Activated naïve γδ T cells accelerate deep molecular response to BCR-ABL inhibitors in patients with chronic myeloid leukemia.

Tyrosine kinase inhibitors (TKIs) that target BCR-ABL are the frontline treatments in chronic myeloid leukemia (CML). Growing evidence has shown that TKIs also enhance immunity. Since gamma-delta T (γδT) cells possess the potent anticancer capability, here we investigated the potential involvement of γδT cells in TKI treatments for CML. We characterized γδT cells isolated from chronic-phase CML patients before and during TKI treatments. γδT expression increased significantly in CML patients who achieved major molecular response (MMR) and deep molecular response (DMR). Their Vδ2 subset of γδT also expanded, and increased expression of activating molecules, namely IFN-γ, perforin, and CD107a, as well as γδT cytotoxicity. Mechanistically, TKIs augmented the efflux of isopentenyl pyrophosphate (IPP) from CML cells, which stimulated IFN-γ production and γδT expansion. Notably, the size of the IFN-γ+ naïve γδT population in TKI-treated CML patients was strongly correlated with their rates to reach DMR and with the duration on DMR. Statistical analysis suggests that a cutoff of 7.5% IFN-γ+ naïve subpopulation of γδT in CML patients could serve as a determinant for MR4.0 sustainability. Our results highlight γδT cells as a positive regulator for TKI responses in CML patients.

KIR3DL1 Allotype-Dependent Modulation of NK Cell Immunity against Chronic Myeloid Leukemia.

Tyrosine kinase inhibitor (TKI)-treated chronic myeloid leukemia (CML) patients with increased NK cell number have a better prognosis, and thus, NK cells may suppress CML. However, the efficacy of TKIs varies for reasons yet to be fully elucidated. As NK cell activity is modulated by interactions between their killer cell Ig-like receptors (KIRs) and HLAs of target cells, the combination of their polymorphisms may have functional significance. We previously showed that allelic polymorphisms of KIR3DL1 and HLAs were associated with the prognosis of TKI-treated CML patients. In this study, we focus on differential NK cell activity modulation through KIR3DL1 allotypes. KIR3DL1 expression levels varied according to their alleles. The combination of KIR3DL1 expression level and HLA-Bw4 motifs defined NK cell activity in response to the CML-derived K562 cell line, and Ab-mediated KIR3DL1 blocking reversed this activity. The TKI dasatinib enhanced NK cell activation and cytotoxicity in a KIR3DL1 allotype-dependent manner but did not significantly decrease effector regulatory T cells, suggesting that it directly activated NK cells. Dasatinib also enhanced NK cell cytotoxicity against K562 bearing the BCR-ABL1 T315I TKI resistance-conferring mutation, depending on KIR3DL1/HLA-Bw4 allotypes. Transduction of KIR3DL1*01502 into the NK cell line NK-92 resulted in KIR3DL1 expression and suppression of NK-92 activity by HLA-B ligation, which was reversed by anti-KIR3DL1 Ab. Finally, KIR3DL1 expression levels also defined activation patterns in CML patient-derived NK cells. Our findings raise the possibility of a novel strategy to enhance antitumor NK cell immunity against CML in a KIR3DL1 allotype-dependent manner.

Antibodies against apoB100 peptide 210 inhibit atherosclerosis in apoE-/- mice.

Atherosclerotic plaques are characterized by an accumulation and subsequent oxidation of LDL, resulting in adaptive immune responses against formed or exposed neoepitopes of the LDL particle. Autoantibodies against native p210, the 3136-3155 amino acid sequence of the LDL protein apolipoprotein B-100 (apoB100) are common in humans and have been associated with less severe atherosclerosis and decreased risk for cardiovascular events in clinical studies. However, whether apoB100 native p210 autoantibodies play a functional role in atherosclerosis is not known. In the present study we immunized apoE-/- mice with p210-PADRE peptide to induce an antibody response against native p210. We also injected mice with murine monoclonal IgG against native p210. Control groups were immunized with PADRE peptide alone or with control murine monoclonal IgG. Immunization with p210-PADRE induced an IgG1 antibody response against p210 that was associated with reduced atherosclerotic plaque formation in the aorta and reduced MDA-LDL content in the lesions. Treatment with monoclonal p210 IgG produced a similar reduction in atherosclerosis as immunization with p210-PADRE. Our findings support an atheroprotective role of antibodies against the apoB100 native p210 and suggest that vaccines that induce the expression of native p210 IgG represent a potential therapeutic strategy for lowering cardiovascular risk.

