Graft versus self (GvS) against T-cell autoantigens is amechanism of graft–host interactionNora Mirzaa,b, Manfred Zierhutc, Andreas Kornd, Antje Bornemanne, Wichard Vogela, Barbara Schmid-Horchf,Wolfgang A. Bethgea, Stefan Stevanovi�cb, Helmut R. Saliha,g, Lothar Kanza, Hans-Georg Rammenseeb,and Sebastian P. Haena,b,1

aAbteilung II fuer Onkologie, Haematologie, Immunologie, Rheumatologie, und Pulmologie, Medizinische Universitaetsklinik, D-72076 Tuebingen,Germany; bAbteilung Immunologie, Interfakultaeres Institut fuer Zellbiologie, D-72076 Tuebingen, Germany; cUniversitaetsaugenklinik, D-72076Tuebingen, Germany; dKlinik fuer diagnostische und interventionelle Neuroradiologie, D-72076 Tuebingen, Germany; eInstitut fuer Neuropathologie,D-72076 Tuebingen, Germany; fZentrum fuer klinische Transfusionsmedizin, D-72076 Tuebingen, Germany; and gDepartment for Internal Medicine II, ClinicalCollaboration Unit Translational Immunology, German Cancer Consortium and German Cancer Research Center, Partner site Tuebingen, D-72076Tuebingen, Germany

Edited by Harvey Cantor, Dana-Farber Cancer Institute, Boston, MA, and approved October 14, 2016 (received for review June 6, 2016)

Graft-versus-host disease (GVHD) represents the major nonrelapsecomplication of allogeneic hematopoietic cell transplantation.Although rare, the CNS and the eye can be affected. In this study,manifestation in the retina as part of the CNS and T-cell epitopesrecognized by the allogeneic T cells were evaluated. In 2 of 6 patientswith posttransplantation retina diseases and 6 of 22 patients withoutocular symptoms, antigen-specific T-cell responses against retina-specific epitopes were observed. No genetic differences betweendonor and recipient could be identified indicating T-cell activationagainst self-antigens (graft versus self). Transplantation of a pre-existing immunity and cross-reactivity with ubiquitous epitopeswas excluded in family donors and healthy individuals. In sum-mary, an immunological reaction against retina cells represents amechanism of graft-versus-host interaction following hematopoi-etic cell transplantation.

allogeneic hematopoietic cell transplantation | graft-versus-host disease |autoimmunity | T-cell epitope | retina

Hematopoietic cell transplantation (HCT) can be the onlycurative treatment option for patients with hematologic

malignancies. Besides direct treatment-associated complications,graft-versus-host disease (GVHD) is the major complication (1).Today, graft–host interaction is thought to be mediated by two

pathomechanisms. (i) Allogeneic T cells recognize differences be-tween donor and recipient in HLAs and the respective expressedself-peptides (2, 3). (ii) Genetic polymorphisms, especially non-synonymous SNPs, lead to expression of proteins with alternateamino acid sequences whose fragments can be presented onMHC molecules functioning as minor histocompatibility anti-gens (miHAGs) (4, 5).Although not one of the main manifestation organs, GVHD

may also affect the CNS, even if thought to occur rarely andevidence being limited to single cases (6–10). Because GVHD ofthe CNS is difficult to distinguish from other complications suchas relapse, infections, and toxicity related to therapy, a thoroughcharacterization of CNS GVHD is challenging (7, 11).The retina constitutes the only part of the CNS that can be

examined directly. Although ocular involvement of the anteriorsegment such as keratoconjunctivitis sicca, corneal epitheliopathy,and pseudomembranous conjunctivitis is observed frequently (12,13), posterior segment (PS) involvement is rare and mostly attrib-uted to the toxicity of irradiation or immunosuppression includingdiseases like central serous chorioretinopathy (14) and ischemicretinopathy (15) and can manifest with cotton wool spots, retinalbleeding, and edema (16). Only a very limited body of evidence hasdescribed retina manifestations linked to GVHD (17, 18).Several proteins have been described to be involved in retinal

inflammation and degeneration comprising the membrane-bound retinal guanylate cyclase (retGC; gene GUCY2D), anenzyme involved in phototransduction and predominantly expressedin the cones (19). Mutations cause autosomal dominant inherited

cone-rod degeneration (20, 21) and Leber’s congenital amaurosis(22). The guanylate cyclase activating proteins 1 and 2 (GCAP1,gene GUCA1A; GCAP2, gene GUCA1B) are involved in thenegative regulation of the retGC and are important for photore-ceptor recovery (23, 24). Mutations in the GUCA1A gene havebeen associated with inherited cone, cone-rod and macula dystrophy(24, 25). Fourth, the retinoid binding protein (RBP3) is importantfor the transport of retinoids between the retinal pigment epi-thelium and photoreceptors. Mutations of the RBP3 protein areassociated with retinitis pigmentosa (26).In this study, we characterize the development of donor T-cell

responses against MHC-restricted retina protein-derived peptides.

