我们感谢 Reata 制药公司提供 CDDO DFPA。我们也承认, 简和李塞德曼主席在儿科癌症创新 (约翰 Letterio) 的支持。这项工作得到了国防部的支持 [W81XWH-12-1-0452];安吉福勒青少年和青年癌症研究倡议在案例综合癌症中心;琼斯卡拉汉基金会的卡拉汉研究生奖学金。

所有的研究都是按照西方储备大学医学院的机构动物护理和使用委员会批准的程序进行的。
1. 制备骨髓源性树突状细胞 (BMDCs)
<ol><li>通过热处理消毒所有手术器械, 并在第二类生物安全柜中进行实验, 并进行安全程序。</li>
	<li>弄死 C57BL/6 小鼠 8-10 周的年龄通过使用 CO2室。将鼠标放在解剖板上, 用70% 乙醇冲洗。使用外科剪刀将胫骨-腓骨和股骨骨放在10厘米的培养皿中, 将70% 乙醇放在其中。</li>
	<li>使用手术刀片和镊子解剖组织尽可能多地在10厘米培养皿的盖子上, 隔离胫骨和股骨在6厘米培养皿中放置70% 乙醇。</li>
	<li>使用手术刀片修剪两端的胫骨和股骨。使用3毫升 PBS 在3毫升注射器与23G 针, 以冲洗的内容骨髓从一个端骨到一个圆锥管包含12毫升 PBS。对每个骨骼的末端重复此步骤3x。</li>
	<li>离心机细胞悬浮在 300 x g 为5分钟。</li>
	<li>取出上清液, 用1毫升 ACK 株溶藻缓冲器并用重悬细胞颗粒, 清除红细胞。</li>
	<li>添加9毫升 PBS 稀释 ACK 株溶藻缓冲和离心机在 300 x g 5 分钟。</li>
	<li>去除上清和并用重悬10毫升培养基 (RPMI-1640 加 l-谷氨酰胺, 10% 血清, 1% 非必需氨基酸 (100x), 10 毫米 HEPES, 50 nM β巯基乙醇, 5% 青霉素/链霉素)。 注: 血清内毒素水平必须少于0.1 欧盟/毫升。</li>
	<li>通过40微米细胞过滤器的细胞悬浮, 并采取20µl 的细胞悬浮和混合80µl 的台盼蓝, 以计数的活细胞的数量, 通过使用。</li>
	<li>将单元格数调整为 1 x 106单元格/ml, 其 15 ng/毫升为 GM-CSF 和 10 IL-4/毫升。</li>
	<li>板3毫升 1 x 106细胞/毫升在每井6井板和孵化细胞在 37°c, 5% CO2和95% 湿气在 CO2孵化器。 注: 一只老鼠的骨髓细胞约为 3-5 x 107细胞, 表明它可以从2到 3 6-井板中放置。</li>
	<li>在3天, 从每个井中移除所有3毫升培养基, 在每个井中加2毫升新鲜的 PBS, 然后轻轻地旋转盘子以确保去除所有非黏附的细胞。</li>
	<li>将3毫升新鲜培养基与 15 IL-4 和 10 ng/毫升在每个井中更换。在 co2孵化器中孵化37°c、5% co2和95% 湿度的单元格。</li>
	<li>在5天, 直接添加另外3毫升新鲜培养基与 15 ng/毫升基因脑脊液和 10 ng/毫升 IL-4 在每个井。孵育细胞在 37°c, 5% co2,和95% 湿度 co2孵化器。 注: 每个井的总容积现在是6毫升。</li>
	<li>7天, 把整个盘子放在冰上10分钟。轻轻地将每个井中的培养基注入, 将松散附着的 BMDCs 作为 iDCs 进入悬浮。 注: 粘附巨噬细胞仍然附着在盘子上。低温温和的程序是避免 DC 活化影响进一步实验的关键。</li>
	<li>离心细胞悬浮在 300 x g 为5分钟和并用重悬以新鲜培养基为进一步实验。 注: BMDCs 可以通过流式细胞仪识别荧光标记的 CD11c 抗体, 我们在图 1中展示了 BMDCs 的浇口策略以进行进一步的实验。</li>
</ol>2. TolDC 基因和蛋白质剖面特征
<ol><li>板2毫升 1 x 106 BMDCs/毫升每井在6毫微米 CDDO-DFPA 的存在或不存在 100-400 nM 的存在或不存在, 为孵化 1 h 在 37°c, 5% co2和95% 湿气在 CO2孵化器。 注: CDDO-DFPA, 合成萜, 是一个核因子 (红衍生 2) 样-2 因子 (Nrf2) 诱导剂和核因子 nf-κb (NF nf-κb) 抑制剂。应用其他制剂诱导 TolDCs 从 iDCs, 如 IL-10, 维生素 D3, 地塞米松或海湾 11-7085, 并修改这一步骤, 以最佳条件为每个代理。</li>
	<li>添加10或 100 ng/毫升的 LPS 孵化4-24 小时诱导 mDCs (孵化时间是不同的, 由于 mRNA 或蛋白质测量)。</li>
	<li>用1毫升的吸管轻轻地将培养基在每一个井中, 并收获细胞悬浮。离心机以 300 x g 为5分钟, 分别收集细胞和上清液。</li>
