Brief Report

Design and Validation of Conditional Ligands for HLA-B*08:01, HLA-B*15:01, HLA-B*35:01, and HLA-B*44:05 Thomas Mørch Frøsig,1,2,3 Jiawei Yap,4,5 Tina Seremet,1 Rikke Lyngaa,1,3 Inge Marie Svane,1,6 Per Thor Straten,1 Mirjam H. M. Heemskerk,7 Gijsbert M. Grotenbreg,3,4 Sine Reker Hadrup1,3*

1

Department of Hematology, Center for Cancer Immune Therapy, Herlev University Hospital, Copenhagen, Denmark

2

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

3

Section for Immunology and Vaccinology, National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark

4

Department of Microbiology, National University of Singapore, Singapore

5

Department of Biological Sciences and the Immunology Program, National University of Singapore, Singapore

6

Department of Oncology, Herlev University Hospital, Copenhagen, Denmark

7

Department of Hematology, Leiden University Medical Centre, Leiden, The Netherlands

Received 19 December 2014; Revised 25 March 2015; Accepted 23 April 2015 Grant sponsors: Danish Cancer Society, University Hospital Herlev Research Council, The Jochum Foundation, The Carlsberg Foundation. Additional Supporting Information may be found in the online version of this article. *Correspondence to: Sine Reker Hadrup, Section for Immunology and Vaccinology, Technical University of Denmark, National Veterinary Institute, B€ ulowsvej 27, 1870 Frederiksberg C, Denmark. E-mail: [email protected]

Cytometry Part A  87A: 967 975, 2015

 Abstract We designed conditional ligands restricted to HLA-B*08:01, 2B*35:01, and 2B*44:05 and proved the use of a conditional ligand previously designed for HLA-B*15:02 together with HLA-B*15:01. Furthermore, we compared the detection capabilities of specific HLA-B*15:01-restricted T cells using the HLA-B*15:01 and HLA-B*15:02 major histocompatibility complex (MHC) multimers and found remarkable differences in the staining patterns detected by flow cytometry. These new conditional ligands greatly add to the application of MHC-based technologies in the analyses of T-cell recognition as they represent frequently expressed HLA-B molecules. This expansion of conditional ligands is important to allow T-cell detection over a wide range of HLA restrictions, and provide comprehensive understanding of the T-cell recognition in a given context. VC 2015 International Society for Advancement of Cytometry  Key terms CD8 T cells; MHC multimer; HLA-B molecules; flow cytometry; conditional ligands

THE tracking of specific T cells by use of fluorochrome-conjugated major histocompatibility complex (MHC) multimers for flow cytometry has increased our understanding in all areas of T-cell immunology (1–3). Staining of cells for binding to MHC multimers, combined with a set of antibodies to identify the CD8 T-cell population, represents an attractive way to quantitatively and phenotypically measure the CD8 T cells with certain specificities in a complex cell sample. It is thus possible to directly investigate the frequency and phenotype of these peptide-MHC (pMHC) multimer-specific T cells. The design and use of conditional ligands for the generation of MHC monomers was introduced in 2006 and revolutionized our ability to generate MHC reagents in a high-throughput fashion (4). These are HLA-binding peptides containing an UV-labile bond next to a 2-nitrophenylglycine or 3-amino-3(2-nitrophenyl)-propionic acid residue (denoted “J”) in their amino acid sequence, and can be integrated into the standard refolding procedure. Upon short-term UV light (366 nm) exposure, the amide bond next to the “J” residue is cleaved, and the fragmented peptide can be substituted with another ligand. The rescue of the MHC Class I complex after UV light exposure is dependent on the binding affinity between the ligand and the MHC molecule. Thus, this technique allows high-throughput affinity estimation using an MHC ELISA (5,6) and the generation of large libraries of pMHC complexes for the determinations of antigen-specific T cells using MHC Class I multimers in flow (7,8) and mass cytometry (9). The UV exchange technology has been extended to several MHC Class I variants (2,6,10), but conditional ligands has previously not been defined for some of the most frequently expressed HLA-B alleles. HLA-B*08:01, 2B*15:01, and 2B*35:01 are among the most frequently observed

Brief Report

Abbreviations: Conditional ligand in complex with MHC molecule, p*MHC; Peptide in complex with MHC molecule, pMHC.

Published online 1 June 2015 in Wiley Online Library (wileyonlinelibrary.com)

Present address of Gijsbert M. Grotenbreg: 121Bio LLC, 700 Main Street, 02139, Cambridge MA, USA.

DOI: 10.1002/cyto.a.22689 C 2015 International Society for Advancement of Cytometry V

HLA-B molecules in the Caucasian population (11), and our identification of conditional ligands for these tissue type molecules and a conditional ligand suggested to be pan-specific for HLA-B*44:02/03/05, is expected to contribute greatly to future immune monitoring and epitope discovery studies in all fields of immunology.

denotes a conditional ligand) complex concentration and the presence of MHC multimers after streptavidin-assisted multimerization was tested by HPLC on a Biosuite size-exclusion column (125 A˚, 4 lm, 4.6 3 300 mm) with detection at 254 nm (Waters, Hedehusene, Denmark) and analyzed by use of the Empower computer program.

MATERIALS AND METHODS

UV-Mediated Peptide Exchange and MHC ELISA Conditional ligands were cleaved and substituted with peptides of interest upon 1 h of exposure to UV light (366 nm) and the residual pMHC complex was analyzed with size-exclusion HPLC or MHC ELISA as described previously (5). Briefly, 25 lg/mL of MHC monomers was generated, diluted to 5 or 10 nM, added to 96-well plates (Corning Costar, BD Biosciences, Albertslund, Denmark), precoated with streptavidin (Life Technologies, Naerum, Denmark), and left for 1 h incubation at 378C. On top of the streptatividinbounded MHC monomer complexes, horseradish peroxidaseconjugated b2-microglobulin antibody (Lifespan Biosciences, Seattle, WA) was added, and the mix was left for 1-h incubation at 378C. The color reaction was developed with ABTS peroxidase substrate and blocked after 15 min with ABTS peroxidase Stop Solution (both from KPL, Gaithersburg, MD). Finally, the absorbance was measured at 405 nm on an Epoch spectrophotometer and analyzed by use of the Gen5 1.10 program (BioTek, Winooski, VT).

Generation of MHC Complexes MHC Class I complexes were produced as described previously (12). Briefly, plamids coding HLA Class I heavy chains or b2-microglobulin were coded into pET-28a(1) (HLAA*01:01, HLA-A*02:01, HLA-B*15:02, and b2-microglobulin), pTCF (HLA-B*08:01), pJA44 (HLA-B*15:01), or pET-3d vectors (HLA-B*35:01 and HLA-B*44:05), using NcoI (CCATGG) and BamHI (GGATCC) restriction sites. Plasmids coding for HLA-A*0101, -A*0201, or b2-microglobulin were kindly provided by Ton N. Schumacher, Netherlands Cancer Institute; the HLA-B*15:02 plasmid was from our own stock (10); the HLA-B*08:01 and 2B*15:01 plasmids were kindly provided by Donielle Bell, NIAID tetramer core facility, Atlanta, GA, USA, and the HLA-B*35:01 and -B*44:05 plasmids were kindly provided by Stefan Stevanovic, University of T€ ubingen, Germany. All plasmids were transfected and overexpressed in BL21DE3pLysS Competent Escherichia coli cells (VWR–Bie & Berntsen, Herlev, Denmark) in Auto Induction Media LB Broth Base Including Trace Elements (Formedium, Hunstanton, United Kingdom) with appropriate antibiotics. Cells were centrifuged, frozen, lysed, and washed to obtain inclusion bodies. These were dissolved in 8 M of urea or a mix of 3 M of guanidine hydrochloride, 10 mM of NaAc, and 10 mM of EDTA. In brief, 1 lM of MHC Class I heavy chain, 2 lM of b2-microglobulin light chain, and 60 lM of peptide were refolded in the presence of 1 mM of phenylmethanesulfonyl fluoride at 48C in the dark for at least 3 days; during this period, additional heavy and light chains were added every day to reach the specified concentrations. Buffer exchange was conducted over a 30-kD centrifugal filter (Millipore, Hellerup, Denmark) before biotinylation of the C-terminal BirAsequence through an enzymatic reaction with BirA (Avidity LLC, Aurora, CO). Biotinylated complexes were purified on a BioSuite size-exclusion high-performance liquid chromatography (HPLC) column (125 A˚, 13 lm, 21.5 3 300 mm) the absorbance detected at 254 nm (column, HPLC instrument, and spectrophotometer from Waters, Hedehusene, Denmark) and the data were analyzed by use of the Empower computer program. Streptavidin–APC (BioLegend, Nordic Biosite, Copenhagen, Denmark) was added to the MHC monomers to multimerize these. The determination of the p*MHC (p* 968

Detection of pMHC Multimer-Binding T Cells by Flow Cytometry For flow cytometry-based measures, pMHC complexes were generated as described for MHC ELISA, but in 25–100 lg/mL based on the MHC monomer concentration. These pMHC complexes were encoded in dual color codes with fluorochrome-coupled streptavidin conjugates of PE, APC, PE-Cy7 (all from BioLegend, Nordic Biosite, Copenhagen, Denmark), Quantum Dot (Qdot) 605, Qdot655, or Qdot705 (all from Invitrogen Life Technologies, Naerum, Denmark) as described earlier (7,13). T-cell clones and peripheral blood mononuclear cells (PBMCs) from healthy donors were stained with MHC multimers for flow cytometry testing either directly ex vivo or after a peptide prestimulation step. FurtherR more, the cells were stained with LIVE/DEADV Fixable NearIR Dead Cell Stain Kit for 633 or 635 nm excitation (Invitrogen, Life Technologies, Naerum, Denmark), CD8-PerCP (Invitrogen, Life Technologies, Naerum, Denmark), and FITCcoupled antibodies to CD4, CD14, CD16, and CD19 (all from BD Pharmingen, Albertslund, Denmark), and CD40 (AbD Serotec, Puchheim, Germany). PBMCs or T-cell clones were stained with pMHC multimers generated with T-cell epitopes derived from virus-related proteins and data were acquired Development of New Conditional Ligands

Brief Report Table 1. Peptides used for refolding with MHC Class I heavy and b2-microglobulin light chains PEPTIDE

HLA MOLECULE

ORIGIN (PDB-ID)

AUCCONDITIONAL LIGAND/AUCNATIVE PEPTIDE

FLRGRAYGL FLRGRA-J-GL

HLA-B*08:01

1M05 (Ref. 14) 1M05, modified version

1.47

KPIVVLHGY KPIVVL-J-GY APENAYQAY

HLA-B*35:01

2CIK (Ref. 15) 2CIK, modified version EBV BZLF1

0.84 1.43

EEFGAAFSF EEFGAA-J-SFa EFFWDANDI

HLA-B*44:05

3L3G (Ref. 16) 3L3G, modified version CMVpp65

1.14 1.63

ILGP-J-GSVY (Ref. 10)

HLA-B*15:01

Ubiquitin-conjugating enzyme-E2l

NA

J denotes the UV-labile amino acid substitution. AUCconditional ligand/AUCnative peptide denotes the average ratio obtained between the areas under the curve measured at 254 nm and the retention time 9.0 min after size-exclusion HPLC, 100 lL was injected from a 10-mL refolding. NA: not applicable. a Designed as a shared HLA-B*44:02, 03, and 05 UV-sensitive ligand based on the crystal structure for HLA-B*44:02 (Ref. 16)), but tested only in relation to HLA-B*44:05.

