Identiﬁcation of Biological Relevant Minor ... · For analysis of IFNg and granulocyte macrophage colony-stimulating factor (GM-CSF) production, 5,000 T cells were stimulated with

Biology of Human Tumors

Identification of Biological Relevant MinorHistocompatibility Antigens within theB-lymphocyte–Derived HLA-Ligandome Using aReverse Immunology ApproachPleun Hombrink1, Chopie Hassan2, Michel G.D. Kester1, Lorenz Jahn1, Margot J. Pont1,Arnoud H. de Ru2, Cornelis A.M.van Bergen1, Marieke Griffioen1, J.H. Frederik Falkenburg1,Peter A. van Veelen2, and Mirjam H.M. Heemskerk1

Abstract

Purpose: T-cell recognition of minor histocompatibility anti-gens (MiHA) not only plays an important role in the beneficialgraft-versus-leukemia (GVL) effect of allogeneic stem cell trans-plantation (allo-SCT) but also mediates serious GVH complica-tions associated with allo-SCT. Using a reverse immunologyapproach,we aim todevelop amethod enabling the identificationof T-cell responses directed against predefined antigens, with thegoal to select those MiHAs that can be used clinically in combi-nation with allo-SCT.

Experimental Design: In this study, we used a recently devel-opedMiHA selection algorithm to select candidate MiHAs withinthe HLA-presented ligandome of transformed B cells. From theHLA-presented ligandome that predominantly consisted ofmonomorphic peptides, 25 polymorphic peptides with a clini-

cally relevant allele frequency were selected. By high-throughputscreening, the availability of high-avidity T cells specific for theseMiHA candidates in different healthy donors was analyzed.

Results: With the use of MHC multimer enrichment, analysesof expanded T cells by combinatorial codingMHCmultimer flowcytometry, and subsequent single-cell cloning, positive T-cellclones directed to two new MiHA: LB-CLYBL-1Y and LB-TEP1-1S could be demonstrated, indicating the immunogenicity ofthese two MiHAs.

Conclusions: The biologic relevance of MiHA LB-CLYBL-1Ywas demonstrated by the detection of LB-CLYBL-1Y–specificT cells in a patient suffering from acute myeloid leukemia (AML)that experienced an anti-leukemic response after treatment withallo-SCT. Clin Cancer Res; 21(9); 2177–86. �2015 AACR.

IntroductionAllogeneic HLA-matched hematopoietic stem cell transplanta-

tion (allo-SCT) and subsequent donor lymphocyte infusion (DLI)to eradicate persistent or relapsed malignant cells are consideredan effective curative treatment for patients with high-risk hema-tologicmalignancies (1). The curative potential of this therapy hasbeen attributed to the recognition of malignant cells by donor Tcells. Detailed analyses of T-cell immune responses in patientsresponding to DLI have demonstrated that donor T cells canrecognize minor histocompatibility antigens (MiHA) presented

by HLAmolecules onmalignant cells. MiHA are peptides derivedfrom polymorphic proteins that differ between donor and recip-ient due to single-nucleotide polymorphism (SNP; refs. 2–5).Previously, it has been demonstrated that T cells directed againstMiHA that are ubiquitously expressed can mediate life-threaten-ing GVH (6), whereas T cells directed against MiHA with hemato-poietic restriction may mediate graft-versus-leukemia (GVL)response in absence of GVHD (7).

Although significant strides inMiHAdiscovery have beenmade(8, 9), a major limitation for clinical implementation is the smallnumber of identified MiHA derived from genes that are exclu-sively expressed in hematopoietic cells. To allow the selectiveanalysis of hematopoietic-restricted MiHA, we and others haveused reverse immunologic approaches in which predicted poly-morphic peptides are the starting point andpeptide candidates aresubsequently screened for their capacity to induce a specific T-cellresponse. This approach has the potential to screen for T cellsrecognizingMiHA that are exclusively expressed byhematopoieticcells. However, it has been reported that when such peptidepredictions are solely based on computer algorithms that predictpeptide–HLA binding affinity and proteolytic cleavage, thedetected T-cell responses are often directed against epitopes thatare not naturally processed and therefore fail to lyse malignanttarget cells (10–12). To circumvent this peptide selection prob-lem, we previously introduced mass spectrometry (MS)-basedHLA-ligandomes as a reliable source for naturally processed and

1Department of Hematology, Leiden University Medical Center,Leiden, the Netherlands. 2Department of Immunohematology andBlood Transfusion, Leiden University Medical Center, Leiden, theNetherlands.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

P. Hombrink and C. Hassan contributed equally to this article.

P.A. van Veelen and M.H.M. Heemskerk share senior authorship.

Corresponding Author: Mirjam H.M. Heemskerk, Department of Hematology,Leiden University Medical Center, 2300 RC Leiden, the Netherlands. Phone:31-71-5262271; Fax: 31-71-5266755; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-2188

�2015 American Association for Cancer Research.

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presented peptides and developed an algorithm that can beexploited to identify T cells specific for potential clinically relevantMiHA (13, 14).

In this study, we selected the 25 most promising MiHAcandidates from a large set of B lymphocyte–derived HLA classI eluted peptides (15). To validate these MiHA candidates, it isessential to demonstrate their immunogenic potential by iso-lation of high avidity–specific T cells. We therefore screened theT-cell receptor (TCR) repertoire of 16 unrelated donors for thepresence of T cells specific for these MiHA candidates. MiHA-specific MHC multimer–positive T cells were isolated fromperipheral blood by magnetic-activated cell sorting (MACS).Subsequently, functional testing of MHC multimer sorted andexpanded T-cell clones demonstrated the immunogenic poten-tial of LB-CLYBL-1Y, LB-TEP1-1S, and 2 previously describedMiHA. For one of the newly defined MiHA, we were able toconfirm the biologic relevance by demonstrating MHC multi-mer–positive T cells in a patient suffering from acute myeloidleukemia (AML) after treatment with allo-SCT and subsequentDLI. Our data illustrate that with this reverse immunologyapproach, biologically relevant MiHA can be identified as wellas MiHA that are not frequently induced in vivo but canpotentially be used for immunotherapeutic strategies.

Materials and MethodsCell collection and culture conditions

Peripheral blood was obtained from different individuals afterinformed consent (Sanquin Reagents, Amsterdam; and LeidenUniversity Medical Center, Leiden, The Netherlands). All experi-ments were approved by the local medical ethics committees.Blood samples were HLA-typed by high-resolution genomic DNAtyping. Peripheral bloodmononuclear cells (PBMC)were isolatedby ficoll gradient separation and cryopreserved for further use. Tcells were cultured in T-cellmedium consisting of IscoveModifiedDulbecco Media (IMDM; Lonza) supplemented with 5% humanserum (HS), 5% FBS, 100 IU/mL IL2 (Proleukin), 2 mmol/L

L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin(Invitrogen). Epstein-Barr virus–transformed lymphoblastoid Bcell lines (EBV-LCL) and phytohemagglutinine (PHA; MurexDiagnostics) blasts were generated using standard procedures.After generation, stock ampules are frozen for which the geneticidentity is confirmed by in-house WGA profiling (IlluminaHuman1M-duo array) as previously described (9). The T2 cellline was cultured in IMDM with penicillin/streptomycin and 5%FCS. Cell lines are no longer cultured then 3 months.

Peptide library of HLA class I eluted peptidesThe peptides used for this study are derived from a newly

established large peptide library that has been recentlydescribed by Hassan and colleagues (15). In short, peptideelution, reverse-phase high-performance liquid chromatogra-phy (RP-HPLC), and tandem mass spectrometry (MS/MS) werecarried out as previously described (16, 17). Briefly, 60 � 109

HLA-A�0201 and HLA-B�0702–positive EBV-LCL were washedwith ice-cold PBS and pellets were stored at �80�C until use.Peptide HLA class I complexes were purified from cell lysate byaffinity chromatography. Subsequently, peptides were elutedfrom HLA molecules and separated from the HLA heavy chainfragments and b2-microglobulin by size filtration. The peptidemixture was separated by various first-dimension separationtechniques, after which the peptides were measured by on-linenanoHPLC-MS/MS.

Selection of MiHA candidates from a set of eluted peptidesMiHA-candidates were selected on the basis of our recently

developed MiHA selection algorithm from the recently estab-lished peptide elution library (13). Briefly, the tandem massspectra of eluted peptides were submitted to the HSPVdb (18),a database optimized for finding polymorphic peptides. Toselect for MiHA candidates within this set, we evaluated thepolymorphic peptides using strict threshold scores for massspectrometry defined sequence reliability (BMI � 30, ppm �2.0), peptide length (8–11 amino acids), predicted peptide–HLA affinity (<500 nmol/L), allele frequencies of the SNPencoding polymorphism (0.05%–0.7%), and specific geneexclusion criteria (no extreme polymorphic genes). After con-firming their integrity by comparing the tandemmass spectra ofthe synthetic peptides with that of the eluted counterparts, thetop 25 MiHA candidates, with the highest threshold scores,were selected for further analysis.

Generation of peptide–MHC complexesAll peptides were synthesized in-house using standard Fmoc

chemistry. Recombinant HLA-A�0201 and HLA-B�0702 heavychain and b2m were in-house produced in Escherichia coli. MHCclass I refolding was performed as previously described withminor modifications (19). MHC class I complexes were purifiedby gel filtration HPLC in PBS and stored at 4�C. The peptideHLA-A�0201 or HLA-B�0702 binding affinity was assessed bysubjecting prefolded UV-sensitive peptide–MHC complexes(100 mg/mL) to 366 nm UV light (Camag) for 1 hour in thepresence of the peptide of interest (200 mmol/L; 20). As controls,theCMVPP65NLVPMVATV,CMVPP65TPRVTGGAM,modifiedMART1 ELAGIGILTV, and wild-type MART1 AAGIGILTV peptidewere used. After exchange, samples were spun at 16,000� g for 5minutes, and supernatants were used to assess HLA monomerrescue using a bead assay as previously described (21).

Translational Relevance

T-cell recognition of minor histocompatibility antigensplays an important role in the graft-versus-leukemia effect ofallogeneic stem cell transplantation (allo-SCT). This studypresents the first T-cell antigen identification approach, imple-menting mass spectrometry–based HLA-ligandomes into areverse immunology that enables the identification of T-cellresponses directed against predefined antigens. The clinicalrelevance of these antigens was demonstrated by the detectionof MHC multimer–positive T cells in patients with leukemiaafter treatment with allo-SCT. In addition, the isolated T-cellclones were able to recognize primary hematopoietic malig-nant cells in a MiHA-restricted pattern but also recognizednon-hematopoietic cells after interferon pretreatment. Ourmethod allows the identification of MiHA-specific T cells thatare not frequently induced in vivo and thereforemay bemissedby forward immunology approaches. These results can be ofvalue for the identification of MiHA or other T-cell epitopesand should have general applicability in peptide vaccinationor adoptive T-cell therapies.

Hombrink et al.

