多发性骨髓瘤患者外周血T淋巴细胞亚九和CD^＋38细胞的变化及…

为探讨多发性骨髓瘤患者细胞免疫功能和外周血肿瘤前体细胞的变化及意义，应用间妆免疫荧光法对２６例ＭＭ患者外周血Ｔ淋巴细胞亚群和携带浆细胞相关抗原－ＣＤ＾＋３８的淋巴细胞进行了检测。结果表明：（１）ＭＭ患者ＣＤ＾＋３细胞，ＣＤ＾＋＿４细胞，ＣＤ＾＋４／ＣＤ＾＋８比值下降，而ＣＤ＾＋８细胞无明显变化。（２）ＭＭ患者外周血ＣＤ＾＋３８细胞明显增高，化疗后ＣＤ＾＋３８细胞数量明显下降。     

紫外线照射自血回输对脑梗塞患者外周血淋巴细胞亚群和血浆纤维连…

对２４例脑梗塞患者紫外线照射自血回输（ＵＢＩＯ）治疗前、后外周血淋巴细胞亚群及血浆纤维连接蛋白（Ｆｎ）含量变化进行了观察。结果ＵＢＩＯ治疗前脑梗塞患者外周血中总Ｔ细胞（ＣＤ３＾＋）、Ｔ抑制细胞（ＣＤ８＾＋）数均较正常人明显减少，ＣＤ４／ＣＤ８比值上升；Ｂ＾＋和人类白细胞抗原（ＨＬＡ－ＤＲ＾＋）细胞数增多；白细胞介素ＩＩ受体（ＩＬ－２Ｒ＾＋）和Ｔ辅助（ＣＤ＾＋）细胞数则与正常人无差异。血浆Ｆｎ含量低     

脐血CD34^＋细胞和外周血单核细胞两种来源的树突状细胞特性？…

目的 体外大量扩增和纯化具有典型表型、形态和功能的树突状细胞（ＤＣ）、以进行相关基础研究和临床应用。方法 采用免疫磁珠江分离脐血ＣＤ３４＾＋细胞及外周血去Ｂ、去Ｔ淋巴细胞的单个核细胞（单核细胞）,然后以ＧＭ－ＣＳＦ、ＩＬ－４、ＴＮＦα、Ｆｌｔ３配基（ＦＬ）、ＳＣＦ等不同的细胞因子配伍分别诱生ＤＣ,通过流式细胞仪、电镜、光镜分析其特性,同时检测其刺激同种Ｔ细胞增殖的能力。结果 脐民外周血诱生ＤＣ的方     

应用SCF＋IL—2等细胞因子从脐血CD34^＋细胞扩增T细胞

肿瘤和艾滋病（ＡＩＤＳ）的基因治疗或免疫治疗的研究需要大量的Ｔ细胞克隆。尽管Ｔ细胞来源于ＣＤ３４＾＋细胞，但由于需要胸腺组织的参与，使其在体外扩增大量细胞克隆非常困难。本研究用脐血单个核细胞作为辅助细胞，在ＳＣＦ＋ＩＬ－２等细胞因了的作用下，人脐血ＣＤ３４＾＋在此培养条件下，不断产生ＣＤ４＾＋或ＣＤ８＾＋Ｔ细胞至少持续３周。在培养扩增过程中，发现ＣＤ４＾＋和ＣＤ８＾＋Ｔ细胞的比例有一个不断变化的动     

肠内营养支持对ICU患者细胞免疫的影响

目的：研究肠内营养（ＥＮ）支持对ＩＣＵ患者的细胞免疫功能的影响。方法：对１１例健康人和１２例ＩＣＵ患者经ＥＮ支持前后的细胞免疫功能进行检测。结果：ＩＣＵ患者的免疫功能明显低于健康,表现为自然杀伤（ＮＫ）细胞活性和Ｔ细胞亚群ＣＤ４,ＣＤ４／ＣＤ８明显降低。经一段时期的ＥＮ支持后,细胞免疫功能有回升,表现为ＣＤ３,ＣＤ４,ＣＤ４／ＣＤ８和ＮＫ细胞活性明显升高,白介素－α（ＩＬ－α）浓度及ＩＬ－２分泌细     