on an LSR II flow cytometer. As positive controls for the staining procedure, we included HLA-A1 and HLA-A2 pMHC multimers generated as above from previously described p*MHC complexes (4,6) and exchanged to hold virus-derived T-cell epitopes. To identify specific T-cell populations, we used the following gating strategy: from the combination of gates enumerating the lymphocytes, the single cells and the living cells we gated on the CD81CD31 or CD81dump2 cells (dump channel detecting cells expressing CD4, CD14, CD16, CD19, or CD40), identified pMHC multimer-positive cells for each fluorochrome used, and showed in subsequent plots the CD8 cells negative for all MHC multimer-associated colors or positive for exactly two MHC multimer-associated colors. This gating strategy is shown in Supporting Information Figure S1 and further described in Ref. 13). Detailed information on the flow cytometric analysis according to the MiFlowCyt requirements is shown in Supporting Information Figure S1 and Table S1. Peptide Synthesis The peptides used in this study were synthesized based on Fmoc (9-fluorenylmethoxy carbonyl) chemistry, either inhouse (HLA-B*15-restricted peptides and conditional ligands) or purchased from KJ Ross-Petersen ApS, Klampenborg, Denmark (remaining peptides). As the photo-labile amino acid substitution building block “J,” we used 3-amino-3-(2-nitrophenyl)-phenyl propionic acid. Peptide Stimulation PBMCs were pulsed with 100 lM of each peptide for 2 h at room temperature and cultured for 8 days in X-vivo 15 (Lonza, Vallensbaek, Denmark) with 5% of human serum (Sigma-Aldrich, Broendby, Denmark), and 40 U/mL of IL-2 (Proleukin, Novartis, Copenhagen, Denmark), prior to being used for MHC multimer staining of peptide-responsive T cells. Cytometry Part A  87A: 967 975, 2015

Patient and Healthy-Donor PBMCs and T-Cell Clones PBMCs were collected from healthy donors and cancer patients. Blood samples from patients were drawn a minimum of 4 weeks after the termination of any kind of anticancer therapy. PBMCs were isolated using Lymphoprep (Stemcell Technologies, Grenoble, France) separation, HLA-typed (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark), and frozen in fetal calf serum (from Gibco, Life Technologies, Naerum, Denmark) with 10% of dimethylsulfoxide (Sigma-Aldrich, Broendby, Denmark). The protocol was approved by the Scientific Ethics Committee for The Capital Region of Denmark and conducted in accordance with the provisions of the Declaration of Helsinki. Written informed consent from the patients was obtained before study entry. The T-cell clones were generated from PBMCs by using autologous antigen-presenting cells loaded with peptide.

RESULTS To develop conditional ligands for HLA-B*08:01, HLAB*35:01, and HLA-B*44 (shared between B*44:02, 03, and 05), we examined www.iedb.org/bb_structure.php for the crystal structures of pMHC complexes, which enabled us to qualitatively select the protruding amino acids for the substitution with the photo-labile amino acid substitution (J) residue. We modified the native HLA ligands (14–16) to FLRGRA-J-GL (HLA-B*08:01), KPIVVL-J-GY (HLAB*35:01), and EEFGAA-J-SF (HLA-B*44). The shared HLA-B*44 UV-sensitive ligand was tested only in relation to HLA-B*44:05, because the refolding of the other HLA-B*44 subtypes was unsuccessful. The insertion of “J” did not impair the refolding efficiency compared to the native peptide or a Tcell epitope derived from a virus-related protein (the latter only for HLA-B*35:01 and HLA-B*44:05) (Table 1). We previously published a conditional ligand for HLA-B*15:02, ILGPPG-J-VY (10), and found that this conditional ligand 969

