白血病细胞和正常细胞生成树突状细胞的比较研究

目的体外诱导髓系白血病原代细胞分化生成树突状细胞(dendritic cell，DC)．并与正常的DC比较。方法分离初诊24例急性髓细胞性白血病和10例慢性髓细胞性白血病患的骨髓单个核细胞以及5例正常人外周血的单个核细胞，用rhGM—CSF 1000U／ml、rhIL-4500U／ml和TNF—α 50U／ml联合培养10天；形态学(Wright染色、倒置显微镜、透射电镜)、免疫学(CD80、CD86、CD83、CD1a、HLA—DR)鉴定，RT—PCR检测白血病克隆以及混合淋巴细胞反应(MLR)检测抗原递呈功能。结果细胞因子诱导后，正常细胞和白血病细胞均出现典型形态的树突状细胞，CD80、CD86、CD83、CD1a的表达明显上调(正常和CML—DC的HLA—DR也明显上调)，急性和慢性髓细胞性白血病以及正常细胞来源的树突状细胞具有不同程度的刺激异基因T淋巴细胞增殖的能力。结论无论是急性和慢性白血病细胞均能诱导分化成DC，并且特征完全相似；白血病与正常骨髓的DC在形态和免疫学表达方面相似，但功能较弱。     

白血病细胞来源的树突状细胞在治疗白血病中的应用

树突状细胞(DC)广泛分布于体内,是机体功能最强的抗原递呈细胞(APC),在免疫应答中起着桥梁作用.最近有文献报道,白血病细胞经诱导后能分化为DC,从而启动特异性的机体抗白血病免疫应答,使白血病的治疗取得了可喜进展.将白血病细胞诱导成为具有递呈肿瘤抗原特性的DC,激发自体特异性细胞毒性T淋巴细胞的产生,对白血病细胞产生杀伤作用,表明白血病细胞来源的DC的诱导方案,其细胞生物学特性及白血病的治疗具有广泛的应用前景.     

IFN-α对CML树突状细胞表型及功能的影响

临床观察表明IFN-α治疗早期慢性期CML患者有效,并且IFN-α治疗CML达到部分细胞遗传学缓解以上的患者较之IFN-α治疗无效者其生存期明显延长.IFN-α治疗CML有效的机制尚不清楚,可能与其增强DC的表型及功能有关.DC是体内唯一能激活初始型T细胞的抗原递呈细胞,在机体免疫系统中处于中心地位.我们采用CML患者骨髓单个核细胞,体外有血清培养体系中观察IFN-α对CML-DCs的分化及其功能的影响.     

脂质体RNA负载慢性粒细胞白血病来源树突状细胞的性能比较

目的研究人慢性粒细胞白血病(CML)总RNA经过脂质体负载后体外转染自体慢性粒细胞白血病 树突状细胞（CML-DC）,并诱导特异性细胞毒T淋巴细胞（CTL）免疫反应。方法CML患者骨髓单个核细胞（CML-BMMNC）在rhIL-4、rhGM-CSF、rhTNF-α细胞因子联合培养诱导出CML-DC。采用Trizol法提取CML-BMMNC的总RNA;反复冻融法制备CML BMMNC抗原。于CML-DC培养第5天,加入脂质体转染的CML总RNA、CML总RNA、CML肿瘤冻融抗原及不加抗原。倒置显微镜观察DC形态学变化;流式细胞仪器检测免疫表型变化;染色体G显带技术检测其染色体核型及逆转录聚合酶链反应（RT-PCR）检测Bcr-abl融合基因的表达;用MTT法检测CTL作用。结果CML BMMNC诱导成CML-DC, CD_1α、CD₈₃表型表达较诱导前明显上调,形态学有典型的DC形态,且均存在Bcr-abl基因带和Ph₁染色体,提示CML-DC为白血病源性。总RNA经脂质体转染CML-DC、CML肿瘤冻融抗原负载CML-DC、总RNA不经脂质体转染CML-DC、未负载抗原CML-DC分别致敏的CTL及IL-2培养的T淋巴细胞在效：靶比为20∶1时的杀伤效率依次降低。结论CML BMMNC来源的 DC既具有CML白血病源性,又具有DC细胞的特性,能诱导特异性CTL杀伤作用;总RNA经脂质体转染的CML-DC诱导的CTL杀伤性对CML细胞的杀伤作用最强。     