Novel therapeutic approaches in chronic myeloid leukemia.

The tyrosine kinase inhibitors (TKIs) have revolutionized the management of chronic myeloid leukemia (CML) and BCR-ABL1 inhibitors form the mainstay of CML treatment. Although patients with CML generally do well under TKI therapy, there is a subgroup of patients who are resistant and/or intolerant to TKIs. In these group of patients, there is the need of additional treatment strategies. In this review, we provide an update on the current knowledge of these novel treatment approaches that can be used alone and/or in combination with TKIs.

Multiparameter flow cytometry applications in the diagnosis of mixed phenotype acute leukemia.

Mixed phenotype acute leukemias (MPALs) represent a rare subgroup of acute leukemias with a poor prognosis. Proper diagnosis and classification of MPAL is extremely important for patients' outcome. Morphology and flow cytometry recognize two types of MPAL: the "bilineal" MPAL with the coexistence of two blast populations of different lineage and truly "biphenotypic" MPAL coexpressing markers of more than one lineage in a homogenous blast population, respectively. The WHO 2008 classification further delineated three categories: associated with t(9;22)/BCR-ABL1 fusion gene, associated with KMT2A (mixed lineage leukemia) rearrangements, and nonotherwise specified. These categories remained unchanged in the WHO2016 update. Molecular studies have further underlined the heterogeneity of MPAL. In this review, rules for the correct assignment of acute leukemia to the MPAL category are discussed, including both flow cytometry and immunohistochemistry on bone marrow or other tissues biopsies. Comparison of the immunophenotypic classification proposals is provided outlining the explorations mandatory for definitive diagnosis. An extensive review of published data summarizes the reported cytogenetic and molecular anomalies. New developments in the understanding of the early stages of hematopoiesis provide clues to the possible etiopathology of these diseases. Finally, current treatment recommendations are summarized and referenced for clinical use, pointing out that allogeneic hematopoietic stem cell transplantation at an early stage should be considered (at least in adult patients). © 2019 International Clinical Cytometry Society.

The HLA ligandome landscape of chronic myeloid leukemia delineates novel T-cell epitopes for immunotherapy.

Antileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry-based approach to identify naturally presented HLA class I- and class II-restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL- and ABL-BCR-derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell-based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients.

The impact of BCR-ABL1 transcript type on tyrosine kinase inhibitor responses and outcomes in patients with chronic myeloid leukemia.

Although the majority of patients with chronic myeloid leukemia do well with treatment with tyrosine kinase inhibitors (TKIs), some patients still have inferior outcomes. There are many factors that might play a part, including the different BCR-ABL1 transcript types at baseline. The current study was performed to determine the possible impact of different transcripts on the treatment responses and outcomes of patients with chronic myeloid leukemia who are receiving TKI therapy. The authors performed a systematic literature search by using the terms "b2a2/b3a2," "e13a2/e14a2," or "transcript type." e14a2 was the more common transcript type. The majority of the studies demonstrated no significant difference regarding age, sex, leukocyte counts, and hemoglobin levels between patients with the e13a2 and e14a2 transcripts. However, in approximately one-half of the studies, the e14a2 transcript was associated with higher platelet counts. Almost no studies demonstrated a significant association between disease risk scores and transcript types. In the majority of studies, having the e14a2 transcript was associated with earlier, deeper, and higher molecular response rates. Although better event-free survival was observed in patients with the e14a2 transcript in some of the studies, the majority demonstrated that transcript type did not have an impact on progression-free and overall survival. Treatment-free remission currently is a topic of much interest, and to the authors' knowledge there are limited data with conflicting results regarding the possible effects of transcript types on the outcomes of patients after discontinuation of TKIs. Because having the e14a2 transcript appears to be related to a favorable outcome, choosing second-generation TKIs for frontline therapy might be a convenient approach in patients with chronic myeloid leukemia with the e13a2 transcript. The authors believe this finding warrants further investigation.

Specific Monoclonal Antibody Against Bcr/Abl Out-of-Frame Alternative Proteins as Diagnostic Tool in Chronic Myelogenous Leukemia Patients.