	<li>用流式细胞仪分析细胞颗粒的表面配体, 如刺激配体: CD40、CD80、CD86、MHC II、OX40L、ICOSL 或抑制配体: PD-L1、PD-L2、ILT3、ILT4。</li>
	<li>用定量实时 pcr (qRT pcr) 和 ELISA 法分别从细胞颗粒中分离 rna 和清液样品, 并对细胞因子、基因和蛋白质水平进行分析。 注: 例如, 炎症细胞因子: TNF α、IFNγ、EDN-1、IL-6、IL-12 和 IL-23, 或抗炎性细胞因子: IL-4、IL-10、IL-15、TGF-β1和 HO-1。</li>
</ol>3. 评估 TolDCs体外和体内的功能
<ol><li>
T 细胞自体增殖试验
	<ol>
<li>通过热处理消毒所有手术器械, 并在第二类生物安全柜中进行实验, 并进行安全程序。</li>
		<li>用0.5% 牛血清白蛋白 (BSA) 和2毫米 EDTA 在500毫升 PBS 中制备 mac 缓冲液。通过过滤0.2 µm 过滤器来杀菌缓冲。 注意: 在下面的实验中, 把缓冲器放在冰上。</li>
		<li>使用 CO2室获得 CD4+ t 细胞, 弄死 t 细胞受体 (TCR) 转基因小鼠8-10 周龄。将鼠标放在解剖板上, 用70% 乙醇冲洗。用手术剪刀和镊子将脾脏从腹部左侧分离出来。</li>
		<li>将脾脏与2毫升 PBS 放在6厘米的培养皿中, 使用3毫升注射器的后推, 通过40微米细胞过滤器将脾脏剁碎。</li>
		<li>收集细胞悬浮液和离心机在 300 x g 5 分钟。</li>
		<li>并用重悬在 400 ul 的 mac 缓冲细胞颗粒。</li>
		<li>添加 100 ul CD4+ T 细胞生物素抗体鸡尾酒在 4°c 5 分钟。 注: 抗体鸡尾酒结合其他细胞类型, 除了 CD4+ T 细胞, 如 CD8a, CD11b, CD11c, CD19, CD25, CD45R (B220), CD49b (DX5), CD105, 抗 MHC 类 II, Ter-119 和 TCRγ/δ。</li>
		<li>添加 300 ul 的 mac 缓冲器和 200 ul 的抗生物素珠 (材料表) 在 4°c 10 分钟。</li>
		<li>将铁磁球 (材料表) 和预分离过滤器组成的柱体放在磁场中, 并用3毫升的 mac 缓冲器冲洗。</li>
		<li>在细胞中添加9毫升的 mac 缓冲液, 离心机在 300 x g 处, 5 分钟。</li>
		<li>并用重悬3毫升的 mac 缓冲器中的细胞颗粒, 并应用到该柱上。收集包含 CD4+ T 单元格的流。</li>
		<li>用另一3毫升的 mac 缓冲器洗涤柱, 同时收集流经的流量。</li>
		<li>利用 CO2室, 弄死 C57BL/6 小鼠8-10 周龄, 获得脾泛 DCs。将鼠标放在解剖板上, 用70% 乙醇冲洗。用手术剪刀和镊子将脾脏从腹部左侧分离出来。将脾脏放在含有2毫升胶原酶 d 溶液的6cm 培养皿中 (2 毫克/毫升胶原酶 d 溶于含钙、镁的 HBSS)。</li>
		<li>用1毫升注射器和25G 针注射1毫升的胶原酶溶液到脾脏两次。用小剪刀把脾脏切成小块。</li>
		<li>在室温下摇动和孵化25分钟。</li>
		<li>在室温下添加 500 ul 0.5 米 EDTA, 5 分钟。 注: 3.1. 13-3. 1.14 的步骤对于提高 DCs 的产量至关重要。</li>
		<li>现在使用3毫升注射器的背部, 通过一个40微米细胞过滤器, 并收集细胞悬浮在 300 x g 离心5分钟, 以剁碎脾浆。</li>
		<li>并用重悬在 350 ul 的 mac 缓冲器, 50 ul 的 FcR 阻断试剂, 100 ul 的 Pan 树突状细胞生物素抗体鸡尾酒在4°c 为10分钟。 注: 抗体鸡尾酒对抗原不是由 dc 表达。</li>
		<li>通过添加9毫升的 mac 缓冲器和离心机在 300 x g 5 分钟内冲洗细胞。</li>
		<li>并用重悬在 800 ul 的 mac 缓冲细胞颗粒, 并添加 200 ul 的抗生物素珠在 4°c 10 分钟。</li>
		<li>重复步骤从 3.1. 9-3. 1.11 收集 DCs。</li>
		<li>再用3毫升的 mac 缓冲器清洗一列, 同时收集流量。</li>
		<li>将 2 x 105 DCs/ml 处理为1小时, 在37°c 存在或不存在 100-400 nM CDDO DFPA. 分别, 将 CD4+ T 单元格 (1 x 107/毫升) 标记为15分钟, 在37°c 使用1微米 CFSE, 用 PBS 清洗, 并重新调整音量以获得 2 x 106 T 细胞/毫升的最终浓度。</li>
