Abstract

BACKGROUND AND OBJECTIVES: There is an urgent need for the development of leukemia-targeted immunotherapeutic approaches using defined leukemia-associated antigens that are preferentially expressed by most leukemia subtypes and absent or minimally expressed in vital tissues. M-phase phosphoprotein 11 protein (MPP11) is extensively overexpressed in leukemic cells and therefore is considered an attractive target for leukemia T cell therapy. We sought to identify potential CD8+ cytotoxic T lymphocytes that specifically recognised peptides derived from the MPP11 antigen.

MATERIALS AND METHODS: A computer-based epitope prediction program SYFPEITHI, was used to predict peptides from the MPP11 protein that bind to the most common HLA- A*0201 molecule. Peptide binding capacity to the HLA-A*0201 molecule was measured using the T2 TAP-deficient, HLA-A*0201-positive cell line. Dendritic cells were pulsed with peptides and then used to generate CD8+ cytotoxic T lymphocytes (CTL). The CML leukemic cell line K562-A2.1 naturally expressing the MPP11 antigen and engineered to express the HLA-A*0201 molecule was used as the target cell.
RESULTS: We have identified a potential HLA-A*0201 binding epitope (STLCQVEPV) named MPP-4 derived from the MPP11 protein which was used to generate a CTL line. Interestingly, this CTL line specifically recognized peptide-loaded target cells in both ELISPOT and cytotoxic assays. Importantly, this CTL line exerted a cytotoxic effect towards the CML leukemic cell line K562-A2.1.
CONCLUSION: This is the first study to describe a novel epitope derived from the MPP11 antigen that has been recognized by human CD8+ CTL.

Despite major advances in molecular biology and targeted therapy of leukemia, current treatment strategies induce adverse side effects and fail to achieve and maintain remission in many patients. In acute myeloid leukemia (AML), the most deadly form of leukemia,^1,2 the majority of adult patients younger than 60 years achieve a complete remission following consolidation therapy. However, only approximately 30% to 40% maintain durable remission.³ Furthermore, the complete remission rate and duration attained by older AML patients is less, with an overall survival rate of 20% to 40%.3 Therefore, there is an urgent need for the development of leukemia-targeted immunotherapy designed to eliminate residual leukemic cells thus enhancing the graft-versus-leukemia (GVL) effect observed after allogeneic hematopoietic stem cell transplantation (HSCT) and/or prolonging a complete remission achieved by chemotherapy.^4,5

Leukemia cells express unique antigens or overexpress normal cellular antigens.^6,7 The overexpressed normal antigens constitute attractive targets for the development of leukemia immunotherapy.⁶ Among this group of antigens are the M-phase phosphoprotein 11 (MPP11) proteins. MPP11 was discovered by Matsumoto-Taniura et al^8,9 using an MPM2 monoclonal antibody, which recognizes several important mitosis phosphoproteins found to be present during mitosis.^8-10 Recently, it has been reported that MPP11 functions as a ribosome-associated molecular chaperone.^11,12 MPP11 maps to the region of chromosome 7q22-31.1, which is a critical common position in human cancer.^10,13 MPP11 has been detected by serological analysis of cDNA expression libraries (SEREX) in solid tumours and hematological malignancies including melanoma, breast, renal cell carcinoma, small cell lung cancer and leukemia.^14,15 The humoral immune responses to MPP11 have been detected in patients with acute (AML) and chronic myeloid leukemia (CML), but not in healthy donors or patients with autoimmune diseases.15 Moreover, this protein has been found to be expressed extensively in AML and CML patients as compared to normal controls.^15-18

Immunohistochemical staining of primary tumor sections and Western blot analysis of head and neck squamous cell cancer (HNSCC) cell lines revealed tumor-specific overexpression of MPP11 protein.^13,15 Additionally, fluorescence in situ hybridization analysis carried out on HNSCC cell lines showed an increase in the copy number of MPP11 along with chromosome 7 suggesting an oncogenic role for MPP11.¹³ The specific overexpression of MPP11 by tumor tissues (with the exception of very low expression in testis, kidney, and lung) makes it an attractive target for cancer immunotherapies.^15,17

We sought to identify potential CD8+ cytotoxic T lymphocyte (CTL) epitopes in the leukemia-associated MPP11 antigen that bind to the most common HLA-A*0201 molecule and provoke specific CTL responses. Although various antigenic epitopes derived from leukemia antigens have been identified, progress in targeting specific immunotherapeutic approaches depends on the recognition of a wider range of leukemia antigens. This would allow the use of these peptides in leukemia immunotherapy by either expanding ex vivo tumor-reactive specific CTLs for adoptive T cell therapy or actively inducing leukemia antigen-specific T-cell immunity in vivo by vaccinating patients with well-defined leukemia-associated antigens.

Methods

Leukocyte-rich buffy coats not older than eight hours were obtained from healthy volunteers from the blood bank. All samples were obtained with informed consent at King Faisal Specialist Hospital and Research Centre (KFSHRC). Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from heparinized blood by density gradient centrifugation over Ficoll-Paque PLUS (Amersham Biosciences) according to the manufacturers' instructions. PBMCs were suspended in 90% Fetal Calf Serum (FCS) + 10% dimethyl sulfoxide (DMSO), aliquoted and cryopreserved in liquid nitrogen until further processing. The HLA typing of these samples was carried out at the routine immunopathology laboratory.