用活化B细胞,CD3mAb,IL—2共刺激诱导膀胱TIL特异性抗瘤 …

基于Ｔ细胞完全激活依赖ＴＣＲ产生的第一信号和ＣＤ２８与Ｂ７分子作用产生的第二信号，我们用活化Ｂ细胞提供共刺激信号，联合ＣＤ３ｍＡｂ，ＩＬ－２诱导扩增了人膀胱ＴＩＬ。结果显示，在该培养体系中ＴＩＬ快速扩增，培养第１０天扩增了１１４倍，显著高于ＣＤ３ｍＡｂ／ＩＬ－２组（Ｐ〈０．０１）。ＣＤ８＾＋细胞的扩增大大快于ＣＤ４＾＋细胞，第１０天时阳性率达９２％，扩增１２５倍。ＣＤ４＾＋细胞仅扩增２１倍，膜ＩＬ     

头颈癌病人外周血T淋巴细胞亚群和血清可溶性白细胞介素2受体…

目的 观察头颈癌病人细胞免疫功能的改变及其临床意义。方法 对３２例头颈癌病人进行外周血Ｔ淋巴细胞亚群和血清可溶性白细胞介素２受体的检测并与正常对照线比较。结果 头颈癌病人外周血ＣＤ４＾＋细胞降低、ＣＤ８＾＋细胞升高、ＣＤ４＾＋／ＣＤ８＾＋比值降低、血清ｓＩＬ－２Ｒ水平升高（ｔ＝４．６１￣１２．８８，Ｐ均〈０．０１），Ⅲ、Ⅳ期头颈癌病人软Ⅰ，Ⅱ期者ＣＤ４＾４／ＣＤ８＾＋比值的ｘ－２ｓ为下限，血清ｓＩ     

多发性骨髓瘤患者外周血B细胞激活状态及其对白细胞介素4的反应

用间接免疫荧光染色流式细胞仪分析技术，检测了２８例多发性骨髓瘤（ＭＭ）患者以及２０名健康对照者外周血Ｂ细胞（ＣＤ２０＾＋）以及Ｂ，Ｔ细胞中ＨＬＡ－ＤＲ＾＋细胞比率。发现ＭＭ患者外周血中Ｂ细胞比率（９．３８±２．８３％）以及Ｂ，Ｔ细胞中ＨＬＡ－ＤＲ＾＋细胞比率（２６．０４±６．８０％和１２．５０±４．２２％）明显低于对照组中Ｂ细胞的比率（１２．２０±２．９２％）及Ｂ，Ｔ细胞中ＨＬＡ－ＤＲ＾＋细胞...     

脐血CD34+细胞和外周血单核细胞两种来源的树突状细胞特性的比较研究

目的　体外大量扩增和纯化具有典型表型、形态和功能的树突状细胞（ＤＣ）,以进行相关基础研究和临床应用。方法　采用免疫磁珠法分离脐血ＣＤ３４ ＋ 细胞及外周血去Ｂ、去Ｔ淋巴细胞的单个核细胞（ 单核细胞） ,然后以ＧＭＣＳＦ、ＩＬ４、ＴＮＦα、Ｆｌｔ３ 配基（ＦＬ）、ＳＣＦ等不同的细胞因子配伍,分别诱生ＤＣ,通过流式细胞仪、电镜、光镜分析其特性,同时检测其刺激同种Ｔ细胞增殖的能力。结果　脐血与外周血诱生ＤＣ的方案不同,由脐血ＣＤ３４＋ 细胞诱导ＤＣ 时,ＧＭＣＳＦ＋ ＴＮＦα＋ ＳＣＦ＋ ＦＬ 组合可使ＣＤ１ａ＋ 细胞比例增至（２７ ．１８ ±１－５６）％ ,明显高于单独应用ＧＭＣＳＦ组［（０．６５±０ ．３８）％ ］ 。外周血单核细胞诱导的ＤＣ,则ＧＭＣＳＦ＋ 高剂量ＩＬ４（１０００ Ｕ／ｍｌ） 组合效率最高,诱生的ＣＤ１ａ ＋ 细胞可达（２１ ．８０ ±０－３２） ％ 。两种来源的ＤＣ在表型及形态上差异无显著性,两者同样具有刺激同种异体淋巴细胞增殖的能力。结论　从脐血和外周血均可诱生ＤＣ,根据应用目的的不同对其进行选择,这为ＤＣ用于临床治疗选择不同细胞来源提供了实验基础。     

