白血病抗原冲击致敏的树突状细胞诱导的抗肿瘤免疫反应

目的：研究树突状细胞(DC)的体外培养扩增及诱导特异性的抗肿瘤免疫反应。方法：使用mGM-CSF加mIL-4培养诱导骨髓细胞分化，采用反复冻融法制备L7212白血病细胞的冻融抗原(TAA)，在DC培养的第3天加入TAA，将TAA冲击致敏的DC与T淋巴细胞共培养，获得肿瘤特异性细胞毒性T淋巴细胞(CTL)，采用MTT法检测CTL对L7212细胞的杀伤作用及其对野生型L7212细胞再攻击的免疫保护作用。结果：MTT检测发现CTL对L7212细胞有特异性杀伤抑制作用，L7212抗原冲击致敏的DC能显著提高和延长野生型L7212细胞再攻击小鼠的生存率和存活期。结论：mGM-CSF和mIL-4配伍可有效地从小鼠骨髓细胞中诱导出大量的成熟的功能性DC，L7212白血病TAA冲击致敏的DC可诱导机体产生较强的抗肿瘤免疫反应。     

不同肝癌细胞抗原致敏树突状细胞体外诱导CTL活性的研究

目的比较不同方法提取的肝癌细胞(SMMC-7721)抗原致敏树突状细胞(Dendritic cell DC)后,对特异性细胞毒性T细胞(CTL)的诱导作用。方法分离脐带血单个核细胞,加入重组粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)诱导扩增出脐血DC;SMMC-7721细胞经反复冻融,直接超声破碎及热休克处理后再超声破碎细胞三种方法提取肿瘤抗原,然后分别致敏DC,以MTT法检测脐血DC诱导T细胞增殖及其特异性CTL杀伤活性。结果热休克处理后再超声破碎法提取的抗原致敏DC,能诱导更强的刺激T细胞增殖的能力,并且可以诱导更强的CTL活性。结论加热处理后再超声破碎细胞提取的肿瘤抗原致敏DC,在体外可诱导强大的抗肿瘤免疫保护效应。     

K562冻融抗原负载的树突状细胞诱导抗慢性粒细胞白血病的免疫效应

目的研究K562冻融抗原负载的健康供者来源的树突状细胞(DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤慢性粒细胞白血病(CML)细胞的毒性效应。方法利用健康供者外周血单个核细胞诱导分化为DC,采用反复冻融法从K562中提取的可溶性相关抗原负载DC;流式细胞学检测负载抗原前后DC表面分子表达的变化;ELISA法检测DC上清中IL-12和IFNγ的含量;混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力;乳酸脱氢酶法检测K562冻融抗原负载DC诱导的抗原特异性CTL对CML细胞的杀伤作用。结果与未经抗原负载的DC相比,经K562抗原负载的DC表面分子表达明显上调,CD1a(27·40±5·00)%、(15·40±2·34)%,CD80(61·35±5·35)%、(42·00±2·77)%,CD83(93·30±3·48)%、(25·15±4·02)%,CD86(85·25±4·39)%、(37·25±3·20)%,CD40(89·80±7·18)%、(35·95±4·06)%,HLA-DR(49·50±5·45)%、(17·15±3·61)%,DC分泌IL-12和诱导T细胞分泌IFNγ的能力增加(P<0·05),具有很强的刺激T细胞增殖能力,且刺激强度在24h最强,48h降低,冻融抗原负载DC后激活的CTL在体外对K562的杀伤率为77·35%,显著高于未经抗原负载的DC(P=0·001)。结论经K562细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤CML细胞的作用。     