T lymphocyte epitope prediction and peptide synthesis
The SYFPEITHI prediction algorithm is a database comprising more than 4500 peptide sequences known to bind class I and class II MHC molecules. The scoring system of the SYFPEITHI program is based on the presence of certain amino acids in certain positions along the MHC-binding groove.¹⁹ Peptides qualified as positive if the score ranked ≥22. The amino acid sequences of MPP11 were entered into the specified program and candidate epitopes were selected based on their predicted ability to bind to the HLA- A*0201 molecule. Four 9-mer peptides derived from MPP11 were selected as potential epitopes for the generation of CTL responses. The four peptides were: MPP-1N=MMP11-421-429 (QLLIKAVNL), MPP-2=MP11-419-427 (DLQLLIKAV), MPP-3=MPP11-313-321 (AIMLLLPSA), and MPP-4=MPP11-437-445 (STLCQVEPV). In addition to the predicted peptides, we modified the sequence of the first predicted peptide derived from MPP11 by introducing a Y at its N-terminus (wild type MPP-1N= QLLIKAVNL, analog MPP-1Y=MPP11-1Y(YLLIKAVNL) according to our recent study.²⁰ This approach enhanced both the binding and the immunogenicity of the modified peptide. The amino acid sequences of the nonameric peptides with a purity of ≥90% were synthesized and purified by Alta Bioscience, Birmingham, UK.

T2 binding assay
Peptide binding capacity to the HLA-A*0201 molecule was measured using the TAP-deficient, HLA-A*0201-positive cells as described previously.21 Briefly, T2 cells were washed three times, suspended at 106 cells/mL and incubated for 18 hours at 37ºC in serum-free medium containing 30 µM of each peptide and 1 mg/mL b2-microglobulin (Sigma). Cells were then washed twice with cold FACS buffer (phosphate buffered saline containing 2% FCS). Purified rabbit IgG (Sigma) was added to the cell suspension and incubated for 15 minutes on ice in order to block the FC receptors. FITC-conjugated mouse anti-HLA-A2 antibody was added to the cell suspension (1 µg/10⁶ cells), with a control sample labelled with isotype-matched monoclonal antibody and incubated for 30 min at 4ºC in the dark. The cells were washed twice with cold FACS buffer, and fixed with 300 µL of PBS/4% paraformaldehyde. The level of HLA-A2 expression was analyzed using fluorescence-activated cell sorter (FACS) scan (Becton & Dickinson, Immunocytometry Systems, CA, USA). HLA-A2 expression was quantified as fluorescence index (FI) according to the following formula: fluorescence index = (mean fluorescence intensity with peptide-mean fluorescence intensity without peptide)/mean fluorescence intensity without peptide. All T2 binding assays were carried out in duplicate.

Cell lines
The mutant TAP-deficient cell line T2 (ATCC number CRL-1992) and the HLA-class I and II negative human CML cell line K-562 (ATCC number CCL-243) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). K-562 cells transfected with the HLA-A*0201 gene22 were kindly provided by Dr. Wolfgang Herr (University of Mainz, Germany). The T2 and K562 cells were maintained in complete medium (CM) consisting of RPMI 1640 medium (Sigma, MO, USA) supplemented with 10% FCS (Cambrex Bio Science, MD, USA), 2 mM L-glutamine, 100 IU/mL penicillin and 100 mg/mL streptomycin (Sigma). K562/ A*0201 were cultured in CM containing 0.5 mg/mL geneticin (G418, Sigma). B-lymphoblastoid cell lines (LCLs) were established from normal donors by transformation of B cells using Epstein-Barr virus (EBV) according to a standard technique.23 Briefly, PBMCs (107 cells) were incubated with the virus (EBV virus stock, B95.8) for an hour at 37ºC. The cells were cultured in CM containing 1µg/mL PHA (PHA-P, Sigma) and 0.1 ug/mL cyclosporin A, and established LCLs lines were used after 3-4 weeks of culture.

Generation of DC2d dendritic cells (Fast-DC)
Monocytes (CD14+ cells) were isolated from PBMCs of healthy HLA-A*0201 positive donors by plastic adherence. PBMCs were cultured at 5X10⁶ cells/mL/well of a 6-well plate (Sigma) in X-vivo 15 medium (DC-medium), and allowed to adhere in a 5% CO2 incubator at 37ºC for 90-min. Non-adherent cells and media were removed and fresh DC-medium was added to the cells. After a second incubation period, non-adherent cells and media were removed and the adherent cells were washed carefully with pre-warmed medium. The DC2d were generated according to previously published protocols^24,25 with minor modifications. Briefly, DC-medium supplemented with 100 ng/mL rhGM-CSF and 50 ng/mL rhIL-4 (R&D Systems) was added to monocytes cultured in 6-well plates. Maturation cytokines, 10 ng/mL rhTNFa, 10 ng/mL rhIL-1b, 10 ng/mL rhIL-6 (R&D Systems) and 1 mM PGE2 (Sigma), were added to the cultures on the next day. Cultures were incubated for an additional day before mature DC2d were harvested.

Generation of anti-peptide specific CD8+ T lymphocytes
CD8+ T lymphocytes were negatively separated from PBMCS obtained from healthy HLA-A*0201 positive donors using MACS CD8+ T Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer's protocols. Some of the CD8+ T lymphocytes lines were generated using a protocol adapted from previous studies.^21,26

Generation of T lymphocyte clones
T lymphocytes lines were cloned by limiting dilution as described before^25,27 with minor modifications. T lymphocytes were seeded at 1, 2 and 5 cells/well/200 µL T lymphocytes culture medium in 96-well U-bottomed plates. Allogeneic PBMCs were X-irradiated (2500 rads) and used as a feeder layer (25X10³ cells/well). T2 cells were incubated separately in X-Vivo 15 medium (5X10⁶ cells/mL) with 5 µg/mL b2-microglobulin and 50 µM/mL of MPP11 peptides for 2 hours at 37ºC. T2 cells were then irradiated (7500 rads), washed once and used for T lymphocytes stimulation (50X10³ cells/well) in the presence of 130 IU/mL of rhIL-2.5 ng/mL rhIL15 (R&D Systems), and 30 ng/mL OKT3 (R&D Systems), were also added to the wells. The plates were incubated for 2-4 weeks. Cultures were re-fed with fresh medium containing rhIL-2 (130 IU/mL) every 3-4 days. Wells with positive signs of growth were selected for expansion. Growing clones were transferred to 96-well flat-bottomed plates, and each clone was stimulated with X-irradiated peptide-pulsed T2 cells. The activation cycle was repeated after 7-10 days of culture at T lymphocyte: T2 ratios of 20:1 to 5:1. Clones were screened for their lytic activity against T2-pulsed with or without peptide using chromium release assay.

Cytotoxicity assay (chromium release assay)
Cytotoxicity assays were performed 5 days post in vitro stimulation. Target cells were removed from culture, washed in RPMI1640 serum-free medium, re-suspended in a minimal volume (± 50 µL) of RPMI1640 and incubated with 51Cr (100 µCi) per target for 90 min. Target cells labelled with peptide were concurrently incubated with the appropriate peptide at 50 µM/mL. The cells were then washed and placed in 96 V-bottomed wells at 103 cells/100 µL/well. The effector T cells were washed, and added in triplicate at varying quantities to the target cells, to give varying effector to target ratios (E:T ratios) in a final volume of 200 µL/well. The plates were spun for 5 min (300 g) and incubated for 4 hours at 37ºC and 5% CO2 before 100 µL supernatants were removed and transferred to 1450 Microbeta Plus Wallac plates (Wallac, Turku, Finland). 150 µL scintillation fluids, Optiphase HiSafe 2 (Wallac), were added to each well and the plates were heat-sealed using a Microsealer system (Wallac). Briefly, T2 cells were washed three times in serum-free medium and incubated with each peptide at concentrations of 50 µM/mL and 1 µg/mL b2-microglobulin at 37 °C, 5% CO2 for 2 hours. The peptide-pulsed T2 cells were then X-irradiated (7500 rads), washed and added to freshly purified CD8+ T lymphocytes (T lymphocytes:T2 ratio of 5:1). After 7-10 days of stimulation, the second stimulation was performed and on the following day, 20 IU/mL of rhIL-2 (R&D Systems) was added. The cultured T lymphocytes were maintained by re-stimulations with peptide-pulsed T2 cells. After the third in vitro stimulation, the generated T lymphocytes were tested for their specificity and cytotoxic activity using enzyme-linked immunospot assay (ELISPOT) and chromium release assays. DC2d-loaded peptides were also used to in vitro prime some of the purified CD8+ T lymphocytes. Briefly, mature DC2d cells were harvested, washed in serum-free medium and incubated with the peptides at concentration of 50 µM/mL and 5 µg/mL b2-microglobulin for 2 hours at 37°C, 5% CO2. The peptide loaded DCs were then X-irradiated (2500 rads), washed once and added to the CD8+ T lymphocytes at 20:1 to 5:1 T lymphocytes: DCs ratio. rhIL-7 (20 ng/mL) and rhIL-12 (100 pg/mL) (R&D Systems) were added to each culture. The cells were incubated in 2 mL T lymphocyte culture medium in 24-well plates. After 7-10 days, T lymphocytes were harvested, washed and re-stimulated with peptide loaded DC2d as previously described and rhIL-2 (20 IU/mL) was added the following day. The cultured T lymphocytes were maintained by re-stimulation with autologous peptide-loaded adherent monocytes and feeding with rhIL-2 the next day. T lymphocytes lines against the different peptides were generated. Responder T lymphocytes were tested for their peptide specificity and cytotoxic activity using enzyme-linked immunospot assay (ELISPOT) and chromium release assay against different targets.

Chromium release was assessed by a liquid scintillation counter (Wallac). Target cells were also incubated with 0.2% Tween 20 or medium alone to assess the maximum and minimum (spontaneous) release of the chromium respectively. Spontaneous release was never exceeded 20% of the maximum release. The percentage of specific lysis was calculated as: % specific lysis = (experimental release-spontaneous release)/(maximal release-spontaneous release)_100. In some of the experiments 10 µg/mL anti-class I ABC blocking antibody (clone: W6/32, AbD Serotec) was added to target cells before T lymphocytes addition.

