes were produced for each expected to have little effect on T cell analysis. of the 10 different Ags by UV-mediated peptide exchange (7, 24). The To analyze the ability of this set of subtype variants to detect Ag- set of CD8+ T cell clones was then stained with all pMHC multimer specific T cell populations, we first established UV-induced peptide subtypes, loaded either with cognate peptide or with a control peptide. exchange technology for each of them. In prior work, UV-induced Representative flow cytometry dot plots of an HLA-A*02:01– + peptide exchange was developed for HLA-A*02:01 (24), HLA- restricted CMVNLV-specific CD8 T cell clone are shown in Fig. A*02:06, HLA-A*02:07, and HLA-A*02:11 (7). Based on the 2A. As expected, high-intensity staining (mean fluorescence in- observation that the (predicted) peptide-binding motifs of these tensity [MFI], 2.6 3 103) of this CD8+ T cell clone was achieved HLA-A*02 subtypes are highly similar (23), the previously de- when subtype-matched (HLA-A*02:01) pMHC multimers were veloped conditional ligands were used to refold the HLA*A- used. A similarly high level of staining was achieved when using

02:02, HLA-A*02:05, HLA-A*02:71, and HLA-A*02:77 alleles. HLA-A*02:71 (MFI, 3.0 3 103) and HLA-A*02:77 (MFI, 1.6 3 Downloaded from For all new subtypes, this yielded a conditional ligand that led to 103) pMHC multimers. In contrast, for all four other HLA-A*02 efficient refolding of HLA class I, and that could be exchanged for subtypes, signal did not exceed background (MFI range, 0.7–1.1 3 2 peptides of interest by UV light exposure (data not shown). 10 ). To determine whether a failure to detect these CMVNLV- specific T cells could have been predicted on the basis of pMHC Influence of pMHC multimer subtype variation on detection of binding to these HLA-A*02 subtypes, we performed pMHC binding HLA-A*02:01–restricted T cell clones ELISAs for HLA-A*02:01, HLA-A*02:02, HLA-A*02:05, HLA- http://www.jimmunol.org/ To subsequently determine the effect of polymorphic differences A*02:07, HLA-A*02:11, HLA-A*02:71, and HLA-A*02:77. Tech- within the a1 and a2 domains of pMHC multimers on their ability nical limitations precluded pMHC ELISA for the HLA-A*02:06 by guest on September 28, 2021

FIGURE 2. pMHC multimer staining of HLA-A*02:01–restricted T cell clones is highly influenced by HLA-A*02 micropolymorphisms. (A) Representative dot plots of an HLA-

A*02:01–restricted CMVNLV-specific CD8+ T cell clone stained with the indicated HLA-A*02 subtype multi- mers coupled to allophycocyanin (APC; x-axis) and PE (y-axis) (17). Numbers in the upper-right quadrants indicate the percentage of T cells with a signal intensity above background. (B) Half off-set histograms of 10 dif- ferent HLA-A*02:01–restricted T cell clones stained with the indicated HLA-A*02 multimers. Filled histo- grams depict the signal obtained using the subtype-matched HLA-

