Identification of HLA-A*0201-Presented T Cell EpitopesDerived from the Oncofetal Antigen-Immature LamininReceptor Protein in Patients with Hematological Malignancies

Sandra Siegel,* Andreas Wagner,* Birte Friedrichs,* Anneke Wendeler,* Lena Wendel,*Dieter Kabelitz,† Jorg Steinmann,† Adel Barsoum,§ Joseph Coggin,§ James Rohrer,§

Peter Dreger,‡ Norbert Schmitz,* and Matthias Zeis1*

The oncofetal Ag immature laminin receptor (OFA-iLR) is a potential target molecule for immunotherapeutic studies in severaltumor entities, including hematological malignancies. In the present study, we characterize two HLA-A*0201-presented epitopeseliciting strong OFA-iLR peptide-specific human cytotoxic T cell (CTLs) responses in vitro. Both allogeneic HLA-A*0201-matchedand autologous CTLs recognized and killed endogenously OFA-iLR-expressing tumor cell lines and primary malignant cells frompatients with hemopoietic malignancies in an MHC-restricted fashion but spared nonmalignant hemopoietic cells. SpontaneousOFA-iLR peptide-specific T cell reactivity was detectable in a significant proportion of leukemia patients. Interestingly, in patientswith chronic lymphocytic leukemia and multiple myeloma but not in those with acute myeloid leukemia, significant frequenciesof OFA peptide-specific CTLs could be detected in an early stage of disease but disappeared in patients with progressive disease.The identification of OFA-iLR-derived peptide epitopes provides a basis for tumor immunological studies and therapeutic vac-cination strategies in patients with OFA-iLR-expressing malignancies. The Journal of Immunology, 2006, 176: 6935–6944.

T he oncofetal Ag-immature laminin receptor protein(OFA-iLRP)2 is a 37-kDa evolutionary conserved proteinthat has been detected in several types of human tumor

entities such as breast, ovary, prostate, lung, renal cancer, and he-matological malignancies (1–6). Expression of OFA-iLR was alsofound in embryos and the earlier stage of fetuses but not in termfetus, neonate, or adult differentiated tissues (1, 5). OFA-iLR ap-pears to dimerize after acylation to form the high-affinity mature67-kDa laminin receptor protein. Although the mature 67-kDa pro-tein, which is present on many normal cells, is nonimmunogenic,the immature form can be specifically recognized by the adaptiveimmune system (1, 2, 4, 7, 8). Using dendritic cells (DCs) trans-fected with OFA-iLRP-coding RNA, we have shown previouslythat tumor-specific T cell responses against hemopoietic targetcells can be elicited both in vitro and in vivo (6). Thus, OFA-iLRPcan be regarded as a potential target molecule for immunothera-peutic approaches toward human and rodent cancers (1, 4).

In the present study, we identified and characterized two distinctHLA-A*0201-specific OFA-iLR peptide-epitopes using Ag-spe-

cific CTLs that were generated by in vitro priming with peptides-pulsed monocyte-derived DCs. We demonstrate that OFA-iLRPpeptide-specific CTLs obtained from healthy donors and patientswith acute myeloid leukemia (AML) and chronic lymphocytic leu-kemia (CLL) elicited Ag-specific HLA-A*0201-restricted cyto-lytic activity against several hematological tumor cell lines andfreshly isolated AML and CLL tumor cells. Furthermore, by de-tecting OFA-iLR-specific T lymphocytes in patients with CLL andmultiple myeloma (MM) in an early stage of disease but not inpatients with progressive disease, OFA-iLR-specific CTLs canplay a role in controlling OFA-iLR� tumor cells.

Materials and MethodsPatients and healthy donors

PBMCs and bone marrow (BM) cells from healthy donors and malignantcells from patient with AML, CLL, and MM were obtained by Ficoll den-sity gradient centrifugation after informed consent and approval by ourinstitutional review board. Patient characteristics are reported in Tables Iand II. PBMCs obtained from healthy individuals and patients were stainedwith the anti-HLA-A*0201-FITC mAb (Biologo) and analyzed by flowcytometry and CellQuest software (FACScan; BD Biosciences). HLA-A2-positivity was confirmed by genotyping of the HLA-A2*0201 allele.

Human tumor cell lines and nonmalignant cell subsets

K562 (erythroleukemia), U-266 and IM-9 (plasmacytoma), Karpas-422,Balm-3, REH, Ramos (B lymphoblastic lymphoma), and T2 cells wereobtained from American Type Culture Collection. MEC-1 (chronic lympho-cytic leukemia) was a gift from Dr. F. Caligaris-Cappio (University of Torino,Torino, Italy). CD34� peripheral blood progenitor cells were obtained us-ing MACS technology (Miltenyi Biotec), following the manufacturer’s in-structions. The cell purity was verified by FACS analysis (FACScan; BDBiosciences) and reached � 90%.