细胞因子对脐血树突状细胞体外诱导分化及扩增作用的研究

目的:树突状细胞是初始免疫反应中最重要的抗原提呈细胞。本研究目的是分析脐带血的细胞组成,研究加入细胞因子培养前后脐血树突状细胞的变化,以脐血为来源探索体外诱导、扩增树突状细胞的方法并进行表型鉴定。方法:脐血12例,分离单个核细胞。在脐血单个核细胞中加入细胞因子粒-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-3(IL-3)、造血干细胞生长因子(SCF)和促红细胞生成素(EPO),培养4周。应用流式细胞仪,使用CD4、CD8、CD19、CD34、CD38、CD1a、CD11c和CDw123单克隆抗体,测定培养前及培养后1、2、3、4周脐血细胞的细胞表面抗原变化及树突状细胞情况。结果:新鲜脐血单个核细胞中CD34 造血干细胞0.22×108/L、CD1a 细胞0.27×108/L、CD11c 细胞5.87×108/L、CD83 细胞1.94×108/L、CDw123 细胞2.73×108/L。加入细胞因子GM-CSF、IL-3、SCF、EPO后培养1~4周的脐血单个核细胞分化为CD1a 、CD11c 、CD83 、CDw123 树突状细胞,在培养的2~4周,脐血树突状细胞数量明显增多,此后逐渐减少。通过培养,树突状细胞数量增加达CD1a 细胞11.02×108/L、CD11c 细胞28.24×108/L、CD83 细胞10.57×108/L、CDw123 细胞18.7×108/L。结论:在培养液中加入细胞因子GM-CSF、IL-3、SCF和EPO后培养2~4周,脐血单个核细胞可分化为CD1a 、CD11c 、CD83 、CDw123 树突状细胞。这些树突状细胞作为抗原提呈细胞在肿瘤免疫治疗上将起到重要作用。     

负载胰腺癌抗原的树突状细胞疫苗诱导T淋巴细胞抗肿瘤效应的研究

培养树突状细胞(DC),反复冻融法裂解培养的负载胰腺癌细胞(PC-3),提取细胞抗原,致敏DC,获得负载胰腺癌抗原的DC疫苗后诱导特异性细胞毒性T淋巴细胞(CTL)的生成,MTT法检测CTL对不同肿瘤细胞的杀伤作用.发现负载PC-3细胞抗原的DC疫苗能诱导产生肿瘤特异性的CTL,其对PC-3细胞具有明显地杀伤效应,而对人乳腺癌细胞MCF-7、人肝癌细胞7721细胞杀伤作用弱.认为负载胰腺癌抗原的DC疫苗能够诱导高效而特异地CTT杀瘤活性,为将来DC疫苗在胰腺癌的免疫治疗中提供了实验依据.     

HBV抗原基因修饰树突状细胞疫苗体外抗HBV的作用

目的：探讨腺病毒载体介导HBV抗原基因修饰的树突状细胞(DCs)诱导抗HBV特异性CTL反应方法：制备携带HBsAg、HBeAg和HBcAg基因的3种重组腺病毒Ad-HBs,Ad-HBe,Ad- HBc,分别转染自脐带血体外诱导培养的DCs,观察腺病毒转染DCs效率和DCs中HBV抗原的表达;混合淋巴细胞反应(MLR)测定HBV抗原基因修饰DCs刺激同种异体T淋巴细胞增殖能力;乳酸脱氢酶释放法检测特异性CTL细胞对HepG_222．1．5靶细胞的杀伤能力．结果：腺病毒载体能够高效介导HBV三个抗原基因在DCs中表达,90%以上DCs表达示踪基因EGFP,且DCs细胞形态完整：感染后72 h HBsAg和HBeAg含量分别为0．919和0．328(吸光度A值)．MLR实验显示,HBV抗原基因修饰DCs仍然具有刺激同种异体T细胞的增殖能力,Ad-HBs转染DCs组、Ad-HBe转染DCs组、Ad-HBc转染DCs组和未转染DCs组之间刺激T细胞的增殖水平无明显差异(F=1．194,P=0．389);在E：T比例为2：1,10：1和25：1时,Ad-HBs转染DC组、Ad-HBe转染DCs组和Ad-HBc转染DCs组对HepG_222．1．5细胞的杀伤率均明显高于未转染DCs组(P＜0．001);以Ad- HBc转染DC组对HepG_222．1．5细胞杀伤率最高．结论：HBV抗原基因修饰DCs疫苗具有刺激同种异体T细胞增殖能力,同时能增强抗HBV特异性CTL反应的能力,可能发展为一种新型抗病毒疫苗．     

非淋巴细胞白血病原代细胞诱导成树突状细胞的研究

目的：用白血病细胞诱导树突状细胞,为白血病的免疫治疗提供新途径。方法：分离人的非淋巴细胞白血病原代细胞,体外流体培养体系中加入ＧＭ－ＣＳＦ、ＴＮＦ－α及ＩＬ－４,培养１２ｄ后,与培养前的细胞在形态、ＣＤ１ａ、ＨＬＡ－ＤＲ及ＣＤ８０的表达情况进行比较。结果：经１２ｄ诱导,细胞形态表现典型的树突状细胞的特征;ＣＤ１ａ、ＨＬＡ－ＤＲ及ＣＤ８０的表达明显增高。结论：可将非淋巴细胞白血病原代细胞诱导成树突状     