Clin Cancer Res; 21(9) May 1, 2015 Clinical Cancer Research2178




Isolation of MHC multimer–positive T cells by MHC multimerpull down

Before isolation, PBMC samples were stained with phycoery-thrin (PE)-coupled MHC multimers for 1 hour at 4�C. Subse-quently, cells were washed and incubated with anti-PE beads(Miltenyi Biotec). PE-positive T cells were isolated by MACS,according to the manufacturer's protocol. Positive fraction wascultured with irradiated autologous feeder cells in T-cell mediumsupplemented with 5 ng/mL IL15 (Biosource) and anti-CD3/CD28 Dynabeads (Invitrogen). After 2 weeks, T-cell cultures wereanalyzed by MHC multimer combinatorial coding flow cytome-try. Data acquisition was performed on an LSR-II flow cytometer(BDBiosciences), andMHCmultimer–positive T cell populationswere single-cell sorted on a FACSAria (BD) into round-bottomed96-well plates containing 100 mL T-cell medium supplementedwith 800 ng/mL PHA and 1 � 105 irradiated feeder cells. TCR-bvariable chain (TCR-Vb) usagewas investigated by flow cytometryusing specific monoclonal antibodies as included in the TCR-Vbrepertoire kit (Beckman Coulter).

Cytokine secretion assayFor analysis of IFNg and granulocyte macrophage colony-

stimulating factor (GM-CSF) production, 5,000 T cells werestimulated with 20,000 EBV-LCL or 10,000 fibroblasts loadedwith different concentrations of peptides in round-bottomed 96-well plates. Before stimulation, fibroblasts were either pretreatedwith IFNg (100 IU/mL) or not for 5 days. For recognition ofprimary malignant cells, 1,000 T cells were stimulated with 5,000malignant cells in a 384-well plate. After 18 hours, supernatantswere harvested, and the concentration of IFNg and GM-CSF weremeasured by an ELISA (Sanquin Reagents). An arbitrary detectionlimit was set to 100 pg/mL for both cytokines.

SNP genotyping and microarray gene expression analysisA panel of 100 HLA-typed EBV-LCL was selected for SNP

screening as previously described (9). Briefly, gDNA was isolatedof 5� 106 EBV-LCL cells usingGentra Puregene Cell Kit (Qiagen),and PCR-free whole-genome amplification (WGA) was per-formed. The DNA samples were hybridized to IlluminaHuman1M duo arrays. For gene expression analysis, total RNAwas isolated using small and micro-scale RNAqueous isolationkits (Ambion) and amplified using the TotalPrep RNA Amplifi-cation Kit (Ambion). After preparation using the whole-genomegene expression direct hybridization assay (Illumina), cRNAsamples were dispensed onto Human HT-12 v3 ExpressionBeadChips (Illumina) according to the manufacturer's protocol.Sample collection was performed as previously described(22–24). Microarray gene expression data were analyzed usingR 2.15.

ResultsSelection of high-affinity HLA-A�0201 and B�0702 bindingMiHA candidates from a set of HLA class I eluted peptides

We have recently reported a MiHA selection algorithm to beable to selectMiHA candidates from a library of HLA class I elutedpeptides (13). This MiHA selection algorithm comprises severalevaluation steps that are summarized in Material and Methods.This algorithmwas used to screen our newly established library ofeluted HLA class I peptides derived from multiple HLA-A�0201and B�0702–positive EBV-LCLs, to select for potential MiHA

candidates (15). To validate this newly established library ofapproximately 16,000 eluted HLA class I peptides comprisingmainly monomorphic peptides, we first screened for the presenceof known MiHA. Peptide sequences or their relevant lengthvariants were identified for 10 of 13 MiHA that were expressedby the EBV-LCLs as revealed by SNP genotyping (SupplementaryTable SI; refs. 3, 8, 9), illustrating the high quality of this peptideelution library.

In the next step, we selected a set of 25 MiHA candidates usingthe MiHA selection algorithm, including 22 novel MiHA candi-dates as well as the previously reported LB-NISCH-1A, LB-ERAP1-1R and LB-GEMIN4-1VMiHA (Supplementary Table SII).We firstanalyzed the capacity of the 25 MiHA candidates to stabilize apeptide–HLA complex in an HLA binding assay that is based onUV-induced conditional ligand cleavage as described previously(10, 21, 25). After UV exchange, the HLA-binding affinity of thetested peptides was normalized to the high-affinity control pep-tides CMV-PP65-NLV and CMV-PP65-TPR for HLA-A�0201 andB�0702, respectively (Supplementary Fig. S1A and S1B). MiHAcandidates were selected when their HLA-binding affinityexceeded that of the MART1-WT-AAG peptide, which binds withlow affinity to HLA-A�0201. HLA-binding affinity as measuredwith HLA rescue scores exceeded that of the MART1-WT-AAGcontrol for 8 HLA-A�0201 and 13 B�0702 binding MiHA candi-dates. Peptide sequences and allele frequencies of the MiHA areshown in Table 1.

Isolation of peripheral blood–derivedMHCmultimer–positiveT cells by MHC multimer pull-down

To validate the 21 peptides asMiHAwith immunogenic poten-tial, we generated MHC multimers and analyzed the T-cell rep-ertoire of 16 healthy HLA-A�0201 and B�0702–positive donorsforMHCmultimer reactive T-cells. MiHA-specific T cell lines weregenerated by incubating 100 � 106 PBMC with a specific set ofMHC multimers, followed by enrichment of MHC multimer–positive cells on a magnetic column. To allow the isolation ofhigh-avidity T-cell populations, the set of MHC multimers wasspecifically adjusted for each PBMC sample to cover only thoseMiHA for which the encoding SNP was screened homozygousnegative in the respective donor, as in this individual's T-cellrepertoire, high-avidity MiHA-specific T cells will not have beendeleted because of negative selection. The set of PBMCdonorswasspecifically adjusted to cover asmany applicable donors perMiHAcandidate. Unfortunately, no homozygous negative donors werefound for the SCRIB-1L MiHA candidate. The MHC multimer–enriched T cells were expanded for 14 days in presence of aCD3/28 beads, IL2 and IL15. To increase the frequency of MHCmultimer–positive T cells, a second pull-down was performed atday 14 using the identical initial set of MiHA candidate–specificMHC multimers. After both rounds of MHC multimer enrich-ment, we analyzed the expanding T cell lines for the presence ofMHC multimer–positive T cells by FACS. A representative FACSanalysis after 2 rounds of MHC multimer enrichment is demon-strated in Fig. 1A in which 4 MHC multimer–positive T-cellpopulations specific for HLA-B�0702 binding MiHA candidatesare detected in donor OMH. After the first MHC multimer pulldown, MHC multimer–positive T-cell populations were detectedspecific for 11 of the 20 testedMiHA candidates in one or more T-cell cultures. MHC multimer–positive T-cell frequencies variedbetween 0.01% and 5.0% of total CD8þ T cells (SupplementaryTable SIII). These low frequencies are most likely due to the very