抗白血病树突状细胞疫苗的制备

目的探索抗白血病异体树突状细胞疫苗的制备方法.方法从健康者外周血中诱生DC,用不同方法负载K562抗原(化学融合法,冻融抗原冲击法,肿瘤与DC共培养法),比较这些DC疫苗在递呈抗原激活T细胞增殖及杀伤功能方面的差异.结果从健康者外周血中可诱生出具有正常免疫表型和抗原递呈功能的DC.不同抗原负载方法致敏的DC均能激活T细胞特异性杀伤K562细胞.冻融抗原负载的DC能有效刺激T细胞增殖,其活化的CTL对K562的杀伤毒性最强.结论异体DC可有效递呈肿瘤抗原,激活T细胞杀伤肿瘤,冻融抗原负载的DC激活T细胞对肿瘤的杀伤最强,为今后临床DC疫苗的选择提供了实验依据.     

树突细胞联合β-榄香烯对小鼠胰腺癌治疗的实验研究

背景与目的：树突细胞（Dendritic cells，DC）是目前发现的功能最强大的肿瘤抗原专职免疫递呈细胞，DC疫苗是目前最具临床应用潜能的治疗性疫苗。肿瘤细胞凋亡小体被DC的MHC—1分子提呈并诱导的特异性CTL免疫反应抗癌作用显著。本研究探讨了DC负载凋亡癌细胞抗原后诱导的免疫应答联合β-榄香烯对小鼠胰腺癌治疗作用。方法：制备C57BL／6小鼠骨髓源性DC并鉴定，负载肿瘤抗原后制备成疫苗。分别观察DC诱导的CTL和β-榄香烯对胰腺癌细胞的杀伤作用。体内观察DC疫苗联合β-榄香烯对小鼠胰腺癌的抑制作用。结果1经电镜及流式细胞鉴定可获得典型的具有抗原加工及递呈作用的DC，利用TNF-α可使成熟的树突状细胞比例增加，MHC-Ⅱ、CD83、CD86等表面分子的表达较单纯DC组显著增强。负载凋亡抗原后DC诱导的特异性CTL体外对胰腺癌细胞有显著杀伤作用，效靶比30：1时，诱导的CTL对胰腺癌细胞抑制率达93％。β-榄香烯对癌细胞增殖抑制明显，DC疫苗与β-榄香烯联合治疗可明显抑制荷瘤小鼠肿瘤的生长，使其生存期延长，联合治疗组生存期为49．4天，与单纯β-榄香烯组（24．8天）、DC疫苗（38．5天）、DC组（20．7天）及对照组（17．5天）相比有显著差异（P〈0．05）。结论：经凋亡肿瘤细胞致敏的DC回输荷瘤小鼠可激发宿主针对特异肿瘤的Th1及CTL免疫应答，以DC、介导的免疫反应联合中药的治疗方法可成为抗肿瘤的崭新且有效手段。     