体外负载冻融抗原的脐血树突状细胞诱导的抗肝癌效应

目的评价体外应用肝癌细胞冻融抗原负载的脐血树突状细胞（DC）所诱导的抗肝癌效应。方法采集健康足月剖宫产孕妇胎盘端脐血,分离脐血单个核细胞（CBMNC）及T淋巴细胞,用GM-CSF、IL-4及TNF-α联合诱导CBMNC分化为DC,观察形态学变化并以流式细胞术鉴定,选培养的第3天以肝癌细胞冻融抗原负载的CBMNC-DC,以负载抗原的DC刺激自体淋巴细胞活化为自体细胞毒性T淋巴细胞（CTL）,并用CTL对肝癌细胞进行杀伤,MTT法测定活化的自体淋巴细胞的相对数量和CTL对肝癌细胞的杀伤率。结果体外负载肿瘤冻融抗原的脐血DC可诱导显著的自体效应淋巴细胞增殖及抗肝癌效应。结论体外负载抗原的脐血DC可诱导显著的抗肝癌效应,是具有临床应用前景的肝癌疫苗。     

体外诱导白血病源性树突状细胞及其对T细胞杀伤活性影响的研究

目的 :探讨体外培养白血病源性树突状细胞 (DC)及其对T细胞杀伤活性的影响。方法 :从慢性髓细胞性白血病 (CML)患者外周血中分离单个核细胞 ,加入 1× 10 6U/L粒 巨噬细胞集落刺激因子 (GM CSF)、1×10 6U/L白细胞介素 (IL) 4和 5 0× 10 3 U/L肿瘤坏死因子 (TNF α) ,每 3～ 4天换液 1次 ,连续培养 14天 ,获得DC。取外周血单个核细胞加入 5 0 0× 10 3 U/LIL 2 ,培养至第 7天 ,把细胞分成两组 ,其中一组加入培养的DC ,两组细胞继续培养 3～ 4天 ,然后测定T细胞杀伤活性。结果 :DC具有典型的树状突起 ,高表达CD1a,具有慢粒特异性染色体t(9;2 2 ) ,能增强T细胞对白血病细胞的杀伤活性。结论 :在体外利用细胞因子可以把CML细胞诱导分化成具有刺激T细胞产生细胞毒性反应的白血病源性DC。     

日本血吸虫抗原负载树突状细胞的表型研究

目的 研究负载日本血吸虫谷胱甘肽S-转移酶(glutathione S-transferase,GST)和可溶性虫卵抗原(soluble egg antigen,SEA)的树突状细胞的表型. 方法 骨髓来源的细胞经白介素-4(interleukin-4,IL-4)、粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)诱导培养,获得树突状细胞,体外经GSI、SEA抗原刺激.用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的抗GST单克隆抗体染色法检测GST的负载情况.流式细胞仪检测血吸虫抗原负载后树突状细胞表面CD40、CD80、CD86、CD11c分子的表达情况,并与脂多糖(lipopolysaccharide,LPS)、PBS刺激组作比较. 结果 GST负载后在荧光显微镜下可观察到抗GST的特异荧光,表明抗原已被细胞摄取.与LPS相比较,GST、SEA抗原负载后,树突状细胞表面分子CD40、CD86上调不显著,而更类似于PBS刺激组. 结论 日本血吸虫抗原负载后,树突状细胞的表型类似于未成熟表型.     

Induction of cytotoxic T lymphocytes by dendritic cells pulsed with murine leukemic cell RNA

Peptide-pulsed dendritic cells can stimulate T cells showing specific cytotoxicity in chronic myelogenous leukemia. We tried to induce a specific cytotoxic T-cell response stimulated by RNA-pulsed dendritic cells in acute myelogenous leukemia. The total RNA of WEHI-3BD+, a myelomonocytic leukemia cell line derived from BALB/c mice, was transfected into dendritic cells induced from bone marrow nucleated cells of BALB/c mice with granulocyte macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) using liposome. RNA-pulsed dendritic cells were injected into the peritoneal cavity of BALB/c mice, and splenic T cells were isolated for antigen-stimulated proliferation and leukemia-specific cytotoxicity assay. Cultured bone marrow nucleated cells expressed dendritic cell markers including MHC class II antigen, CD80, CD86, and CD11c. T cells stimulated by RNA-pulsed dendritic cells showed enhanced proliferation than those stimulated by unpulsed dendritic cells (P = 0.05) and showed dose-dependent specific cytotoxicity against WEHI-3BD+ cells. We concluded total RNA-pulsed dendritic cells could induce a specific T-cell cytotoxicity in acute myelogenous leukemia.     