More recently, alternative splicing of specific genes are investigated for their therapeutic potential. In particular, we reported the existence of BCR-ABL alternative splicing isoforms, in about 80% of Philadelphia-positive patients, which lead to the expression of aberrant proteins. These fusion proteins are characterized by an orphan initial and correct Bcr portion attached to a 112 amino acid sequence, arising from the impairment in the reading frame (reading of ABL exon 4 and 5). We demonstrated that these Abl-out-of-frame (OOF) isoforms could have an immunological role with therapeutic implications. The aim of this study was to characterize a new monoclonal antibody (MAb) specific for Abl-OOF protein portion, for diagnostic use, to detect this biomarker in Philadelphia chromosome-positive chronic myelogenous leukemia (CML) patients and to generate novel approaches in the immunotherapy. 5F11G11 MAb recognizes the OOF protein portion of the native full-length Bcr/Abl-OOF protein expressed in cells transiently transfected, as demonstrated by immunoprecipitation and immunofluorescence. In addition, we demonstrate the MAb's ability to recognize the alternative hybrid Bcr/Abl fusion protein expressed in leukemic cells from CML patients, to support the possible use of 5F11G11 MAb as a diagnostic tool to select patients with Philadelphia chromosome-positive CML that could be eligible for an immunotherapeutic approach with this new antigen.

BCR-ABL-specific T-cell therapy in Ph+ ALL patients on tyrosine-kinase inhibitors.

Although the emergence of bone marrow (BM)-resident p190BCR-ABL-specific T lymphocytes has been correlated with hematologic and cytogenetic remissions in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) undergoing maintenance tyrosine-kinase inhibitor treatment, little is known about the possibility of culturing these cells ex vivo and using them in T-cell therapy strategies. We investigated the feasibility of expanding/priming p190BCR-ABL-specific T cells in vitro by stimulation with dendritic cells pulsed with p190BCR-ABL peptides derived from the BCR-ABL junctional region and alternative splicing, and of adoptively administering them to patients with relapsed disease. We report on the feasibility of producing clinical-grade BCR-ABL-specific cytotoxic T lymphocytes (CTLs), endowed with antileukemia activity, from Ph+ ALL patients and healthy donors. We treated 3 patients with Ph+ ALL with autologous or allogeneic p190BCR-ABL-specific CTLs. No postinfusion toxicity was observed, except for a grade II skin graft-versus-host disease in the patient treated for hematologic relapse. All patients achieved a molecular or hematologic complete remission (CR) after T-cell therapy, upon emergence of p190BCR-ABL-specific T cells in the BM. Our results show that p190BCR-ABL-specific CTLs are capable of controlling treatment-refractory Ph+ ALL in vivo, and support the development of adoptive immunotherapeutic approaches with BCR-ABL CTLs in Ph+ ALL.

Immunological Analyses of Leukemia Stem Cells.

Traditionally, the intracellular localization and expression levels of specific proteins in CML Leukemia stem cells (LSCs) have been evaluated by fluorescence immunohistochemistry (FIHC). More recently, Duolink(®) in situ PLA technology has opened up a new and more quantitative way to evaluate signal transduction, posttranslational modification, and protein-protein interaction at the single-stem-cell level. This novel methodology, which employs two antibody-based probes, has already increased our understanding of the biology of the rare CML LSC population. In the future, the use of this approach may contribute to the development of novel therapeutics aimed at eradicating CML LSCs in CML patients.

The Rho-ROCK pathway as a new pathological mechanism of innate immune subversion in chronic myeloid leukaemia.

CD1d-restricted invariant natural killer T (iNKT) cells are believed to play a key role in cancer immune surveillance, and are functionally deficient in patients with chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CD1d or its mRNA transcripts, consistent with intracellular retention. In vitro treatment of CML mDCs with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 partially restored both cell surface CD1d expression and CD1d-mediated antigen presentation, whereas it had no effect on HD mDCs. An inhibitor of BCR-ABL tyrosine kinase (TK), imatinib mesylate (IM), had no such activity. Similar recovery of CD1d expression occurred with fasudil, another ROCK inhibitor that is commonly used in clinical trials. Our data support the conclusion that BCR-ABL-dependent ROCK, but not TK, is involved in CD1d downregulation. We propose that ROCK, which is most likely activated by the DH/PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CD1d expression on CML mDCs. Our study reveals the ROCK-mDC axis as a new potential target to restore immune surveillance in patients with CML, offering new therapeutic perspectives for CML treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

The Chromatin Regulator CHD8 Is a Context-Dependent Mediator of Cell Survival in Murine Hematopoietic Malignancies.