紫外线照射自血回输对脑梗塞患者外周血淋巴细胞亚群和血浆纤维连接蛋白的影响

对２４例脑梗塞患者紫外线照射自血回输（ＵＢＩＯ）治疗前、后外周血淋巴细胞亚群及血浆纤维连接蛋白（Ｆｎ）含量变化进行了观察．结果ＵＢＩＯ治疗前脑梗塞患者外周血中总Ｔ细胞（ＣＤ）、Ｔ抑制细胞（ＣＤ）数均较正常人明显减少，ＣＤ４／ＣＤ８比值上升；Ｂ＋和人类白细胞抗原（ＨＬＡ－ＤＲ＋）细胞数增多；白细胞介素Ⅱ受体（ＩＬ－２Ｒ＋）和Ｔ辅助（ＣＤ＋）细胞数则与正常人无差异。血浆Ｆｎ含量低于正常人。提示脑梗塞患者存在免疫功能紊乱．ＵＢＩＯ治疗后，外周血中ＣＤ、ＣＤ细胞和Ｆｎ水平上升，ＣＤ４／ＣＤ８比值下降，Ｂ细胞数下降，ＨＬＡ－ＤＲ＋细胞数无明显变化．提示ＵＢＩＯ具有免疫调节作用．     

Dendritic cells cultured in anti-CD40 antibody-immobilized plates elicit a highly efficient peptide-specific T-cell response

The function of dendritic cells (DCs), antigen-presenting cells that can initiate and regulate cellular and humoral responses, is highly influenced by their level of maturation. Immature DCs may be harmful in anti-tumor immunotherapy, because they can induce immunotolerance rather than immunostimulation. In this study, the authors sought to determine the optimal culture conditions for obtaining fully mature DCs. When DCs were cultured in agonistic anti-CD40 monoclonal antibody-immobilized plates, they showed a higher expression of the maturation marker CD83 than DCs cultured without CD40 ligation or those cultured in medium supplemented with anti-CD40 monoclonal antibody. In addition, when interferon-gamma (IFN-gamma) was added to the medium, additive up-regulation of CD83 expression was observed. These DCs treated with both maturation signals showed a higher secretion of interleukin-12. To evaluate the capacity of antigen presentation, specific cytotoxic T lymphocytes were generated using autologous DC pulsed with a human lymphocyte antigen-A24-restricted peptide epitope derived from carcinoembryonic antigen. Interferon-gamma-secreting CD8+ T cells were analyzed by flow cytometry using the cellular affinity matrix technology. Dendritic cells, matured with CD40 ligation and IFN-gamma, were more efficient at eliciting an antigen-specific T-cell response in vitro than DCs stimulated with anti-CD40 monoclonal antibody or IFN-gamma alone. A cytotoxicity assay using carcinoembryonic antigen-expressing tumor cell lines also showed that DCs matured with both signals were more efficient at inducing cytotoxic T lymphocytes. These results demonstrate that DC culture in an anti-CD40 monoclonal antibody-immobilized plate in medium supplemented with IFN-gamma has a positive impact on DC maturation and may be optimal for eliciting an antigen-specific T-cell response without the need for CD4+ T-helper epitopes.     

Dendritic cells loaded with killed allogeneic melanoma cells can induce objective clinical responses and MART-1 specific CD8+ T-cell immunity

Dendritic cells (DCs) loaded with killed allogeneic tumors can cross-prime tumor-specific naive CD8 T cells in vitro, thereby providing an option to overcome human leukocyte antigen restriction inherent to loading DC vaccines with peptides. We have vaccinated 20 patients with stage IV melanoma with autologous monocyte-derived DCs loaded with killed allogeneic Colo829 melanoma cell line. DCs were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (granulocyte macrophage-colony stimulating factor) and interleukin (IL-4) and activated by additional culture with tumor necrosis factor and CD40 ligand. A total of 8 vaccines were administered at monthly intervals. The first patient was accrued December 2002 and the last November 2003. Fourteen patients were alive at 12 months, 9 patients were alive at 24 months, and 8 patients are alive as of January 2006. The estimated median overall survival is 22.5 months with a range of 2 to 35.5 months. Vaccinations were safe and tolerable. They induced, in 2 patients who failed previous therapy, durable objective clinical responses, 1 complete regression (CR) and 1 partial regression (PR) lasting 18 and 23 months, respectively. Three out of 13 analyzed patients showed T-cell immunity to melanoma antigen recognized by autologous T cells (MART-1) tissue differentiation antigen. Two of 3 patients showed improved immune function after vaccinations demonstrated by improved secretion of interferon (IFN)-gamma or T-cell proliferation in response to MART-1 derived peptides. In one of these patients, vaccination led to elicitation of CD8 T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. Thus, the present results justify the design of larger follow-up studies to assess the clinical response to DC vaccines loaded with killed allogeneic tumor cells in patients with metastatic melanoma.     

Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation

Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the development of both Th1 and Th2 effector cells depending on the dose of antigen. In general, high antigen doses induced Th1 cell development whereas low antigen doses induced Th2 cell development. Both cultured and ex vivo splenic plasmacytoid-derived DCs enhanced CD4(+) T cell proliferation and induced strong Th1 cell development when activated with the Toll-like receptor (TLR)9 ligand CpG, and not with the TLR4 ligand lipopolysaccharide (LPS). The responsiveness of plasmacytoid pDCs to CpG correlated with high TLR9 expression similarly to human plasmacytoid pDCs. Conversely, myeloid DCs generated with granulocyte/macrophage colony-stimulating factor enhanced Th1 cell development when stimulated with LPS as a result of their high level of TLR4 expression. Polarized Th1 responses resulting from high antigen dose were not additionally enhanced by stimulation of DCs by TLR ligands. Thus, the net effect of antigen dose, the state of maturation of the DCs together with the stimulation of DCs by pathogen-derived products, will determine whether a Th1 or Th2 response develops.     

Effect of ex vivo culture duration on phenotype and cytokine production by mature dendritic cells derived from peripheral blood monocytes

BACKGROUND: To generate clinical-grade dendritic cells (DCs) ex vivo for immunotherapy trials, peripheral blood monocytes are typically cultured in granulocyte-macrophage–colony-stimulating factor (GM-CSF) and interleukin (IL)-4 and then matured using one or more agents. Duration of the initial DC culture is one important variable that has not been systematically evaluated for its effect on the characteristics of the final mature DC product.
STUDY DESIGN: DCs were generated from elutriated peripheral blood monocytes by incubation in medium containing 2000 units per mL each of GM-CSF and IL-4 for 3 to 7 days, followed by maturation with lipopolysaccharide and interferon-γ (IFN-γ). DC yield, viability, flow cytometric phenotype, and cytokine production were evaluated.
RESULTS: The percentage yield and viability of mature DCs were similar after GM-CSF/IL-4 culture for 3 or 7 days. In either case, mature DCs expressed abundant CD80, CD86, CD83, and CCR7, but 3-day DCs expressed these antigens in a more consistent and homogeneous manner. Mature 3-day DCs produced much more IL-12 and less IL-10 after restimulation with CD40L-LTK than 7-day DCs. The former were also more effective in presenting immunogenic peptides to CD8 T cells. Analogous changes in cytokine production were observed in mature DCs prepared using lower concentrations of GM-CSF/IL-4 or when the alternative maturation cocktails poly(I:C)/IFN-γ and soluble CD40L/IFN-γ were used.
CONCLUSION: Extended initial culture of DCs in GM-CSF/IL-4 does not affect yield or viability of subsequently matured DCs, but can adversely affect their ability to homogeneously express high levels of functionally important surface molecules such as CD83 and CCR7 and to produce IL-12.     