慢性髓性白血病来源的树突状细胞诱导的CTL体外作用研究

 目的　研究慢性髓性白血病来源的树突状细胞的免疫功能。方法　分离慢性期CML患者外周血单个核细胞 (PBMNC) ,用细胞因子GM CSF及IL 4在体外诱导培养DC ,检测细胞表型 ,并观察其诱导的CTL体外抗肿瘤效应。用酶联免疫 (ELISA)方法测定CML DC混合淋巴细胞反应 (MLR)上清液中IL 12及IFN γ的量 ,并与正常DC进行比较。结果　联合GM CSF及IL 4可诱导CML细胞分化为CML DC ,CML DC的CD1a、CD80、CD83表达率均低于正常DC ;CML DC混合淋巴细胞反应上清IL 12及IFN γ含量均低于正常DC ;CML DC能诱导出对自身CML细胞有特异性杀伤作用的CTL。结论　CML DC具有抗原提呈细胞的免疫功能 ,能诱导抗白血病CTL反应。     

流感疫苗对慢性粒细胞白血病源性树突状细胞功能影响的研究

 目的 探讨流感疫苗对白血病源性树突状细胞（DC）功能的影响。方法 分离慢性粒细胞白血病（CML）患者骨髓单个核细胞,加入GM-CSF和IL-4诱导培养7 d,将所得细胞分为4组：完整失活流感疫苗（WIV）组;裂解流感疫苗（SIV）组;TNF-α组;对照组,用不同培养方法再培育24 h后,流式细胞仪分析DC免疫表型,ELISA法检测上清液中IL-12浓度。结果 CML源性DC细胞表面分子表达、上清液中IL-12浓度在WIV及SIV组较TNF-α和对照组显著增高,差异有统计学意义（P＜0.05）;其中WIV组高于SIV组（P＜0.05）,而TNF-α组与对照组差异无统计学意义（P＞0.05）。结论 流感疫苗具有刺激CML源DC成熟的功能。     

脐血、健康人及慢粒患者外周血来源的DC介导的抗白血病免疫效应研究

目的：观察脐血、健康人及慢性柱细胞性白血病(CML)患者外周血3种不同来源的树突状细胞(DC)及其抗白血病效应。方法：分离3种来源的单个核细胞(MNC)并在细胞因子(GM-CSF IL-4 TNF-α)作用下诱导培养DC。观察DC形态，流式细胞技术检测DC免疫表型(CD1α、CD80、CD83、HLA-DR)；将所得DC经CML细胞可溶性抗原负载后和其同源T细胞混合诱导抗原特异性CTL，MTT法检测其杀伤活性。结果：3种来源MNC在细胞因子作用下均可获得形态典型的DC，且高表达CD1α、CD80、CD83、HLA-DR，经抗原负载后的DC诱导的CTL对CML靶细胞的杀伤活性也较强。结论：脐血、健康人及CML患者外周血均为理想的DC来源；有较好的诱导CTL杀伤白血病细胞效应；应用DC瘤苗免疫治疗白血病必将成为一种崭新的肿瘤免疫治疗方法，有十分诱人的发展前景。     

树突状细胞疫苗与慢性粒细胞白血病

树突状细胞（DC）是体内最重要的抗原提呈细胞,在抗肿瘤免疫中起着重要的作用。慢性粒细胞白血病（CML）是起源于多能造血干细胞恶性克隆性疾病．负载CML的肿瘤细胞可溶性抗原、bcr/abl抗原肽于DC形成DC疫苗,在体外诱导出抗CML细胞毒作用,用于清除微小残留病。现综述DC疫苗在CML免疫治疗的研究进展。     

Treatment options for chronic myeloid leukemia: imatinib versus interferon versus allogeneic transplant

PURPOSE OF REVIEW: This review considers recent developments in the treatment of chronic myeloid leukemia with attention to current data evaluating the relative roles of imatinib mesylate, interferon-alpha, and allogeneic blood or marrow transplantation. Additionally, the review discusses advances in the basic understanding of the mechanisms by which these three different therapies function against chronic myeloid leukemia. RECENT FINDINGS: Long-term follow-up has found that interferon-alpha was able to produce complete cytogenetic remission in15 to 25% of patients, with some of these patients achieving a molecular remission. Some patients who achieve a complete cytogenetic remission also achieve long-term disease-free survival and possibly cure. Imatinib has produced remarkable hematologic and cytogenetic responses in newly treated and in interferon-alpha-refractory patients, yet there are no long-term survival data at this point. Some laboratory findings imply that imatinib may primarily affect mature chronic myeloid leukemia progenitors and not chronic myeloid leukemia stem cells, leaving doubt that the improved rate of complete cytogenetic remissions will result in increased overall survival. Other clonal cytogenetic abnormalities have also been reported in the Philadelphia chromosome-negative cells present in complete cytogenetic remissions to imatinib. The use of donor lymphocytes infusion (DLI) continues to treat relapsed chronic myeloid leukemia effectively after allogeneic blood or marrow transplantation whereas the use of nonmyeloablative therapy has effectively reduced transplant-related mortality. SUMMARY: Patients with chronic myeloid leukemia now have several potential treatment options from which to choose. Imatinib mesylate currently provides excellent hematologic and cytogenetic response rates with minimal toxicity. However, long-term data of efficacy is lacking. Emerging evidence that imatinib rarely leads to molecular complete remission and that many patients are still at risk of relapse and other clonal disorders is concerning and suggest the possibility that imatinib's early high response rates may not translate into survival advantage. Interferon-alpha continues to be effective therapy for many patients and, along with blood or marrow transplantation, is proved to prolong survival. The impacts of both are in part limited because of their toxicity profiles. Ongoing laboratory investigations and clinical trials remain paramount to providing the best treatment approach for our patients with chronic myeloid leukemia.     

Vaccination with leukemia-loaded dendritic cells eradicates residual disease and prevent relapse

We have previously demonstrated that TNF-alpha gene therapy with myeloid progenitor cells inhibits the progression of 32Dp210 myeloid leukemia in mice. Because TNF-alpha has been shown to induce the activation and maturation of dendritic cells (DCs), we investigated the efficacy of DC-based leukemia vaccine for eradication of residual disease when administered following cytoreductive therapy. Immunization with DC cells loaded with 32Dp210 myeloid leukemia cells (32Dp210 vaccine) was far more effective in preventing the development of leukemia compared to immunization with irradiated leukemia cells alone. The resistance to leukemia could be adoptively transferred to na?ve mice with the spleen cells of mice immunized with DC-32Dp210 vaccine, and splenic cells responsible for adoptive transfer of resistance were identified as CD90 + T lymphocytes. Development of immunity in vaccinated mice was associated with the generation of leukemia specific cytotoxic T lymphocytes (CTLs) and secretion of cytokines TNF-alpha and IFN-gamma. Further, immunization with DC-32Dp210 vaccine following cytoreductive therapy with Cytoxan was effective in eradicating residual disease in approximately 50 percent of the animals. However, eradication of residual disease was significantly improved (approximately 74%) when animals were treated with DC-32Dp210 vaccine in which DCs were activated with TNF-alpha prior to loading of 32Dp210 leukemia cells. Cured mice were in molecular remission since Bcr/Abl oncogene could not be amplified from the DNA isolated from the marrow, spleen, or liver of cured mice. Taken together, these data demonstrate the efficacy of DC-based leukemia vaccine for eradication of residual disease and prevention of relapse.     