MiHA Identification by Reverse Immunology

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low frequency of MHC multimer–positive T cells in the na€�verepertoire. After the second MHC multimer pull down, MHCmultimer–positive T-cell populationswere detected specific for anincreased number of 16 of the 20 tested MiHA candidates withfrequencies up to 85%of total CD8þT cells (Supplementary TableSIII). As demonstrated in Fig. 1B, T cells reactive with the CLYBL-1Y and LB-GEMIN4-1V MHC multimer were frequently detectedin 8 of the 16 and 6 of the 9 enriched T cell lines, respectively. Incontrast, T cells specific for the other MiHA appeared morerestricted to a few donors.

Detection of high-avidity T-cell clones by screening for MiHA-specific IFNg and GM-CSF production

Next, a total number of 806 MHC multimer–positive T-cellclones representing all detected T-cell populations were generatedby FACS. To demonstrate recognition of potential MiHA candi-dates, all 806 T-cell clones were stimulated with specific antigen,and cytokine production was determined as a measure for anti-gen-specific reactivity. Because the T-cell clones were most likelyderived from the na€�ve T-cell repertoire and have presumably notall acquired the capacity to produce IFNg upon antigen encounter,we first screened all T-cell clones for their potential to secrete IFNgafter aCD3/28 stimulation. As demonstrated in Fig. 2A, thegenerated T-cell clones demonstrated a broad range of IFNgsecretion. To investigate whether GM-CSF was of additional valueto improve the screening efficiency, the GM-CSF production ofpart of the T-cell clones with variable IFNg secretion potential wasmeasured after aCD3/28 stimulation. The results demonstratethat a substantial number of T-cell clones with poor intrinsic IFNgproductionwere able to produce pronouncedGM-CSF levels (Fig.2B). Therefore, by using both IFNg and GM-CSF as a readout, wecould increase the number of MHC multimer–positive T-cellclones that could be screened. By setting an arbitrary detectionlimit to 100pg/mL for both cytokines,we increased the number ofT-cell clones that could subsequently be screened for MiHA-specific reactivity from approximately 85% (IFNg only) to 98%

(IFNg and GM-CSF). By including GM-CSF release as selectioncriteria, 108 more T-cell clones could be screened for MiHA-specific reactivity.

To demonstrate the capacity of the isolated MHC multimer–positive T-cell clones to recognize specific MiHA peptides, westimulated all T-cell clones with HLA-A�0201 and B�0702–pos-itive T2 target cells loaded with titrated concentrations of specificpeptide. MiHA-specific T-cell reactivity was observed for 8 of 16testedMiHA candidates, and either IFNg or GM-CSF was detectedafter stimulation with CLYBL-1Y, ERAP1-1R, GEMIN4-1V,NISCH-1A, HMMR-1V, C18orf21-1A, APOBEC3H-1K, andTEP1-1S MiHA peptides. For each donor, the MiHA-specific T-cell clone that demonstrated the highest peptide avidity is shown(Fig. 3A–E). MiHA-specific T-cell clones demonstrated variablepeptide avidity and half maximum cytokine production (IC50)varied between an IC50 of approximately 100pmol/L for the high-avidity T-cell clone K156 specific for GEMIN4 (Fig. 3C) and anIC50 of approximately 62.5 nmol/L for the low-avidity T-cell cloneK337 specific for C18orf21 (Fig. 3E). For the CLYBL-1Y MiHAcandidate, MHCmultimer–positive T-cell clones were successful-ly isolated from 4 different donors. High-avidity CLYBL-1Y–specific T-cell clones were isolated from donor ABM, whereasonly low-avidity or nonreactive T cells were isolated from donorUDN, EPP, and FHT, respectively (Fig. 3A). For the previouslydescribed MiHA ERAP1-1R, both high- and low-avidity T-cellclones were isolated from the 2 donors that demonstrated MHCmultimer–positive T-cell populations after pull down. For thepreviously described MiHA GEMIN4-1V, high-avidity T-cellclones were only isolated from 4 of 6 donors that demonstratedMHCmultimer–positive T-cell populations after pull down (Fig.3B and C; low-avidity T-cell clones not shown). The NISCH-1A,HMMR-1V, C18orf21-1A, and APOBEC3H-1K–specific T-cellclones were each successfully isolated from one donor andexhibited high (HMMR-1V) to low peptide avidity (NISCH-1A,C18orf21-1A, and APOBEC3H-1K; Fig. 3D). For the TEP1-1SMiHA candidate, high-avidity T-cell clones were isolated from

Table 1. Validated MiHA candidates

HLA Peptide Sequencea Gene RS number SNP Allele Frequency

A�02:01 P1 GLLGQEGLVEI PARP10 rs11136343 L/P 0.66P2 ALAPAPAEV NISCHb rs887515 A/V 0.17P3 AMLERQFTV FAM119A rs2551949 T/I 0.19P4 FLSSANEHL GLRX3 rs2274217 S/P 0.25P5 MMYKDILLL HNF4G rs1805098 M/I 0.40P6 SLAAYIPRL CLYBL rs17577293 Y/D 0.05P7 SLQEKVAKA HMMR rs299295 V/A 0.20P8 VLQNVAFSV BCL2A1 rs1138358 N/K 0.69