A*02:01 multimers. A*02:01irr,HLA- A*02:01 pMHC multimers loaded with control peptide HIVSLY.Data shown are representative of three independent experiments. The Journal of Immunology 645 allele, and for this allele binding affinity predictions were there- not identified. We next wished to assess whether a set of HLA- fore performed using netMHCpan (12) (Supplemental Table I). A*02:01 multimers loaded with previously described epitopes for The CMVNLV peptide was shown/predicted to bind to seven of this HLA-A*02 subtype could correctly describe T cell reactivity eight HLA-A*02 subtypes (Fig. 3, Supplemental Table I), in- in a patient expressing a related HLA-A*02 subtype. To this pur- cluding four of the five alleles that failed to detect the CMVNLV- pose, we created a collection of HLA-A*02:01 pMHC multimers specific T cells. Thus, the absence of T cell staining for these loaded with a set of 145 HLA-A*02:01–restricted melanoma- alleles cannot simply be predicted on the basis of pMHC binding associated epitopes. Subsequently, CD8+ T cell responses against analysis of the epitope. The same analysis was subsequently re- this set of epitopes were analyzed in TIL cultures from an HLA- peated for each CD8+ T cell clone (Fig. 2B). In all cases, staining A*02:06+ melanoma patient (HLA-A*02:01 and HLA-A*02:06 with the subtype-matched HLA-A*02:01 multimers (filled histo- differ at position 9, F9Y) by combinatorial coding analysis (16). grams) yielded a high-intensity staining. In contrast, staining with By performing the same analysis for this collection of epitopes any of the seven subtype-mismatched pMHC multimers was highly complexed with HLA-A*02:06 multimers, it was possible to directly variable, with some clones being stained by many pMHC multi- compare T cell reactivity as measured by use of subtype-matched and mers (EBVGLC), whereas others were detected by only a few subtype-mismatched pMHC multimers. These experiments revealed subtype-mismatched pMHC multimers (MAGE-C2KVL). Also for that when subtype-matched (HLA-A*02:06) pMHC multimers were these epitopes, pMHC binding analysis could not explain the ab- used, four different T cell responses were detected, reactive with sence of multimer staining in most cases. For those alleles for the melanoma Ags gp100KTW, tyrosinaseYMD,MART-1ELA,and which pMHC binding measurements were feasible (i.e., excluding GnTVVLP (Fig. 4). Importantly, when the same analysis was per-

HLA-A*02:06), absence of T cell staining could be explained by formed using subtype-mismatched (HLA-A*02:01) pMHC multi- Downloaded from impaired pMHC binding in only 3 of 35 cases (Fig. 3; CMVNLV, mers, only the gp100KTW-specific T cell response was detected at + FLUGIL,andMAGE-C2ALK in the HLA-A*02:07 panel; the a similar magnitude (0.199 and 0.236% of CD8 T cells using HLA- MART-1ELA peptide is ignored in this matter, as the Ag-specific A*02:06 and HLA-A*02:01, respectively). Specifically, the magni- T cell clone is HLA-A*02:06 restricted and peptide binding could tude of two of the four T cell responses (tyrosinaseYMD and MART- not be assessed for this allele). As a side note, the pMHC binding 1ELA) was underestimated by 2- to 3-fold. Furthermore, the T cell measurements confirm the structural integrity of the vast majority response specific for the GnTVVLP Ag was missed entirely when http://www.jimmunol.org/ of the pMHC complexes. using HLA-A*02:01 pMHC multimers (Fig. 4), even though this peptide constitutes a bona fide HLA-A*02:01–restricted epitope (25). Influence of HLA subtype variation on MHC-based To also investigate the reactivity of these HLA-A*02:06–re- high-throughput immunomonitoring stricted T cell populations with the other HLA-A*02 subtypes, the In the above analyses, pMHC multimers were generated using GnTVVLP- and MART-1ELA–specific T cell populations present in HLA-A*02 subtypes for which these 10 epitopes were originally this TIL product were isolated using HLA-A*02:06–restricted by guest on September 28, 2021

FIGURE 3. pMHC binding. Bar graphs of pMHC binding of 11 different peptides to 7 different HLA-A*02 subtypes. The black dotted line reflects twice the highest background measurement for that allele and is used as a threshold to define binders/nonbinders. Combined data of two experiments are shown. Data were normalized to a positive control (KLIHLTNAL) and are shown as means 6 SD of 2 3 two replicate measurements. A, negative control 1

(CTELKLSDY); B, negative control 2 (IVTDFSVIK); 1, CMVNLV;2,EBVGLC;3,EBVYVL;4,FluGIL;5,GnTVVLP;6,HA2YIG;7,MAGEA10GLY; 8, MAGE C2ALK;9,MAGEC2KVL;10,MELOE-1TLN; 11, MART-1ELA. Black-filled bars depict negative controls; open bars depict pMHC combinations that did not allow detection of HLA-A*02:01–restricted Ag-specific T cells to the same extent as subtype-matched pMHC multimers. Gray bars indicate pMHC combinations that allowed detection of HLA-A*02:01–restricted Ag-specific T cells with a comparable sensitivity as subtype-matched pMHC multimers(Fig.