Enzyme-linked immunospot assay (ELISPOT)
ELISPOT assay was performed using IFN-gamma, granzyme B, and perforin kits (Mabtech, Mariemont, OH) according to the manufacturer's protocols. 5_103 to 5_104 T lymphocytes/well and 2_104 to 105 cells/well of different stimulators were seeded in Multiscreen 96-well plates (Millipore, MA) pre-coated overnight (4ºC) with catching-antibody. The plates were then blocked with T lymphocytes culture medium. After 40 hours incubation (37ºC, 5% CO2), cells were removed and after washing, biotinylated monoclonal antibodies specific for IFN-_, granzyme B, or perforin were added and incubated for 3 hours at room temperature (RT). After washing, Streptavidin-alkaline phosphatase28 or Streptavidin-horseradish peroxidase (HRP) were added to each well and incubated at RT for 2 hours. After washing, the appropriate substrate (BCIP/NBT in case of ALP and AEC in case of HRP) was added to each well and incubated at RT until color developed according to the manufacturer's instructions. Spots were counted using an automated ELISPOT reader (AID, Strasberg, Germany). Antigen-specific T lymphocytes frequencies were considered to be significantly higher if they were at least two-fold higher than in the control wells.

Results

Selection of potential CTL epitopes derived from the MPP11 protein
The amino acid sequences of MPP11 were entered into the computer program and potential candidate epitopes were selected, synthesized and tested for their capacity to bind to the HLA-A*0201 molecule and/or generate cytotoxic T lymphocytes responses. We selected four native peptides derived from MPP11 protein using the Syfpeithi server available at www.syfpeithi.de. These peptides showed high theoretical binding affinity to the HLA-A*0201 molecule with binding scores ranging from 22 to 25. In addition to the predicted peptides, we have carried out tyrosine amino acid substitution in the P1 position of the first predicted epitope MPP-1Y (QLLIKAVNL). The binding score of the modified epitope, MPP-1Y (YLLIKAVNL), was enhanced from 25 to 27 as assessed by the Syfpeithi program. The sequences of the peptides as well as their binding scores are presented in Table 1.

We used the standard T2 binding assay to evaluate the ability of MPP11 candidate peptides to bind and stabilize the HLA-A*0201 molecule using the well-characterized A2-binding peptides WT1-187 as a positive control (WT1 derived peptide known to effectively bind to the HLA-A*0201 molecule.20,29 The human processing-defective T2 cell line express empty HLA class I molecules. The binding assay is based on the stabilization of HLA class I molecules on the cell surface by the addition of peptides exogenously. The T2 cells are incubated with peptides overnight and if the peptide binds to the HLA-A*0201, the surface expression will be stabilized and there will be an upregulation of this molecule. Changes in its expression were assessed by staining HLA-A*0201 and quantifying the fluorescence intensity by flow cytometry analysis. The peptides are assumed to bind if their fluorescence ratio is greater than 1. All T2 binding assays were done in duplicate. As shown in Figure 1, the MPP-3 and MPP-4 peptides (Fluorescence Index [FI] =1.1 and 2.5 respectively) stably bind to the HLA*A-0201 molecule. The affinity of the MPP-4 peptide to the molecule was comparable with that observed for the positive control WT1-187 epitope (FI=2.6). Because of their high binding affinities, we selected MPP-3 and MPP-4 peptides for the generation of peptide-specific T lymphocytes lines. On the other hand, the peptides MPP-1N, MPP1-Y, and MPP-2 were excluded from testing for immunogenicity since they did not show any binding or only very weak binding to the HLA-A*0201 molecule.

Generation of MPP11-specific T lymphocytes lines
We tested the ability of the MPP11-derived peptides to induce specific cytotoxic T lymphocytes (CTLs). We used PBMCs obtained from four different HLA-A*0201 positive healthy volunteers to generate CTLs specific to the selected peptides. The four donors were designated as BC-21, BC-32, BC-37 and BC-41. PBMCs from each donor were used as the source of the responder cells and antigen presenting cells. The responder cells were CD8+ T cells purified by MACS negative selection and stimulated in vitro with antigen presenting cells loaded with peptide for 7-10 days. Initially, we used T2 cells pulsed with peptides to prime and activate T lymphocytes raised against MPP11-derived peptides. However, we did not observe any specific CD8+ T lymphocyte responses to any of the peptides tested including the WT1-126N positive control peptide. Therefore, we used another protocol for the generation of CD8+ T lymphocyte lines where peptide-pulsed DC2d were used to prime the CD8+ T lymphocytes and peptide-pulsed monocytes were used for subsequent T lymphocyte stimulations. To investigate whether the MPP11 synthetic peptides could stimulate peptide-specific CTLs, the generated T cell lines were tested against different HLA-A*0201+ targets in the presence or absence of autologous peptide using a chromium release assay and an ELISPOT assay after 3 to 8 rounds of stimulations.

Cytotoxic T cell lines generated against MPP-4 (STLCQVEPV) peptide produced specific IFN-gamma
After the third round of stimulation, the specificity of in vitro primed CTLs was first tested for specific IFN-_ release by stimulation with the corresponding peptides using an ELISPOT assay. The CTLs generated against the MPP-4 (STLCQVEPV) peptide produced specific IFN-_  spots when stimulated with T2-loaded peptide whereas non-specific production was seen in case of CTLs generated against the MPP-3 (AIMLLLPSA) peptide as shown in Figure 2A and B.
Because CD8+ T lymphocytes are known to use cytotoxic factors such as granzyme B and/or perforin to induce killing of their target cells, so we tested the MPP-4- CTL for the release of granzyme B and/or perforin after 5 rounds of stimulation. MPP-4 pulsed T2 or autologous LCLs were used as stimulators. There was no specific production of granzyme B and/or perforin to the peptide MPP-4 due to very high background responses seen when T lymphocytes were used alone in the assay (Figures 2c and 2d).

T lymphocytes generated against the MPP-4 peptide specifically lyse MPP-4-pulsed target cells and leukemic cells
The expanded MPP11 T lymphocyte lines generated against the MPP-4 and MPP-3 peptides were tested for their specific cytotoxicity against T2 cells and autologous LCLs using a chromium release assay. Specific lysis of 17.8% against the T2 cells loaded with the MPP-4 peptide compared to 0% in the absence of peptide was recorded for TCL21 MPP-4 at effector to target ratio (E:T) of 50:1 (Figure 3A). In addition, 35% specific lysis for the same T lymphocytes line was recorded against autologous LCL loaded with the MPP-4 peptide compared to 6% against autologous LCL in the absence of the peptide at E: T ratio of 50:1 (Figure 3b).

The cytotoxic activity of the MPP-4 T lymphocytes line against the leukemic K562-A2.1 cell line was also measured (K562-A2.1 cells which naturally express the MPP11 antigen was transfected with the HLA-A*0201 molecule). Interestingly, the MPP-4 CTLs exerted a lytic activity of 14% against this leukemia cell line at an E:T ratio of 50:1, whereas only 7% lysis towards the wild type K-562 (lacking the HLA-A2 expression) was recorded at the same E:T ratio (Figure 3c). Similar to the ELISPOT results, the T lymphocytes line generated against the MPP-3 peptide did not show any specific cytotoxic activity (Figure 3d).

Discussion

CD8+ CTLs recognize endogenously processed peptides^3,30 consisting of 8-11 amino acids, most commonly 9 amino acids long, presented on the cell surface of target cells in the context of major MHC class I molecules. These cells are believed to play an important role in identifying and eliminating tumor cells.³¹ To date, several CD8+ CTL epitopes derived from leukemia antigens have been identified, and some of them have been investigated in clinical trials.^32-34 Importantly, the graft-versus-leukemia (GVL) effect often seen after allogeneic hematopoietic stem cell transplantation (AHSCT) has been found to be mediated mostly by donor-derived CD8+ T lymphocytes recognizing peptides derived from leukemia antigens and/or minor histocompatibilty antigens (mHAs) presented on recipient cells.^35-37 Overexpressed antigens such as WT1, PR-3 and MUC1 represent possible targets of specific GVL reactions.^38-40
 
Previous studies have reported the existence of a humoral response against the MPP11 protein as well as the overexpression of this protein in patients with myeloid leukemias.^15-17 However, the identification of CD8+ CTL epitopes derived from this protein is a crucial contribution to the field of leukemia immunotherapy. In this study, we investigated potential CD8+ CTL epitopes derived from the MPP11 antigen using a reverse immunology approach.⁴¹ The reverse immunology approach permits the identification of class I as well as class II candidate epitopes derived from known tumor antigens on the basis of HLA-binding motifs.^42,43 Using this approach, numerous epitopes were identified in leukemia^21,29,44,45 as well as in solid tumours.^46-51 We first examined the amino acid sequences derived from the MPP11 protein for the presence of peptides containing binding motifs for one of the most frequent A*0201 class I alleles,52 using a computer-based epitope prediction program. We identified four MPP11-derived peptides predicted to bind with high affinity to the HLA-A*0201 molecule. The peptides were named MPP-1N, MPP-2, MPP-3 and MPP-4. Using the T2 binding assay, we tested the actual binding capacity of the selected peptides. The MPP11-derived peptides MPP-3 and MPP-4 recorded binding scores lower than MPP-1N, and MPP2 scores as determined by the SYFPEITHI software. However, in the actual T2 binding assay, MPP-3 and MPP-4 were found to have higher binding affinities compared to the two other peptides. This is probably due to a very poor dissolution of the MPP-1N and MPP-2 peptides since more hydrophobic amino acids are contained within their sequence. In addition to the four predicted peptides, we modified the amino acid sequence of the highest scoring peptide i.e., MPP-1N (QLLIKAVNL). We²⁰ and others^53,54 have shown that introducing a tyrosine amino acid residue at the first position (P1Y) of a peptide enhances epitope immunogenicity. Therefore, a tyrosine amino acid residue was introduced in P1 of the MPP-1N peptide to boost its binding affinity and/or immunogenicity. The modification of MPP-1N peptide enhanced the binding affinity of the modified MPP-1Y peptide using the algorithm prediction but in the actual binding assay, the analogue peptide could not stabilize the HLA-A*0201 molecule which is again may be due to its poor dissolution.