人脾脏树突状细胞大量培养的新方法

目的：建立人脾脏单核细胞体外培养生成大量树突状细胞的新方法。方法：人脾脏细胞悬液培养2h获得贴壁的单核细胞,加入重组人粒细胞－巨噬细胞集落刺激因子（rhGM-CSF)100μg/L或rhGM-CSF100μg/L 重组人白细胞介素－4（rhIL-4)500kU/L,体外培养7天,收集悬浮细胞,以流式细胞仪分析细胞表型及抗原内蚕能力,体外混合淋巴细胞反应检测细胞抗原呈递功能。结果：以细胞因子培养7天生成的悬浮细胞,20％－80％表达树突状细胞（DC）特异性标志CD1a,表达高水平的HLA－DR、B7－1、B7－2分子,具有较强的抗原内吞能力,体外可以强烈刺激同种T细胞增生,其形态,表型与人外周血单核细胞获得的DC相似。结论：从人脾脏获得的贴型的单核细胞,体外以GM-CSF rhIL-4培养,可以获得极其大量的DC（＞10^9细胞／个脾脏）为进一步研究和应用打下了基础。     

Activation of autologous leukemia-specific T cells in acute myeloid leukemia: monocyte-derived dendritic cells cocultured with leukemic blasts compared with leukemia-derived dendritic cells

In acute myeloid leukemia (AML) blasts can be differentiated into dendritic cell (DC) like cells (AML-DC). These cells have a mature DC-like phenotype, are strong stimulators in mixed leukocyte reactions and can be used to generate leukemia-specific cytotoxic T cells. However, recent reports about naturally existing leukemic DC with immunoregulatory dysfunctions in peripheral blood of AML patients caused concerns about the use of AML-DC for therapeutic purposes. Systematic intra-individual comparisons between AML-DC and non-leukemic DC derived from monocytes (MoDC) in AML patients are missing. Thus, we investigated the ability to generate MoDC from peripheral blood of 17 AML patients in first remission and their functional integrity to stimulate leukemia-specific T cells by simple coculture with leukemic blasts. Phenotypic analysis of AML-DC and MoDC from the same individual patients revealed that MoDC exhibit a more homogenous mature DC phenotype. Additionally, functional analysis demonstrated the ability of remission MoDC to activate autologous leukemia-specific T cells in 11 of 12 patients, whereas AML-DC led to a specific T cell activation in four of eight patients. The presented findings might have impact on the design of further therapeutic studies using autologous antigen-presenting cells.     

Extensive flow cytometric characterization of plasmacytoid dendritic cell leukemia cells

OBJECTIVES: Accumulating evidence suggests that non-T, non-B cell CD4+CD56+ neoplasms with lymphoblastic morphology include clinically and immunophenotypically diverse entities. Although their cells of origin or classification are still controversial several entities clearly represent a distinct type of neoplasms that are clinically aggressive. METHODS: In this work we present the immunophenotypic and genotypic features of bone marrow (BM), peripheral blood (PB), lymph node and skin lymphocytes from a patient diagnosed as plasmacytoid dendritic cell leukemia involving the skin, BM, PB, lymph nodes, liver and spleen. For determination of immunophenotypic characteristics of malignant plasmacytoid dendritic cells 73 monoclonal antibodies detecting lineage markers, chemokine receptors, cytokine receptors, activation, and co-stimulatory molecules were used. RESULTS AND CONCLUSION: The malignant cells proved to express CD4+, CD56+ lineage negative leukemia phenotype characteristically positive for CD36, CD38, CD40, CD45, CD45RA, CD68, CD123, CD184, HLA-DR, BDCA2, and granzyme-B corresponding to the preplasmacytoid dendritic cell developmental stage. The presence of CD11a/CD18, CD84, CD91, CD95, alphavbeta5, CDw197, and the absence of CD52 and CD133 in this case can be regarded as additional features of malignant cells. Completing the immunophenotypes with multidrug resistance function can provide additional information for characterizing pDC leukemia.     

Functional profile of activated dendritic cells in unstable atherosclerotic plaque

Background Unstable atherosclerotic plaque typically contains an infiltrate of activated macrophages and activated T cells. This study established a functional profile of plaque-residing dendritic cells (DC) to examine whether they can function as Ag-presenting cells to facilitate in situ T-cell activation. Methods Carotid artery plaque tissues were collected from 19 asymptomatic and 38 symptomatic patients undergoing endarterectomy. Matched samples of normal coronary artery wall, stable nonruptured plaque, and eroded unstable plaque were harvested from patients with fatal myocardial infarction. Quantitative PCR and immunohistochemistry were used to analyze the tissues for markers of DC activation (CD83, CD86, CCL19,CCL21) and correlate them with T-cell activation (IFN-γ,TNF-α). Results Carotid artery plaques from patients with ischemic symptoms compared to asymptomatic patients were characterized by the presence of high amount of T-cells (P < 0.01) and tissue production of high levels of the T-cell cytokines IFN-γ (P = 0.001) and TNF-α (P = 0.006). Plaque tissues from patients with ischemic complications contained elevated levels of CD83 (P < 0.001), a marker of DC activation, and the DC chemokines CCL19 (P = 0.001) and CCL21 (P < 0.02). Unstable coronary artery plaques were similarly correlated compared to carotid plaques from symptomatic patients with the accumulation of T cells (P = 0.001) and the production of T cell chemokines IFN-γ (P = 0.001) and TNF-α (P = 0.002). Immunohistochemistry confirmed the presence of CD83⁺ DC in the shoulder region of unstable plaques, where they produced the T cell-attracting chemokines CCL19 and CCL21. Mapping of activated DC demonstrated close contact between mature DC and T cells expressing the activation marker CD40 ligand (CD40L). Conclusion Activated and fully mature DC are represented in the inflammatory infiltrate characteristic for unstable carotid and coronary atheroma. Such DC produce chemokines, and thus can regulate the cell traffic into the lesion. Through the expression of the costimulatory ligand CD86, plaque-residing DC can augment T-cell stimulation and provide optimal stimulation conditions for T lymphocytes, resembling the microenvironment in organized lymphoid tissues.     

参附注射液对人慢性髓性白血病细胞来源的树突状细胞诱导生成的影响

目的研究参附注射液（SFI）对慢性髓性白血病细胞系K-562细胞诱导生成树突状细胞（DCs）的影响。方法用不同浓度的SFI、粒—巨噬细胞集落因子（GM-CSF）/白细胞介素4（IL-4）及GM-CSF/IL-4联合不同浓度SFI 3种组合在体外诱导培养K-562细胞。结果体外单用SFI不能诱导K-562细胞分化为DCs。单用GM-CSF/IL-4可诱导K-562细胞分化为DCs,并具有DCs的特异性标志及功能,但不成熟。不同浓度的SFI联合GM-CSF/IL-4对K-562 DCs有促进成熟和抑制增殖的双向调节作用,其最佳诱导浓度为75 mg/ml。结论适当浓度的SFI联合GM-CSF/IL-4在体外能更有效地诱导K-562细胞分化为DCs,并促进DCs成熟,增强DCs功能,且75 mg/mlSFI与GM-CSF/IL-4联合有协同作用。     

Strategies for loading dendritic cells with hepatitis C NS5a antigen and inducing protective immunity

Summary.  Dendritic cell (DC)-based vaccination strategies are promising for the treatment of cancers and infectious diseases including hepatitis C virus (HCV). As the induction of T cell-mediated immune responses by DC vaccination is highly dependent on efficient antigen loading of the DCs, the purpose of this study was to identify an optimal nonviral DC loading strategy for HCV NS5a. Furthermore, the efficacy of immunization with the NS5a-loaded DCs in comparison to plasmid encoding NS5a and NS5a protein was evaluated. Transfection of DCs with mRNA was most efficient with close to 100% of DCs expressing NS5a, whereas approximately 10% of protein-pulsed DCs and <1% of plasmid-transfected DCs expressed NS5a, suggesting remarkably different loading efficiencies. Vaccination of mice with NS5a mRNA-transfected DCs or NS5a protein-pulsed DCs resulted in significantly stronger CD4⁺ and CD8⁺ T-cell responses and protection from challenge with vaccinia virus expressing NS3/NS4/NS5, in comparison to vaccination with NS5a DNA-transfected DCs, plasmid encoding NS5 or rNS5a protein formulated with alum. Furthermore, vaccination with NS5a mRNA-transfected DCs was superior to vaccination with rNS5a-pulsed DCs. These data have important clinical implications, with mRNA-transfected DCs providing a safe and effective vaccination strategy against hepatitis C and possibly other pathogens.     

Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-derived immature dendritic cells

The hypothesis that productive infection of monocyte-derived immature dendritic cells (DCs) by the human cytomegalovirus (HCMV) is associated with decreased immunostimulatory capacity was tested in this study. DCs were infected with 60-80% efficiency by HCMV strain TB40/E. Infected versus uninfected cells were analysed by fluorescence-activated cell sorting and by immunocytochemistry for surface expression of major histocompatibility complex (MHC) and co-stimulatory molecules as well as cytokine secretion during the 3 d after infection. The immunostimulatory capacity of these cells was measured by mixed leucocyte reaction. In spite of the fact that HCMV infection of DCs induced an increased release of tumour necrosis factor-alpha (TNF-alpha) and a decreased interleukin 10 (IL-10) production, expression of MHC class I and II, as well as CD40 and CD80 molecules, were downregulated on infected DCs. The mixed leucocyte reaction showed significantly reduced immunostimulatory capacity of infected DC cultures. Simultaneous detection of MHC antigens and virus antigens by double immunofluorescence revealed that downregulation occurred only on infected cells, but not on uninfected bystander cells. These findings demonstrate on a single cell level, together with the marked downregulation of MHC and co-stimulatory molecules in the presence of high TNF-alpha and low IL-10 levels, a direct inhibitory effect of HCMV on antigen presentation by immature DCs independent of soluble mediators.     

Ex vivo generation of human cytomegalovirus-specific cytotoxic T cells by peptide-pulsed dendritic cells

Adoptive transfer of donor-derived human cytomegalovirus (HCMV)-specific T-cell clones can restore protective immunity after stem cell transplantation. Ex vivo induction of HCMV-specific T cells using HCMV-infected fibroblasts as stimulator cells confines this approach to HCMV-seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV-specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV-specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide-pulsed dendritic cells as antigen-presenting cells. HCMV-specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV-seropositive and 2 out of 11 HCMV-seronegative donors. The CTLs recognized HCMV-infected autologous fibroblasts. No lysis was observed with either non-infected autologous or HLA-mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptide-specific T cells of up to 28% and > 90% of CD8(+) T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of > 1 x 10(9) HCMV-specific T cells was possible after 6--7 weeks when cultures were initiated with 1--5 x 10(6) responder cells. Thus, the approach with peptide-pulsed DCs to generate HCMV-specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.     

Platelet concentrates modulate myeloid dendritic cell immune responses

Platelet transfusion has been reported to modulate the recipients’ immune system. To date, the precise mechanism(s) driving poor patient outcomes (e.g., increased rate of mortality, morbidity, infectious complications and prolonged hospital stays) following platelet transfusion are largely undefined. To determine the potential for platelet concentrates (PC) to modulate responses of crucial immune regulatory cells, a human in vitro whole blood model of transfusion was established. Maturation and activation of human myeloid dendritic cells (mDC) and the specialized subset blood DC antigen (BDCA)3⁺ DC were assessed following exposure to buffy-coat derived PC at day (D)2 (fresh) and D5 (date-of-expiry). In parallel, to model recipients with underlying viral or bacterial infection, polyinosinic:polycytidylic acid or lipopolysaccharide was added. Exposure to PC had less of an impact on mDC responses than BDCA3⁺ DC responses. PC alone downregulated BDCA3⁺ DC expression of co-stimulatory molecules CD40 and CD80. In the model of viral infection, PC downregulated expression of CD83, and in the bacterial model of infection, PC downregulated CD80, CD83, and CD86. PC alone suppressed mDC production of interleukin (IL)-8, IL-12 and tumor necrosis factor (TNF)-α and BDCA3⁺ DC production of IL-8, IL-12, and IL-6. In the model of viral infection, production of IL-12 and interferon-gamma inducible protein (IP)-10 was reduced in both DC subsets, and IL-8 was reduced in BDCA3⁺ DC following PC exposure. When modeling bacterial infection, PC suppressed mDC and BDCA3⁺ DC production of IL-6 and IL-10 with a reduction in TNF-α evident in mDC. This study assessed the impact of PC “transfusion” on DC surface antigen expression and inflammatory mediator production and provided the first evidence that PC transfusion modulates blood mDC and BDCA3⁺ DC maturation and activation, particularly in the models of infection. Results of this study suggest that patients who receive PC, particularly those with underlying infectious complications, may fail to establish an appropriate immune response precipitating poor patient outcomes.