抗原冲击致敏树突状细胞诱导特异性CTL杀伤Jurkat细胞实验研究

本研究以健康人外周血单核细胞为前体细胞，体外诱导其为树突状细胞（DC），分别负载Jurkat细胞冻融抗原及WT1多肽抗原，产生特异性细胞毒性T淋巴细胞（CTL），以探讨其对白血病Jurkat细胞的杀伤作用。联合应用rhGM—CSF、rhIL-4、rhTNF-α及rhsCD40L等细胞因子，密度梯度离心法、贴壁法从外周血获取单核细胞并进行诱导扩增，培养出DC，分别用冻融抗原及WT1多肽抗原冲击致敏DC。实验分4组：冻融抗原致敏DC组为实验组A，WT1多肽致敏DC组为实验组B，未致敏DC组为对照组A，单核细胞组为对照组B，观察CTL对Jurkat细胞的杀伤作用。结果表明：培养出了具有典型特征的DC，它高表达CD40、CD80、CD1a及CD86等表面标志，能体外诱导强烈的同种异基因混合淋巴细胞增殖反应。实验组显示高水平杀伤率，与对照组比较均具有统计学意义（P〈0.01），实验组A、B间比较无统计学意义。结论：Jurkat细胞冻融抗原及WT1多肽抗原冲击致敏DC能有效诱导T细胞抗白血病作用，为临床研制DC疫苗提供了实验依据。     

Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients

Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10-specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.     

硒与树突状细胞对T细胞杀伤白血病细胞活力的影响

本研究探讨经亚硒酸钠（Na2SeO3）处理、K562细胞裂解物冲击致敏的外周血衍生的树突状细胞（DC）的生物学特性和体外诱导抗原特异性细胞毒性T淋巴细胞（CTL）应答的能力。健康人外周血单个核细胞（PBMNC）于体外在含3种细胞因子（rhGM—CSF、rhIL-4、TNF-α）的RPMI1640＋10％FBS培养液中培养4天，收获贴壁细胞，实验分4组：DCⅠ组：仅含DC；DCⅡ组：DC＋Se（0．5μmol／L）；DCⅢ组：DC＋K562细胞裂解液；DCⅣ组：在DCⅢ组中加入Se（0．5μmol／L）。在培养的第7天于倒置显微镜下进行活细胞观察。用流式细胞术（FCM）检测细胞表型CD1a、CD40、CD83、CD86。乳酸脱氢酶（LDH）释放试验检测CTL效应。用酶联免疫吸附试验（ELISA）测定IL-12含量。结果表明：各组DC均具有典型树突状细胞形态，均较培养前集落增多。DCⅠ组和DCⅡ组的细胞形态及数量无明显差异，DCⅢ组和DCⅣ组的细胞集落数量增加，悬浮细胞比例增加。各组DC细胞的CD1a、CD40、CD83、CD86表达率较PBMNC明显增高（P〈0．01），各组间DC细胞的CD1a和CD40的表达率无明显差异，DCⅢ组和DCⅣ组的CD83和CD86的表达率均高于DCⅠ组和DCⅡ组（P〈0.01），DCⅠ和DCⅡ以及DCⅢ和DCⅣ两组之间CD83和CD86表达率均无明显差异。在效靶比例为25：1时，各组DC致敏的T淋巴细胞对K562细胞的杀伤率为15．3±2．3％、26．3±3．7％、28．2±4.5％和36．2±3．7％，均明显高于未经DC致敏的单独T淋巴细胞组（5．9±2．4％）（P〈0．01），DCⅣ组的CTL效应最强，高于DCⅠ、Ⅱ、Ⅲ组（P〈0．01），DCⅡ和DCⅢ组的CTL效应也高于DCⅠ组（P〈0．01），而DCⅡ和DCⅢ两组间CTL效应程度无明显差异（P〉0.05）；各组DC与T淋巴细胞共培养的上清液中IL-12水平为256．96±64．2、328．12±43.9、322．98±53．5和353.85±46．2pg／ml，均显著高于未经DC致敏的单独T淋巴细胞组（35?     

Immunomodulatory dendritic cells generated from nonfractionated bulk peripheral blood mononuclear cell cultures induce growth of cytotoxic T cells against renal cell carcinoma