Peptides and loading of T2 cells

For binding assay and generation of peptide-specific CTLs, the peptidefrom influenza A virus matrix protein M1, FluM158–66 (GILGFVFTL, pos-itive control), HIV-Pol476–484 (ILKEPVHGV, positive control), msurv33(LYLKNYRIA, murine survivin peptide epitope specific for H-2d, negativecontrol), iLR159–68 (LLAARAIVAI, Rammensee score: 18), iLR2146–154

*General Hospital St. Georg, Department of Hematology, Hamburg, Germany; †In-stitute of Immunology, University of Kiel, Kiel, Germany; ‡Department of Stem CellTransplantation, University of Heidelberg, Heidelberg, Germany; and §Department ofMicrobiology and Immunology, University of South Alabama College of Medicine,Mobile, AL 36602

Received for publication August 11, 2005. Accepted for publication February27, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Matthias Zeis, Department ofHematology, General Hospital St. Georg, Lohmuhlenstrasse 5, D-20099 Hamburg,Germany. E-mail address: mzeis47@hotmail.com2 Abbreviations used in this paper: OFA/ILRP, oncofetal Ag-immature laminin re-ceptor; DC, dendritic cell; AML, acute myeloid leukemia; CLL, chronic lymphocyticleukemia; BM, bone marrow; MM, multiple myeloma; iDC, immature DC; DOTAP,dioleoyloxy-trimethylammonium-propane-methylsulfate; GVHD, graft-vs-hostdisease.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

(ALCNTDSPL, Rammensee score: 22), iLR360–68 (LAARAIVAI, Ram-mensee score: 23), iLR458–66 (LLLAARAIV, Rammensee score: 26),iLR57–15 (VLQMKEEDV, Rammensee score: 22), iLR650–58 (NLKRTWEKL, Rammensee score: 21), iLR766–74 (VAIENPADV, Rammenseescore: 21), iLR8139–147 (YVNLPTIAL, Rammensee score: 21),iLR9177–185 (MLAREVLRM, Rammensee score: 21), iLR10249–257 (SEGVQVPSV, Rammensee score: 19), iLR1118–26 (FLAAGTHLG, Ram-mensee score: 18), iLR1257–66 (KLLLAARAIV, Rammensee score: 25),iLR1367–76 (AIENPADVSV, Rammensee score: 23), and iLR14173–182

(LMWWMLAREV, Rammensee score: 23) were selected by prediction tobind to the HLA-A*0201 molecule using the computer programs PAProC(�www.uni-tubingen.de/uni/kxi�) and SYFPEITHI (�www.syfpeithi.de�).The peptides, which were purchased from Biosynthan, were provided with�90% purity and were analyzed by HPLC and mass spectometry. TheHLA-A2-positive mutant cell line T2 was incubated with peptides for 2 hat 37°C and 5% CO2 at various concentrations, washed three times withPBS (Biochrom), and used as targets in 51Cr release or T2-binding assays.

MHC-binding and stabilization assay

Evaluation of HLA-A2-binding affinity was performed by flow cytometryanalysis based on the use of the HLA-A*0201 TAP-deficient T2 lymphomacell line using a modified protocol adopted from Casati et al. (9). T2 cells(2.5 � 106/ml) were seeded in RPMI 1640 (Invitrogen Life Technologies)with 2.5 �g/ml �2-microglobulin (Sigma-Aldrich). Peptides were added at

various concentrations. Cells were incubated overnight in a water-saturatedatmosphere with 5% CO2 at 37°C. After incubation, cells were washedwith cold PBS with 0.05% BSA (Sigma-Aldrich) to remove unbound pep-tides. To determine surface expression of the HLA-A2 molecule, cells wereresuspended in 100 �l of PBS/0.05% BSA and incubated with 10 �l ofanti-HLA-A*0201-FITC mAb for 30 min at 4°C. Afterward, cells werewashed twice with cold PBS/0.05% BSA. Fluorescence intensity was mea-sured by FACS analysis. Controls consisted of T2 cells cultured withFluM1 (positive control), msurv33, or with no peptide (negative controls).

To determine the stability of the peptide/HLA-A2 complex, 2 � 106 T2cells were incubated overnight at 27°C in 2 ml of RPMI 1640 supple-mented with 10% FCS and 2.5 �g/ml �2-microglobulin. Peptides wereadded at a concentration of 10 �M. Cells were then washed and incubatedat 37°C for 0, 2, 4, 6, or 8 h. HLA-A2 expression on these cells wasdetermined, as described above. Results are expressed as percentage (overcontrol) of the remaining BB7.2 mean fluorescence intensity. Controls arethe BB7.2 mean fluorescence intensity of T2 cells at the onset of incubationat 37°C.

Western blot analysis

Whole cell protein extracts were prepared from 1 � 107 cells using a lysisbuffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NonidetP-40 (Sigma-Aldrich), 2 mM EDTA (pH 8.0), and Complete mini (proteaseinhibitor; Roche). Cell lysates were separated by 12% gels in SDS-PAGE

Table II. iLR1- and iLR2-specific CTL responses postallotransplanta

Pat.No.

Age(years)

Gender(male/female)

PreparatoryRegimen

TransplantType

Sampling Time(day post-PBPCT)

Maximal T Cell ResponseSpots/105 PBMCs GVHD

Status at LastFollow-Up

1 61 M Flu/Cy/ATGb MUDPBPC

d�56 post DLI iLR1: 134 iLR2: 56 Acute skin II Alive and CR

2 55 M Flu/Cy/ATG MUDPBPC

d�98 iLR1: 68 iLR2: 44 No Alive and CR

3 53 F Flu/Bu/Cy/ATG MUDPBPC

d � 554 iLR1: 56 iLR2: 43 No Alive and CR

4 65 F Clad/Cy/ATG Sib. PBPC d �132 iLR1: 102 iLR2: 67 No Alive and CR5 44 F Flu/Cy/ATG MUD

PBPCd �67 iLR1: 0 iLR2: 0 No Dead

6 43 M Flu/Cy/ATG Sib. PBPC d �105 iLR1: 56 iLR2: 39 No Alive and CR7 53 M Flu/Cy/ATG MUD

PBPC� �3, 5yrs iLR1: 0 iLR2: 0 Chronic skin Alive and CR

8 45 M Flu/Cy/ATG MUDPBPC

d �351 iLR1: 0 iLR2: 0 Chronic skin Alive and CR

9 M Flu/Cy/ATG MUDPBPC

d �88 iLR1: 0 iLR2: 0 Chronic skin Dead

10 59 F Flu/Cy/ATG MUDPBPC

d �96 iLR1: 122 iLR2: 59 No Alive and CR

a Postallogenic stem cell transplantation PBMCs from 10 HLA-A-*0201-positive patients were analyzed by IFN-� ELISPOT for specific T cell responses against iLR1 andiLR2 peptides.

b Flu, fludarabine; Cy, cyclophosphamide; ATG, antitymocyteglobulin; Bu, busulfan; Clad, cladiribine; MUD, matched unrelated donor; PBPC, peripheral blood progenitorcell; DLI, donor lymphocyte infusion; Sib, sibling.

Table I. Clinical characteristics and spontaneous OFA peptide-specific CTLs of the patient material at testing time

B-CLL Myeloma AML

Total 21 10 15Males/females 13/8 7/3 8/7Age (years)

Range/median 43–78/63.5 55–82/65.3 32–69/42.5Stage of disease/OFA-specific CTLsa

Binet A: 14; 8/14b,c Stage I: 5; 5/5d,e At diagnosis: 6; 2/6f

Binet B: 2; 1/2 Stage II: 2; 1/2 CR: 9; 4/9Binet C: 5; 0/5 Stage III: 5; 0/5

TreatmentPreviously untreated 100% 100% 40%Previously treated 0% 0% 60%g

a No. of patients with significant frequencies of iLR1- and iLR2-specific T cells detected in IFN-� ELISPOT assay.b CLL staging as defined in Rai et al. (19).c OFA-specific CTLs: CLL-Binet A � B vs Binet C: p � 0.001;d Myeloma staging as defined in Durie et al. (20).e Myeloma stage I vs stage II/III: p � 0.001.f At diagnosis vs CR (complete remission): not significant.g The majority of patients had received daunorubicine and cytosine arabinosid.

6936 DETECTION OF ANTI-OFA/iLR-SPECIFIC IMMUNE RESPONSES

under reducing conditions for 60 min at 140 V. Proteins were transferredonto a nitrocellulose membrane (Macherey & Nagel) in 25 mM Tris base,0.2 M glycine, and 20% methanol for 60 min at 100 V and blocked in 20mM Tris (pH 8.0), 137 mM NaCl, and 0.1% Tween 20 (TBST), and 5%powdered milk overnight. Immunoblottings were performed using themouse anti-OFA-iLRP mAb at a concentration of 0.75 �g/ml (in TBST and5% BSA) as primary and horseradish phosphatase-linked goat anti-mouseIgG (Dianova) (diluted to 1/5000 v/v in TBST and powdered milk) as

secondary Ab. All immunoblots were performed using Enhanced Che-moluminescence solution (ECL; Amersham Biosciences), following themanufacturer’s instructions.

ELISPOT assay

PBMCs (106/ml) obtained after Ficoll density gradient centrifugation fromHLA-A*0201 patients with AML, CLL, and MM were cultured in AIMV

FIGURE 1. Detection of OFA/iLRP on or in ma-lignant and normal hemopoietic cells. OFA/iLRPexpression on the surface of two distinct B-CLLsamples (A and B) was determined by flow cytom-etry using an anti-OFA/iLRP mAb (see Materialsand Methods). Western blot analysis was performedto examine the OFA/iLR protein expression in celllysates of several normal and malignant hemopoieticcell samples (C).

FIGURE 2. Peptide binding and titrationassay. HLA-A2 binding of 14 iLR peptideswas measured using HLA-A*0201-positiveTAP-deficient T2 cells and conformation-de-pendent HLA-A2-specific Abs. Binding ef-ficiency of iLR peptides was compared withthat of the known HLA-A2-binding peptideFluM1. Surv33 served as a HLA-A2-non-binding negative control (A and B). To de-termine the stability of the peptide/HLA-A2complex, HLA-A2 stabilization assay (C)was performed, as described in Materialsand Methods. Peptide titration experimentswere performed using iLR1- and iLR2-spe-cific CTLs directed against T2 cells priorloaded with increasing amounts of the cog-nate peptides. The HLA-A2-binding HIV-Pol476–484 peptide served as a negative con-trol. The cytotoxic potential of OFA/iLRpeptide-specific CTLs were measured in51Cr release assays (D).

6937The Journal of Immunology

FIGURE 3. Spontaneous T cell responsesagainst OFA/iLR-derived candidate peptidesas measured by IFN-� ELISPOT. PBMCsfrom 15 healthy donors, 21 patients withCLL, 15 patients with AML, and 10 patientswith MM were analyzed. All individualswere HLA-A-*0201 positive. The peptidesiLR1, iLR2, iLR3, iLR4, iLR9, and iLR14were examined.

6938 DETECTION OF ANTI-OFA/iLR-SPECIFIC IMMUNE RESPONSES

medium (Invitrogen Life Technologies) with 5% heat-inactivated humanAB serum (CC-Pro) and 10 �g of peptide for 10 days. After 24 h, IL-2(CellConcepts) was added at a concentration of 20 IU/ml. Nitrocelluloseplates (96-well; MAHA S4510; Millipore) were coated overnight with 100�l/well of 10 �g/ml anti-human IFN-� mAb (Mabtech) in PBS by 4°C.Afterward, plates were washed three times with 150 �l/well PBS for 5 minand blocked with AIMV medium for 1 h in 5% CO2 at 37°C. After block-ing, 1 � 105 precultured cells in a volume of 50 �l/well were seeded, andpeptides at a concentration of 10 �g/ml in 50 �l of AIMV medium wereadded to cells. Plates were incubated for 18–20 h by 37°C with 5% CO2.Plates were then washed six times for 3 min with TBS (Bio-Rad) and0.05% Tween 20 (Sigma-Aldrich), and wells were incubated with 100�l/well of biotinylated mouse anti-human IFN-� mAb (Mabtech) at a con-centration of 2 �g/ml in TBS for 2 h by 37°C and 5% CO2. After washingsix times, ExtrAvidin alkaline phosphatase conjugate (1 �g/ml in TBS;Sigma-Aldrich) was added for 1 h at room temperature. Next, plates werewashed three times with TBS/0.05% Tween20 and five times with TBSonly. Development of the spots was performed by adding 100 �l/well of5-bromo-4-chloro-3-indolyl phosphate/NBT substrate for 10–30 min indarkness. IFN-�-secreting T cells were counted using the automated imageanalysis system ELISPOT Reader (AID). Each experiment was performedin duplicate.

Pentamer staining

To determine the frequency of OFA-iLR-specific CD8� T cells in HLA-A2�individuals, FluM158–66-pentamer-PE (positive control),HIV-Pol476–484-pentamer-PE (negative control), iLR159–68-pentamer-PE,and iLR2146–154-pentamer-PE were used (Proimmune). Peptide-prestimu-lated PBMCs (see ELISPOT assay) were incubated with 10 �l of pentamerfor 30 min in darkness. Cells were washed and resuspended in 100 �l ofwash buffer. PBMCs were incubated with FITC-conjugated anti-CD8 mAb(Caltag Laboratories) for 25 min by 4°C and washed twice with washbuffer. To exclude dead cells, propidium iodide (Sigma-Aldrich) was addedat a concentration of 1 �g/ml. Fluorescence intensity was determined byFACS analysis. The percentage of peptide-specific T cells was determinedafter gating the CD8� T cells inside the T cell population. Analysis wasperformed using the CellQuest Pro software.

Generation of mature DCs and loading with peptides

Mature DCs from healthy HLA-A*0201 donors were generated followingthe protocol of Feuerstein et al. (10). A concentrated leukocyte fraction wasgenerated through a 2-h restricted peripheral blood leukapheresis process-ing 6–8 liters of blood with each collection. A total of 2 � 108 PBMCs/mlobtained after Ficoll density gradient centrifugation was incubated in 30 mlof serum-free AIMV medium (Invitrogen Life Technologies) for 2 h in awater-saturated atmosphere with 5% CO2 at 37°C. The nonadherent T cell-enriched fraction was cryoconserved and used later as a source of CTLgeneration. The adherent monocytes were cultured for 5 days with 500U/ml human IL-4 and 800 U/ml human GM-CSF (CellConcepts) to obtainimmature DCs (iDCs). Maturation of DCs was performed in AIMV me-dium (Invitrogen Life Technologies) by adding human IL-1�, humanTNF-� (10 ng/ml; CellConcepts), human IL-6 (1000 U/ml; CellConcepts),and human PGE2 (1 �g/ml; Sigma-Aldrich) for 2 additional days at 37°Cwith 5% CO2.

Mature DCs from patients with AML and CLL were generated by sep-arating PBMCs from 100 ml of whole blood donation, following the pro-tocol described above. Loading of the mature DCs with peptide was per-formed by incubation of 1 � 106/ml mature DCs with 50 �g/ml peptide for2 h at 37°C with 5% CO2.

Isolation of human OFA-iLRP coding RNA and EGFP-RNA

Preparation of an OFA-iLRP expression vector was done as described pre-viously (6). The vector was transformed into Escherichia coli JM83 by heatshock transformation. After ampicillin selection, plasmid isolation (Plas-mid Maxi kit; Qiagen) was performed, and the correct size of the plasmidwas checked on a 1% agarose gel. OFA-iLRP sequence was verified byautomatic sequencing (ABI 310; PerkinElmer). Resulting plasmid DNAwas linearized, and in vitro transcription was performed using the T7RiboMAX Large Scale Transcription kit (Promega) as described previ-ously (6). EGFP vector was provided by Dr. M. Marget (Institute of Im-munology, University of Kiel, Kiel, Germany). Quantity and purity of theRNA was determined by UV spectrophotometry.

OFA-iLR-RNA transfection and maturation of human DCs

Transfection of iDCs with in vitro-transcribed iLRP-RNA was performedby using the liposomal transfection reagent dioleoyloxy-trimethylammo-nium-propane-methylsulfate (DOTAP; Roche). DOTAP (20 �l) and invitro-transcribed RNA (10 �g) were mixed in 500 �l of Opti-MEM (In-vitrogen Life Technologies) and incubated for 20 min at room temperature.

FIGURE 4. Peptide titration assay using iLR-specific CTLs derivedfrom two patients with CLL treated with an allogeneic stem cell transplant(patients 1 and 6, see Table II). As target cells, T2 cells already loaded withincreasing amounts of the cognate peptides were used. The HLA-A2-bind-ing HIV-Pol476–484 peptide served as a negative control. The cytotoxicpotential of OFA/iLR peptide-specific CTLs was measured in 51Cr releaseassays.

FIGURE 5. Pentamer staining for determining the frequency of iLR-specific CD8� T cells by flow cytometry. PBMCs from a CLL patient wereprestimulated with iLR1 (upper left), iLR2 (upper right) peptide, a nega-tive control HIV-Pol476–484 (lower left), and a positive control FluM1 pep-tide (lower right) as described in Materials and Methods. The percentageof peptide-specific T cells was evaluated after gating for CD8� T cellswithin the PBMCs. The experiment was conducted with samples from fivedifferent CLL patients.

6939The Journal of Immunology

The RNA-lipid complex was added to 1 � 106 iDCs/ml (in Opti-MEM)and incubated at 37°C for 3 h. Maturation of the transfected iDCs wasperformed by culturing for 2 days in serum-free medium supplementedwith GM-CSF, IL-4, IL-1�, TNF-� (10 ng/ml; CellConcepts), IL-6 (1000U/ml; CellConcepts), and PGE2 (1 �g/ml; Sigma-Aldrich). Flow cytomet-ric analysis revealed that �30% of iLRP-RNA-transfected and maturatedDCs expressed iLRP and high levels of CD80, CD83, CD86, MHC classI, and MHC class II molecules.

Generation of CTLs

CTLs were generated, as described previously (11). A total of 2 � 107

cryoconserved nonadherent PBMCs was cultured with 2 � 106/ml autol-ogous OFA-iLR peptide-pulsed DCs in 10 ml of AIMV medium (Invitro-gen Life Technologies) supplemented with 20 U/ml human IL-2 and 10ng/ml human IL-7 (CellConcepts). Cells were weekly restimulated withpeptide-loaded DCs.

For the induction of autologous peptide-specific CTLs obtained frompatients with AML and CLL, CD8� T cells were separated from PBMCsusing immunomagnetic beads (MACS beads; Miltenyi Biotec), followingthe manufacturer’s instructions. Cells were cultured with autologous OFA-iLR peptide-pulsed DCs and weekly restimulated with autologous peptide-pulsed PBMCs in the presence of IL-2 (1 ng/ml; CellConcepts). After threeto four restimulations, �90% of cells in each culture were CD3� lympho-cytes, of which �90% were CD8� and 3.5–4.8% were CD4�. Culturescontained �1.5% CD14� and 3.5–4.5% CD56� cells. In another set ofexperiments, peptide-specific CTLs were enriched from CLL patients usingthe IFN-� secretion assay (Miltenyi Biotec), as described previously (6).CTL reactivity was determined in a conventional 4-h 51Cr release assay.

51Cr release assay, cold target inhibition assay, and Ab-blocking experiments

Standard 4-h 51Cr release assay was performed, as described previously (11).Specificity of tumor cell lysis was determined in a cold target inhibition assayby analyzing the capacity of unlabeled HLA-A2� iLRP-negative T2 cells,

OFA-iLR peptide (iLR1 � iLR2)- and msurv33 peptide-labeled T2 cells, andOFA-iLR-positive leukemic blasts from an HLA-A*0201 AML patient(AML-01) to block lysis of tumor cells at a ratio of 20:1 (inhibitor:target ratio).

For Ab-blocking experiments, human CTLs were generated as describedabove. Tumor cells were incubated with 10 �g/ml anti-HLA-A2 mAb(BB7.2; BD Biosciences), and CTLs were incubated with anti-CD8 (T8),anti-Pan-TCR (BMA 031), and anti-CD4 (T4) isotype-matched controlsMAM-6 and HMFG-1 (Beckman Coulter) for 30 min. Cell suspensionswere washed twice with complete medium and tested in a 51Cr releaseassay.

Statistics

Student’s t test was performed to evaluate the significance of the results.

ResultsOFA-iLRP is expressed in hematological malignancies

The expression of OFA-iLRP in or on malignant and nonmalignantcells was assessed by Western blot and FACS analyses using thepreviously described anti-OFA-iLRP mAb. All hematological tu-mor samples used in this study showed a strong up-regulation ofOFA-iLRP expression. In Fig. 1, some representative samples aredemonstrated. As expected, we could not detect any OFA-iLRPexpression in nonmalignant hemopoietic cell subtypes (CD34�

progenitor cells, bone marrow cells, B cells, and activated B cells).

Identification of HLA-A2-binding peptides in OFA-iLRP

To identify T cell-binding epitopes deduced from the OFA-iLRprotein, 14 peptides were synthesized that were predicted by thecomputer programs PAProC (�www.uni-tuebingen.de/uni/kxi/

FIGURE 6. Induction of peptide-specific CTL re-sponses by peptide-pulsed DCs. Cytotoxic potential ofiLR1- and iLR2-specific CTLs against untransfectedDCs and DCs loaded with FluM1 peptide, either en-hanced GFP- or iLRP-RNA-transfected DCs. iLR1- andiLR2-specific CTL reactivity against OFA�/iLR�

HLA-A2� and HLA-A2� hematological cell lines (Cand D).

6940 DETECTION OF ANTI-OFA/iLR-SPECIFIC IMMUNE RESPONSES

[Lang]) and SYFPEITHI (�www.syfpeithi.de�) to bind to the HLA-A*0201 molecule (see Materials and Methods). In an in vitro re-constitution assay using the TAP-deficient T2 cell line, twopeptides (iLR1 and iLR2) showed similar binding affinity to HLA-A*0201 (Fig. 2A) as the positive control Flum1. In addition, asillustrated in Fig. 2C, the HLA-A2/iLR1 and HLA-A2/iLR2 com-plexes showed intermediate complex stability when comparedwith the Flum1 peptide.

In the presence of fully matured DCs pulsed with either theiLR1 or iLR2 peptide, CTL lines could be generated from healthyHLA-A*0201� donors. To assess the avidity of the induced CTLlines, T2 cells were pulsed with titrated amounts of the peptidesiLR1, iLR2, or HIV-Pol476–484 used as a negative control. Usingan E:T ratio of 40:1, peptide titration curves demonstrated thatiLR1- and iLR2-specific CTLs were of high affinity to the peptide/MHC complex (Fig. 2D).

Detection of OFA-iLR peptide-specific T cells in patients withhemopoietic malignancies

In a next set of experiments, iLR1 and iLR2 peptides were testedfor their potential to elicit specific ex vivo T cell responses inhealthy volunteers and in patients with CLL, AML, and MM. Toextend the sensitivity of the ELISPOT assay, PBMCs were stim-ulated with OFA-iLR peptides once in vitro before analysis. Asdepicted in Fig. 3, in 9 of 21 (43%) and 10 of 21 (48%) of CLLpatients, significant frequencies of iLR1 (Fig. 3A)- and iLR2 (Fig.3B)-specific T cell responses were detected, whereas no spontane-ous T cell responses against both peptide epitopes occurred inperipheral blood samples of 15 HLA-A2*0201-positive healthyindividuals. In patients with AML, 6 of 15 (40%) mounted a CTLresponse against iLR1 or iLR2 peptide, respectively. We also an-alyzed PBMCs from 12 HLA-A*0201-positive patients with MMand detected in 6 samples (50%) a reactivity against iLR1 andiLR2. Additionally, in PBMCs of several leukemia and myelomapatients tested, we also found spontaneous T cell reactivity againstthe peptides iLR3, iLR4, iLR9, and iLR14 (Fig. 3, C–F). Interest-

ingly, significant frequencies of OFA-iLR peptide-specific CTLswere only found in blood samples of CLL and MM patients in anearly stage of disease. In 8 of 14 patients with a Binet A CLL,iLR1- and iLR2-specific T cell reactivity was detected, whereas inCLL patients with progressive disease (Binet C), no OFA-iLR-specific T cell responses occurred (Table I). Similar results wereobtained in MM patients.

We next examined whether OFA-iLR peptide-specific T cellswere also present in PBMCs from CLL patients treated with anallogeneic stem cell transplant. Whereas no OFA-iLR-specific Tcell responses were observed in the ELISPOT assay before trans-plantation, 6 of 10 allograft recipients developed significant T cellfrequencies against both iLR1 and iLR2 peptides (Table II). Toanalyze whether iLR-specific CTLs obtained from a patient withacute graft-vs-host disease (GVHD) of the skin (patient 1) providehigher avidity responses than CTLs from a patient lacking clini-cally overt GHVD, peptide titration assays with T2 cells pulsedwith the cognate peptide were performed. Fig. 4 illustrates thatCTLs from patients 1 and 6 do not differ in their potential to rec-ognize and kill peptide-pulsed T2 cells. In one transplant patientwith high numbers of iLR1 and iLR2-specific T cells (patient 6),responding cells were selected by the IFN-� secretion assay. Themajority of the iLR1- and iLR2-specific T lymphocytes wasCCR7�CD45RA� memory effector cells (data not shown).

Pentamer staining of CD8� T lymphocytes recognizing thepeptides iLR1 and iLR2 in the context of HLA-A2

In five CLL patients with significant OFA-iLR responses in theELISPOT assay, we also determined the frequency of iLR1- andiLR2-specific CD8� T lymphocytes within the PBMCs by per-forming pentamer staining. Two-color flow cytometry with anti-CD8 mAb and HLA-A2/iLR1- and HLA-A2/iLR2-pentamericcomplexes revealed a frequency of CD8� T cells recognizing theiLR1 and iLR2 peptides, respectively, ranging from 0.6 to 2.5%for iLR1 peptide and 0.5 to 1.5 for iLR2 peptide. In contrast, in the

FIGURE 7. ILR peptide-specific CTLsrecognize and kill primary malignant cellsfrom AML and CLL patients. ILR1- andiLR2-induced CTLs from healthy donorslysed freshly isolated malignant cells fromHLA-A2� AML patients but not nonmalig-nant HLA-A2�CD34� progenitor cells andbone marrow cells. HLA-A2� tumor cellswere not recognized (A and B). OFA/iLR-specific CTLs from healthy donors lysedfreshly isolated leukemia cells from HLA-A2� CLL patients, whereas nonmalignantHLA-A2� B cells were not lysed (C and D).

6941The Journal of Immunology

presence of the HIV peptide (negative control) or the Flum1 (pos-itive control), we detected frequencies between 0.01 to 0.04% and5.4 to 23.3%, respectively (one representative experiment shownin Fig. 5).

OFA-iLR peptide-specific CTLs efficiently lyse tumor cellsendogenously expressing OFA-iLR

We next analyzed the ability of the in vitro-induced CTLs to lysetumor cells that express the OFA-iLR protein. Both CTL linesspecific for iLR1 (Fig. 6A) or iLR2 (Fig. 6B) elicited strong cyto-lytic activity against the HLA-A*0201�/OFA-iLR� hematologicaltumor lines Karpas-422, Balm-3, IM-9, U266, REH, and Ramos.There was no recognition of the HLA-A2-negative tumor linesMEC-1 and K562. In addition, iLR1- and iLR2-specific T celllines recognized and efficiently killed HLA-A2�/OFA-iLR� pri-mary malignant AML blasts (Fig. 7, A and B) and CLL cells (Fig.7, C and D) but spared HLA-A2-negative targets and normal he-mopoietic cells (Fig. 7).

Cold target inhibition assays and Ab-blocking experiments re-vealed an MHC class I-restricted killing induced by peptide-spe-cific CD8� T lymphocytes (Fig. 8, A and B).

OFA-iLR peptide-specific CTLs can lyse autologous DCstransfected with OFA-iLR-coding RNA

To test the presentation of OFA-iLR-specific T cell epitopes upontransfection with OFA-iLR-coding RNA, we used autologousDCs, generated from the same PBMCs that were used for CTLinduction, as target cells. As illustrated in Fig. 5, C and D, ILR1-and iLR2-specific CTLs efficiently lysed autologous DCs trans-

fected with the OFA-iLR-coding RNA, whereas DCs pulsed withthe Flum1 peptide or the enhanced EGFP-RNA were not recog-nized. These findings demonstrate that the identified OFA-iLRpeptides iLR1 and iLR2 are processed and presented after trans-fection of DCs with the OFA-iLR-RNA.

OFA-iLR peptide-specific CTLs lysed autologous malignanthemopoietic cells

In one patient with CLL (Fig. 9, A and B) and in another with AML(Fig. 9, C and D), we were able to generate autologous CTL linesthat were specific for the peptides iLR1 and iLR2. These CTLswere capable of efficiently lysing autologous and allogeneic HLA-A2-matched malignant cells endogenously expressing OFA-iLRprotein, whereas HLA-A2-negative tumor cells were not recog-nized and killed. To provide more direct evidences that patient-derived iLR-specific CTLs are functionally active, CD8� T lym-phocytes specifically recognizing the iLR1 and iLR2 epitopesseparately were highly enriched from peripheral blood samplesobtained from a CLL patient (CLL-02). As depicted in Fig. 9, Eand F, both iLR1- and iLR2-specific epitopes CTLs were capableof recognizing autologous tumor cells, whereas allogeneic CLLcells were not attacked.

DiscussionOFA-iLRP is a tumor-associated protein, which is widely ex-pressed and detectable in the majority of tumor entities such assarcomas and carcinomas, including hemopoietic malignancies(1–6, 12). Several studies documented the immunogenicity ofOFA-iLR protein specifically recognized by T and B lymphocytes

FIGURE 8. Cold target inhibitionand Ab-blocking experiments. Thespecificity of OFA/iLR-specific CTLlines was tested in a 4-h 51Cr releaseassay in the presence of unlabeledcold targets, T2 cells, iLR1, iLR2,and mSurv peptide-pulsed T2 cellsand primary malignant blasts from aHLA-A2� patient (AML-01). Coldtargets were added at an inhibitor totarget ratio of 20:1 (A and C). To ex-amine MHC-dependent cytotoxicity,Ab-blocking experiments were per-formed in a 4-h 51Cr release assay (Band D). One of two representative ex-periments is shown.

6942 DETECTION OF ANTI-OFA/iLR-SPECIFIC IMMUNE RESPONSES

(1, 7–9, 13–15). In a previous report, we were able to demonstratethat OFA-iLRP-specific CTLs generated in the presence of fullymatured autologous OFA-iLRP-RNA-transfected DCs were capa-ble of eliminating both tumor cell lines and malignant cells ofAML and CLL patients (6).

In the current study, we screened the OFA-iLR protein for HLA-A2-binding peptides using the computer-assisted software SYF-PEITHI. In a standard T2-binding assay, 2 of 14 candidatepeptides, iLR1 and iLR2, exhibited a high binding affinity to theHLA-A*0201 molecule. To analyze whether these epitopes arepresented by tumor cells endogenously expressing OFA-iLR pro-tein, we generated OFA-iLR peptide-specific CTLs in the presenceof monocyte-derived autologous fully matured DCs that had beenloaded with iLR1 and iLR2, respectively. After several rounds ofrestimulations, T cell lines were tested for their cytotoxic capacityagainst OFA-iLR-expressing hematological tumor lines and pri-mary malignant cells from CLL and AML patients. The in vitro-induced peptide-specific CTLs not only recognized and killed tu-mor lines but also freshly isolated patient-derived leukemic cells.The specificity of the lytic activity was confirmed by Ab-blockingexperiments and cold target inhibition assays.

As shown in Fig. 3, spontaneous T cell responses were alsodetectable against the OFA-derived peptides iLR3, iLR4, iLR9,

and iLR14 having a lower binding affinity to the HLA-A2 mole-cule than iLR1 or iLR2, as determined in the T2-binding assay.Whereas iLR360–68 (LAARAIVAI), iLR458–66 (LLLAARAIV),and iLR159–68 (LLAARAIVAI) share the identical core aminoacid sequence, the peptides iLR9177–185 (MLAREVLRM) andiLR14173–182 (LMWWMLAREV) might represent another poten-tial distinct T cell epitope. Immunological studies are currentlyunderway in our lab to analyze these peptides further.

We further analyzed the specificity of in vitro-generated CTLlines recognizing iLR1 and iLR2 epitopes by using autologousDCs as target cells loaded with the cognate peptides or transfectedwith the OFA-ILR-coding RNA. ILR1 and iLR2 peptide-specificCTLs were capable of efficiently lysing both peptide-pulsed DCsand DCs transfected with OFA-ILR-RNA. These data clearly dem-onstrate that the peptides used for CTL induction are endogenouslyprocessed and presented by OFA-iLR RNA-transfected DCs. Intwo patients with hematological malignancies, we were able togenerate autologous iLR1 and iLR2 peptide-specific CTLs recog-nizing and killing autologous OFA-iLR-expressing leukemic cellsin an MHC-restricted and Ag-specific manner. These findings, to-gether with the ELISPOT analyses and the pentamer stainings,demonstrate that OFA-iLR-specific CTLs can be detected and gen-erated in patients with hemopoietic malignancies.

FIGURE 9. Lysis of autologous and allogeneicleukemic cells by iLR1- and iLR2-specific CTLs.Cells from HLA-A2� patients with CLL and AMLwere stimulated with iLR1- and iLR2-pulsed autol-ogous DCs: cytotoxicity against autologous and al-logeneic freshly isolated CLL cells and AML blastscells was measured in a 4-h 51Cr release assay (Aand B). One of two experiments is depicted here.Using IFN-� secretion assay, autologous iLR-CTLswere enriched and tested against autologous (CLL-01) and allogenic (CLL-06) target cells (E and F).

6943The Journal of Immunology

The induction of autoimmunity in individuals treated with tu-mor-associated vaccines represents a potential risk and thus meritscareful consideration (16). Our previous studies (6) and the find-ings described in the present study provide some evidence that theautoimmunogenic potential of OFA-iLR-derived peptides seems tobe low. First, by using ELISPOT assay and pentamer staining, wedetected OFA-iLR peptide-specific T lymphocytes in a significantproportion of patients without the occurrence of a clinical mani-festation of autoimmune diseases; second, iLR1 and iLR2 peptide-specific CTLs did not recognize and lyse normal HLA-A2-matched hemopoietic cell subpopulations, including B cells,activated B cells, DCs, CD34� progenitor cells, and bone marrowcells; third, OFA-iLR epitopes were not recognized by T lympho-cytes from healthy donors; fourth, iLR-specific CTLs obtainedfrom a patient with a clinically overt autoimmune disease (GVHD)displayed no differences in their functional avidity than CTLs froma patient lacking GVHD; and fifth, treatment of mice with synge-neic bone marrow-derived DCs transfected with the murine OFA-iLR-coding RNA resulted in significant antitumor activity againsta B cell lymphoma without inducing any macroscopic or histologicsigns of autoimmune reactivity (6). We have shown previously thatOFA-iLR-specific regulatory CD8� T cell clones secreting IL-10can be identified both in mice bearing an OFA/iLR� tumor (12,17) and in patients with advanced breast carcinomas (17, 18). In asmall series of CLL patients with early (Binet A, n 4) andprogressive disease (Binet C, n 5), no relevant IL-10-secretingT cells specific for the iLR1 or iLR2 peptide were detectable (datanot shown). However, as another important result of our study, wewere able to detect significant frequencies of OFA-iLR-specificIFN-�-secreting CTLs in the PBMCs of early-stage patients withCLL and MM when compared with patients with progressive dis-ease (see Table I). We hypothesize that OFA-iLR-specific CTLscan play a role in controlling OFA-iLR� tumor cells in the earlyphase of disease but were deleted in an advanced stage, possibly asa result of a tumor escape mechanism. We are currently involvedwith experiments to elucidate more precisely a possible relation-ship between the occurrence of anti-OFA/iLRP-specific immuneresponses (humoral and T cell-mediated) and the stage of diseasein patients with CLL and MM.

Taken together, we identified for the first time two distinctHLA-A*0201-specific peptide epitopes derived from the OFA-iLRprotein. These peptides represent useful tools for both conductingtumor immunological studies and vaccination strategies in OFA/iLRP-expressing malignancies.

DisclosuresThe authors have no financial conflict of interest.

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