透毒复方对急性髓系白血病完全缓解患者CD+34细胞源树突状细胞生物学效应的影响

 目的　研究透毒复方青蒿鳖甲汤对急性髓系白血病完全缓解期（AML-CR）患者骨髓CD+34 细胞来源树突状细胞（DC）生物学效应的影响。方法　分离纯化骨髓CD+34 细胞,体外不同浓度透毒中药含药血清与细胞因子联合诱导分化为DC,观察DC形态特征,流式细胞术检测DC表面分子CD80、CD83、CD86的表达,将DC分别与自体、异体外周血T细胞共同培养,四甲基偶氮唑蓝（MTT）比色法检测激活的T细胞不同效靶比对人类白血病细胞株（K562细胞）的杀伤效应。结果　青蒿鳖甲汤联合细胞因子能促进AML-CR患者骨髓CD+34 分化为形态典型DC,不同剂量含药血清均能上调DC表面特征性分子及共刺激分子CD80、CD83、CD86比例,较单纯细胞因子组比较差异有统计学意义（P＜0.01）,且含药血清组较单纯细胞因子更能激发外周血T淋巴细胞对K562细胞的杀伤作用,两者间比较差异有统计学意义（P＜0.01）。结论　青蒿鳖甲汤能促进髓系微小残留白血病患者CD+34 细胞向DC转化,提高DC的生物学效应。     

Calcium ionophore activation of chronic myelogenous leukemia progenitor cells into dendritic cells is mediated by calcineurin phosphatase

We have previously demonstrated that Ph+ myeloid progenitor cells of patients with chronic myeloid leukemia (CML) can acquire characteristics of mature dendritic cells (DC) following calcium mobilization with calcium ionophore (A23187, CI). In this study we characterize the intracellular signaling pathway by which CI induces the acquisition of DC features in these leukemic cells. CI-induced activation of CML cells is attenuated by the calcineurin phosphatase inhibitor cyclosporin A (CsA) as well as the calmodulin (CaM) antagonist W-7. These cause ablation of both the CI-induced immunophenotypic expression of DC markers and immunostimulatory properties in mixed leukocyte responses (MLR). Minimal blocking effect was observed when Ca(2+)/CaM kinase II (281-301) inhibitor was added to the cultures. These findings suggest a Ca(2+)-dependent mechanism for the CI-induced activation of CML cells into antigen-presenting cells (APC), which is primarily mediated through the CaM/calcineurin pathway.     

Circulating myeloid dendritic cell directly isolated from patients with chronic myelogenous leukemia are functional and carry the bcr-abl translocation

Leukemic bcr-abl positive dendritic cells (DCs) are likely to be present in vivo in chronic myelogenous leukemia (CML) patients, but no data are available on their functional qualities. We analyzed the circulating BDCA-1+ myeloid DC compartment in 15 chronic phase CML patients. Phenotypic features of CML DCs were comparable with that of normal DCs, except for the CD80 and CD40 antigens, significantly under-represented in CML patients. Nonetheless, no differences were found between normal samples and leukemic DCs in the allostimulatory ability, as well as in the production of cytokines and polarization of T cell responses. CML DCs were analyzed by fluorescence in situ hybridization (FISH) and found positive for the bcr-abl translocation. However, when bcr-abl+ DCs were tested for their ability to stimulate an autologous T-cell response in vitro, we could not detect a specific recognition. We conclude that an apparently normal circulating DC compartment carrying the Ph+ chromosome can be identified in CML patients; however, these cells appear unable to trigger a protective anti-leukemic immune response in autologous T cells.     

Association between C1236T Genetic Variant of ABCB1 Gene and Molecular Response to Imatinib in Indonesian Chronic Myeloid Patients

Objective: Imatinib is the first-line drug used for the treatment of chronic myeloid leukemia (CML) patients due to high molecular response and overall survival rate. However, some patients develop resistance to imatinib even after attaining a response. Mutation in ABCB1 efflux transporters is one of the known mechanisms of resistance to imatinib in chronic myeloid leukemia patients. This study was aimed to investigate the association of ABCB1 C1236T polymorphism in Indonesian chronic myeloid patients with molecular response to imatinib treatment. Methods: We analyzed 120 samples from chronic myeloid leukemia patients in the chronic phase, who had been on imatinib treatment for at least 12 months. We analyzed the C1236T variant of the ABCB1 gene using PCR, followed by direct sequencing, and associate them with the achievement of major molecular response (MMR). Results: The major molecular response was achieved in 28% of patients. The frequencies of the SNPs were CC (40%), CT (46%), and TT (14%). Our result showed that there was a lack of association between polymorphism of ABCB1 C1236T and imatinib response in Indonesian patients, with OR = 0.646 (95% CI: 0.283, 1.471; p>0.05). Conclusion: There was no association between ABCB1 C1236T variants with the major molecular response in Indonesian chronic myeloid leukemia patients receiving imatinib treatment.     