2B). In the case of the MART-1ELA T cell clone, the restriction element is HLA-A*02:06. The restriction element of all other T cell clones is HLA-A*02:01. 646 HLA MICROPOLYMORPHISMS STRONGLY AFFECT pMHC-BASED ANALYSIS

FIGURE 4. Effect of HLA micropolymorphism on pMHC-based high-throughput immunomonitoring. Dot plots of Ag-specific CD8+ T cell responses + (MART-1ELA, gp100KTW, GnTVVLP, and tyrosinaseYMD, respectively) in expanded TIL populations from an HLA-A*02:06 patient when stained with Downloaded from either HLA-A*02:01 or HLA-A*02:06 subtype pMHC multimers. CD8+ pMHC multimer2 cells are indicated in gray; CD8+ pMHC multimer+ cells are indicated in green. Cells indicated in blue are CD8+ cells positive for one of the other fluorochrome combinations used. Numbers in the upper-right quadrants indicate the percentage of T cells with a signal intensity above background. Identified Ag-specific T cell responses were in all cases confirmed in an independent stain using a different fluorochrome combination.

pMHC multimers. Subsequently, both T cell populations were pMHC multimers (MFI range, 0.9–5.8 3 102, Fig. 5A), even though http://www.jimmunol.org/ stained with the panel of HLA-A*02 subtype multimers (represen- all mismatched HLA-A*02 subtypes were shown to bind the tative dot plots shown in Fig. 5A). High-intensity staining (MFI, 2.0 3 GnTVVLP peptide (Fig. 3). When the same analysis was performed 3 10 ) of GnTVVLP-specific T cells was achieved when subtype- for the MART-1-HLA-A*02:06–reactive T cell population, a highly matched (HLA-A*02:06) pMHC multimers were used. However, variable staining with the seven other HLA-A*02 subtypes was for the other HLA-A*02 subtype multimers, staining patterns were likewise observed (Fig. 5B). pMHC multimer titration experiments again highly variable and never reached the sensitivity of matched (Supplemental Fig. 1) demonstrated that the ability or inability to by guest on September 28, 2021

FIGURE 5. Effect of HLA-A*02 micropolymorphism on detection of HLA-A*02:06–restricted T cell clones. (A) Representative dot plots of an

HLA-A*02:06–restricted GnTVVLP- specific CD8+ T cell clone stained with the indicated HLA-A*02 sub- type multimers, coupled to allophy- cocyanin (APC; x-axis) and PE (y-axis). Numbers in the upper-right quadrants indicate the percentage of T cells with a signal intensity above background. (B) Half off-set histo- grams for the PE (y-axis) channel for

GnTVVLP- and MART-1ELA-specific HLA-A*02:06–restricted T cell clones stained with the indicated HLA-A*02 subtype pMHC multimers. Filled his- tograms depict the signal obtained using the subtype-matched HLA-