Consistent with other groups^21,26,29 who have succeeded in identifying CD8+ CTL epitopes using a simple method that employs T2 cells as antigen-presenting cells, we could not generate specific immune responses using this method to both MPP11-derived peptides as well as WT1-126N peptide which was included as a positive control. Since MPP11 and WT1 are self proteins, the majority of high avidity CD8+ T lymphocytes may have been deleted during thymic maturation.^5556,57 Therefore, we used DCs as antigen-presenting cells for the generation of anti-MPP11 CTL responses. DCs are known as the professional antigen presenting cells because of their unique ability to stimulate naïve T lymphocyte responses. The conventional protocol for generating monocyte-derived DCs included 7-9 days of culture, however, Dauer et al,²⁴ described a rapid protocol for the generation of mature DCs from monocytes within only two days of in vitro culture. Such cells, termed fast DC or DC2d, were found to be capable of inducing tumor-specific CD8+ CTL responses as effectively as DCs generated according to the conventional 7-day protocol.^25,58 Moreover, CTL lines primed with the DC2d cells expanded more effectively and showed greater lytic activity than lines stimulated with the DC7d cells.^25,58 Using the DC2d protocol, we were able to detect antigen-specific responses by the CTL-21 line generated against the MPP-4 peptide. MPP-4-CTLs selectively recognized peptide-loaded T2 cells and specifically responded to the targets by releasing IFN-g in an ELISPOT assay. Importantly, these T lymphocytes specifically killed T2 cells and autologous LCLs when loaded with the MPP-4 peptide. Moreover, they were capable of recognizing the leukemia cell line K562-A2.1 expressing the MPP11 protein. Even though T2 cells were not efficient at priming and generating specific-CD8+ CTL immunity, these cells were very useful as targets when conducting functional assays such as cytotoxicity and ELISPOT assays.

We have attempted to generate MPP-4-specific CTLs from four healthy HLA-A*0201 positive individuals; however, specific responses could only be observed in one CTL line established from one donor (BC-21). The failure to obtain specific CTL responses to tumor antigens in all donors tested was observed in a previous study was thought to be due to the low frequency of peptide-specific CD8+ T lymphocyte precursors in healthy individuals.⁵⁹ We aimed to clone the MPP-4 CTL line before testing its capability of recognizing fresh A*0201-positive leukemic cells that overexpress the MPP11 protein. Unfortunately, regardless of our attempts to generate MPP-4-specific clones, we could not generate any specific clones. Standard techniques for cloning T cells employ stimulation with antigen loaded-APCs in the presence of IL-2 and allogeneic feeder cells in limiting dilution cultures followed by repeated re-stimulations to maintain growth and specificity. In the first attempt, the MPP-4 CTL line was cloned by stimulation with peptide pulsed-T2 cells in the presence of allogeneic feeder cells in the presence of IL-2, IL-15 and OKT3. IL-2 is the cytokine frequently used to promote the survival and expansion of cultured T cells.⁶⁰ IL-2 and IL-15 share the beta-chain and the common gamma-chain receptor (IL-2/IL-15 Rbetagammac), but each cytokine has its unique alpha-chain receptor.^61,62 IL-15 was initially identified by its capability to promote the development of NK cells and the survival of CD8+ memory T lymphocytes in vivo.^63-65 However, it has been reported that antitumor-CTLs expanded and survived for relatively long time periods in vitro in the presence of IL-15 whereas CTLs maintained in IL-2 died at earlier stages.⁶⁶ Furthermore, IL-15-maintained CTLs retained their effector function as CTLs and did not switch into memory cells.66 For TCR triggering, we used OKT3 as reported previously.25 Because all of the generated clones were not specific, we conducted another attempt to clone the line but without the addition of either IL-15 or OKT3 antibody. The generated T lymphocytes clones were very few and died at early stage perhaps as a result of an old T lymphocyte culture.

In summary, our data suggests that CD8+ CTL reactive to the MPP-4 peptide could be generated from the T lymphocytes repertoire of HLA-A*0201+ healthy individuals. More importantly, these CTLs were able to specifically lyse the K562-A2.1 leukemic cells naturally expressing the MPP11 antigen. Thus it is more difficult to generate CTL responses from the remaining low avidity-naïve precursors because their activation requires more stringent costimulatory requirements.

Acknowledgments
We are very grateful to the administration of the Research Centre and the Research Advisory Council (RAC) for their support. This work was sponsored by RAC (proposal # 2040010). We would like to thank Dr. Wolfgang Herr (University of Mainz, Germany) for providing the K-562 cells transfected with the HLA-A*0201 gene.

References

1. Bozzone DM. Leukemia. 1st ed: Chelsea House, New York. 2009.
2. Leukemia Facts and Statistics. Leukemia and Lymphoma Society, 2009.
3. Tallman MS, Gilliland DG, Rowe JM. Drug therapy for acute myeloid leukemia. Blood. 2005;106:1154-1163.
4. Guglielmelli P, Zini R, Bogani C, et al. Molecular profiling of CD34+ cells in idiopathic myelofibrosis identifies a set of disease-associated genes and reveals the clinical significance of Wilms' tumor gene 1 (WT1). Stem Cells. 2007;25:165-173.
5. Boon T, Old LJ. Cancer Tumor antigens. Curr Opin Immunol. 1997;9:681-683.
6. Dermime S, Armstrong A, Hawkins RE, Stern PL. Cancer vaccines and immunotherapy. Br Med Bull 2002;62:149-162.
7. Dermime S, Gilham DE, Shaw DM, et al. Vaccine and antibody-directed T cell tumour immunotherapy. Biochim Biophys Acta. 2004;1704:11-35.
8. Matsumoto-Taniura N, Pirollet F, Monroe R, Gerace L, Westendorf JM. Identification of novel M phase phosphoproteins by expression cloning. Mol Biol Cell. 1996;7:1455-1469.
9. Davis FM, Tsao TY, Fowler SK, Rao PN. Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci USA. 1983;80:2926-2930.
10. Zenklusen JC, Conti CJ. Cytogenetic, molecular and functional evidence for novel tumor suppressor genes on the long arm of human chromosome 7. Mol Carcinog. 1996;15:167-175.
11. Hundley HA, Walter W, Bairstow S, Craig EA. Human Mpp11 J protein: ribosome-tethered molecular chaperones are ubiquitous. Science. 2005;308:1032-1034.
12. Otto H, Conz C, Maier P, et al. The chaperones MPP11 and Hsp70L1 form the mammalian ribosome-associated complex. Proc Natl Acad Sci USA. 2005;102:10064-10069.
13. Resto VA, Caballero OL, Buta MR, et al. A putative oncogenic role for MPP11 in head and neck squamous cell cancer. Cancer Res. 2000;60:5529-5535.
14. Gure AO, Stockert E, Scanlan MJ, et al. Serological identification of embryonic neural proteins as highly immunogenic tumor antigens in small cell lung cancer. Proc Natl Acad Sci USA. 2000;97:4198-4203.
15. Greiner J, Ringhoffer M, Taniguchi M, et al. Characterization of several leukemia-associated antigens inducing humoral immune responses in acute and chronic myeloid leukemia. Int J Cancer. 2003;106:224-231.
16. Schmitt M, Li L, Giannopoulos K, et al. Chronic myeloid leukemia cells express tumor-associated antigens eliciting specific CD8+ T-cell responses and are lacking costimulatory molecules. Exp Hematol. 2006;34:1709-1719.
17. Greiner J, Ringhoffer M, Taniguchi M, et al. mRNA expression of leukemia-associated antigens in patients with acute myeloid leukemia for the development of specific immunotherapies. Int J Cancer. 2004;108:704-711.
18. van der Geld YM, Limburg PC, Kallenberg CG. Proteinase 3, Wegener's autoantigen: from gene to antigen. J Leukoc Biol 2001;69:177-190.
19. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenet. 1999;50:213-219.
20. Al Qudaihi G, Lehe C, Negash M, et al. Enhancement of lytic activity of leukemic cells by CD8+ cytotoxic T lymphocytes generated against a WT1 peptide analogue. Leuk Lymphoma. 2009;50:260-269.
21. Molldrem J, Dermime S, Parker K, et al. Targeted T-cell therapy for human leukemia: cytotoxic T lymphocytes specific for a peptide derived from proteinase 3 preferentially lyse human myeloid leukemia cells. Blood. 1996;88:2450-2457.
22. Britten CM, Meyer RG, Kreer T, Drexler I, Wolfel T, Herr W. The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8(+) T lymphocytes in IFN-gamma ELISPOT assays. J Immunol Methods. 2002;259:95-110.
23. Neitzel H. A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum Genet. 1986;73:320-326.
24. Dauer M, Obermaier B, Herten J, et al. Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors. J Immunol. 2003;170:4069-4076.
25. Ho WY, Nguyen HN, Wolfl M, Kuball J, Greenberg PD. In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naive repertoire. J Immunol Methods. 2006;310:40-52.
26. Houbiers JG, Nijman HW, van der Burg SH, et al. In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53. Eur J Immunol. 1993;23:2072-2077.
27. Dermime S, Bertazzoli C, Marchesi E, et al. Lack of T-cell-mediated recognition of the fusion region of the pml/RAR-alpha hybrid protein by lymphocytes of acute promyelocytic leukemia patients. Clin Cancer Res. 1996;2:593-600.
28. Clark WH, Jr., Elder DE, Guerry Dt, et al. Model predicting survival in stage I melanoma based on tumor progression. J Natl Cancer Inst. 1989;81:1893-1904.
29. Oka Y, Elisseeva OA, Tsuboi A, et al. Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms' tumor gene (WT1 ) product. Immunogenet. 2000;51:99-107.
30. Tallman MS. New strategies for the treatment of acute myeloid leukemia including antibodies and other novel agents. Hematology Am Soc Hematol Educ Program. 2005:143-150.
31. Melief CJ, Kast WM. T-cell immunotherapy of tumors by adoptive transfer of cytotoxic T lymphocytes and by vaccination with minimal essential epitopes. Immunol Rev. 1995;145:167-177.
32. Heslop HE, Stevenson FK, Molldrem JJ. Immunotherapy of hematologic malignancy. Hematology Am Soc Hematol Educ Program. 2003:331-349.
33. Qazilbash MH, Wieder E, Rios R, et al. Vaccination with the PR1 Leukemia-Associated Antigen Can Induce Complete Remission in Patients with Myeloid Leukemia. ASH Annual Meeting Abstracts 2004;104:259.