Aberrant chromatin regulation is a frequent driver of leukemogenesis. Mutations in chromatin regulators often result in more stem-like cells that seed a bulk leukemic population. Inhibitors targeting these proteins represent an emerging class of therapeutics, and identifying further chromatin regulators that promote disease progression may result in additional drug targets. We identified the chromatin-modifying protein CHD8 as necessary for cell survival in a mouse model of BCR-Abl+ B-cell acute lymphoblastic leukemia. This disease has a poor prognosis despite treatment with kinase inhibitors targeting BCR-Abl. Although implicated as a risk factor in autism spectrum disorder and a tumor suppressor in prostate and lung cancer, the mechanism of CHD8's activity is still unclear and has never been studied in the context of hematopoietic malignancies. Here we demonstrate that depletion of CHD8 in B-ALL cells leads to cell death. While multiple B cell malignancies were dependent on CHD8 expression for survival, T cell malignancies displayed milder phenotypes upon CHD8 knockdown. In addition, ectopic expression of the Notch1 intracellular domain in a T cell malignancy partially alleviated the detrimental effect of CHD8 depletion. Our results demonstrate that CHD8 has a context-dependent role in cell survival, and its inhibition may be an effective treatment for B lymphoid malignancies.

Adaptive Immunity to Leukemia Is Inhibited by Cross-Reactive Induced Regulatory T Cells.

BCR-ABL(+) acute lymphoblastic leukemia patients have transient responses to current therapies. However, the fusion of BCR to ABL generates a potential leukemia-specific Ag that could be a target for immunotherapy. We demonstrate that the immune system can limit BCR-ABL(+) leukemia progression although ultimately this immune response fails. To address how BCR-ABL(+) leukemia escapes immune surveillance, we developed a peptide: MHC class II tetramer that labels endogenous BCR-ABL-specific CD4(+) T cells. Naive mice harbored a small population of BCR-ABL-specific T cells that proliferated modestly upon immunization. The small number of naive BCR-ABL-specific T cells was due to negative selection in the thymus, which depleted BCR-ABL-specific T cells. Consistent with this observation, we saw that BCR-ABL-specific T cells were cross-reactive with an endogenous peptide derived from ABL. Despite this cross-reactivity, the remaining population of BCR-ABL reactive T cells proliferated upon immunization with the BCR-ABL fusion peptide and adjuvant. In response to BCR-ABL(+) leukemia, BCR-ABL-specific T cells proliferated and converted into regulatory T (Treg) cells, a process that was dependent on cross-reactivity with self-antigen, TGF-ß1, and MHC class II Ag presentation by leukemic cells. Treg cells were critical for leukemia progression in C57BL/6 mice, as transient Treg cell ablation led to extended survival of leukemic mice. Thus, BCR-ABL(+) leukemia actively suppresses antileukemia immune responses by converting cross-reactive leukemia-specific T cells into Treg cells.

Early cytotoxic lymphocyte expansion contributes to a deep molecular response to dasatinib in patients with newly diagnosed chronic myeloid leukemia in the chronic phase: results of the D-first study.

Dasatinib is one of the key treatment options for chronic myeloid leukemia (CML) patients. Increase in lymphocyte counts has been known to be predictive of a good treatment response under dasatinib treatment as a second line therapy. However, clinical significance of lymphocyte dynamics in the upfront setting has yet to be clarified. To investigate the significance of lymphocyte dynamics in newly diagnosed chronic phase (CP)-CML, patient data of D-First study (ClinicalTrials.gov NCT01464411) were analyzed. Fifty-two CML-CP patients enrolled to this study were treated with dasatinib (100 mg day(-1) ) and all were followed-up for 18 months. The incidence of lymphocyosis was observed in 14 (27%), but it was not associated with deep molecular response achievement. However, natural killer (NK) cell or cytotoxic T lymphocyte (CTL) counts at 1 month were significantly higher in patients with deep molecular response (DMR) by 18 months compared to those without DMR. When the patients were divided into two groups according to those calculated thresholds by receiver operating characteristic curve (407/µL for NK cells and 347/µL for CTLs), the cumulative DMR rates by 18 months were significantly better in higher value group compared to lower value group. In contrast, regulatory T cell counts were significantly lower at 12 and 15 months in patients achieved DMR. These results suggest the presence of dual effects of dasatinib on immune system through the cytotoxic lymphocytes activation and Treg deregulation in different periods in newly diagnosed CML-CP.

Haploidentical hematopoietic stem cell transplantation without in vitro T cell depletion for the treatment of philadelphia chromosome-positive acute lymphoblastic leukemia.

The role of haploidentical related allogeneic hematopoietic stem cell transplantation (allo-HSCT) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) is not clear. We aimed to investigate the long-term survival of Ph(+) ALL patients who underwent haploidentical donor (HID)-HSCT and to analyze the factors influencing relapse and survival after allo-HSCT. The study population included Ph(+) ALL patients who underwent haploidentical related allo-HSCT. Additionally, Ph(+) ALL patients who underwent HLA-matched related donor (MRD) transplants during the same period were included to compare outcomes. BCR-ABL transcript levels were analyzed by using real-time quantitative reverse transcription PCR. Clinical data from 139 Ph(+) ALL patients who received allo-HSCT in our center were analyzed. Of these patients, 101 received HID transplants and 38 received MRD transplants. At a median follow-up of 36 months, 5-year disease-free survival (DFS) and overall survival (OS) rates in the HID transplant group were 65.8% and 74.0%, respectively. The 5-year cumulative incidence of relapse (CIR) and nonrelapse mortality (NRM) rates for the HID transplant group were 20.3% and 15.6%, respectively. In addition, there were no differences in OS, DFS, CIR, and NRM between the HID and MRD groups. Multivariate analysis showed that imatinib resistance was a significant factor influencing DFS and CIR in HID transplant patients. Haploidentical HSCT for the treatment of Ph(+) ALL achieves promising long-term survival, which is comparable with that of HLA-MRD HSCT. Imatinib resistance is a negative predictor of relapse and DFS after allo-HSCT.

Patients with Philadelphia-positive leukemia with BCR-ABL kinase mutations before allogeneic transplantation predominantly relapse with the same mutation.

Despite the successes of tyrosine kinase inhibitors (TKIs) in improving outcomes in patients with chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL), allogeneic hematopoietic stem cell transplantation (HSCT) continues to be an important and potentially curative option for selected patients with either disease. After HSCT, TKIs are increasingly being used to treat or prevent disease relapse, and practice patterns suggest that these TKIs are often chosen empirically without regard to pre-HSCT mutation status. We investigated whether ABL kinase domain mutations persist after transplantation and, thus, whether pre-HSCT mutation status should inform the selection of post-HSCT TKIs in these patients. We retrospectively analyzed adults who underwent allogeneic HSCT for CML and Ph + ALL at our institution between 2000 and 2010, and we identified subjects who had detectable BCR-ABL transcripts by polymerase chain reaction (PCR), as well as available RNA for Sanger sequencing of the ABL kinase domain, in both the pre- and post-HSCT settings. In total, 95 CML and 20 Ph + ALL patients with positive PCR transcripts were identified, of which 10 (10.5%) and 4 (20.0%), respectively, were found to have pre-HSCT ABL kinase mutations known to confer TKI resistance. In 9 (64.2%) of these 14 patients, the same kinase mutation was also detectable at an average time of 191 days after HSCT. Seven (50.0%) of the 14 harboring mutations had relapsed/refractory disease by last follow-up, of which, in retrospect, 6 had received a predictably ineffective TKI within the first 100 days after transplantation based on our mutation analysis. These data support the idea that pre-existing mutations in the ABL kinase domain, frequently associated with resistance to TKIs and prevalent in a transplantation population, are persistently detectable in the majority of patients after transplantation. We propose that such resistance patterns should be considered when selecting TKIs in the post-HSCT setting, including clinical trials of post-HSCT TKI prophylaxis.

Development and immunological evaluation of HLA-specific chronic myeloid leukemia polyepitope vaccine in Chinese population.

BACKGROUND: BCR/ABL and Wilms' tumor 1 (WT1) are an ideal tumor associated antigens which can be used to develop a potential chronic myeloid leukemia (CML) dentritic cell (DC) vaccine. Here, we constructed a novel polyepitope vaccine which used recombinant lentiviral vector carrying BCR/ABL and WT1 genes, and determined the immunological effects of this vaccine in vitro. METHODS: The DC vaccine was constructed using lentiviral vector transduced DCs. T lymphocytes were stimulated with DC vaccine and then co-cultured in vitro with peripheral blood mononuclear cells (PBMCs) from CML or ALL patients, respectively. The cytotoxicity of proliferous cytotoxic T lymphocytes (CTLs) was determined by the LDH assay. The IFN-γ production of CTLs was detected using ELISPOT assay. RESULTS: We constructed an lentiviral vector encoding 50 different epitopes from BCR/ABL and WT1 antigens, and transferred it into DCs to prepare the DC vaccine successfully. The in vivo stimulation of CTLs with this DC vaccine were proved to show strong cytotoxicity and produce high level of IFN-γ. CONCLUSIONS: The novel recombinant lentiviral polyepitope DC vaccine is a promising candidate for clinical trials and may be an effective approach for CML immunotherapy.