Brief Report and ILGP-J-GSVY had similar UV-induced exchange performances for HLA-B*15:02 (G. M. G., unpublished). In this study, we investigated the functionality of ILGP-J-GSVY in complex with HLA-B*15:01. The UV light-induced cleavage of the conditional ligands was investigated through the surrogate measure of loss of folded MHC Class I molecules after UV light exposure. We analyzed this by measuring the 254-nm absorbance combined with size-exclusion HPLC separation for the remaining complex. We compared the amount of folded MHC complex after the following treatments: (1) no UV light exposure, (2) 1 h of UV light exposure without the addition of a rescue peptide, and (3) 1 h of UV light exposure with the addition of a rescue peptide (Fig. 1). After 1 h of UV light exposure, we observed considerable degradation of the complexes if no peptide was present and significant rescue after the addition of peptide (HLA ligand), suggesting that the conditional ligand had been substituted for all p*MHC Class I complexes (p* denotes a conditional ligand). To further investigate the peptideexchange efficiency, we performed MHC ELISA. The presence of peptide in the MHC-binding pocket is necessary to stabilize the complex, and thus by measuring the amount of correctly folded pMHC Class I complex in a sandwich ELISA upon exposure to UV light and addition of a rescue peptide, an estimate of the binding affinity between this peptide and the MHC Class I complex is given (5,6). The exchange efficiency and ability to evaluate this parameter by the MHC ELISA was investigated using a collection of previously described HLA ligands from virus-related proteins (further details are provided in Table 2). The MHC ELISA-based measure of exchange efficiency using these ligands was tested as opposed to negative controls with either no rescue peptide or an irrelevant HLA-mismatch ligand. HLA-B*08:01, 2B*15:01, and 2B*44:05 p*MHC complexes showed the ability to exchange high- and low-affinity peptides and provided a window for an MHC ELISA-based measure of the affinity difference between the peptides as shown in Figure 2. We were not able to obtain a valuable MHC ELISA-based readout for the HLA-B*35:01 p*MHC complex owing to a very high background signal. We proceeded to validate the use of all four conditional ligands for the generation of pMHC complexes multimerized with fluorochrome-conjugated streptavidin for the detection of antigen-specific T-cell populations by flow cytometry (7). Pretesting of donor PBMCs by ELISPOT revealed a response to HLA-B*08:01/CMV IE1ELR, and this response was confirmed with dual-color-encoded pMHC multimers as shown in Figure 3A (the gating strategy is shown in Supporting Information Fig. S1). To detect HLA-B*15:01-, 2B*35:01-, and 2B*44:05-restricted T cells, we generated a small library of dual color-coded MHC Class I multimers with different Tcell epitopes derived from virus-related proteins (Table 2). We screened PBMCs from 17 donors and detected HLA-B*15:01/ EBV EBNA3BGQG-specific T cells in three out of the seven HLA-B*15:01-positive donors analyzed (a representative example, Fig. 3B). No responses were detected against the HLA-B*35- and HLA-B*44-restricted T-cell epitopes; instead, we obtained T-cell clones restricted to these HLA molecules 970

and generated pMHC multimers with the appropriate peptides via UV-induced peptide exchange. We spiked the T-cell clones into PBMCs from a healthy donor and used the pMHC multimers to detect HLA-B*35:01/CMVpp65IPS- and HLAB*44:05/ACC-2KEF-specific T cells as shown in Figures 3C and 3D. The HLA-B*44:05-detected T-cell clone originates from an HLA-B*44:02 donor, but was previously investigated for binding to HLA-B*44:05 (M. H. M. H., unpublished data). To investigate HLA-B*15 subtype staining differences, we also included HLA-B*15:02 pMHC multimers holding the same peptide library as that used for HLA-B*15:01. No responses were detected using HLA-B*15:02 pMHC multimers in three patients although we did detect responses with the HLA-B*15:01 pMHC multimers (Figs. 4A and 4B and Supporting Information Table S2). To enhance the frequency of peptide-responsive T cells, we included a peptide prestimulation using the EBV EBNA3BGQG peptide (full sequence, Table 2). In one patient, after peptide prestimulation, EBV EBNA3BGQG-specific T cells could be detected with both HLAB*15:01 and HLA-B*15:02 MHC multimers although in very different magnitudes (22.7 and 0.8%, respectively) of the CD8 cells (Figs. 4C and 4D). In the additional two patients, no HLA-B*15:02 pMHC multimer-responsive T cells could be detected despite the prestimulation procedure (Supporting Information Table S2). This finding indicates that only a minor fraction of the T cells in the HLA B*15:01 donors are crossreactive between the B*15:01 and the HLA-B*15:02 molecules.

DISCUSSION In this study, we have developed conditional ligands restricted to HLA-B*08:01, 2B*15:01, 2B*35:01, and 2B*44:05 for the generation of MHC Class I monomers using UV-induced peptide exchange. We were able to show their functional capabilities in different assays and expect their use to be of value in future investigations of T-cell recognition in individuals holding these HLA molecules. The knowledge of conditional ligands for these four HLA molecules add to the previous findings and enables high-throughput screening of pMHC affinity and generation of pMHC complexes for monitoring of specific T-cell responses in any immunological context (5,7–9,13). It is not possible to directly determine the full degradation of the conditional ligand upon UV light exposure using MHC complex destabilization as the read-out (here measured by HPLC or MHC ELISA) as the MHC complex may, to some extent, remain conformational intact although the conditional ligand is fully cleaved. The molecule, however, is receptive to an incoming exchange ligand in this situation. It has previously been shown by both mass spectrometry (4) and acid elution of the remaining p*MHC complex after UV light exposure from a reverse-phase HPLC column (6), that the conditional ligand is indeed fully cleaved and absent from the binding groove although a residual peak is present in the HPLC chromatogram. Of interest, we found a significant difference in the number of measured MHC Class I multimer-positive cells upon staining with HLA-B*15:01 and HLA-B*15:02 pMHC multimers in HLA-B*15:01 Development of New Conditional Ligands

Brief Report

Figure 1. UV light-mediated cleavage and rescue of the folded pMHC Class I complexes measured by size-exclusion separation HPLC and Abs254 nm. The folded complex not exposed to UV light (in black), the residual complex upon UV light exposure degradation (in gray), and the complex partly rescued by adding a rescue peptide in parallel to the UV light exposure (dotted line) are shown. (A) p*HLA-B*08:01, rescue peptide CMV IE1ELR (a slight difference in the retention time of the fully folded complexes not affected by UV light owing to a change in the pressure between the two runs has been adjusted for). (B) p*HLA-B*15:01, rescue peptide MTB ESAT-6-like protein esxBNIR. (C) p*HLA-B*35:01, rescue peptide Influenza MatrixASC. (D) p*HLA-B*44:05, rescue peptide HLA-DPa chainEEFGR. The measurement of the p*MHC complexes not exposed to UV light was taken in duplicates, whereas the remaining measurements were taken in singlets (except for the HLA-B*08:01 for which we measured all samples in duplicates and HLA-B*15:01 for which we measured all samples in singlets) owing to low yields from the individual refoldings. Data representative for multiple refoldings for each HLA molecule are shown.

Cytometry Part A  87A: 967 975, 2015

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Brief Report Table 2. Peptides used for MHC ELISA and flow cytometrya HLA RESTRICTION

HLA-A1 HLA-A2 HLA-B35

HLA-B8

HLA-B15

HLA-B44

PEPTIDE NO.

SOURCE

SEQUENCE

IEDB-ID

NA NA NA NA NA NA NA NA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

CMV pp65 CMV pp50 CMV pp65 CMV IE1 Influenza matrix EBV EBNA3B EBV BZLF1 CMVpp65 CMV IE1 Influenza NP EBV BZLF1 EBV EBNA3A EBV EBNA3A MTB ESAT-6-like protein esxB (75–83) EBV EBNA3A nuclear protein EBV EBNA3B (EBNA4A) latent protein EBV EBNA3C latent protein EBV nuclear antigen EBNA-3C Influenza polymerase PB1 Influenza polymerase PB1 EBV EBNA3C latent protein EBV EBNA3C latent protein EBV nuclear antigen EBNA-3C EBV nuclear antigen EBNA-3C EBV EBNA3C CMVpp65 HLA-DPa chain Cytochrome P450 ACC-2

YSEHPTFTSQY VTEHDTLLY NLVPMVATV VLEETSVML ASCMGLIY AVLLHEESM APENAYQAY IPSINVHHY ELRRKMMYM ELRSRYWAI RAKFKQLL QAKWRLQTL FLRGRAYGL NIRQAGVQY LEKARGSTY GQGGSPTAM LLDFVRFMGV QNGALAINTF TQIQTRRSF KMARLGKGY LDFVRFMGV LLDFVRFMG QNGALAINT NGALAINTF EENLLDFVRF EFFWDANDI EEFGRAFSF EEFGAAFSF KEFEDDIINW

75718 71290 44920 69452 4349 5439 3537 28061 13257 13263 53128 50298 16878 44327 35533 21870 37153 51685 65880 32289 35162 37153 for LLDFVRFMGV 51685 for QNGAKAINTF NA 11804 11994 95009 134907 238653

a Peptides indicated in italics were included only in the MHC ELISA screening and peptides indicated in bold were included only in flow cytometry screening. Remaining peptides were present in both screenings. NA: not applicable. IEDB-ID numbers were found at http:// www.iedb.org/.

Figure 2. UV light-mediated cleavage and rescue of the folded p*MHC Class I complexes as measured by MHC ELISA by HLA ligands from virus-related proteins; absorbance values relative to the pMHC complex with highest absorbance are given. Exchange peptides are listed in Table 2, and numbered accordingly. (A) p*HLA-B*08:01, (B) p*HLA-B*15:01, and (C) p*HLA-B*44:05. Each data point is the mean of triplicate measurements (except for HLA-B*15:01 for which each point represents the mean of duplicate measurements). The bars indicate SD and representative data from two experiments are shown.

972

Development of New Conditional Ligands

Brief Report

Figure 3. The detection of antigen-specific T cells by flow cytometry using pMHC Class I multimers generated by UV-induced peptide exchange of the here described conditional ligands. The dot plots show CD8 cells (gray) and antigen-specific T cells (black) selected based on binding to the pMHC multimers coded with two different fluorochromes. Flow cytometry results from direct ex vivo staining of PBMCs with different pMHC Class I multimers, (A) HLA-B*08:01 pMHC multimers: 0.86% HLA-B*08:01/CMV IE1ELR-specific T cells in 180,000 CD8 cells. (B) HLA-B*15:01 pMHC multimers: 0.09% HLA-B*15:01/EBV EBNA3BGQG-specific T cells in 65,000 CD8 cells, (C) HLA-B*35:01 pMHC multimers: 21.6% HLA-B*35:01/CMVpp65IPS-specific T cells of 44,000 CD8 cells, (D) HLA-B B*44:05 pMHC multimers: 4.2% HLA-B*44:05/ ACC-2KEF-specific T cells of 43,000 CD8 cells. Representative data from two experiments for each tissue type are shown. The gating strategy is shown in Supporting Information Figure S1.

individuals. These data indicate a very limited overlap between the T-cell recognition presented by HLA-B*15:01 and HLA-B*15:02 and point to the importance of detailed tissue typing and use of matching HLA molecules when Tcell immunity is measured via staining with MHC Class I multimers. A similar difference among HLA-A*02:06 and HLA-A*02:01 subtypes was recently described although the HLA-A*02:06 heavy chain differs only by a single amino acid from that of HLA-A*02:01 (17). The HLA-B*15:01 and 2B*15:02 heavy chains, on the contrary, differ at five amino acid positions, including three functional residues expected Cytometry Part A  87A: 967 975, 2015

to participate in the T-cell receptor binding (18). HLAB*44:02, 03, and 05 differ only in one amino acid position, hold the same anchor residues, and present a very similar set of peptides (11,19); however, the T-cell recognition among these subtypes may still vary substantially. It has been previously shown that HLA-peptide binding affinity is important for the T-cell-mediated killing, and this parameter has been directly correlated to the relapse or eradication of an induced tumor in an experimental mouse model by Engels et al. (20). As it was outlined in a comment to this study, the binding affinity range of the interactions between the 973

