Therapeutic effects of haematopoietic stem cell transplantation are not limited to maximal chemoradiotherapy and subsequent bone marrow regeneration, but include specific as well as unspecific immune reactions known as graft-versus-leukaemia (GvL) effects. Specific immune reactions are likely to be particularly relevant to the long-term treatment of diseases, such as chronic myeloid leukaemia (CML), in which residual cells may remain quiescent and unresponsive to cytotoxic and molecular therapies for long periods of time. Specific GvL effects result from the expression on leukaemic cells of specific tumour-associated antigens (TAAs) in the context of HLA proteins. As human leukocyte antigen (HLA) types vary widely, the development of broadly applicable tumour vaccines will require the identification of multiple TAAs active in different HLA backgrounds. Here, we describe the identification of NM23-H2 as a novel HLA-A32-restricted TAA of CML cells and demonstrate the presence of specifically reactive T cells in a patient 5 years after transplantation. As the NM23 proteins are aberrantly expressed in a range of different tumours, our findings suggest potential applications beyond CML and provide a new avenue of investigation into the molecular mechanisms underlying CML.
Chronic myeloid leukaemia (CML) is treated with a steady improvement in outcome having been achieved in recent years both by the introduction of specific inhibitors of the oncogenic BCR/ABL fusion protein and by improving the outcome of haematopoietic cell transplantation (HCT).1, 2, 3 Both therapeutic options have different underlying mechanisms, one being a molecular approach using tyrosine kinase inhibitors and the other an immunological approach with a more or less intensive cytostatic component using HCT. For a stem cell disease such as CML, in which residual populations of tumour cells may remain in a quiescent state unresponsive either to cytotoxic/radiation therapy or to specific BCR/ABL inhibitors,4, 5, 6 it is very likely that immune surveillance by the donor-derived immune system plays a decisive role in the long-term control of the disease.
Graft-mediated immune reactions, however, are not restricted to tumour cells (graft-versus-leukaemia reaction) but are also directed to normal host cells (graft-versus-host disease).7 There is growing interest in harnessing the antitumour potential of the incoming graft to maximize reactions against tumour cells while limiting those against normal host tissues. The molecular basis for the antigen-specific immune reaction lies in the presentation of a repertoire of short peptides derived from the degradation of intrinsic proteins. These peptides are routinely displayed by human leukocyte antigen (HLA) molecules as ‘self-antigens’ on the surface of normal cells and/or on the surface of tumour cells.8 Either mutation (neoantigens) or aberrant expression (self-antigens) of a particular protein in tumour cells can result in the display of such antigens in the context of HLA molecules and in tumour-specific cytotoxic T-cell reaction. Alloantigens restricted to tumour cells or to a specific cell system (for example, haematopoietic cells) might be interesting for GvL effects without GvHD. Clearly, the ultimate aim is not only to identify the target structures of GvL reactions but also to develop vaccination protocols that maximize specific reactions against tumour-specific antigens (to increase GvL) while minimizing those against normal tissues (to control GvH).
Here, we describe, for the first time, the identification of non-metastasis protein 23-H2 (NM23-H2) as an immunogenic tumour-associated protein in Ph+ cells and demonstrate the detection of specific ex vivo T-cell reactivity in a patient 5 years after transplant.
Materials and methods
In 1979, a 25-year-old patient was diagnosed with Philadelphia chromosome-positive (Ph+) CML. A HCT with minimal conditioning was performed in 2001 from his HLA-identical sister. The patient was treated with fludarabin (30 mg/m2, days −4 to −2) and total body irradiation of 2 Gy, followed by immunosuppression with cyclosporine A and mycofenolat mofetil. Leukaemic peripheral blood mononuclear cells (PBMC) were collected before HCT during the chronic phase of the disease. At the time of writing, more than 6 years post-HCT, the patient is in complete molecular remission.
Cell lines and antigen-presenting cells
An Epstein–Barr virus (EBV)-transformed lymphoblastoid cell line (EBV-LCL) was established by culturing 5 × 106 PBMC isolated from the patient pre-transplant in 500 μl culture supernatant from the EBV producer line B95-8 and 1 μg/ml cyclosporine A (Sandimmun; Novartis, Basel, Switzerland). The EBV-LCL was maintained thereafter in RPM1640 (Biochrom AG, Berlin, Germany), 10% fetal calf serum (Invitrogen, Carlsbad, CA, USA), 1% glutamine and penicillin/streptomycin (Invitrogen). Fluorescence in situ hybridization revealed the EBV-LCL to be 100% Ph+.