Dendritic cells (DCs) loaded with tumor antigens have the potential to become a powerful tool for clinical cancer treatment. Recently, the authors showed that a tumor-specific immune response can be elicited in culture via stimulation with autologous renal tumor lysate (Tuly)-loaded DCs that were generated from cytokine-cultured adherent peripheral blood mononuclear cells (PBMCs). Here, the authors show that immunomodulatory DCs can be generated directly from nonfractionated bulk PBMC cultures. Kinetic studies of DC differentiation and maturation in PBMC cultures were performed by monitoring the acquisition of DC-associated molecules using fluorescence-activated cell sorting analysis to determine the percentage of positive immunostained cells and the mean relative linear fluorescence intensity (MRLFI). Compared with conventional adherent CD14+ cultures, which have mostly natural killer, T, and B cells removed before cytokine culture, bulk PBMC cultures exhibited an early loss of CD14+ cells (day 0 = 78.8%, day 2 = 29.6% versus day 0 = 74%, day 2 = 75%) with an increase in yield of mature DCs (DC19- CD83+) (day 5 = 17%, day 6 = 21%, day 7 = 22% versus day 5 = 11%, day 6 = 15%, day 7 = 23%). Although a comparable percentage of DCs expressing CD86+ (B7-2), CD40+, and HLA-DR+ were detected in both cultures, higher expression levels were detected in DCs derived from bulk culture (CD86 = MRLFI 3665.1 versus 2662.1 on day 6; CD40 = MRLFI 1786 versus 681.2 on day 6; HLA-DR = MRLFI 6018.2 versus 3444.9 on day 2). Cytokines involved in DC maturation were determined by polymerase chain reaction demonstrating interleukin-6 (IL-6), IL-12, interferon-gamma, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha mRNA expression by bulk culture cells during the entire 9-day culture period. This same cytokine mRNA profile was not found in the conventional adherent DC culture. Autologous renal Tuly (30 micrograms protein/10(7) PBMCs) enhanced human leukocyte antigen expression by DCs (class I = 7367.6 versus 4085.4 MRFLI; class II = 8277.2 versus 6175.7 MRFLI) and upregulated cytokine mRNAs levels. Concurrently, CD3+ CD56-, CD3+ CD25+, and CD3+ TCR+ cell populations increased and cytotoxicity against autologous renal cell carcinoma tumor target was induced. Specific cytotoxicity was augmented when cultures were boosted continuously with IL-2 (20 U/mL biological response modifier program) plus Tuly stimulation. These results suggest that nonadherent PBMCs may participate in enhancing DC maturation. Besides the simplicity of this culture technique, bulk DC cultures potentially may be used with the same efficiency as conventional purified DCs. Furthermore, bulk culture-derived DCs may be used directly in vivo as a tumor vaccine, or for further ex vivo expansion of co-cultured cytotoxic T cells to be used for adoptive immunotherapy.     

CTLA4 blockade maximizes antitumor T-cell activation by dendritic cells presenting idiotype protein or opsonized anti-CD20 antibody-coated lymphoma cells

CTLA4 is a negative regulator of the costimulatory signals induced by the interaction of CD28 on T cells and B7 on dendritic cells (DCs). Antibodies (Abs) against CTLA4 can block its function and increase the activation of T cells primed to recognize antigens. The effect of CTLA4 blockade on the cross-presentation of tumor antigens by DCs to T cells was examined. Immune T cells and DC precursors were collected from patients receiving idiotype protein-pulsed DC vaccines, exposed to antigen, and examined for antitumor activity by measuring intracellular cytokine production by FACS. Idiotype-specific activation occurred in CD8+ and CD4+ T-cell populations and was up to 58 fold higher with CTLA4 blockade. These T cells could be expanded quickly and maintained tumor cytolytic activity. T-cell responses to whole tumor cell-pulsed DCs were then examined. DCs contain Fc receptors and efficiently phagocytose lymphoma cells when coated with opsonizing anti-CD20 Abs. Within a few hours, DCs ingested tumor cells and labeled proteins were observed in the cytoplasm. When anti-CD20 Ab-coated tumor-pulsed DCs were used in combination with CTLA4 blockade, up to 15 fold higher activation of Id-specific CD8+ and 3 fold higher CD4+ T cells resulted. Thus, CTLA4 blockade can enhance the measurement of Ag-specific T-cell responses and the expansion of T cells for clinical studies. In addition, the combination of CTLA4 blockade and Ab targeting of tumor to DCs is an effective method for the cross-presentation of tumor cell antigens.     

A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma by an IL-12-independent but CD70-dependent mechanism in vivo

Interferon (IFN)-gamma, a cytokine critical for resistance to infection and tumors, is produced by CD4(+) helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12-independent pathway whereby DCs induce IFN-gamma-secreting T helper (Th)1 CD4(+) T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-gamma in vivo but required IL-12, not CD70. Isolated CD205(+) DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205(+) DC function in vivo. Detection of the IL-12-independent IFN-gamma pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205(+) DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms.