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The Journal of Immunology, 2006, 176: 6935-6944.
Copyright © 2006 by The American Association of Immunologists

Identification of HLA-A*0201-Presented T Cell Epitopes Derived from the Oncofetal Antigen-Immature Laminin Receptor Protein in Patients with Hematological Malignancies

Sandra Siegel*, Andreas Wagner*, Birte Friedrichs*, Anneke Wendeler*, Lena Wendel*, Dieter Kabelitz{dagger}, Jörg Steinmann{dagger}, Adel Barsoum§, Joseph Coggin§, James Rohrer§, Peter Dreger{ddagger}, Norbert Schmitz* and Matthias Zeis1,*

* General Hospital St. Georg, Department of Hematology, Hamburg, Germany; {dagger} Institute of Immunology, University of Kiel, Kiel, Germany; {ddagger} Department of Stem Cell Transplantation, University of Heidelberg, Heidelberg, Germany; and § Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36602


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The oncofetal Ag immature laminin receptor (OFA-iLR) is a potential target molecule for immunotherapeutic studies in several tumor entities, including hematological malignancies. In the present study, we characterize two HLA-A*0201-presented epitopes eliciting strong OFA-iLR peptide-specific human cytotoxic T cell (CTLs) responses in vitro. Both allogeneic HLA-A*0201-matched and autologous CTLs recognized and killed endogenously OFA-iLR-expressing tumor cell lines and primary malignant cells from patients with hemopoietic malignancies in an MHC-restricted fashion but spared nonmalignant hemopoietic cells. Spontaneous OFA-iLR peptide-specific T cell reactivity was detectable in a significant proportion of leukemia patients. Interestingly, in patients with chronic lymphocytic leukemia and multiple myeloma but not in those with acute myeloid leukemia, significant frequencies of 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 vaccination strategies in patients with OFA-iLR-expressing malignancies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The oncofetal Ag-immature laminin receptor protein (OFA-iLRP)2 is a 37-kDa evolutionary conserved protein that has been detected in several types of human tumor entities such as breast, ovary, prostate, lung, renal cancer, and hematological malignancies (1, 2, 3, 4, 5, 6). Expression of OFA-iLR was also found in embryos and the earlier stage of fetuses but not in term fetus, neonate, or adult differentiated tissues (1, 5). OFA-iLR appears to dimerize after acylation to form the high-affinity mature 67-kDa laminin receptor protein. Although the mature 67-kDa protein, which is present on many normal cells, is nonimmunogenic, the immature form can be specifically recognized by the adaptive immune system (1, 2, 4, 7, 8). Using dendritic cells (DCs) transfected with OFA-iLRP-coding RNA, we have shown previously that tumor-specific T cell responses against hemopoietic target cells can be elicited both in vitro and in vivo (6). Thus, OFA-iLRP can be regarded as a potential target molecule for immunotherapeutic approaches toward human and rodent cancers (1, 4).

In the present study, we identified and characterized two distinct HLA-A*0201-specific OFA-iLR peptide-epitopes using Ag-specific CTLs that were generated by in vitro priming with peptides-pulsed monocyte-derived DCs. We demonstrate that OFA-iLRP peptide-specific CTLs obtained from healthy donors and patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) elicited Ag-specific HLA-A*0201-restricted cytolytic activity against several hematological tumor cell lines and freshly isolated AML and CLL tumor cells. Furthermore, by detecting OFA-iLR-specific T lymphocytes in patients with CLL and multiple myeloma (MM) in an early stage of disease but not in patients with progressive disease, OFA-iLR-specific CTLs can play a role in controlling OFA-iLR+ tumor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients and healthy donors

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


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

 

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

 
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 were obtained from American Type Culture Collection. MEC-1 (chronic lymphocytic leukemia) was a gift from Dr. F. Caligaris-Cappio (University of Torino, Torino, Italy). CD34+ peripheral blood progenitor cells were obtained using MACS technology (Miltenyi Biotec), following the manufacturer’s instructions. The cell purity was verified by FACS analysis (FACScan; BD Biosciences) and reached > 90%.