34. Schmitt M, Schmitt A, Rojewski MT, et al. RHAMM-R3 peptide vaccination in patients with acute myeloid leukemia, myelodysplastic syndrome, and multiple myeloma elicits immunologic and clinical responses. Blood. 2008;111:1357-1365.
35. Schetelig J, Kiani A, Schmitz M, Ehninger G, Bornhauser M. T cell-mediated graft-versus-leukemia reactions after allogeneic stem cell transplantation. Cancer Immunol Immunother. 2005;54:1043-1058.
36. Wu CJ, Ritz J. Induction of tumor immunity following allogeneic stem cell transplantation. Adv Immunol. 2006;90:133-173.
37. Appelbaum FR. Haematopoietic cell transplantation as immunotherapy. Nature. 2001;411:385-389.
38. Kapp M, Stevanovic S, Fick K, et al. CD8+ T-cell responses to tumor-associated antigens correlate with superior relapse-free survival after allo-SCT. Bone Marrow Transpl. 2009;43:399-410.
39. Molldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med. 2000;6:1018-1023.
40. Rezvani K, Grube M, Brenchley JM, et al. Functional leukemia-associated antigen-specific memory CD8+ T cells exist in healthy individuals and in patients with chronic myelogenous leukemia before and after stem cell transplantation. Blood. 2003;102:2892-2900.
41. Viatte S, Alves PM, Romero P. Reverse immunology approach for the identification of CD8 T-cell-defined antigens: advantages and hurdles. Immunol Cell Biol. 2006;84:318-330.
42. Stevanovic S. Identification of tumour-associated T-cell epitopes for vaccine development. Nat Rev Cancer. 2002;2:514-520.
43. Paschen A, Eichmuller S, Schadendorf D. Identification of tumor antigens and T-cell epitopes, and its clinical application. Cancer Immunol Immunother. 2004;53:196-203.
44. Greiner J, Li L, Ringhoffer M, et al. Identification and characterization of epitopes of the receptor for hyaluronic acid-mediated motility (RHAMM/CD168) recognized by CD8+ T cells of HLA-A2-positive patients with acute myeloid leukemia. Blood. 2005;106:938-945.
45. Asemissen AM, Keilholz U, Tenzer S, et al. Identification of a highly immunogenic HLA-A*01-binding T cell epitope of WT1. Clin Cancer Res. 2006;12:7476-7482.
46. Alves PM, Faure O, Graff-Dubois S, et al. EphA2 as target of anticancer immunotherapy: identification of HLA-A*0201-restricted epitopes. Cancer Res. 2003;63:8476-8480.
47. Ayyoub M, Stevanovic S, Sahin U, et al. Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma. J Immunol. 2002;168:1717-1722.
48. Fujie T, Tahara K, Tanaka F, Mori M, Takesako K, Akiyoshi T. A MAGE-1-encoded HLA-A24-binding synthetic peptide induces specific anti-tumor cytotoxic T lymphocytes. Int J Cancer. 1999;80:169-172.
49. Kawashima I, Hudson SJ, Tsai V, et al. The multi-epitope approach for immunotherapy for cancer: identification of several CTL epitopes from various tumor-associated antigens expressed on solid epithelial tumors. Hum Immunol. 1998;59:1-14.
50. Kessler JH, Beekman NJ, Bres-Vloemans SA, et al. Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis. J Exp Med. 2001;193:73-88.
51. Neumann F, Wagner C, Preuss KD, et al. Identification of an epitope derived from the cancer testis antigen HOM-TES-14/SCP1 and presented by dendritic cells to circulating CD4+ T cells. Blood. 2005;106:3105-3113.
52. Player MA, Barracchini KC, Simonis TB, et al. Differences in frequency distribution of HLA-A2 subtypes between North American and Italian white melanoma patients: relevance for epitope specific vaccination protocols. J Immunother Emphasis Tumor Immunol. 1996;19:357-363.
53. Tourdot S, Scardino A, Saloustrou E, et al. A general strategy to enhance immunogenicity of low-affinity HLA-A2. 1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000;30:3411-3421.
54. Valmori D, Fonteneau JF, Lizana CM, et al. Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. J Immunol. 1998;160:1750-1758.
55. Starr TK, Jameson SC, Hogquist KA. Positive and negative selection of T cells. Annu Rev Immunol. 2003;21:139-176.
56. Janeway CA, Jr., Bottomly K. Signals and signs for lymphocyte responses. Cell 1994;76:275-285.
57. Swain SL, Croft M, Dubey C, et al. From naive to memory T cells. Immunol Rev. 1996;150:143-167.
58. Dauer M, Schad K, Herten J, et al. FastDC derived from human monocytes within 48 h effectively prime tumor antigen-specific cytotoxic T cells. J Immunol Methods. 2005;302:145-155.
59. Ohminami H, Yasukawa M, Fujita S. HLA class I-restricted lysis of leukemia cells by a CD8(+) cytotoxic T-lymphocyte clone specific for WT1 peptide. Blood. 2000;95:286-293.
60. Smith KA. Interleukin-2: inception, impact, and implications. Science 1988;240:1169-1176.
61. Carson WE, Giri JG, Lindemann MJ, et al. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med. 1994;180:1395-1403.
62. Grabstein KH, Eisenman J, Shanebeck K, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science. 1994;264:965-968.
63. Colucci F, Caligiuri MA, Di Santo JP. What does it take to make a natural killer? Nat Rev Immunol. 2003;3:413-425.
64. Zhang X, Sun S, Hwang I, Tough DF, Sprent J. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immun. 1998;8:591-599.
65. Schluns KS, Lefrancois L. Cytokine control of memory T-cell development and survival. Nat Rev Immunol. 2003;3:269-279.
66. Lu J, Giuntoli RL, 2nd, Omiya R, Kobayashi H, Kennedy R, Celis E. Interleukin 15 promotes antigen-independent in vitro expansion and long-term survival of antitumor cytotoxic T lymphocytes. Clin Cancer Res. 2002;8:3877-3884.