Brief Report

Figure 4. Major differences in the detection of HLA-B*15:01/EBV EBNA3BGQG-specific T cells upon staining with HLA-B*15:01 and HLAB*15:02 pMHC Class I multimers. (A) HLA-B*15:01 pMHC multimers, directly ex vivo: 0.09%-specific T cells in 65,000 CD8 cells (identical to Fig. 3B); (B) Staining with HLA-B*15:02 pMHC multimers, directly ex vivo: no specific cells in 27,000 CD8 cells. After a peptide prestimulation: (C) Staining with HLA-B*15:01 pMHC multimers reveals 22.7% specific T cells in 17,800 CD8 cells (right); and (D) Staining with HLAB*15:02 pMHC multimers reveals 0.80% specific T cells in 19,000 CD8 cells (right). The data from one patient (Patient 3) is shown, data from additionally two patients tested in parallel with HLA-B*15:01 and HLA-B*15:02 MHC multimers are shown in Supporting Information Table S2.

peptides and the MHC Class I molecules is far larger than the range of the binding affinity between the T-cell receptor and the pMHC complex (21). Therefore, the pMHC affinity should be considered when clinical strategies for immune enhancements are designed. The conditional ligands presented here provide an effective tool for measuring this interaction in a high-throughput manner for HLA-B*08:01, 2B*15:01, and 2B*44:05 by means of the MHC ELISA method. We were not able to establish the MHC ELISA method for HLA-B*35:01 owing to a very high background signal. This could be indicative for enhanced stability of the HLA-B*35:01 molecule after conditional ligand cleavage under these experimental condi974

tions although previous data relating to the assembly efficiency for HLA-B*35 do not support this (22). It could be speculated if the remaining six amino acids after UV light induced cleavage would stabilize the HLA-B*35:01 to retain the conformation even when no exchange peptide is added. However, the photo-labile subunit is localized at the same position also for the HLA-B*15:01 and B*44:05 conditional ligands, for which alleles the ELISA-based method do work. Additionally, for these alleles, compared to HLA-B*35, the native peptide sequences that form the backbone of the conditional ligands are even predicted to form more stable complexes with their HLA (23). Thus, there is currently more explanation of the increased Development of New Conditional Ligands

Brief Report background in the HLA-B*35 ELISA and this may be subjected to further optimization processes. The identification of new T-cell epitopes restricted to these HLA molecules will lead to a broader coverage in the monitoring of patients treated with immunotherapeutic interventions. These four HLA-B molecules are among the most frequently observed in the Caucasian population (11), and the identification of conditional ligands for these will greatly contribute to epitope discovery studies and monitoring of patients treated with immunotherapeutic interventions.

ACKNOWLEDGMENTS The authors acknowledge Mads Duus Hjortsø for identifying the HLA-B8/CMV IE1ELR T-cell response by ELISPOT. Further, they thank Danielle Bell from the NIAID Tetramer Core Facility, Emory University, Atlanta, GA, USA, for kindly providing the plasmid constructs for HLA-B*08:01 and HLAB*15:01, Stefan Stevanovic of the University of T€ ubingen, Germany, for kindly providing the plasmid constructs for HLAB*35:01 and HLA-B*44:05 and Ton N. Schumacher of The Netherlands Cancer Institute, Amsterdam, The Netherlands, for kindly providing the HLA-A1, HLA-A2, and b2microglobulin plasmid constructs.

AUTHOR CONTRIBUTIONS T. M. F. designed and performed experiments, analyzed data, and wrote the article; J. Y. designed and performed experiments and analyzed data; T. S. performed experiments and analyzed data; R. L. helped with the experiments and data analysis; I. M. S. provided healthy donor and patient blood samples; P. t. S. provided financial support and laboratory facilities; M. H. M. H. provided T-cell clones; G. M. G. designed experiments, conceived the approach, and analyzed data; S. R. H. conceived the approach, designed experiments, analyzed data, and cowrote the article.

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