Peptides and loading of T2 cells

For binding assay and generation of peptide-specific CTLs, the peptide from influenza A virus matrix protein M1, FluM158–66 (GILGFVFTL, positive control), HIV-Pol476–484 (ILKEPVHGV, positive control), msurv33 (LYLKNYRIA, murine survivin peptide epitope specific for H-2d, negative control), iLR159–68 (LLAARAIVAI, Rammensee score: 18), iLR2146–154 (ALCNTDSPL, Rammensee score: 22), iLR360–68 (LAARAIVAI, Rammensee score: 23), iLR458–66 (LLLAARAIV, Rammensee score: 26), iLR57–15 (VLQMKEEDV, Rammensee score: 22), iLR650–58 (NLKRTWEKL, Rammensee score: 21), iLR766–74 (VAIENPADV, Rammensee score: 21), iLR8139–147 (YVNLPTIAL, Rammensee score: 21), iLR9177–185 (MLAREVLRM, Rammensee score: 21), iLR10249–257 (SEGVQVPSV, Rammensee score: 19), iLR1118–26 (FLAAGTHLG, Rammensee 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 to bind to the HLA-A*0201 molecule using the computer programs PAProC (<www.uni-tübingen.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. The HLA-A2-positive mutant cell line T2 was incubated with peptides for 2 h at 37°C and 5% CO2 at various concentrations, washed three times with PBS (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 cytometry analysis based on the use of the HLA-A*0201 TAP-deficient T2 lymphoma cell line using a modified protocol adopted from Casati et al. (9). T2 cells (2.5 x 106/ml) were seeded in RPMI 1640 (Invitrogen Life Technologies) with 2.5 µg/ml beta2-microglobulin (Sigma-Aldrich). Peptides were added at various concentrations. Cells were incubated overnight in a water-saturated atmosphere with 5% CO2 at 37°C. After incubation, cells were washed with cold PBS with 0.05% BSA (Sigma-Aldrich) to remove unbound peptides. To determine surface expression of the HLA-A2 molecule, cells were resuspended in 100 µl of PBS/0.05% BSA and incubated with 10 µl of anti-HLA-A*0201-FITC mAb for 30 min at 4°C. Afterward, cells were washed twice with cold PBS/0.05% BSA. Fluorescence intensity was measured by FACS analysis. Controls consisted of T2 cells cultured with FluM1 (positive control), msurv33, or with no peptide (negative controls).

To determine the stability of the peptide/HLA-A2 complex, 2 x 106 T2 cells were incubated overnight at 27°C in 2 ml of RPMI 1640 supplemented with 10% FCS and 2.5 µg/ml beta2-microglobulin. Peptides were added at a concentration of 10 µM. Cells were then washed and incubated at 37°C for 0, 2, 4, 6, or 8 h. HLA-A2 expression on these cells was determined, as described above. Results are expressed as percentage (over control) of the remaining BB7.2 mean fluorescence intensity. Controls are the BB7.2 mean fluorescence intensity of T2 cells at the onset of incubation at 37°C.

Western blot analysis

Whole cell protein extracts were prepared from 1 x 107 cells using a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40 (Sigma-Aldrich), 2 mM EDTA (pH 8.0), and Complete mini (protease inhibitor; Roche). Cell lysates were separated by 12% gels in SDS-PAGE under reducing conditions for 60 min at 140 V. Proteins were transferred onto 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 20 mM Tris (pH 8.0), 137 mM NaCl, and 0.1% Tween 20 (TBST), and 5% powdered milk overnight. Immunoblottings were performed using the mouse anti-OFA-iLRP mAb at a concentration of 0.75 µg/ml (in TBST and 5% BSA) as primary and horseradish phosphatase-linked goat anti-mouse IgG (Dianova) (diluted to 1/5000 v/v in TBST and powdered milk) as secondary Ab. All immunoblots were performed using Enhanced Chemoluminescence solution (ECL; Amersham Biosciences), following the manufacturer’s instructions.

ELISPOT assay

PBMCs (106/ml) obtained after Ficoll density gradient centrifugation from HLA-A*0201 patients with AML, CLL, and MM were cultured in AIMV medium (Invitrogen Life Technologies) with 5% heat-inactivated human AB serum (CC-Pro) and 10 µg of peptide for 10 days. After 24 h, IL-2 (CellConcepts) was added at a concentration of 20 IU/ml. Nitrocellulose plates (96-well; MAHA S4510; Millipore) were coated overnight with 100 µl/well of 10 µg/ml anti-human IFN-{gamma} mAb (Mabtech) in PBS by 4°C. Afterward, plates were washed three times with 150 µl/well PBS for 5 min and blocked with AIMV medium for 1 h in 5% CO2 at 37°C. After blocking, 1 x 105 precultured cells in a volume of 50 µl/well were seeded, and peptides at a concentration of 10 µg/ml in 50 µl of AIMV medium were added 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) and 0.05% Tween 20 (Sigma-Aldrich), and wells were incubated with 100 µl/well of biotinylated mouse anti-human IFN-{gamma} mAb (Mabtech) at a concentration of 2 µg/ml in TBS for 2 h by 37°C and 5% CO2. After washing six times, ExtrAvidin alkaline phosphatase conjugate (1 µg/ml in TBS; Sigma-Aldrich) was added for 1 h at room temperature. Next, plates were washed three times with TBS/0.05% Tween20 and five times with TBS only. Development of the spots was performed by adding 100 µl/well of 5-bromo-4-chloro-3-indolyl phosphate/NBT substrate for 10–30 min in darkness. IFN-{gamma}-secreting T cells were counted using the automated image analysis system ELISPOT Reader (AID). Each experiment was performed in 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-prestimulated PBMCs (see ELISPOT assay) were incubated with 10 µl of pentamer for 30 min in darkness. Cells were washed and resuspended in 100 µl of wash buffer. PBMCs were incubated with FITC-conjugated anti-CD8 mAb (Caltag Laboratories) for 25 min by 4°C and washed twice with wash buffer. To exclude dead cells, propidium iodide (Sigma-Aldrich) was added at a concentration of 1 µg/ml. Fluorescence intensity was determined by FACS analysis. The percentage of peptide-specific T cells was determined after gating the CD8+ T cells inside the T cell population. Analysis was performed using the CellQuest Pro software.

Generation of mature DCs and loading with peptides

Mature DCs from healthy HLA-A*0201 donors were generated following the protocol of Feuerstein et al. (10). A concentrated leukocyte fraction was generated through a 2-h restricted peripheral blood leukapheresis processing 6–8 liters of blood with each collection. A total of 2 x 108 PBMCs/ml obtained after Ficoll density gradient centrifugation was incubated in 30 ml of serum-free AIMV medium (Invitrogen Life Technologies) for 2 h in a water-saturated atmosphere with 5% CO2 at 37°C. The nonadherent T cell-enriched fraction was cryoconserved and used later as a source of CTL generation. The adherent monocytes were cultured for 5 days with 500 U/ml human IL-4 and 800 U/ml human GM-CSF (CellConcepts) to obtain immature DCs (iDCs). Maturation of DCs was performed in AIMV medium (Invitrogen Life Technologies) by adding human IL-1beta, human TNF-{alpha} (10 ng/ml; CellConcepts), human IL-6 (1000 U/ml; CellConcepts), and human PGE2 (1 µg/ml; Sigma-Aldrich) for 2 additional days at 37°C with 5% CO2.

Mature DCs from patients with AML and CLL were generated by separating PBMCs from 100 ml of whole blood donation, following the protocol described above. Loading of the mature DCs with peptide was performed by incubation of 1 x 106/ml mature DCs with 50 µg/ml peptide for 2 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 previously (6). The vector was transformed into Escherichia coli JM83 by heat shock transformation. After ampicillin selection, plasmid isolation (Plasmid Maxi kit; Qiagen) was performed, and the correct size of the plasmid was checked on a 1% agarose gel. OFA-iLRP sequence was verified by automatic sequencing (ABI 310; PerkinElmer). Resulting plasmid DNA was linearized, and in vitro transcription was performed using the T7 RiboMAX Large Scale Transcription kit (Promega) as described previously (6). EGFP vector was provided by Dr. M. Marget (Institute of Immunology, University of Kiel, Kiel, Germany). Quantity and purity of the RNA was determined by UV spectrophotometry.

OFA-iLR-RNA transfection and maturation of human DCs

Transfection of iDCs with in vitro-transcribed iLRP-RNA was performed by using the liposomal transfection reagent dioleoyloxy-trimethylammonium-propane-methylsulfate (DOTAP; Roche). DOTAP (20 µl) and in vitro-transcribed RNA (10 µg) were mixed in 500 µl of Opti-MEM (Invitrogen Life Technologies) and incubated for 20 min at room temperature. The RNA-lipid complex was added to 1 x 106 iDCs/ml (in Opti-MEM) and incubated at 37°C for 3 h. Maturation of the transfected iDCs was performed by culturing for 2 days in serum-free medium supplemented with GM-CSF, IL-4, IL-1beta, TNF-{alpha} (10 ng/ml; CellConcepts), IL-6 (1000 U/ml; CellConcepts), and PGE2 (1 µg/ml; Sigma-Aldrich). Flow cytometric analysis revealed that ~30% of iLRP-RNA-transfected and maturated DCs expressed iLRP and high levels of CD80, CD83, CD86, MHC class I, and MHC class II molecules.

Generation of CTLs

CTLs were generated, as described previously (11). A total of 2 x 107 cryoconserved nonadherent PBMCs was cultured with 2 x 106/ml autologous OFA-iLR peptide-pulsed DCs in 10 ml of AIMV medium (Invitrogen Life Technologies) supplemented with 20 U/ml human IL-2 and 10 ng/ml human IL-7 (CellConcepts). Cells were weekly restimulated with peptide-loaded DCs.

For the induction of autologous peptide-specific CTLs obtained from patients with AML and CLL, CD8+ T cells were separated from PBMCs using immunomagnetic beads (MACS beads; Miltenyi Biotec), following the 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 three to four restimulations, >90% of cells in each culture were CD3+ lymphocytes, of which >90% were CD8+ and 3.5–4.8% were CD4+. Cultures contained <1.5% CD14+ and 3.5–4.5% CD56+ cells. In another set of experiments, peptide-specific CTLs were enriched from CLL patients using the IFN-{gamma} 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 assay by analyzing the capacity of unlabeled HLA-A2+ iLRP-negative T2 cells, OFA-iLR peptide (iLR1 + iLR2)- and msurv33 peptide-labeled T2 cells, and OFA-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 described above. Tumor cells were incubated with 10 µg/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 controls MAM-6 and HMFG-1 (Beckman Coulter) for 30 min. Cell suspensions were washed twice with complete medium and tested in a 51Cr release assay.

Statistics

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
OFA-iLRP is expressed in hematological malignancies

The expression of OFA-iLRP in or on malignant and nonmalignant cells was assessed by Western blot and FACS analyses using the previously described anti-OFA-iLRP mAb. All hematological tumor samples used in this study showed a strong up-regulation of OFA-iLRP expression. In Fig. 1, some representative samples are demonstrated. As expected, we could not detect any OFA-iLRP expression in nonmalignant hemopoietic cell subtypes (CD34+ progenitor cells, bone marrow cells, B cells, and activated B cells).


Figure 1
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  		FIGURE 1. Detection of OFA/iLRP on or in malignant and normal hemopoietic cells. OFA/iLRP expression on the surface of two distinct B-CLL samples (A and B) was determined by flow cytometry using an anti-OFA/iLRP mAb (see Materials and Methods). Western blot analysis was performed to examine the OFA/iLR protein expression in cell lysates of several normal and malignant hemopoietic cell samples (C).

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

To identify T cell-binding epitopes deduced from the OFA-iLR protein, 14 peptides were synthesized that were predicted by the computer programs PAProC (<www.uni-tuebingen.de/uni/kxi/[Lang]) and SYFPEITHI (<www.syfpeithi.de>) to bind to the HLA-A*0201 molecule (see Materials and Methods). In an in vitro reconstitution assay using the TAP-deficient T2 cell line, two peptides (iLR1 and iLR2) showed similar binding affinity to HLA-A*0201 (Fig. 2A) as the positive control Flum1. In addition, as illustrated in Fig. 2C, the HLA-A2/iLR1 and HLA-A2/iLR2 complexes showed intermediate complex stability when compared with the Flum1 peptide.


Figure 2
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  		FIGURE 2. Peptide binding and titration assay. HLA-A2 binding of 14 iLR peptides was measured using HLA-A*0201-positive TAP-deficient T2 cells and conformation-dependent HLA-A2-specific Abs. Binding efficiency of iLR peptides was compared with that of the known HLA-A2-binding peptide FluM1. Surv33 served as a HLA-A2-nonbinding negative control (A and B). To determine the stability of the peptide/HLA-A2 complex, HLA-A2 stabilization assay (C) was performed, as described in Materials and Methods. Peptide titration experiments were performed using iLR1- and iLR2-specific CTLs directed against T2 cells prior loaded with increasing amounts of the cognate peptides. The HLA-A2-binding HIV-Pol476–484 peptide served as a negative control. The cytotoxic potential of OFA/iLR peptide-specific CTLs were measured in 51Cr release assays (D).

 
In the presence of fully matured DCs pulsed with either the iLR1 or iLR2 peptide, CTL lines could be generated from healthy HLA-A*0201+ donors. To assess the avidity of the induced CTL lines, T2 cells were pulsed with titrated amounts of the peptides iLR1, iLR2, or HIV-Pol476–484 used as a negative control. Using an E:T ratio of 40:1, peptide titration curves demonstrated that iLR1- 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 with hemopoietic malignancies

In a next set of experiments, iLR1 and iLR2 peptides were tested for their potential to elicit specific ex vivo T cell responses in healthy volunteers and in patients with CLL, AML, and MM. To extend the sensitivity of the ELISPOT assay, PBMCs were stimulated with OFA-iLR peptides once in vitro before analysis. As depicted in Fig. 3, in 9 of 21 (43%) and 10 of 21 (48%) of CLL patients, significant frequencies of iLR1 (Fig. 3A)- and iLR2 (Fig. 3B)-specific T cell responses were detected, whereas no spontaneous T cell responses against both peptide epitopes occurred in peripheral blood samples of 15 HLA-A2*0201-positive healthy individuals. In patients with AML, 6 of 15 (40%) mounted a CTL response against iLR1 or iLR2 peptide, respectively. We also analyzed PBMCs from 12 HLA-A*0201-positive patients with MM and detected in 6 samples (50%) a reactivity against iLR1 and iLR2. Additionally, in PBMCs of several leukemia and myeloma patients tested, we also found spontaneous T cell reactivity against the peptides iLR3, iLR4, iLR9, and iLR14 (Fig. 3, C–F). Interestingly, significant frequencies of OFA-iLR peptide-specific CTLs were only found in blood samples of CLL and MM patients in an early stage of disease. In 8 of 14 patients with a Binet A CLL, iLR1- and iLR2-specific T cell reactivity was detected, whereas in CLL patients with progressive disease (Binet C), no OFA-iLR-specific T cell responses occurred (Table I). Similar results were obtained in MM patients.


Figure 3
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  		FIGURE 3. Spontaneous T cell responses against OFA/iLR-derived candidate peptides as measured by IFN-{gamma} ELISPOT. PBMCs from 15 healthy donors, 21 patients with CLL, 15 patients with AML, and 10 patients with MM were analyzed. All individuals were HLA-A-*0201 positive. The peptides iLR1, iLR2, iLR3, iLR4, iLR9, and iLR14 were examined.

 
We next examined whether OFA-iLR peptide-specific T cells were also present in PBMCs from CLL patients treated with an allogeneic stem cell transplant. Whereas no OFA-iLR-specific T cell responses were observed in the ELISPOT assay before transplantation, 6 of 10 allograft recipients developed significant T cell frequencies against both iLR1 and iLR2 peptides (Table II). To analyze whether iLR-specific CTLs obtained from a patient with acute graft-vs-host disease (GVHD) of the skin (patient 1) provide higher avidity responses than CTLs from a patient lacking clinically overt GHVD, peptide titration assays with T2 cells pulsed with the cognate peptide were performed. Fig. 4 illustrates that CTLs from patients 1 and 6 do not differ in their potential to recognize and kill peptide-pulsed T2 cells. In one transplant patient with high numbers of iLR1 and iLR2-specific T cells (patient 6), responding cells were selected by the IFN-{gamma} secretion assay. The majority of the iLR1- and iLR2-specific T lymphocytes was CCR7–CD45RA+ memory effector cells (data not shown).


Figure 4
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  		FIGURE 4. Peptide titration assay using iLR-specific CTLs derived from 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 with increasing amounts of the cognate peptides were used. The HLA-A2-binding HIV-Pol476–484 peptide served as a negative control. The cytotoxic potential of OFA/iLR peptide-specific CTLs was measured in 51Cr release assays.

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

In five CLL patients with significant OFA-iLR responses in the ELISPOT assay, we also determined the frequency of iLR1- and iLR2-specific CD8+ T lymphocytes within the PBMCs by performing pentamer staining. Two-color flow cytometry with anti-CD8 mAb and HLA-A2/iLR1- and HLA-A2/iLR2-pentameric complexes revealed a frequency of CD8+ T cells recognizing the iLR1 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 presence of the HIV peptide (negative control) or the Flum1 (positive control), we detected frequencies between 0.01 to 0.04% and 5.4 to 23.3%, respectively (one representative experiment shown in Fig. 5).


Figure 5
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  		FIGURE 5. Pentamer staining for determining the frequency of iLR-specific CD8+ T cells by flow cytometry. PBMCs from a CLL patient were prestimulated with iLR1 (upper left), iLR2 (upper right) peptide, a negative control HIV-Pol476–484 (lower left), and a positive control FluM1 peptide (lower right) as described in Materials and Methods. The percentage of peptide-specific T cells was evaluated after gating for CD8+ T cells within the PBMCs. The experiment was conducted with samples from five different CLL patients.

 
OFA-iLR peptide-specific CTLs efficiently lyse tumor cells endogenously expressing OFA-iLR

We next analyzed the ability of the in vitro-induced CTLs to lyse tumor cells that express the OFA-iLR protein. Both CTL lines specific for iLR1 (Fig. 6A) or iLR2 (Fig. 6B) elicited strong cytolytic activity against the HLA-A*0201+/OFA-iLR+ hematological tumor lines Karpas-422, Balm-3, IM-9, U266, REH, and Ramos. There was no recognition of the HLA-A2-negative tumor lines MEC-1 and K562. In addition, iLR1- and iLR2-specific T cell lines recognized and efficiently killed HLA-A2+/OFA-iLR+ primary malignant AML blasts (Fig. 7, A and B) and CLL cells (Fig. 7, C and D) but spared HLA-A2-negative targets and normal hemopoietic cells (Fig. 7).


Figure 6
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  		FIGURE 6. Induction of peptide-specific CTL responses by peptide-pulsed DCs. Cytotoxic potential of iLR1- and iLR2-specific CTLs against untransfected DCs and DCs loaded with FluM1 peptide, either enhanced GFP- or iLRP-RNA-transfected DCs. iLR1- and iLR2-specific CTL reactivity against OFA+/iLR+ HLA-A2+ and HLA-A2– hematological cell lines (C and D).

 

Figure 7
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  		FIGURE 7. ILR peptide-specific CTLs recognize and kill primary malignant cells from AML and CLL patients. ILR1- and iLR2-induced CTLs from healthy donors lysed freshly isolated malignant cells from HLA-A2+ AML patients but not nonmalignant HLA-A2+CD34+ progenitor cells and bone marrow cells. HLA-A2– tumor cells were not recognized (A and B). OFA/iLR-specific CTLs from healthy donors lysed freshly isolated leukemia cells from HLA-A2+ CLL patients, whereas nonmalignant HLA-A2+ B cells were not lysed (C and D).

 
Cold target inhibition assays and Ab-blocking experiments revealed an MHC class I-restricted killing induced by peptide-specific CD8+ T lymphocytes (Fig. 8, A and B).


Figure 8
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  		FIGURE 8. Cold target inhibition and Ab-blocking experiments. The specificity of OFA/iLR-specific CTL lines was tested in a 4-h 51Cr release assay in the presence of unlabeled cold targets, T2 cells, iLR1, iLR2, and mSurv peptide-pulsed T2 cells and primary malignant blasts from a HLA-A2+ patient (AML-01). Cold targets were added at an inhibitor to target ratio of 20:1 (A and C). To examine MHC-dependent cytotoxicity, Ab-blocking experiments were performed in a 4-h 51Cr release assay (B and D). One of two representative experiments is shown.

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

To test the presentation of OFA-iLR-specific T cell epitopes upon transfection with OFA-iLR-coding RNA, we used autologous DCs, generated from the same PBMCs that were used for CTL induction, as target cells. As illustrated in Fig. 5, C and D, ILR1- and iLR2-specific CTLs efficiently lysed autologous DCs transfected with the OFA-iLR-coding RNA, whereas DCs pulsed with the Flum1 peptide or the enhanced EGFP-RNA were not recognized. These findings demonstrate that the identified OFA-iLR peptides iLR1 and iLR2 are processed and presented after transfection of DCs with the OFA-iLR-RNA.

OFA-iLR peptide-specific CTLs lysed autologous malignant hemopoietic 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 lines that were specific for the peptides iLR1 and iLR2. These CTLs were capable of efficiently lysing autologous and allogeneic HLA-A2-matched malignant cells endogenously expressing OFA-iLR protein, whereas HLA-A2-negative tumor cells were not recognized and killed. To provide more direct evidences that patient-derived iLR-specific CTLs are functionally active, CD8+ T lymphocytes specifically recognizing the iLR1 and iLR2 epitopes separately were highly enriched from peripheral blood samples obtained from a CLL patient (CLL-02). As depicted in Fig. 9, E and F, both iLR1- and iLR2-specific epitopes CTLs were capable of recognizing autologous tumor cells, whereas allogeneic CLL cells were not attacked.


Figure 9
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  		FIGURE 9. Lysis of autologous and allogeneic leukemic cells by iLR1- and iLR2-specific CTLs. Cells from HLA-A2+ patients with CLL and AML were stimulated with iLR1- and iLR2-pulsed autologous DCs: cytotoxicity against autologous and allogeneic freshly isolated CLL cells and AML blasts cells was measured in a 4-h 51Cr release assay (A and B). One of two experiments is depicted here. Using IFN-{gamma} secretion assay, autologous iLR-CTLs were enriched and tested against autologous (CLL-01) and allogenic (CLL-06) target cells (E and F).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
OFA-iLRP is a tumor-associated protein, which is widely expressed and detectable in the majority of tumor entities such as sarcomas and carcinomas, including hemopoietic malignancies (1, 2, 3, 4, 5, 6, 12). Several studies documented the immunogenicity of OFA-iLR protein specifically recognized by T and B lymphocytes (1, 7, 8, 9, 13, 14, 15). In a previous report, we were able to demonstrate that OFA-iLRP-specific CTLs generated in the presence of fully matured autologous OFA-iLRP-RNA-transfected DCs were capable of eliminating both tumor cell lines and malignant cells of AML and CLL patients (6).

In the current study, we screened the OFA-iLR protein for HLA-A2-binding peptides using the computer-assisted software SYFPEITHI. In a standard T2-binding assay, 2 of 14 candidate peptides, iLR1 and iLR2, exhibited a high binding affinity to the HLA-A*0201 molecule. To analyze whether these epitopes are presented by tumor cells endogenously expressing OFA-iLR protein, we generated OFA-iLR peptide-specific CTLs in the presence of monocyte-derived autologous fully matured DCs that had been loaded with iLR1 and iLR2, respectively. After several rounds of restimulations, T cell lines were tested for their cytotoxic capacity against OFA-iLR-expressing hematological tumor lines and primary malignant cells from CLL and AML patients. The in vitro-induced peptide-specific CTLs not only recognized and killed tumor lines but also freshly isolated patient-derived leukemic cells. The specificity of the lytic activity was confirmed by Ab-blocking experiments and cold target inhibition assays.

As shown in Fig. 3, spontaneous T cell responses were also detectable against the OFA-derived peptides iLR3, iLR4, iLR9, and iLR14 having a lower binding affinity to the HLA-A2 molecule 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 amino acid sequence, the peptides iLR9177–185 (MLAREVLRM) and iLR14173–182 (LMWWMLAREV) might represent another potential distinct T cell epitope. Immunological studies are currently underway in our lab to analyze these peptides further.

We further analyzed the specificity of in vitro-generated CTL lines recognizing iLR1 and iLR2 epitopes by using autologous DCs as target cells loaded with the cognate peptides or transfected with the OFA-ILR-coding RNA. ILR1 and iLR2 peptide-specific CTLs were capable of efficiently lysing both peptide-pulsed DCs and DCs transfected with OFA-ILR-RNA. These data clearly demonstrate that the peptides used for CTL induction are endogenously processed and presented by OFA-iLR RNA-transfected DCs. In two patients with hematological malignancies, we were able to generate autologous iLR1 and iLR2 peptide-specific CTLs recognizing and killing autologous OFA-iLR-expressing leukemic cells in an MHC-restricted and Ag-specific manner. These findings, together with the ELISPOT analyses and the pentamer stainings, demonstrate that OFA-iLR-specific CTLs can be detected and generated in patients with hemopoietic malignancies.

The induction of autoimmunity in individuals treated with tumor-associated vaccines represents a potential risk and thus merits careful consideration (16). Our previous studies (6) and the findings described in the present study provide some evidence that the autoimmunogenic potential of OFA-iLR-derived peptides seems to be low. First, by using ELISPOT assay and pentamer staining, we detected OFA-iLR peptide-specific T lymphocytes in a significant proportion of patients without the occurrence of a clinical manifestation 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 marrow cells; third, OFA-iLR epitopes were not recognized by T lymphocytes from healthy donors; fourth, iLR-specific CTLs obtained from a patient with a clinically overt autoimmune disease (GVHD) displayed no differences in their functional avidity than CTLs from a patient lacking GVHD; and fifth, treatment of mice with syngeneic bone marrow-derived DCs transfected with the murine OFA-iLR-coding RNA resulted in significant antitumor activity against a B cell lymphoma without inducing any macroscopic or histologic signs of autoimmune reactivity (6). We have shown previously that OFA-iLR-specific regulatory CD8+ T cell clones secreting IL-10 can be identified both in mice bearing an OFA/iLR+ tumor (12, 17) and in patients with advanced breast carcinomas (17, 18). In a small series of CLL patients with early (Binet A, n = 4) and progressive disease (Binet C, n = 5), no relevant IL-10-secreting T cells specific for the iLR1 or iLR2 peptide were detectable (data not shown). However, as another important result of our study, we were able to detect significant frequencies of OFA-iLR-specific IFN-{gamma}-secreting CTLs in the PBMCs of early-stage patients with CLL and MM when compared with patients with progressive disease (see Table I). We hypothesize that OFA-iLR-specific CTLs can play a role in controlling OFA-iLR+ tumor cells in the early phase of disease but were deleted in an advanced stage, possibly as a result of a tumor escape mechanism. We are currently involved with experiments to elucidate more precisely a possible relationship between the occurrence of anti-OFA/iLRP-specific immune responses (humoral and T cell-mediated) and the stage of disease in patients with CLL and MM.

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


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Matthias Zeis, Department of Hematology, General Hospital St. Georg, Lohmühlenstrasse 5, D-20099 Hamburg, Germany. E-mail address: mzeis47{at}hotmail.com Back

2 Abbreviations used in this paper: OFA/ILRP, oncofetal Ag-immature laminin receptor; DC, dendritic cell; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; BM, bone marrow; MM, multiple myeloma; iDC, immature DC; DOTAP, dioleoyloxy-trimethylammonium-propane-methylsulfate; GVHD, graft-vs-host disease. Back

Received for publication August 11, 2005. Accepted for publication February 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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