ResultsPatients. The first group (Table S1, left column) comprisedpatients with diseases of the PS after HCT. PS diagnoses wereoptic atrophy of unknown origin (n = 2), in one case combinedwith a selective cone dysfunction, optic neuritis (n = 2), anemicretinopathy (n = 1), and CMV retinitis (n = 1). The medianonset of PS diagnoses occurred at 9 mo after HCT (range 3–25 mo). The second group (Table S1, right column) comprised22 consecutive patients recruited before allogeneic HCT. Forboth groups, characteristics of individual patients are providedin Table S2.

Significance

As the mechanism of graft-versus-host disease (GVHD) afterallogeneic hematopoietic cell transplantation (HCT), recogni-tion of the recipient’s body by donor immune cells was pre-viously believed to be based on genetic and immunologicaldifferences between donor and recipient. However, evidencein murine models and in autologous HCT has shown that alsoautoimmunity contributes to GVHD. In this study, we show thedevelopment of auto-reactivity after human allogeneic HCTand characterize the specific self-epitopes of T cells that maycontribute to mediation of GVHD. Such autoantigens havenever been characterized before. These observations contrib-ute to a better understanding of the immune responses thatare activated following hematopoietic cell transplantation.

Author contributions: L.K., H.-G.R., and S.P.H. designed research; N.M., M.Z., A.B., and S.P.H.performed research; M.Z., A.K., A.B., B.S.-H., and S.S. contributed new reagents/analytictools; N.M., M.Z., A.K., A.B., W.V., W.A.B., S.S., H.R.S., L.K., H.-G.R., and S.P.H. analyzed data;N.M., M.Z., W.A.B., S.S., H.R.S., L.K., H.-G.R., and S.P.H. wrote the paper; and S.P.H.obtained funding.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: sebastian.haen@med.uni-tuebingen.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1609118113/-/DCSupplemental.

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Approach of the Study and Peptide Identification. The retina-spe-cific target proteins (retGC, GCAP1, GCAP2, and RBP3) wereidentified using the swissprot (www.uniprot.org), ensembl (useast.ensembl.org/index.html), and geoprofiles (www.ncbi.nlm.nih.gov/geoprofiles) databases. Candidate T-cell epitopes were identifiedusing two approaches. (i) Peptide pairs for the patient HLA typewere predicted based on published SNP using the internet baseddatabases SYFPEITHI (www.syfpeithi.com) and EpiToolKit (www.epitoolkit.de) (MHC-I; Table S3) or designed as 17-mers with theSNP positioned in the middle of the peptide (MHC-II; TableS3). Gene sequences of patient and donor were confirmed inSanger sequencing. (ii) Recipient and donor DNA was sequencedby Sanger sequencing. Here, peptides were predicted on the basisof identified SNPs (Fig. S1). Variant amino acids are printed inbold throughout the article and in red in the figures.

Retina-Specific T-Cell Responses in Patients with PS Manifestations.Retina-antigen specific T cells were detected in two of six patients(Fig. 1) with PS diseases. Patient 1 [diagnosis of acute lympho-blastic leukemia (ALL)] experienced progressive visual loss 14 moafter HCT (at 14 mo: 20/30 in both eyes; at 25 mo: finger countingin both eyes). Optic atrophy and selective dysfunction of the coneswith normal rod function was diagnosed using fundus photogra-phy (Fig. 1A) and electroretinography (ERG). Stereotactic biopsyof a ventricular lesion revealed a CD8+ vasculitis (Fig. 1B). In-fections and disease relapse were excluded in evaluation of cere-brospinal fluid (no detection of pathogens or malignant cells) andbiopsy specimens. In a sample of peripheral blood mononuclearcells (PBMCs) harvested 40 mo after HCT (Fig. 1C), weak T-cellresponses could be detected by IFN-γ enzyme linked immunospot(ELISpot) after stimulation with two overlapping HLA B*0702-restricted retGC-derived peptides (GUCY2D 46B: QPPALSSVFT,GUCY2D 47B: PPALSSVFT, mean: 19.5 spots/250,000 PBMCs,39-fold of negative control). The T-cell response was not reflectedby a difference in the DNA sequence between donor and recipientbecause the donor was a carrier of the heterozygous SNP leading toamino acid exchange from alanine to serine at position 52 of theGUCY2D (A52S), whereas the recipient carried the major allelecoding for alanine (Fig. 1D).In a second patient [patient 5, diagnosis acute myeloid leu-