Phythaemagglutinin (PHA) blasts were generated by stimulating 0.5 × 106 pre-transplant patient PBMC with 1 μg/ml PHA (Sigma-Aldrich, St Louis, MO, USA) for 72 h in AIM-V medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% pooled human serum (Sigma-Aldrich) and 250 U/ml IL-2 (Chiron-Behring, Marburg, Germany). The cells were further expanded in the same medium without PHA. At day 20, the cells were frozen in aliquots and thawed at the day of the experiment. PHA blasts were Ph− as assessed by reverse transcription-PCR (Table 1).
Monocyte-derived dendritic cells were used as APC. Mononuclear cells (MNCs) from 100 ml blood of the HLA-identical donor were purified by density gradient centrifugation over Biocoll (Biochrom AG, Berlin, Germany). Further isolation of the monocyte fraction was then performed by using a mini-MACS-positive selection system as described by the manufacturer (Miltenyi Biotech, Bergisch Gladbach, Germany). Monocytes were seeded at 7 × 105 cells per ml in CellGro DC medium (CellGenix, Freiburg, Germany) supplemented with 1000 U/ml IL-4 (Cell Concepts, Umkirch, Germany) and 800 U/ml granulocyte/macrophage colony-stimulating factor (Leukomax; Schering-Plough, Kenilworth, NJ, USA) for 6 days. Maturation of immature dendritic cells was promoted by the addition of 10 ng/ml tumour necrosis factor-alpha (TNFα), 1000 U/ml IL-6, 10 ng/ml IL-1β (all from Cell Concepts) and 1 μg/ml prostaglandin E2 (PGE2) (Sigma-Aldrich) for 24–48 h.9
Mixed lymphocyte/leukaemic cell culture and IFN-γ ELISPOT assays
Leukaemia-specific mixed lymphocyte/leukaemic cell culture (MLLC) was performed by co-culturing on 24-well plates 106 donor PBMC and 106 irradiated Ph+ patient PBMC per well in 2 ml AIM-V medium (Gibco BRL) supplemented with 10% human serum and recombinant human IL-2 (from day 4, 250U/ml). MLLC was stimulated on days 7 and 14 with 1 × 106 irradiated EBV-LCL (Ph+) cells per well and 250 U/ml IL-2. CD8+ T lymphocytes were isolated using a CD8+ T-cell Isolation Kit (Miltenyi Biotech). CD8+ MLLC was continued by weekly stimulation of 106 T cells with 106 irradiated EBV-LCL (Ph+) and 2 × 105 irradiated CD8− autologous PBMC. CD8+ MLLC responders were frozen in aliquots on day 20. For functional assays, CD8+ MLLCs were restimulated with irradiated EBV-LCL (Ph+) cells for 4 days after thawing and then tested in 50 μl of AIM-V medium (1 × 104 per well) against pre- and post-transplant recipient and donor PBMC, recipient EBV-LCL (Ph+ leukaemic cells 1 × 105 per well), recipient PHA blasts (Ph− cells 1 × 105 per well), 293T transfectants (2 × 104 per well), donor APC (1 × 104 per well) and subpopulations of cells (1 × 105 per well) in 50 μl of AIM-V medium. Subpopulations were isolated from the blood (CD3+, CD4+, CD8+, CD14+) or bone marrow cells (CD34+) from recipients and third-party patients using a mini-MACS-positive selection system as described by the manufacturer (Miltenyi Biotech). HLA-A*3201-negative as well as HLA-A*3201-positive PBMC from third-party patients with CML were used to test specificity. Assays were incubated for 24 or 48 h (293T transfectants) at 37 °C in 5% CO2 in air and then developed. The Vectastain Elite ABC Kit (Axxora Inc., San Diego, CA, USA) was used for colourimetric detection of spots, which were counted using an automated image analysis ELISPOT reader system (AID, Straβberg, Germany).
HLA expression plasmids
cDNAs encoding HLA-A*3101, HLA-A*3201, HLA-B*4401, HLA-B*6001, HLA-Cw*0301 and HLA-Cw*0501 were cloned by reverse transcription-PCR from patient leukaemic cells into pcDNA3.1 (Invitrogen) as described.10
Construction and screening of the cDNA library
A cDNA library of patient Ph+ leukaemic cells was constructed in the vector pcDNA3.1 using a cDNA construction kit (Invitrogen) and divided into pools of 100 cDNAs. 293T cells (20 000 per well) were then co-transfected with the HLA-cDNA (100 ng per well) and mixed cDNAs prepared from the pools of leukaemic cells (100 pools, 100–200 ng per well) in MultiScreen ELISPOT MAIPSWU10 plates (Millipore, Bedford, MA, USA) using PolyFect transfection reagent (Qiagen, Basel, Switzerland) and tested in an IFN-γ ELISPOT assay after 48 h. Positive cDNA pools, which were recognized by the specific cytotoxic T-cell line (CTL), were then subdivided into 400 pools of 10 colonies each and retested in the same way. Positive pools were finally diluted to 400 single clones each and subjected to a final round of screening.
cDNA fragments and synthetic peptides
Antigen-coding cDNA fragments were amplified by PCR and cloned into pcDNA3.1/V5-His TOPO (Invitrogen). 293T cells were transiently co-transfected with these plasmids together with HLA-A*3201 cDNA and then tested for recognition by T cells. Peptides were synthesized by S Rothemund, University of Leipzig, and then solubilized in PBS or PBS/0.5% dimethylsulphoxide and stored at −20 °C. For experiments with peptide loading, 106 APCs per ml in AIM-V medium without serum were incubated with the indicated concentrations of peptides for 2 h at 37 °C.
Ex vivo testing of CD8+ sorted PBMC before and after HCT
CD8+ cells (0.5–2.0 × 105 per well) of the donor and of the patient before HCT and at different time points after HCT were obtained from PBMC and tested directly in the ELISPOT assay against 2 × 104 293T cells per well transfected with HLA-A*3201 cDNA and either loaded with 10 μM peptide or co-transfected with full-length Nm23-H2 (Figure 6).
Fluorescence in situ hybridization, quantitative reverse transcription-PCR and NM23-H2 protein expression
The percentage of Ph+ interphases was determined by fluorescence in situ hybridization of at least 30 interphases with the LSI bcr/abl ES probe (Vysis, Stuttgart, Germany). For quantitative reverse transcription-PCR, 0.2–8.5 × 105 cells of the patient's PBMC and CD4+, CD8+, CD14+ and CD34+ subpopulations were lysed in guanidine isocyanate solution11 and stored at −20 °C until used. RNA was extracted with the RNeasy Micro Kit (Qiagen) and total RNA yield was reverse transcribed into cDNA with random hexamer primers. The quantitative measurement of bcr/abl fusion gene transcripts was carried out by a TaqMan-based real-time quantitative PCR analysis as described by Gabert et al.12 mRNA levels of NM23-H2 were measured using the QuantiTect Primer Assay (Qiagen). Results were normalized to RPLP0 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Protein expression of NM23-H2 was investigated by immunocytochemistry. A total of 1 × 105 cells per slide were methanol-fixed and stained with the NM23-H2-specific antibody L-16 (Santa Cruz) and β-actin antibody (Sigma-Aldrich). Evaluation was carried out using a confocal laser scanning microscope LSM 510 (Zeiss).
CML-reactive T cells
Peripheral blood mononuclear cells isolated from a CML patient in the chronic phase of the disease shortly before transplantation were irradiated and used to stimulate PBMC from the HLA-identical donor in a MLLC. Fluorescence in situ hybridization revealed the bone marrow mononuclear cells of the patient to be 100% Ph+. On day 14 of the MLLC, reactive CD8+ T cells were purified from the culture using a magnetic bead affinity procedure and expanded for a further 6 days in the continued presence of irradiated target cells. This procedure generated over 2 × 108 T cells that were frozen in aliquots for further studies.13
The expanded donor T cells were highly reactive against PBMC of the recipient before HCT. The specificity and HLA restriction of antigen recognition were assessed by exposure to a range of cell types and subtypes of the original patient, a range of cells from third-party patients with CML and by blocking antibodies to HLA antigens expressed by the patient (Figure 1a). T cells were found to recognize PBMC and EBV-LCL (both Ph+) but not PHA-stimulated blasts (Ph−; see Table 1). Testing of sorted fractions of recipient blood and bone marrow cells pre-transplant revealed reactivity against myeloid (CD34+ and CD14+) but not T lymphoid (CD4+ and CD8+) cells, consistent with the specific recognition of cells affected by the bcr/abl mutation. Although reactivity was efficiently blocked by the W6.32 antibody against all HLA-A, HLA-B and HLA-C heavy chains14 and by the B1.23.2 antibody, which blocks HLA-B and HLA-C, as well as HLA-A31 and HLA-A32,15 the irrelevant HLA-A3 antibody GAP-A316 had no effect. Partial inhibition was achieved with the SFR8-B6 antibody, which recognizes the Bw6 determinant on HLA-B and HLA-C molecules.17 Taken together, the blocking experiments confirmed HLA-class I restriction of the CTL. HLA restriction was confirmed using CML cells from unrelated patients with and without HLA-A*3201. Only HLA-A*3201-positive, bcr/abl-positive cells (PBMC, CD14+) but not bcr/abl-negative cells (CD3+) from the third-party patient were recognized by our CTL (Figure 1b).
A cDNA expression library was constructed using mRNA from the leukaemic population. Approximately 1000 pools of 100 clones each were picked. Each pool was then transfected into 293T cells together with plasmids expressing each of the HLA class I proteins present on patient MNCs (HLA-A*3101, HLA-A*3201, HLA-B*4401, HLA-B*6001, HLA-Cw*0301, HLA-Cw*0501), making a total of 6000 independent transfections. The presence of antigen-coding cDNA was determined by ELISPOT assay (Figure 2a). Among the positive reactions, we analyzed the one restricted by HLA-A*3201 in detail.
This initial round of screening identified 3 pools of 100 clones (B2, E10 and E12), which produced a significant ELISPOT reaction. Each of the pools was then divided into 400 pools of 10 clones each and screened again, resulting in the identification of one positive pool (B2.A1; Figure 2b), which originated from the original pool B2. We prepared and tested 400 single clones of this pool and found 3 of them to be recognized by the MLLC (Figure 2c).
Restriction analysis and sequencing showed all three positively reacting cDNAs to be the same, 600 bp long, and to be identical to NM23-H2.18 As many of the tumour antigens characterized to date result from tumour-specific mutations,19 we performed an intensive mutation screen using multiple NM23-H2-specific primers. All positive clones were sequenced entirely on both strands and were found to contain the whole open reading frame of NM23-H2 without any mutations, compared to the published human sequence (Gene ID: 4831). To confirm this finding, the entire open reading frames of NM23-H2 and NM23-H1 (88% homology to H2) were amplified independently by PCR, cloned into an expression vector and transfected together with HLA-A*3201 cDNA into 293T cells. Spot formation of the MLLC responders clearly confirmed the specific recognition of NM23-H2 (Figure 3).
Identification of the peptide antigen
Although the anchor amino acids for HLA-A32 have yet to be defined, a comparison of the two HLA-A*3201-restricted peptides identified to date revealed a common tryptophan residue at the C terminus of each nonapeptide.20 The NM23-H2 coding region contains three candidate tryptophan residues. In an attempt to identify HLA-A32-restricted antigenic peptides from NM23-H2, nonameric peptides corresponding to the NM23-H2 sequences up to and including each of these tryptophan residues (NM23-H270−78; NM23-H2125−133 and NM23-H2141−149) were synthesized. These peptides were loaded at concentrations of 10 μM separately onto HLA-A*3201-expressing APCs and tested in the ELISPOT assay with the CD8+ MLLC. Under these conditions, significant reactivity was seen only to NM23-H270−78 (SGPVVAMVW, Figure 4).
To obtain a more quantitative measure of the specificity and avidity of the CD8+-CTL for the antigenic NM23-H2 peptides, the ELISPOT was repeated using serial 10-fold dilutions of each peptide from 10 μM down to 1 nM (Figure 5). This revealed a very high level of specificity and avidity for the peptide NM23-H270−78, such that recognition was still 50% at the lowest concentration tested (1 nM). It is not surprising that the maximal recognition reached was 85% of EBV-LCL recognition, as the MLLC showed additional specificities to HLA-B and HLA-C molecules (see blocking experiments, Figure 1a).
NM23-H270−78-reactive T cells ex vivo
Peripheral blood mononuclear cells of the donor and of the patient before and at different time points after HCT were collected and tested directly ex vivo. Interestingly, CD8+ T cells showed low reactivity in the recipient before HCT, but not in the donor and in the patient up to 3 months after HCT against NM23-H270−78. Later on, a distinct reactivity against NM23-H270−78 and endogenously expressed full-length NM23-H2 was found concomitant with bcr/abl negativity (Figure 6).
Expression of NM23-H2 was investigated on the protein and mRNA level in the pre-transplant PBMC of the patient, compared with PBMC of the HLA-identical donor. As shown in Figure 7a, NM23-H2 protein expression was clearly increased in the CML cells of the patient where NM23-H2 was colocalized with β-actin in the cytoplasm. In contrast, there was no upregulation of NM23-H2 at the mRNA level (Figure 7b).
In the present analysis, we identify for the first time an immunogenic peptide derived from NM23-H2 as a tumour-associated antigen in cells from a patient with CML and demonstrate the presence of NM23-H2-reactive T cells after HCT. Ph+ cells (EBV-LCL; CD34+ and CD14+) but not Ph− cells (CD4+ and CD8+) were recognized by the CD8+-CTL. In addition, CML cells from other HLA-A*3201 but not from HLA-A*3201-negative patients were identified by the CTL.
As no mutations were apparent in the antigenic NM23 sequences cloned from the CML cells, we assume that the observed immunogenicity resulted from a tumour cell-specific expression pattern of NM23-H2. An alternative but less likely possibility is that a mutation in donor NM23-H2 is responsible for specific recognition of patient cells by donor T cells. Whatever the basis of the immune recognition, our findings suggest that an immune response to aberrantly expressed NM23-H2 may be involved in the long-term GvL effect following HCT and that the successful stimulation of this reaction in vivo may provide additional protection against disease recurrence.
Non-metastasis protein 23 was first described as a tumour marker associated with reduced metastatic activity in melanoma.21 Aberrant NM23 expression (as opposed to mutation) has since been found to be of clinical significance in a wide range of cancers, although the consequences of overexpression vary widely.22 In melanoma and in breast cancer, for example, high mRNA levels are associated with a reduced likelihood of metastatic progression and a favourable outcome, whereas increased expression in prostate cancer, neuroblastoma or non-Hodgkin's lymphoma is associated with a poor outcome.23, 24, 25, 26 Similarly, the overexpression of either of the closely related nm23-H1 or nm23-H2 genes in AML has been reported to be associated with reduced survival.27, 28 However, the evidence to date for an involvement of NM23-H2 in CML is very scant, with two reports of an increase in mRNA levels during blast crisis.29, 30 Despite the implication of a general involvement of NM23 proteins in tumourigenesis, there is currently no clear consensus concerning the underlying mechanisms. Both NM23-H1 and NM23-H2 are nucleoside diphosphate kinases and are likely to play multiple roles in coordinating the balance of nucleoside diphosphates and triphosphates available for gene expression, DNA synthesis, signalling and metabolism. Furthermore, NM23-H1 and NM23-H2 each interact directly with other regulatory proteins controlling cell fate.22 Specifically, NM23-H1 interacts directly with a wide range of proteins involved in the regulation of signalling, metabolism, apoptosis and cell division,22 whereas NM23-H2 is a transcriptional activator of c-myc,18, 31, 32 which in turn can activate expression of both the nm23-H1 and nm23-H2 genes.33