Identification of precursors of leukemic dendritic cells differentiated from patients with acute myeloid leukemia.

Dendritic cells (DC) can facilitate immune responses that might help in the induction of effective antitumor T cell responses. We reported previously that leukemic blasts from selected patients with acute myeloid leukemia (AML) were able to differentiate in vitro into cells with mature DC features. However, despite the use of a wide variety of cytokine combinations, leukemic DC could not be obtained from all AML patients. In this study, we investigated in a wide range of AML patients (n = 30), the nature and functional characteristics of the blast compartment that can be induced to acquire DC features in vitro. Our results demonstrate that leukemic DC generated in the presence of GM-CSF, IL-4 and matured with CD40L, are composed of two major subsets: DC derived from CD14(+) leukemic cells and leukemic DC derived from in vivo expanded circulating blood myeloid DC (MDC). Leukemic DC of both subsets exhibited DC morphology, had a phenotype of mature DC, and could induce a potent proliferative response of naive CD4(+) T cells. Moreover, both subsets produced large amounts of IL-12p70 and leukemic CD14(+)-derived DC could induce a potent Th1 response. These results can be considered as a prerequisite before the design of vaccine immunotherapy protocols for the adjuvant treatment of AML patients.     

Some aspects of immunological and cytochemical markers in leukemia

Summary The value of immunological and of cytochemical markers for understanding of pathophysiology and for diagnosis in different subtypes of leukemia is discussed.Abbreviations used in this paper AML Acute myeloid leukemia - AMoL acute monocytoid leukemia - ALL acute lymphoid leukemia - ANAE acid -naphtyl acetate esterase - APh acid phosphatase - B-ALL (-CPL,-CLL) B-lymphoid acute (prolymphocytic, chronic) leukemia - C-Ag membrane antigen of common ALL - C-ALL common ALL whose cells react with a specifically absorbed antiserum against ALL of non-T, non-B variety - CLL chronic lymphoid leukemia - CPL chronic prolymphocytic leukemia - E rosettes with sheep erythrocytes - Ia immune response antigen - M-Ag myeloid antigen - POX peroxidase - SmIG surface membrane Ig - T-Ag T-lymphocyte antigen - T-ALL (-CPL,-CLL) T-lymphocyte antigen-positive acute (prolymphocytic chronic) lymphatic leukemia - TdT terminal deoxynucleotidyl transferase - UAL undifferentiated acute leukemia     

Genetic Mechanisms of Chronic Myeloid Leukemia Blastic Transformation

The BCR-ABL1 oncogenic tyrosine kinase can transform pluripotent hematopoietic stem cells and initiate chronic myeloid leukemia in chronic phase (CML-CP), a myeloproliferative disorder characterized by excessive accumulation of mature myeloid cells. Patients in CML-CP usually respond to treatment with ABL1 tyrosine kinase inhibitors (TKIs) such as imatinib, though some patients who respond initially may become resistant later. CML-CP leukemia stem cells (LSCs) are intrinsically insensitive to TKIs and thus survive in the long term. These LSCs or their progeny may at some stage acquire additional genetic changes that cause the leukemia to transform further, from CML-CP to a more advanced phase, which has been subclassified as either accelerated phase (CML-AP) or blastic phase (CML-BP). CML-BP is characterized by a major clonal expansion of immature progenitors, which have either myeloid or lymphoid features. CML-BP responds poorly to treatment and is usually fatal. This review discusses the role of genomic instability leading to blastic transformation of CML and proposes some novel therapeutic approaches.