kemia (AML)] with a CMV retinitis diagnosed by vitreal biopsy(fundus photograph in Fig. 1E), a strong T-cell response wasdetected after stimulation with MHC II-restricted retGC-derivedpeptides (GUCY2D 52A: LLQPPALSAVFTVGVLG, GUCY2D52B: LLQPPALSSVFTVGVLG, 546.5 spots/500,000 PBMCs,121-fold compared with negative control) at 24 mo after HCT(Fig. 1F). DNA sequencing did not reveal a SNP, indicatingrecognition of self-antigens (Fig. 1G).

T-Cell Responses in Patients Irrespective of Ocular Symptoms. In twopatients (Fig. 2: patient 7; Fig. S2: patient 9), strong T-cell re-sponses directed against a predicted HLA A*0301-restrictedGCAP1-derived self-peptide (GUCA1A 47A: NLSPSASQY)were detected by IFN-γ ELISpot (Fig. 2A: patient 7; up to 659spots/500,000 PBMCs, 220-fold of negative control; Fig. S2A:patient 9; up to 300 spots/500,000 PBMCs, 35-fold of negativecontrol) and corresponding intracellular cytokine staining (ICS).No IFN-γ secretion was observed when PBMCs were stimu-lated with the corresponding variant peptide (GUCA1A 47B:NLSLSASQY). In particular, the ICS revealed a CD4+ T cell-mediated TNF and IFN-γ response (Fig. 2B and Fig. S2B). Ofnote, no T-cell responses were observed in PBMC samples

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Fig. 1. T-cell responses after HCT in patients with PS diseases. (A–D) Patient1: fundus photography (A) showing optic nerve atrophy with very narrowvessels. (B) MRI scan (Left) of the brain revealed a periventricular lesionwhere a stereotactic biopsy was taken. Histologic workup (Right) includingimmunohistochemistry showed a CD8+ atypical vasculitis without any evi-dence of meningeal disease relapse. (C) T-cell response after stimulation of250,000 prestimulated (12 d) PBMCs per well with retGC-derived peptides(1 μg/mL) detected by IFN-γ ELISpot and sequencing (D) of donor and

recipient DNA (GUCY2D exon 2). (E–G) Patient 5: fundus photography (E)revealing a massive, centrally located CMV retinitis with detection of vitre-ous cells (cloudiness of the picture). T-cell responses were evaluated (F) afterstimulation of 500,000 prestimulated (12 d) PBMCs per well with retGC-de-rived MHC class II peptides (2.5 μg/mL) detected by IFN-γ ELISpot and se-quencing (G) of donor and recipient DNA (GUCY2D exon 2). CD, cluster ofdifferentiation; OD, oculus dexter; OS, oculus sinister.

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harvested before HCT (Fig. 2A and Fig. S2A). T-cell responsesremained detectable up to 14 mo after HCT. DNA sequencingrevealed no difference between donor and recipient (Fig. 2C andFig. S2C). Hence, the observed T-cell response was specificfor self-peptides.In PBMC samples of patient 7, an additional CD4+ T-cell re-

sponse was observed after stimulation with a HLA A*0101-predictedGCAP2-derived self-peptide (GUCA1B 133A: QTEQGQLLT).Patient 11 displayed a T-cell response after stimulation with

a peptide pool consisting of the GCAP2-derived peptides GUCA1B133A (QTEQGQLLT) and GUCA1B 133B (QTEQDQLLT) 7 moafter HCT in ELISpot (73 spots/500,000 PBMCs, sevenfold ofnegative control; Fig. S2 D and E). Donor and recipient sharedthe same GUCA1B gene sequence, indicating recognition of aself-peptide (Fig. S2F). The recognized peptide sequence could,however, not be further discriminated due to limited availabilityof patient material.In PBMC samples of patient 12, strong T-cell responses

against HLA A*0201-restricted retGC- (13-fold of negativecontrol, 15-fold of donor sample; Fig. S3 A–C), GCAP1- andGCAP2- (21-fold of negative control, 23-fold of donor sample)derived peptides were detected 5 mo after HCT, whereas noIFN-γ secretion was observed before HCT. One month later, theresponse intensity decreased, and a remaining positive T-cellresponse was observed after stimulation with the GCAP2-derivedpeptides (GUCA1B 131A: ELQTEQGQLL, GUCA1B 131B:ELQTEQDQLL; Fig. S3 A–C). Due to limited cell numbers,other time points after HCT could not be separately monitoredby ICS. In this patient, sequencing of donor and recipient DNArevealed a heterozygous missense SNP for the recipient in exon2 of the GUCY2D gene (A52S), whereas the donor was a ho-mozygous carrier of the major allele (Fig. S3D). This sequencedifference in the GUCY2D gene could result in recognition ofthe alloantigen. However, both peptides were recognized reflectingcross-reactivity against either the allo- or the autoantigen. Con-sidering the T-cell responses against the GCAP-2–derived pep-tides in this patient and the other observations in this study, thereactivity is not likely to be based on gene sequence differences.Moreover, analysis of the donor sample revealed no detectableT-cell response after stimulation with the HLA A*0201-restrictedpeptides (Fig. S3 A and B).In patient 20, weak T-cell reactivity was observed by ELISpot

against a pool consisting of four retGC-derived MHC classII-restricted peptides (GUCY2D 52A: LLQPPALSAVFTVGVLG;GUCY2D 52B: LLQPPALSSVFTVGVLG; GUCY2D 782A:DQAPVECILLMKQCWAE; GUCY2D 782B: DQAPVE-CIHLMKQCWAE) 5 mo after HCT (mean: 29 spots/250,000PBMCs, 4.38-fold of negative control). In this patient, the rec-ognized epitopes could not be further assessed due to limitedavailability of T cells. The response was no longer detectable14 and 16 mo after HCT. No response was detected before HCT(Fig. S4 A and B). DNA sequencing revealed no genetic differencebetween donor and recipient (Fig. S4C).In patient 23, a homozygous SNP in exon 2 of the GUCY2D

gene (A52S) was identified for recipient and donor (Fig. S5E).No T-cell response was detected in a sample of this patientharvested before HCT. The observed response was first (4 and

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Fig. 2. T-cell responses after HCT in patient 7. CD4+ T-cell responses afterstimulation of 500,000 prestimulated (12 d) PBMCs per well with the GCAP-1– and the GCAP-2–derived MHC class I predicted peptide detected by (A)IFN-γ ELISpot (peptides at 1 μg/mL) and (B) ICS (peptides at 10 μg/mL). (A) Tcells were analyzed over a period of 17 mo (before HCT until 17 mo afterHCT). IFN-γ ELISpot (Upper) revealed changing of the T-cell reactivity againstone peptide but not against the corresponding variant peptide, which werenot detectable before HCT. Before HCT, only very limited PBMC counts were

available due to the myelodysplastic syndrome. The bar graph presentschanges in the T-cell reactivity (absolute spot counts in ELISpot) normalizedon the negative control (incubation with irrelevant peptide) over time.(B) ICS revealed T-cell activation indicated by TNF (far left and third panelfrom left) and IFN-γ (second panel from left and far right panel) productionon incubation with the A variant peptides (panels first row), but not with thevariant peptide with difference in one amino acid (panels second row). Theresponse was mediated by CD4+ T cells. As controls, incubation with HIV-derived epitopes are presented in the lower two rows for CD4+ (third row)and CD8+ (fourth row) responses. (C) Sequencing of the GUCA1A (Upper)and GUCA1B (Lower) genes for donor (Left) and recipient (Right) showingthe same genetic sequence and, hence, revealing no SNP.

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7 mo after HCT) directed against a non–self-peptide (GUCY2D52A: LLQPPALSAVFTVGVLG); later a strong responseagainst both self- and non–self-peptide (665.5 and 657.5 spots/500,000 PBMCs, 22-fold of negative control, 46-fold of donorsample; Fig. S5 A, C, and D) was observed. Because donor andrecipient were both homozygous carriers of the minor allele, theT-cell responses against the non–self-peptide reflects cross-reactivity against an irrelevant peptide expressed in neither thepatient nor the donor, but likely reflects reactivity against theself-epitope. The observed response was no longer detectable12 mo after HCT. Of note, this decrease was not correlated toany clinical parameters, e.g., escalation of immunosuppression.A limited IFN-γ production by CD4+ T cells (0.0381%) wasdetected after stimulation with the non–self-peptide 12 mo afterHCT (Fig. S5B). No preexisting T-cell responses were observedin ELISpot after stimulation with the GUCY2D 52A and Bpeptides in donor PBMCs (Fig. S5 A and C).In total, T-cell responses against retina-specific self- and non–

self-peptides were observed in 6 of 22 patients (27%). Two patients(7, 9) showed T-cell responses directed against self-peptides,whereas in samples of two other patients (12, 23), T cells rec-ognized both self- and non–self-peptides. In two patients (11,20), T-cell responses could not be further discriminated. Of note,none of the observed T-cell responses was associated with adefined genetic polymorphism.

Clinical Factors Influencing the Development of Autoreactive T Cells.Next, we analyzed patient records and laboratory findings tostudy whether patient- and/or transplantation-related parame-ters could influence the development of the observed antigen-specific T cells (Tables S1 and S2). Patients with PS diseaseswere more likely to have received total body irradiation forconditioning (83% vs. 27%; positive predictive value 1; 95%, CI0.19–1). Due to the small sample size, this difference was notstatistical significant (P = 0.25). If any, there was a slight dif-ference in the occurrence of extensive chronic GVHD (66% vs.27%, P = 0.25; positive predictive value, 0.5; 95% CI, 0.03–0.97). Also, appearance of acute GVHD, GVHD prophylaxis,and graft composition did not contribute to the developmentof PS diseases. No timely association with acute or chronicGVHD or between PS diseases and other GVHD manifes-tations was observed. Overall, no clear parameters for both thedevelopment of PS symptoms and autoreactive T cells couldbe identified.

Antigen-Specific T Cells in Healthy Individuals. To exclude that ourfindings were due to an unspecific response reflecting, e.g., cross-reactivity with viruses, samples from healthy donors (HDs) wereevaluated. Donors were HLA matched to the respective MHC-Irestriction of the peptides. No T-cell responses were found afterstimulation with the MHC-I–restricted peptides in PBMC samplesof all tested HDs (10 per peptide; Table S3).To assess the binding of the nonamer peptides GUCA1A

47A (NLSPSASQY) and GUCA1B 133A (QTEQGQLLT) toMHC class II molecules, the HLA typing of the three res-ponding patients (7, 9, 11) was compared. Responders toNLSPSASQY were found to be DRB1*1501 and DQB1*0602positive, whereas QTEQGQLLT responders were carrierof DRB1*0702 and DRB1*0202 alleles. Six DRB1*15 andDQB1*06, as well as eight DRB1*07 and DQB1*02 positive HDs,were analyzed for cytokine release. No T-cell responses wereobserved herein. As control for every retGC-derived MHC-IIpeptide (GUCY2D 52A, 52B and GUCY2D 782A, 782B), 10HDs were tested for T-cell responses in ELISpot. In one case,a response on stimulation with the GUCY2D 52B peptide wasdetected, which was validated by ICS. Here, T cells recog-nized both peptides (52A: LLQPPALSAVFTVGVLG; 52B:LLQPPALSSVFTVGVLG).Moreover, all peptide sequences were compared with proteins

derived from bacteria, viruses, fungi, and other pathogens (blast.ncbi.nlm.nih.gov/Blast.cgi). No significant homology was identified.

In Vitro Priming of Antigen-Specific T Cells Against Retina Epitopes.To investigate the immunogenicity of the predicted peptides,in vitro priming of HD CD8+ T cells was performed for allMHC-I peptides using at least three different HDs for eachpeptide pair. Thereby, 36 of 55 peptides (Fig. 3 and Table S3,list) were confirmed as T-cell epitopes (HLA A*0101, A*0201,A*0301, A*1101, A*2601, B*0702 restricted) in patients andpriming (n = 6) or in priming experiments only (n = 30). Twopeptides (GUCA1A 47A and GUCA1B 133A) could be confirmedas T-cell epitopes in patients only (n = 2). Seventeen peptidescould not be verified as epitopes (Fig. 3C). The intensity of T-cellreactivity varied with detection of very strong (n = 8; 22%),strong (n = 8; 22%), intermediate (n = 9; 25%), weak (n = 9;25%), or very weak (n = 2; 6%) IFN-γ responses (Fig. 3D).

Epidemiology of the Identified SNP. Sequencing of the GUCY2Dgene resulted in the identification of four different nonsynonymousSNPs (Table S4) resulting in a change of the amino acid sequence.Three (A52S rs61749665 in exon 2, V361M rs186508466 in exon4, and L782H rs8069344 in exon 12) were already published(www.ncbi.nlm.nih.gov/snp), whereas one (S25F in exon 2) waspreviously unknown. For frequency analysis, we sequenced 100persons and found one subject with a heterozygous SNP (S25F).The most frequent missense SNP leading to an amino acidchange from alanine to serine at position 52 is encoded by exon 2of the GUCY2D gene with a reported minor allele frequency(MAF) of 37.7% (rs61749665). Our frequency analysis of 100persons revealed an allele frequency of 30% (11% homozygous,38% heterozygous, and 51% carried the WT alleles). The SNPA361M was unknown when this study was initiated and laterreported to have a MAF of 0.1% (rs186508466). Here, in a co-hort of 200 people, no individual carrying the SNP was identified.The SNP L782H has a reported MAF of 15.5% (rs8069344).

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Fig. 3. In vitro priming of peptide-specific CD8+ T cells. DCs based in vitropriming experiments were performed for all MHC class I peptides andmeasured in ICS. Two exemplified peptide pairs are shown. (A) GUCA1A 144:Weak T-cell reactivity could be induced against the B-variant (DVNGDGEFSL)but not against the A-variant peptide (DVNGDGELSL) as indicated by TNFand IFN-γ production. (B) GUCY2D 18: Very strong T-cell reactivity could beinduced against both peptide variants as indicated by TNF and IFN-γ pro-duction detected by flow cytometry. (C) Summary of identification of retina-specific T-cell epitopes: antigen-specific T cells could be detected in patientsonly (n = 2), patients and in vitro priming (n = 6), and in vitro priming only(n = 30). Seventeen peptides could not be confirmed as epitopes. (D) Clas-sification of the intensity of CD8 T-cell responses into very weak >0.05–0,1%,weak >0.1–0.5%, intermediate >0.5–1%, strong >1–3%, and very strongT-cell responses >3% (percentage is referred to all CD4-negative gated cells).

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No SNPs were found in patient and donor DNA by sequencingthe GUCA1A gene, whereas we observed a heterozygous pub-lished SNP in one patient in exon 3 of the GUCA1B gene,leading to an amino acid exchange from glutamic acid to asparticacid (E155D). This SNP is rare, with a reported MAF of 0.5%(rs139923590).

DiscussionThe cellular mechanisms of graft–host interaction comprise,among others, T-cell recognition of major (MHC) and minor(miHAG) histocompatibility antigens (Fig. S6) (5). However, inmurine models using the transfer of in vivo-generated CD4+ Tcells, the development of autoreactivity has been suggested (27).Of note, these reports did not evaluate the target antigens ofautoreactive T cells. Our data provide evidence in human allo-geneic HCT that genetic differences are not required for allo-geneic T-cell reactivity to develop (Fig. S6, Right) and describethe precise epitopes (autoantigens) of these T cells. In fact, weobserved self-antigen–specific T cells directed against epitopesderived from three highly polymorphic retina proteins in 2 of 6analyzed patients with inflammatory PS complications and in 6of 22 patients without ocular symptoms. Because two of the re-spective proteins (retGC and GCAP1) are predominantly in-volved in cone metabolism (28, 29), slight damage would result inchanges in color vision, which could manifest subclinically (30).The detectable T cells could be also of low avidity and, hence,not capable of inducing retina damage. In line, it has been ob-served that also circulating Melan-A/MART-1–specific naïve-phenotype T cells were not associated with vitiligo (31). Incontrast to these observations, we could not detect any reactivityin healthy volunteers for most peptides. In our study, the avidityof the detected T cells could not be determined because the usedpeptide concentrations were chosen to activate all potential antigen-specific cells (32, 33). Further avidity characterization using peptidetitrations was not possible due to limited sample availability butshould be performed in future studies.Thus, our data provide evidence that reactivity of allogeneic T

cells cannot only be induced by allo-antigens but also by antigenssharing sequences with donor antigens, which implies that graftcells can be activated by self-antigens. This hypothesis is furtherunderlined by the observation that a GVHD can also be presentin patients undergoing autologous HCT where genetic differ-ences cannot contribute to its development (34).The activation or induction of such allogeneic, autoreactive T

cells could be based on several mechanisms including decreasedthymic selection as shown in murine models (27, 35) or an impairedthymic function during allogeneic HCT and GVHD, leading toimpaired immunological reconstitution (36, 37). Also, impairedfunction of regulatory T cells (e.g., through conditioning regimens orimmunosuppression) could play a role (34). However, also trans-plantation of quiescent autoreactive T cells in the grafts could lead tosubsequent activation and amplification of such cells in the absenceof an effective immunosurveillance during transplantation (38, 39).Because we did not detect any autoreactive T cells in donorsamples, the first mechanism seems to be more likely. Due to thelimited sample numbers, the latter can still not be fully excluded.The development of autoinflammatory diseases in patients

with full donor chimerism after HCT is referred to as new autoim-munity. However, the discrimination between classical GVHD, dis-ease relapse, infection, and treatment-related toxicity is difficult. Mostof the reported manifestations are antibody-mediated diseases such asautoimmune hemolytic anemia (40–42), immune thrombocytopenia(41, 43), thyroiditis, and myasthenia gravis (42). As of yet, systematicstudies have evaluated the development of new autoimmunityonly in patients with primary autoimmune diseases being presentbefore autologous and allogeneic HCT (42, 44). No data areavailable about de novo development of new autoimmunity.Notably, the term new autoimmunity is commonly referred to

diseases that share laboratory findings and clinical presentationwith normal autoimmune diseases including detection of diseasespecific antibodies (37). In contrast, alloreactivity denotes recognition

of antigens, based on structural differences between donor andrecipient. According to these definitions, recognition of self-antigens by allogeneic T cells can neither be subsumed as newautoimmunity nor as alloreactivity. Hence, we propose the termgraft-versus-self (GvS) for description of autoantigens recog-nized by donor T cells to reflect our observation.In the pathogenesis of GVHD, endothelial damage and infec-

tions of respective tissues play a crucial role. Hence, the main sitesof manifestation represent organs with higher susceptibility to in-fections (45), but also CNS may be involved (9, 10). With regardto the retina, only indirect evidence was reported because inflam-mation improved or deteriorated alongside changes in immuno-suppression (17). In our study, viral eye infections as observed inpatient 5 also could trigger the development of a tissue antigen-specific GvS reaction. However, further studies are required to es-tablish risk factors for the development of GvS and to evaluate theclinical relevance of such circulating antigen-specific T cells.In summary, our data represent a report of autoantigen-specific T

cells and their precise epitope in human allogeneic HCT and pro-vide evidence that allogeneic T-cell reactivity is by far more complexthan previously believed. Not only genetic differences lead to in-duction of recipient-specific T cells, but also autoantigens can berecognized. Hence, beyond recognition of MHC molecules basedon differences in the MHC locus and MHC-restricted peptides(miHAG) due to genetic differences between donor and recipientleading to variant amino acid sequences of translated proteins, anovel mechanism of the T cell-based graft–host interaction ischaracterized on a molecular basis of T-cell function by our data:the recognition of autoepitopes (GvS) not requiring genetic differ-ences between patient and donor.

MethodsSample Collection. This study was approved by the ethics committee of theUniversity of Tuebingen (Tuebingen, Germany). All patients gave their writteninformed consent before entering the study. Blood samples were obtained be-fore the start of conditioning regimen (second group). After hematologic re-generation, blood samples were obtained until 1 y after HCT. In case of anobserved T-cell response, the period could be extended after reapproval of thepatients. PBMCs were isolated by density gradient centrifugation. DNA wasisolated from blood before (recipient DNA) and after HCT (donor DNA) using theInvitrogen DNA Isolation Kit (Invitrogen). All patients had full donor chimerism atthe time of DNA isolation and immunological evaluation. For patients recruitedafter HCT, autologous DNAwas isolated fromoral mucosa. Frozen donor sampleswere used if not required for quality control purposes. Donors had given theirconsent to scientific use of residual material.

PCR and Sanger Sequencing. PCR and Sanger sequencing for the entire codingsequence were performed for SNP hotspot regions. After PCR using specificprimers (Table S4), reactions were plotted on agarose gels, and bands wereexcised and purified with a DNAGel extraction kit (Promega). Sanger sequencingresults were processed using the National Center for Biotechnology Information(NCBI) blast platform (blast.ncbi.nlm.nih.gov/Blast.cgi).

Synthetic Peptides. Peptides were synthesized by solid-phase Fmoc chemistryusing a peptide synthesizer 433A (Applied Biosystems). Identity and purity ofthe peptides were analyzed by reversed-phase HPLCy (HPLC) and matrix-assisted laser desorption/ionization/time-of-flight MS (Thermo Fischer). Pep-tides were further purified by reversed-phase HPLC to >90% purity.

Priming of T Cells with Peptide-Loaded Dendritic Cells. CD8+ T cells were iso-lated from PBMCs using magnetic cell sorting (MACS) according to themanufacturer’s instructions (Miltenyi Biotec). Autologous monocyte-deriveddendritic cells (DCs) were generated from PBMCs with GM-CSF (50 ng/mL;PeproTech) and IL-4 (20 ng/mL; R&D Systems). DC maturation was induced bylipopolysaccharide (LPS; Sigma-Aldrich). Mature DCs were harvested, loadedwith peptide (20 μg/mL), and added to T cells (ratio 1:3 or 1:5). Mixed lym-phocyte cultures were supplemented with IL-12 (PromoKine). T cells wererestimulated weekly with autologous peptide-pulsed irradiated CD8− cells orDCs. IL-2 (R&D Systems) was added on days 1 and 3 after every restimulation.

Peptide Presensitization of PBMCs. As described previously, T cells were pre-sensitized with peptides for 12 d (1 and 5 μg/mL for MHC classes I and II,respectively) (33). For stimulation with MHC class I-predicted peptides, IL-4

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and IL-7 (PromoKine) were added on days 0 and 1. IL-2 was added on days 3,5, 7, and 9. Cells were harvested on day 12 and further evaluated.

IFN-γ ELISpot Assay. Twelve-day presensitized PBMCs were added to a pre-coated (anti-human IFN-γ antibody 1-D1 K; Mabtech) nitrocellulose plate.Cells were incubated for 24–26 h with peptides. As positive and negativecontrols, a pool of viral epitopes (derived from CMV, EBV, and influenza)and an HIV-derived epitope or human self-epitope was used, respectively(Table S3). Plates were further incubated (2 h) with a biotin-labeled anti–IFN-γantibody (7-B6-1 biotin; Mabtech) followed by an incubation step for 1 hwith streptavidin-alkaline phosphatase (Sigma-Aldrich). Afterward, 5-bromo-4-chlore-3-indolyl-phosphate/nitroblue tetrazolium (Sigma-Aldrich) was added.Spots were automatically counted using the ImmunoSpot S6 Ultra-V AnalyzerELISpot reader (CTL Europe). Duplicate wells were considered positive if(i) there were at least 10 spots per 250,000 PBMCs detectable in either well,and (ii) the mean number of spots was at least threefold of the countedspots in the negative control.

ICS. Twelve-day presensitized PBMCs or primed T cells were incubated withpeptides for 5–6 h or overnight. To inhibit cytokine secretion, GolgiStopsolution (BD Biosciences) was added. Cell membrane molecules were stained

with CD4-APC (BD Biosciences) and CD8-PeCy7–conjugated antibodies (BeckmanCoulter). To label dead cells, PBMCs were stained with AquaLifeDead (Invi-trogen, Life Technologies). After permeabilization (Cytoperm/Cytofix solution;BD Biosciences), intracellular staining was done with IFN-γ-FITC (BD Biosciences)and TNFα-PE (Beckman Coulter) –conjugated antibodies. Cells were measured inFACSCalibur, FACSCanto, and LSR Fortessa flow cytometers (BD Biosciences). Acytokine response was regarded positive when the percentage of the TNF-α– orIFN-γ–producing T-cell population (CD4 or CD8 negative) was at least twofold ofthe negative control. A response was considered specific when at least 0.05% ofthe T cells (CD4 or CD8 negative) produced IFN-γ.

ACKNOWLEDGMENTS.We thank Patricia Hrstiç, Nicole Bauer, and KatharinaGraf for peptide synthesis, as well as Lynne Yakes for editorial support;Cécile Gouttefangeas for expert support in T-cell monitoring; Susanne Kohlfor provision of PCR systems; Dr. Christoph Faul, Ute Schroeder, Grazia Koch,and Erwin Schleicher for provision of patient and anonymous donor samples;Richard F. Spaide, Lawrence A. Yannuzzi, and Yale Fisher of the Vitreous,Retina and Macula Consultants New York for helpful discussion; and Selmaand Austin I. Fink for providing helpful feedback. This project was supported bythe fortüne Programm of the Eberhard Karls Universität Tübingen (Grant 1832-0-1), the Deutsche José Carreras Leukämie Stiftung (Grant DJS 08/04), and theDeutsche Krebshilfe (Grant 110465). All funding was granted to S.P.H.

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