B�07:02 P9 APNTGRANQQM BFAR rs11546303 M/R 0.57P10 LPMEVEKNSTL HDGF rs4399146 L/P 0.40P11 RPRAPTEELAL C14orf169 rs3813563 A/V 0.40P12 APDGAKVASL TEP1 rs1760904 S/P 0.49P13 APAGVREVM AKAP13 rs7162168 M/T 0.37P14 KPQQKGLRL APOBEC3H rs139298 K/E 0.52P15 LPQKKSNAL POP1 rs17184326 N/K 0.10P16 LPQQPPLSL SCRIB rs6558394 L/P 0.64P17 NPATPASKL C18orf21 rs2276314 A/T 0.21P18 SPASSRTDL MTRR rs1532268 S/L 0.68P19 SPSLRILAI LLGL2 rs1671036 R/H 0.50P20 HPRQEQIAL ERAP1b rs34753 R/P 0.31P21 FPALRFVEV GEMIN4b rs1045481 V/E 0.20

NOTE: Non and immunogenic allele indicated by amino acid code, allele frequencies are calculated by quantification in a panel of 100 Dutch individuals using SNPgenotyping array.aSNP underlined.bPublished MiHA epitope or length variant.

Hombrink et al.





donor EPP. T-cell clone K091 demonstrated high peptide–specificGM-CSF production and low IFNg production (Fig. 3E). Nopeptide-specific T-cell clones specific for the other MiHA candi-dates were observed.

All T-cell clones that demonstrated peptide-specific cytokineproduction were stimulated with a panel of 10 SNP-genotypedHLA-A�0201 and B�0702–positive EBV-LCL target cells toscreen for reactivity against endogenously processed and pre-sented peptide. As demonstrated in Fig. 3F, G, H, and J, all high-avidity T-cell clones specific for CLYBL-1Y, TEP1-1S, ERAP1-1R,and GEMIN4-1V demonstrated recognition of all target cellsthat endogenously process and present their respective MiHA,whereas targets that were negative for the MiHA were notrecognized. Surprisingly, the high-avidity T-cell clone K507specific for HMMR-1V did not show a recognition pattern thatcorrelated with MiHA expression. This may be caused by theabsence of endogenously presented MiHA peptide by some ofthe SNP-positive EBV-LCL or by the recognition of allo-HLAmolecules expressed by EBV-LCL. No or only marginal targetcell recognition was observed for the NISCH-1A, C18orf21-1A,and APOBEC3H-1K–specific T-cell clones (Fig. 3I). These results

indicate that the MiHA CLYBL-1Y and TEP1-1S represent poten-tially immunologic relevant MiHA candidates.

Detection of MiHA-specific T-cell responses in patients afterallo-SCT

To validate the biologic relevance of the MiHA candidates, weanalyzed the peripheral blood of patients suffering from varioushematopoietic malignancies that received an allo-SCT and DLIand demonstrated a clinical response revealed by decliningpatient chimerism, for the detection of MiHA-specific T cells.Patients were only screened with MiHA-specific MHC multimerswhen they were positive for the MiHA and received a DLI from adonor who was homozygous-negative for the SNP encodingMiHA (Supplementary Table SIII). Tested patient samples wereobtained during or after the peak response, 5 to 8 weeks aftertreatment with DLI. For the newly identified CLYBL-1Y MiHA,MHC multimer–positive T cells were detected in 1 of the 3screened patients (Fig. 4A) with a frequency of 0.34% of totalCD8þ T cells at day 41 after DLI (AML patient MBF, Fig. 4B). Forthe previously described ERAP1-1R andGEMIN4-1VMiHA,MHCmultimer–positive T cells were detected in 1 of the 2 and 1 of the 4

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Figure 1.Detection of MiHA-specific T cells after MHC multimer enrichment and expansion. FACS analyses were performed to detect MiHA-specific T cells throughcombinatorial coding MHC multimer screening after second pull-down and in vitro expansion. Each peptide–MHC complex was encoded by a uniquecombination of fluorochromes. Two HLA-restricted sets of MHCmultimers were used to screen for detection of all MiHA candidate–specific T cells. A, representativeanalysis of the 2 times enriched T-cell line derived from donor OMH with the HLA-B�0702–restricted MHC multimer set. Total CD8þ T cells are shown. T cellsthat are not stained with any MHC multimer or those that are positive for only 1 or more than 2 MHC multimers, an indication for nonspecific staining, areindicated in black. T cells positive for the MHC–multimer combinations encoding the BFAR-1M, AKAP13-2M, POP1-1N, or C18orf21-1A epitopes are indicated in red,green, dark green, or purple, respectively. All dot plots are shown with biexponential axes and display fluorescence intensity for the fluorochromes indicatedon each axis. Frequencies indicate MHC multimer–positive T cells of total CD8þ cells. B, total MHC multimer–positive T-cell populations detected in 16 tested PBMCdonors after second pull-down by combinatorial coding MHC multimer screen. Bars indicate the number of donors homozygous negative for the indicatedSNP encodingMiHA and applicable for MHCmultimer pull down. The number of MHCmultimer–positive T-cell populations detected per MiHA candidate is indicatedin black. No homozygous-negative donors were found for SCRIB-1L, and therefore SCRIB-1L is not indicated in the figure. At least 2 homozygous-negativedonors were included for the other 20 MiHA candidates.






screened patients [both multiple myeloma (MM) patient CUB atday 86], respectively (Fig. 4A and B). Detected frequencies ofcirculatingMHCmultimer–positive T cells rangedbetween0.04%and 0.34% of total CD8þ T cells. For the other 17 MiHA candi-dates with validated HLA-binding affinity and SNP occurrence,including the newly identified MiHA TEP1-1S, no MHC multi-mer–positive T cellswere detected in the peripheral bloodof 1 to 5screened patients (Fig. 4A and B and Supplementary Table SIII).

To investigate whether the MHC multimer–enriched T cellclones exerted comparable peptide-specific avidity as the in vivogenerated patient-derived MiHA-specific T cells, we generatedCLYBL-1Y–specific T-cell clones by single-cell sorting of CLYBL-1Y MHC multimer–positive T cells from patient MBF. Afterexpansion, the T-cell clones were stained with theMHCmultimerandTCR-Vbmonoclonal antibodies. The T-cell cloneK264,whichwas generated by MHC multimer enrichment from donor ABM,demonstrated similar MHC multimer staining intensity as thepatient-derived K2-339 clone (Fig. 5A), but a difference in TCRVbusage; clones derived from donor ABM expressed TCR Vb22 andpatient MBF–derived clones were TCR-Vb1–positive. ClonesK264 and K2-339 were stimulated with T2 cells loaded withtitrated concentrations of either the specific or the allelic variantpeptide and IFNg production was measured (Fig. 5B). Both T-cellclones demonstrated high CLYBL-1Y–specific peptide reactivity,with IC50 varying between 1 and 4 pmol/L, whereas the allelic

CLYBL-1D variant was not or hardly recognized by both T-cellclones, demonstrating that T-cell clones derived from anunprimed setting can be equally potent as T cells derived froman in vivo primed setting.

CLYBL-1Y- and TEP1-1S–specific T-cell recognition ofhematopoietic malignant cells

To investigate the expression pattern of the CLYBL and TEP1genes, we performed a microarray gene expression array using apanel of primary and cultured malignant (and nonmalignant)hematopoietic and non-hematopoietic cells (Supplementary Fig.S2A and S2B). The data showed that the CLYBL gene is broadlyexpressed in hematopoietic and non-hematopoietic cells. Expres-sion of the TEP1 gene was not significantly measured in themajority of the samples. To investigate whether the CLYBL-1Y-and TEP1-1S–specific T-cell cloneswere able to recognize hemato-poieticmalignant cells, theywere stimulatedwithprimary chronicmyelogenous leukemia (CML), AML, and acute lymphoblasticleukemia (ALL) cells derived from different MiHA-positive and-negative patients who were positive for the restricting HLAmolecule. As a control, T-cell clones were also tested for recog-nition of EBV-LCL generated from the same individuals. Both thehigh-avidity T-cell clones CLYBL K264 and TEP1 K091 demon-strated MiHA-specific recognition of primary hematopoieticmalignant cells (Fig. 5C andE).No reactivitywas observed againstMiHA-negative target cells. These data indicate that the testedMiHA can be presented in the context of HLA at the surface ofleukemic cells and may therefore serve as direct targets of CD8þ Tcells involved in a GVL response. In addition, the potential of theMiHA to serve as target in GVHD was estimated and the MiHA-specific T-cell clones were tested for recognition of non-hemato-poietic fibroblasts. To mimic the proinflammatory cytokinemilieu, early after transplant or during potent GVHD responses,fibroblasts were pretreated with IFNg . Although both the CLYBLK264 and TEP1 K091 T-cell clone poorly recognized nontreatedfibroblasts, they clearly recognized those that were IFNg pre-treated (Fig. 5D and F). The in vivo generated CLYBL K2-339demonstrated a similar recognition pattern of non-hematopoieticcells (data not shown). As patient MBF demonstrated both aclinical response and GVHD after DLI, we speculate that theCLYBL-1Y specific T cells may have recognized both malignantand normal non-hematopoietic cells. These data indicate thatboth CLYBL-1Y and TEP1-1S may be considered as MiHA withpotential therapeutic value under noninflammatory conditions,but they may participate in toxic GVHD responses in a proin-flammatory environment. A feature that renders these MiHA notpreferable for immunotherapy but set them as potential candi-dates for new GVHD biomarkers.

DiscussionIn this study, we demonstrate the immunogenicity of 2 pre-

defined MiHA candidates that were predicted using a reverseimmunology approach. The biologic relevance of at least one ofthem, theCLYBL-1YMiHA, was demonstrated by the detection ofa substantial MHC multimer–positive T-cell population specificfor thisMiHA in a patient suffering fromAMLwho experienced anantileukemic response after treatment with allo-SCT and subse-quent DLI.. These in vivo primed T cells demonstrated to be high-avidity T cells specific forCLYBL-1Y. For theTEP1-1SMiHA, only 2patients could be screened for the presence of TEP1-1S–specific

A

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Figure 2.Isolated T-cell clones vary in production of IFNg and GM-CSF upon aCD3/28stimulation. A, distribution of the intrinsic IFNg secretion capacity of 806MiHA-specific MHCmultimer–positive T-cell clones stimulated with aCD3/28beads. B, production of GM-CSF for 150 selected T-cell clones withvariable IFNg secretion capacity. MiHA-specific MHCmultimer–positive T-cellclones were stimulated for 18 hours in presence of aCD3/28 beads,and GM-CSF and IFNg secretion was measured by standard ELISA.

Hombrink et al.





MHC multimer–positive cells. Therefore, absence of an in vivoinduced immune response against TEP1-1S could either be due tothe low number of patients that could be screened or due tosubdominance of TEP1-1S in immune responses. Our reverseimmunology approach has the advantage to allow identification

of subdominant MiHA, as the T-cell repertoire of patients that arescreened in forward immunology approaches is skewed towardhighly immunodominant MiHA-specific responses. Subdomi-nant MiHA, however, may be of therapeutic interest as they canbe exploited in potential peptide vaccination or adoptive T-cell

0

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HMMR-1VC18orf21-1A

+–––+––++––+–+–++++–

T-cell clone

K441 (APOBEC3H ABM)

APOBEC3H-1K +++++–+++–

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K226 (ERAP1 EPP)K159 (ERAP1 ABM)

K156 (GEMIN4 ABM)K358 (GEMIN4 FHT)K355 (GEMIN4 UHR)K360 (GEMIN4 JDB)

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Figure 3.MHC multimer–positive T-cell clonesdemonstrate MiHA-specificreactivity. Isolated MHC multimer–positive T-cell clones werestimulated with HLA-A�0201 andB�0702–positive T2 cells loadedwithtitrated concentrations of peptidesfor 18 hours [E:T] 5,000:25,000. Foreach donor, the MiHA-specific T-cellclone that demonstrated the highestpeptide avidity is shown for therespective cytokine. MiHA-specificIFNg production was observed for T-cell clones specific for (A) CLYBL-1Y,(B) ERAP1-1R, (C) GEMIN4-1V, (D)NISCH-1A, HMMR-1V, C18orf21-1A,APOBEC3H-1K, and (E) TEP1-1S MiHAcandidates. For the TEP1-1S–specificT-cell clone K091 derived from donorEPP, both the peptide-induced IFNgand GM-CSF secretion are shown.F–J, to measure the reactivity of theMiHA-specific T-cell clones againstendogenously processed andpresented antigens, all MiHA-specificT-cell clones that demonstrated IFNgor GM-CSF production in response tospecific peptide were stimulatedwith a panel of 10 HLA-A�0201 andB�0702–positive EBV-LCL targetcells positive (þ) or negative (�) forthe indicated MiHA for 18 hours.Cytokine secretion was measured bystandard ELISA.






therapies when they demonstrate promising gene expressionpatterns.

A major limitation for the identification of large numbers ofMiHA using a reverse immunology approach may be the lowfrequency of high-avidity MiHA-specific T cells within an indi-vidual's T-cell repertoire. By performing 2 rounds of MHCmultimer enrichments each followed by a 10-day expansionstep, we increased the number of MHC multimer–positive Tcells that was isolated, as we observed that after the secondenrichment round previously undetected MHC multimer–pos-itive T-cell populations were found. In addition, by measuringGM-CSF in addition to IFNg as readout for T-cell reactivity, wewere able to increase the number of MHC multimer–positive T-cell clones that could be screened for functional activity. How-ever, the failure to isolate high-avidity T cells for the previouslyidentified LB-NISCH-1A MiHA indicates that isolation of high-avidity T cells may still be a matter of chance (13). Alternatively,T cells may be tolerant for some MiHA due to molecularmimicry with non-polymorphic epitopes or due to their failureto discriminate between both allelic variants of an SNP encod-ing MiHA (26), explaining why not all MiHA candidates will beof clinical relevance.

For 8 of the 20 MiHA candidates, MHCmultimer–positive T-cell clones were isolated that demonstrated MiHA-specificpeptide reactivity. The different MHC multimer–positive T-cellclones however demonstrated functional heterogeneity. Wehave recently demonstrated that CMV-specific MHC multi-mer–positive T cells isolated from CMV-negative individualsby MHC multimer enrichments also demonstrated a largevariation in functional avidity (27). This heterogeneity in

functional activity of the MHC multimer–positive T-cell popu-lations may be specific for T cells isolated from the na€�verepertoire as memory T cells are skewed toward a high-avidityrange as a result of antigen encounter in vivo. Nonresponsive-ness of MHC multimer–positive T-cell clones has also beenreported by others for T cells derived from the na€�ve repertoire(28, 29). The discrepancy between MHC multimer reactivityand T-cell functionality can most likely be explained by thestaining with multimerized MHC–peptide complexes. BecauseMHC monomers do not stably bind to TCRs, multimerizationis necessary to identify antigen-specific T cells. However, multi-merization of MHC–peptide complexes alters the TCR–MHC–peptide dissociation on- and off-rate kinetics and will result inincreased binding avidity of the multimerized MHC–peptidecomplex to surface TCR. By measuring the strength of TCRbinding to monomeric peptide–MHC complexes using theStreptamer Koff rate assay, we recently demonstrated that thedissociation kinetics correlated with the observed functionalavidity of different MHC multimer–positive T-cell clones andthat T cells demonstrating lower dissociation rates confer sig-nificantly better antigen-specific reactivity than those with fastdissociation rates (27, 30). The integration of such or alterna-tive methodologies which allow the controlled disassembly ofMHC multimers directly after T-cell isolation and subsequentFACS of T cells demonstrating a low TCR–MHC–peptide dis-sociation rate may further increase the efficiency of futureapproaches to identify high-avidity T-cell clones with a pre-defined specificity. In conclusion, our data illustrate that withthe reverse immunology approach presented in this study,biologically relevant MiHA can be identified as well as MiHA

A

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0,3%

MHC-multimer

USF

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Patient PBMC analyzed:MHC‐multimer–positive T cellsNo MHC‐multimer–positive T cells

Figure 4.MHC multimer–positive T-cellpopulations detected in patients afterallo-SCT. A, number of MHCmultimer–positive T-cell populationsdetected by combinatorial codingMHC multimer analysis in peripheralblood samples of 16 patients withvarious hematologic malignanciesthat received an allo-SCT anddemonstrated a clinical response toDLI. Patients were only screenedwhen they were positive for the SNPencoding MiHA and received a DLIfrom a donor that was homozygousnegative for the SNP encoding MiHA.Bars indicate the number of patientsapplicable for MiHA-specific MHCmultimer screening. The number ofMHC multimer–positive T-cellpopulations detected per MiHAcandidate is indicated in black. B,representative FACS analysis of MHCmultimer–positive T-cell populationsdetected in the indicated patients. Alldot plots display fluorescenceintensity for CD8 and specific MHCmultimer staining. Total lymphocytesare shown. Frequencies indicateMiHA-specific T cells of total CD8þ Tcells.