A*02:06 multimers. A*02:06irr,HLA- A*02:06 pMHC multimers loaded with control peptide HIVSLY.Data shown are representative of three independent experiments. The Journal of Immunology 647 detect Ag-specific T cell populations cannot be remedied by an in- The HLA subtype tools developed in this study for HLA-A*02 crease in the pMHC multimer concentration used for T cell staining. should contribute to the development of the personalized immuno- monitoring that is made possible by next generation sequencing of Discussion pathogens and tumors. As a first example, next generation sequencing In this study, we demonstrate that micropolymorphic variation be- of viral quasispecies will make it feasible to follow the dynamic tween HLA subtypes can greatly influence the ability to describe relationship between the T cell repertoire and the viral quasispe- human Ag-specific CD8+ T cell responses by pMHC multimer cies for viruses such as C virus and HIV. As a second staining. We have analyzed this issue for 88 situations in which Ag- example, human tumors contain large numbers of mutations that specific T cells were stained with subtype-mismatched HLA-A*02 lead to altered (non-self) peptide sequences that are unique to each multimers. Of those 88 cases, detection of Ag-specific T cells oc- patient. Because these sequences have not been encountered curred with a comparable sensitivity as with subtype-matched HLA during T cell development (i.e., are “foreign”), it may be specu- multimers in only 35% of cases, and in .55% of cases no T cell lated that T cells recognizing these mutated epitopes are of par- staining was detected altogether. ticular importance in (therapy-induced) T cell–mediated tumor The strong effect of minor sequence variation on pMHC-based regression (37). Recent work by Rosenberg and colleagues (38) immunomonitoring that is described in this study extends earlier and by us (39) has demonstrated how cancer exome sequencing work that demonstrated specific cases in which sequence variation data can be used to reveal T cell responses against patient-specific between HLA subtypes was shown to influence T cell recognition neoantigens in humans, and how such responses can be influenced of target cells. Specifically, in work by Yu et al. (26), it was shown by immunotherapy (39). The development of pMHC-based mon- that the HLA-B*57:01 and HLA-B*57:03 subtypes, which differ itoring technology for large sets of HLA subtypes will be of Downloaded from by only two amino acids, can both present the same HIV-1 Gag substantial importance for such personalized immunomonitor- KF11 epitope, but with T cell recognition only being observed in ing, either by the flow cytometry–based approaches used in this the context of HLA-B*57:01. Likewise, a differential recognition study, or by the high-throughput MHC multimer–based mass of the same EBV epitope by Ag-specific T cells has been observed cytometry recently developed by Newell and colleagues (40). for three of the HLA-B*44 subtypes (27). Structural studies pro- vide strong evidence that the effect of HLA micropolymorphisms Acknowledgments http://www.jimmunol.org/ on TCR interaction can in many cases not be predicted by peptide We thank Can Keşmir (Division of Theoretical Biology, Utrecht University, binding studies. Specifically, variants of the HLA-B*44 subtype Utrecht, The Netherlands) for advice on pMHC predictions, Patrick H. Celie have been shown to present a largely identical epitope repertoire, (Division of Biochemistry, The Netherlands Cancer Institute, Amsterdam, with T cell recognition primarily being influenced by the effect of The Netherlands) for the generation of the three-dimensional molecular mod- the “hidden” polymorphic residue on the conformation of the peptide els of the subtype MHC molecules, Ce´cile Alanio and Matthew L. Albert Ag with the peptide-binding groove (28, 29). Even more striking, (Centre d’Immunologie Humaine, Institut Pasteur, Paris, France) for critical crystallographic analysis has demonstrated that micropolymorphisms reading of the manuscript, and members of the Schumacher and Haanen within the HLA-B*57 allele do not influence the structure of HLA- Laboratories for useful discussions. B*57 complexed with the Gag KF11 epitope in isolation: Only upon by guest on September 28, 2021 TCR ligation, the conformation of the pMHC complex becomes Disclosures different for HLA-B*57:01 and B*57:03. The authors have no financial conflicts of interest. Based on this earlier work on target cell recognition, and based on the observation that the peptides used in the present study were References shown to bind to most of the HLA-A*02 subtype alleles (Fig. 3), 1. Altman, J. D., P. A. Moss, P. J. Goulder, D. H. Barouch, M. G. McHeyzer- we consider it likely that altered TCR recognition of pMHC sub- Williams, J. I. Bell, A. J. McMichael, and M. M. Davis. 1996. Phenotypic type variants also forms the major factor preventing accurate T cell analysis of antigen-specific T lymphocytes. Science 274: 94–96. 2. Davis, M. M., J. D. Altman, and E. W. Newell. 2011. Interrogating the repertoire: detection in the current study. Furthermore, the observation that broadening the scope of peptide-MHC multimer analysis. Nat. Rev. Immunol. 11: for polyclonal Ag-specific T cell populations, subtype-mismatched 551–558. HLA multimers in some cases selectively fail to detect part of the 3. Cobbold, M., N. Khan, B. Pourgheysari, S. Tauro, D. McDonald, H. Osman, M. Assenmacher, L. Billingham, C. Steward, C. Crawley, et al. 2005. Adoptive Ag-specific T cell population (Fig. 5B) also provides evidence for transfer of -specific CTL to transplant patients after altered TCR binding as a contributing mechanism. As such, it will selection by HLA-peptide tetramers. J. Exp. Med. 202: 379–386. 4. Moon, J. J., H. H. Chu, M. Pepper, S. J. McSorley, S. C. Jameson, R. M. Kedl, not be feasible to prevent this issue by pMHC binding studies. and M. K. Jenkins. 2007. Naive CD4+ T cell frequency varies for different The present data clearly demonstrate that human immunomo- epitopes and predicts repertoire diversity and response magnitude. Immunity 27: nitoring requires high-resolution matching between the HLA 203–213. 5. Alanio, C., F. Lemaitre, H. K. Law, M. Hasan, and M. L. Albert. 2010. Enu- alleles of biological samples and the pMHC reagents used for meration of human antigen-specific naive CD8+ T cells reveals conserved pre- monitoring. Pioneering work of Dausset, Bodmer, and others cursor frequencies. Blood 115: 3718–3725. resulted in the first descriptions of HLA Ags by serological typing 6. Andersen, R. S., C. A. Thrue, N. Junker, R. Lyngaa, M. Donia, E. Ellebæk, I. M. Svane, T. N. Schumacher, P. Thor Straten, and S. R. Hadrup. 2012. Dis- (30). Serological typing of the HLA haplotype is experimentally section of T-cell antigen specificity in human melanoma. Cancer Res. 72: 1642– straightforward and is still commonly used in clinical studies that 1650. 7. Chang, C. X., A. T. Tan, M. Y. Or, K. Y. Toh, P. Y. Lim, A. S. Chia, use pMHC-based monitoring (31, 32). Likewise, the use of low- T. M. Froesig, K. D. Nadua, H. L. Oh, H. N. Leong, et al. 2013. Conditional resolution PCR for HLA haplotype analysis, as still frequently ligands for Asian HLA variants facilitate the definition of CD8+ T-cell responses performed (33), does not provide information on the sequence in acute and chronic viral diseases. Eur. J. Immunol. 43: 1109–1120. 8. Rohren, E. M., L. R. Pease, H. L. Ploegh, and T. N. Schumacher. 1993. Poly- variability that is shown in this study to be of major importance. In morphisms in pockets of major histocompatibility complex class I molecules future clinical studies, this issue may be readily addressed by influence peptide preference. J. Exp. Med. 177: 1713–1721. incorporation of high-resolution PCR-based haplotyping. Addi- 9. Robinson, J., J. A. Halliwell, H. McWilliam, R. Lopez, P. Parham, and S. G. Marsh. 2013. The IMGT/HLA database. Nucleic Acids Res. 41(Database tionally, for the recently developed approaches that aim to obtain issue): D1222–D1227. HLA haplotype information from whole exome/RNAseq next 10. Gras, S., X. Saulquin, J. B. Reiser, E. Debeaupuis, K. Echasserieau, A. Kissenpfennig, F. Legoux, A. Chouquet, M. Le Gorrec, P. Machillot, et al. generation sequencing data, it will also be important to ensure that 2009. Structural bases for the affinity-driven selection of a public TCR against high resolution is achieved (34–36). a dominant human cytomegalovirus epitope. J. Immunol. 183: 430–437. 648 HLA MICROPOLYMORPHISMS STRONGLY AFFECT pMHC-BASED ANALYSIS

11. Rodenko, B., M. Toebes, S. R. Hadrup, W. J. van Esch, A. M. Molenaar, induction and differential susceptibility to human immunodeficiency virus type 1 T. N. Schumacher, and H. Ovaa. 2006. Generation of peptide-MHC class I mutational escape associated with a two-amino-acid difference between HLA class complexes through UV-mediated ligand exchange. Nat. Protoc. 1: 1120–1132. I subtypes. J. Virol. 81: 1619–1631. 12. Hoof, I., B. Peters, J. Sidney, L. E. Pedersen, A. Sette, O. Lund, S. Buus, and 27. Archbold, J. K., W. A. Macdonald, S. Gras, L. K. Ely, J. J. Miles, M. J. Bell, M. Nielsen. 2009. NetMHCpan, a method for MHC class I binding prediction R. M. Brennan, T. Beddoe, M. C. Wilce, C. S. Clements, et al. 2009. Natural beyond humans. Immunogenetics 61: 1–13. micropolymorphism in human leukocyte antigens provides a basis for genetic 13. Toebes, M., B. Rodenko, H. Ovaa, and T. N. Schumacher. 2009. Generation of control of antigen recognition. J. Exp. Med. 206: 209–219. peptide MHC class I monomers and multimers through ligand exchange. Curr. 28. Stewart-Jones, G. B., P. Simpson, P. A. van der Merwe, P. Easterbrook, Protoc. Immunol. Chapter 18: Unit 18.16. doi:10.1002/0471142735.im1816s87 A. J. McMichael, S. L. Rowland-Jones, E. Y. Jones, and G. M. Gillespie. 2012. 14. Garboczi, D. N., D. T. Hung, and D. C. Wiley. 1992. HLA-A2-peptide complexes: Structural features underlying T-cell receptor sensitivity to concealed MHC class refolding and crystallization of molecules expressed in Escherichia coli and com- I micropolymorphisms. Proc. Natl. Acad. Sci. USA 109: E3483–E3492. plexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA 89: 3429–3433. 29. Macdonald, W. A., A. W. Purcell, N. A. Mifsud, L. K. Ely, D. S. Williams, 15. Andersen, R. S., P. Kvistborg, T. M. Frøsig, N. W. Pedersen, R. Lyngaa, L. Chang, J. J. Gorman, C. S. Clements, L. Kjer-Nielsen, D. M. Koelle, et al. A. H. Bakker, C. J. Shu, Pt. Straten, T. N. Schumacher, and S. R. Hadrup. 2012. 2003. A naturally selected dimorphism within the HLA-B44 supertype alters Parallel detection of antigen-specific T cell responses by combinatorial encoding class I structure, peptide repertoire, and T cell recognition. J. Exp. Med. 198: of MHC multimers. Nat. Protoc. 7: 891–902. 679–691. 16. Kvistborg, P., C. J. Shu, B. Heemskerk, M. Fankhauser, C. A. Thrue, M. Toebes, 30. Park, I., and P. Terasaki. 2000. Origins of the first HLA specificities. Hum. N. van Rooij, C. Linnemann, M. M. van Buuren, J. H. Urbanus, et al. 2012. TIL Immunol. 61: 185–189. therapy broadens the tumor-reactive CD8+ T cell compartment in melanoma 31. Sauce, D., M. Bodinier, M. Garin, B. Petracca, N. Tonnelier, A. Duperrier, patients. OncoImmunology 1: 409–418. J. V. Melo, J. F. Apperley, C. Ferrand, P. Herve´, et al. 2002. Retrovirus-mediated 17. Hadrup, S. R., A. H. Bakker, C. J. Shu, R. S. Andersen, J. van Veluw, gene transfer in primary T lymphocytes impairs their anti-Epstein-Barr virus P. Hombrink, E. Castermans, P. Thor Straten, C. Blank, J. B. Haanen, et al. 2009. potential through both culture-dependent and selection process-dependent Parallel detection of antigen-specific T-cell responses by multidimensional mechanisms. Blood 99: 1165–1173. encoding of MHC multimers. Nat. Methods 6: 520–526. 32. Shankar, P., M. Russo, B. Harnisch, M. Patterson, P. Skolnik, and J. Lieberman. 18. Linnemann, C., B. Heemskerk, P. Kvistborg, R. J. Kluin, D. A. Bolotin, X. Chen, 2000. Impaired function of circulating HIV-specific CD8+ T cells in chronic K. Bresser, M. Nieuwland, R. Schotte, S. Michels, et al. 2013. High-throughput human immunodeficiency virus infection. Blood 96: 3094–3101. Downloaded from identification of antigen-specific TCRs by TCR gene capture. Nat. Med. 19: 33. Lambert, M., M. Gannage´, A. Karras, M. Abel, C. Legendre, D. Kerob, 1534–1541. F. Agbalika, P. M. Girard, C. Lebbe, and S. Caillat-Zucman. 2006. Differences in 19. Dudley, M. E., J. R. Wunderlich, T. E. Shelton, J. Even, and S. A. Rosenberg. the frequency and function of HHV8-specific CD8 T cells between asymptom- 2003. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive atic HHV8 infection and Kaposi sarcoma. Blood 108: 3871–3880. transfer therapy for melanoma patients. J. Immunother. 26: 332–342. 34. Warren, R. L., G. Choe, D. J. Freeman, M. Castellarin, S. Munro, R. Moore, and 20. Saper, M. A., P. J. Bjorkman, and D. C. Wiley. 1991. Refined structure of the R. A. Holt. 2012. Derivation of HLA types from shotgun sequence datasets. human histocompatibility antigen HLA-A2 at 2.6 A resolution. J. Mol. Biol. 219: Genome Med. 4: 95.

277–319. 35. Boegel, S., M. Lower, M. Schafer, T. Bukur, J. de Graaf, V. Boisguerin, http://www.jimmunol.org/ 21. Matsumura, M., D. H. Fremont, P. A. Peterson, and I. A. Wilson. 1992. O. Tureci, M. Diken, J. C. Castle, and U. Sahin. 2013. HLA typing from RNA- Emerging principles for the recognition of peptide antigens by MHC class I Seq sequence reads. Genome Med. 4: 102. molecules. Science 257: 927–934. 36. Liu, C., X. Yang, B. Duffy, T. Mohanakumar, R. D. Mitra, M. C. Zody, and 22. Sidney, J., B. Peters, N. Frahm, C. Brander, and A. Sette. 2008. HLA class I J. D. Pfeifer. 2013. ATHLATES: accurate typing of human leukocyte antigen supertypes: a revised and updated classification. BMC Immunol. 9: 1. through exome sequencing. Nucleic Acids Res. 41: e142. 23. Rapin, N., I. Hoof, O. Lund, and M. Nielsen. 2008. MHC motif viewer. Im- 37. Kvistborg, P., M. M. van Buuren, and T. N. Schumacher. 2013. Human cancer munogenetics 60: 759–765. regression antigens. Curr. Opin. Immunol. 25: 284–290. 24. Toebes, M., M. Coccoris, A. Bins, B. Rodenko, R. Gomez, N. J. Nieuwkoop, 38. Robbins, P. F., Y. C. Lu, M. El-Gamil, Y. F. Li, C. Gross, J. Gartner, J. C. Lin, W. van de Kasteele, G. F. Rimmelzwaan, J. B. Haanen, H. Ovaa, and J. K. Teer, P. Cliften, E. Tycksen, et al. 2013. Mining exomic sequencing data to T. N. Schumacher. 2006. Design and use of conditional MHC class I ligands. identify mutated antigens recognized by adoptively transferred tumor-reactive Nat. Med. 12: 246–251. T cells. Nat. Med. 19: 747–752.

25. Guilloux, Y., S. Lucas, V. G. Brichard, A. Van Pel, C. Viret, E. De Plaen, 39. van Rooij, N., M. M. van Buuren, D. Philips, A. Velds, M. Toebes, by guest on September 28, 2021 F. Brasseur, B. Lethe´, F. Jotereau, and T. Boon. 1996. A peptide recognized by B. Heemskerk, L. van Dijk, S. Behjati, H. Hilkmann, D. el Atmioui, et al. 2013. human cytolytic T lymphocytes on HLA-A2 is encoded by an intron Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an sequence of the N-acetylglucosaminyltransferase V gene. J. Exp. Med. 183: ipilimumab-responsive melanoma. J. Clin. Oncol. 31: e439–e442. 1173–1183. 40. Newell, E. W., N. Sigal, N. Nair, B. A. Kidd, H. B. Greenberg, and M. M. Davis. 26. Yu, X. G., M. Lichterfeld, S. Chetty, K. L. Williams, S. K. Mui, T. Miura, N. Frahm, 2013. Combinatorial tetramer staining and mass cytometry analysis facilitate M. E. Feeney, Y. Tang, F. Pereyra, et al. 2007. Mutually exclusive T-cell receptor T-cell epitope mapping and characterization. Nat. Biotechnol. 31: 623–629. GnTvVLP