As overexpression of c-Myc protein is closely associated with CML progression,34, 35 alterations in NM23-H2 expression in CML seem likely. It is therefore somewhat surprising that we can find no evidence for a general upregulation of NM23-H2 mRNA in CML cells, including those from which the antigenic sequences were cloned and which are recognized by the specific T cells. This suggests that the CML-specific changes in regulation may be at a post-translational level. Indeed, both the c-myc and nm23-H2 genes are subject to regulation at the level of mRNA translation, with c-myc translation being dependent on signalling through the ras/map kinase pathway, whereas NM23-H2 translation appears to respond to the Akt/mTOR pathway.36, 37, 38 This would be consistent with a recent study indicating that a number of peptides presented predominantly on tumour tissue showed no or only minor changes in mRNA expression levels compared with normal tissue.39
Regulatory networking of the c-myc and nm23 genes involving decisive changes at the translational or post-translational level may help to explain why the correlations made between mRNA levels and prognosis in various cancers are highly context-dependent. For these reasons, we are currently testing the possibility that CML cells accumulate NM23-H2 protein without significant increases in the levels of specific mRNA, and preliminary results support this theory. Although it is possible that BCR/ABL exerts specific effects on NM23-H2 expression by the activation of signalling pathways and c-Myc expression, recent reports demonstrate a broad effect of BCR/ABL activity on tumour antigen expression. Hence, the increased expression of survivin, adipophilin, hTERT, WT-1, Bcl-x1 and Bcl-2 is mediated by BCR/ABL and reversed by imatinib mesylate,40 whereas BCR/ABL-transfected dendritic cells elicit T-cell responses with specificities very similar to those found in CML patients in major molecular remission following imatinib mesylate treatment.40, 41
Regardless of the molecular mechanisms affecting NM23-H2 expression in CML, the detection of a specific T-cell reaction in a transplanted patient implies that NM23 can mediate clinically relevant immunogenicity and may therefore be a candidate leukaemia-specific antigen for therapeutic manipulation of the immune response. A variety of leukaemia-specific antigens have been identified in recent years. One of the most obvious candidates for CML is the BCR/ABL protein itself, and as BCR/ABL-derived peptides have indeed been shown to elicit a cytotoxic T-cell response in vitro,42 their potential as tumour vaccines is currently being assessed.43, 44 Primary granule proteins, which act as autoantigens in the autoimmune reactions of Wenger's Granuloma and related vasculitis, have also been identified as promising candidate for myeloid leukaemia-specific antigens. Two of these, PR3 and NE, have been studied in detail. PR3 was originally identified as an HLA-A0201-restricted peptide that induced myeloid-specific CTL responses. These CTL can be expanded in vitro and are cytotoxic to CML cells.45 Encouraging results in patients with refractory or progressing myeloid leukaemia have recently led to the initiation of immunotherapy studies with PR3 in less advanced patients. Finally, a screen of previously characterized leukaemia-specific antigens with specific relevance to CML has identified further candidates for CML immunotherapy, at least one of which (RHAMM/CD168-R3) appears to have elicited specific T-cell responses in transplanted patients.46 There is, therefore, good reason to believe that immunotherapies based on TAAs could improve long-term outcome in CML patients.47 It should be noted that, whereas the antigenic peptides previously described were identified by ‘reverse immunology’, the NM23 peptide reported here was identified from an MLLC by cDNA cloning.
To assess the ultimate therapeutic potential of peptide vaccines derived from NM23, it will be necessary to determine, firstly, whether or not aberrant NM23-H2 expression is a widespread feature of CMLs and, secondly, whether the protein generates peptides that can act as functional antigens in HLA backgrounds other than HLA-A32. Given the widespread involvement of NM23 proteins in tumourigenesis, it will also be interesting to investigate the potential relevance of NM23-H2 as a therapeutic TAA in other cancers. In the meantime, the regulatory interdependence of NM23-H2 and c-myc provides a basis from which to design specific studies to elucidate the function of NM23 proteins in normal and leukaemic cells, which should contribute to our understanding of the molecular mechanisms underlying the development and progression of CML.
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We thank A Jilo for technical assistance. This work was supported by Grant 70-3344 (TP IV C/D) from the Deutsche Krebshilfe and by Grant D08 from the IZKF Leipzig to DN.
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Tschiedel, S., Gentilini, C., Lange, T. et al. Identification of NM23-H2 as a tumour-associated antigen in chronic myeloid leukaemia. Leukemia 22, 1542–1550 (2008). https://doi.org/10.1038/leu.2008.107
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