Hombrink et al.





that are not frequently induced in vivo but can potentially beused for immunotherapeutic strategies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: P. Hombrink, C. Hassan, M.G.D. Kester, J.H. FrederikFalkenburg, P.A. van Veelen, M.H.M. HeemskerkDevelopment of methodology: P. Hombrink, M.G.D. Kester, P.A. van Veelen,M.H.M. HeemskerkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Hombrink, C. Hassan, M.G.D. Kester, L. Jahn,M.J. Pont, A.H. de Ru, M. GriffioenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Hombrink, C. Hassan, M.G.D. Kester, P.A. vanVeelen, M.H.M. Heemskerk

Writing, review, and/or revision of the manuscript: P. Hombrink, C. Hassan,M.G.D. Kester, J.H. Frederik Falkenburg, P.A. van Veelen, M.H.M. HeemskerkAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Hombrink, M.G.D. Kester, C.A.M. van BergenStudy supervision: P. Hombrink, J.H. Frederik Falkenburg, P.A. van Veelen, M.H.M. Heemskerk

Grant SupportThis project was supported by the Dutch Cancer Society grant no. 07-3825

and the Landsteiner Foundation for Blood Transfusion Research grant no.LSBR0713.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 3, 2014; revisedDecember 22, 2014; acceptedDecember28, 2014; published OnlineFirst January 14, 2015.

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No

mar

ker

MHC-multimer

A

0

1,000

2,000

3,000

4,000

5,000

6,000

7,000

250

nmol

/L

50 n

mol

/L

10 n

mol

/L

2 nm

ol/L

400

pmol

/L

80 p

mol

/L

16 p

mol

/L

3,2

pmol

/L

0,64

pm

ol/L

0,12

pm

ol/L

0,02

pm

ol/L

No

Pep

tide

K264

K2-339

Allelic variant: CLYBL-1D

K264

K2-339

Eluted peptide: CLYBL-1Y

B

CLYBL-1Y K2-339

6,419

CLYBL-1Y K264

6,123

C

0

500

1,000

1,500

FB

446

1F

B +

INF

γE

BV

446

1

FB

315

6F

B +

INF

γE

BV

315

6

FB

585

2F

B +

INF

γE

BV

585

2

FB

705

5F

B +

INF

γE

BV

705

5

FB

287

7F

B +

INF

γE

BV

287

7

FB

335

6F

B +

INF

γE

BV

335

6

FB

517

7F

B +

INF

γE

BV

517

7

FB

476

9F

B +

INF

γE

BV

476

9

0

200

400

600

800

EB

V 4

461

CM

L 44

61

EB

V 7

055

AM

L 70

55

EB

V 3

518

AM

L 35

18

EB

V 0

733

AM

L 07

33

EB

V 3

379

ALL

337

9

EB

V 3

870

AM

L 38

70

0

500

1,000

1,500

FB

315

6F

B +

INF

γE

BV

315

6

FB

585

2F

B +

INF

γE

BV

585

2

FB

517

7F

B +

INF

γE

BV

517

7

FB

476

9F

B +

INF

γE

BV

476

9

FB

705

5F

B +

INF

γE

BV

705

5

FB

287

7F

B +

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BV

287

7

FB

335

6F

B +

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BV

335

6

0

500

1,000

1,500

EB

V 3

518

AM

L 3

518

EB

V 6

204

CM

L 62

04

EB

V 7

055

AM

L 7

055

EB

V 0

733

AM

L 0

733

EB

V 3

379

ALL

337

9

EB

V 3

870

AM

L 3

870

CLYBL-1Y K264

IFN

γ (p

g/m

L)

IFN

γ (p

g/m

L)

IFN

γ (p

g/m

L)

IFN

γ (p

g/m

L)

D CLYBL-1Y K264

E TEP1-1S K091 F TEP1-1S K091

IFN

γ (p

g/m

L)

––––++MiHA:

–+++++MiHA:

––––++MiHA: +

––++MiHA: ++++

Figure 5.Functional avidity of CLYBL-1Y–specific T cells and reactivity toward hematopoietic malignancies. A, FACS analysis of MHC multimer–positive CLYBL-1Y–specific T-cellclones. To prevent doublet formation, CLYBL-1Y–specific T-cell clones were mixed with CD4 T-cell blasts. Dot plots display fluorescence intensity for MHC multimerstaining. Total lymphocytes are shown. B, CLYBL-1Y–specific T-cell clones derived from donor ABM (K264; white) or from patient MBF (K2-339; black) werestimulated with HLA-A�0201–positive T2 target cells loaded with titrated concentrations of CLYBL-1Y peptide (squares) or allelic variant (triangles) for 18 hours [E:T]5,000:25,000. IFNg secretion was measured by standard ELISA. C–F, indicated MHC multimer–positive T-cell clones were screened for recognition of variousprimary hematopoietic malignancies and non-hematopoietic fibroblasts isolated from different individuals. As a control for T-cell recognition, all T-cell clones were alsostimulated with EBV-LCL generated from the same individuals. (C) CLYBL-1Y clone K264 and (E) TEP1-1S clone K091 were stimulated with HLA-A�0201 or B�0702–positive primaryAML, CML, andALL cells either positive (þ) or negative (�) for theMiHA forwhich the tested T-cell clone demonstrated reactivity, directly after isolationof malignant cells for 18 hours [E:T] 1,000:5,000. To measure the recognition of non-hematopoietic cells, (D) CLYBL-1Y clone K264 and (F) TEP1-1S clone K091were stimulated with fibroblasts either pretreated with IFNg (100 IU/mL) or not. MiHA-specific recognition of target cells was measured by standard IFNg ELISA.






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Hombrink et al.




2015;21:2177-2186. Published OnlineFirst January 14, 2015.Clin Cancer Res Pleun Hombrink, Chopie Hassan, Michel G.D. Kester, et al. Reverse Immunology Approach

Derived HLA-Ligandome Using a−Antigens within the B-lymphocyte Identification of Biological Relevant Minor Histocompatibility

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