105 0.001% 0.001% 0.048% 0.082% 0.121% 0.107% 0.113%

104

A*02:06 103

102 0

105 0.002% 0.001% 0.001% 0.001% 0.001% 0.008% 0.007%

104

A*02:01 103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

MART-1ELA

105 0.001% 1.3% 2.96% 4.32% 4.71% 5% 5.19%

104 A*02:06 103

102 0

105 0.001% 0.626% 1.7% 2.65% 3.71% 3.86% 3.55%

104 A*02:01 103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

gp100KTW

105 0.001% 0.035% 0.244% 0.302% 0.357% 0.361% 0.378%

104 A*02:06 103

102 0

105 0.001% 0.204% 0.291% 0.3% 0.316% 0.392% 0.383%

104 A*02:01 103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

tyrosinaseYMD

5 10 0.001% 0.001% 0.003% 0.036% 0.092% 0.133% 0.147% 104

A*02:06 103

102 0

5 10 0.001% 0.001% 0.005% 0.018% 0.070% 0.096% 0.124% 104

A*02:01 103

102 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

625x 125x 25x 5x 1x 5x 25x Supplementary figure 1. Inability to detect antigen-specific T cell populations by subtype- mismatched MHC multimers is not influenced by pMHC-multimer concentration. + Representative dot plots of antigen specific CD8 T cell responses (GnTVVLP, MART-1ELA,

gp100KTW and tyrosinaseYMD, respectively) in TIL populations from an HLA-A*02:06-positive patient when stained with a titration of either HLA-A*02:06 or HLA-A*02:01 subtype pMHC multimers. A 1x pMHC multimer staining condition corresponds to a MHC concentration of 6 μg/mL. CD8+ pMHC- multimer-cells are indicated in red, CD8+ pMHC-multimer+ cells are indicated in blue. Num- bers in the upper-right quadrants indicate the percentage of T cells with a signal intensity above background. Data representative of two independent experiments. Supplementary table 1. Predicted IC50 values of peptide-MHC combinations

A*02:06

CMV 51 NLV EBV 815 GLC EBV 4 YVL FLUGIL 39

GnTVVLP 251

HA-2YIG 5

MageA10GLY 8

MageC2ALK 366

MageC2KVL 14

Meloe1TLN 10

Mart-1ELA 272

1

Supplementary table 1. Predicted IC50 values of peptide-MHC combinations Binding affinities of each peptide MHC combination were predicted using netMHCpan and are given as IC50 values (in nM). Peptides with IC50 values < 50 nM are regarded as strong binders, with IC50 values of 50 > 500 nM as poor binders, and with IC50 values > 500 as non-binders. Peptide-MHC combinations that allowed detection of antigen specific T cells with a comparable sensitivity as subtype matched HLA multimers are marked in green.