		<li>接下来, 在一个96井板块, 共培养 100 ul 的处理树突状细胞具有相同体积的 CFSE 标记 CD4+ T 细胞, 两者都收集从上述步骤获得1:10 的比率。现在添加 100 ng/毫升卵清蛋白 (卵) 肽323-329 每井, 并通过流式细胞术在2-3 天的孵化后测量 T 细胞的 CFSE 强度。 注意: 从我们以前的工作37中优化了 DCs 和 T 单元格的数目和比率。</li>
	</ol>
</li>
	<li>
髓鞘少突胶质糖蛋白 (MOG) 注射 BMDCs 脉冲诱导无源 EAE (35-55)
	<ol>
<li>板2毫升 1 x 106 BMDCs/毫升每井在6个有文化媒介的 CDDO 的存在或没有100-400 毫微米 DFPA 为孵化1小时。</li>
		<li>加入10毫升的 LPS, 孵化24小时。</li>
		<li>加入100微克/毫升的 MOG (35-55) 孵化4小时。</li>
		<li>在 300 x g 的5分钟内收获细胞悬浮液和离心机。</li>
		<li>并用重悬细胞颗粒与 PBS, 计数细胞。</li>
		<li>皮下注射 200 ul 2 x 106细胞到8-10 周老女性 C57BL/6 小鼠 (100 ul 到每条后腿)。</li>
		<li>在 BMDC 注射日和48小时。然后, 在每只老鼠身上注射200的百日咳毒素 (PTX)。</li>
		<li>每周重复步骤3.2、6-3、2.7, 连续4周。</li>
		<li>采用标准标准37 (0.5 跛尾、1跛尾、2中下肢无力、3-严重后肢无力、4-完全后肢麻痹、5-四肢或垂死状态、6-死亡) 评价 EAE 的临床症状。</li>
	</ol>
</li>
</ol>

本文描述了一种有效的协议, 可用于重现性生成 iDCs, 并随后将其分化为 TolDCs, 我们建议, 这可以用于评估新分子靶剂的能力, 以诱导 TolDC表。如本报告所述, 我们遵循了一个序列, 我们首先分析了流式细胞术对表面配体的 TolDC 表达, 然后通过 qRT PCR 和 ELISA 测定对 DC 细胞因子剖面的评价。最后, 利用 MS 的临床前小鼠 EAE 模型, 证明了 TolDCs在体外抑制 T 细胞增殖的能力, 以及在体内抑癌的功效, 证...

树突状细胞 (DCs) 是先天免疫系统不可分割的一部分, 并首次发现和特征的拉尔夫斯坦曼和 Zanvil 科恩在1973年作为主要的专业抗原呈现细胞1。DCs 在免疫活化方面发挥了重要作用, 通过在次级淋巴器官中通过主要的组织相容性复合物 (MHC) 向 T 细胞和 B 细胞呈现经过处理的抗原, 将先天免疫系统和自适应的免疫力结合在一起2。在哺乳动物免疫系统中, 至少有两类 dcs 被描述为髓系 dcs 和浆 dcs (pDCs)3。髓细胞 dcs, 也称为常规 dcs (招揽), 其特点是 CD11c 的表达, 并可区分为未成熟的 dcs (iDCs)体外从骨髓前体细胞或外周血单核单位使用粒细胞-巨噬细胞集落刺激因子 (GM-CSF) 和 IL-4 的小鼠或人类物种, 分别为4。
激活 ' 危险 ' 信号, 如病原体相关的分子模式 (玉兰) 或损伤相关的分子模式 (潮湿), 将推动 iDCs 成熟免疫 dcs 作为成熟的 dcs (mDCs) 通过捆绑各种模式识别受体对DC 曲面5。免疫 DCs 进一步通过上调 MHCII2、刺激配体 (CD80、CD86 和 CD40)6、细胞因子和其他可溶性调解人7, 进一步促进天真的 T 细胞增殖和分化。免疫 DCs 对细胞因子介导的 T 细胞分化至关重要。例如, 干扰素和 IL-12 都是 Th1 分化所必需的8和 IL-1、IL-6 和 IL-23 对于天真的 T 细胞极化对 Th17 细胞9是至关重要的。虽然成熟的 DCs 对外来抗原有反应, 但自抗原控制的 dc 活化可能导致耐受性消融, 并通过生成自身反应性 T 细胞来促进自身免疫性疾病的发展, 其活化导致组织破坏10.
最近的报告提供了直流可塑性的明确证据, 其例证是它们能够在组织微环境中与不同的线索相互作用, 并区分为不同的效应/抑制 dc 子集。抗炎介质 (如 IL-1011、TGF β12和 HO-113 ) 在诱导致耐受性 DCs (TolDCs) 的免疫抑制中发挥了重要作用。这些 TolDCs 获取调节功能并抑制 T 细胞增殖14。此外, DCs 缺乏协同刺激和 TolDCs 的抗炎介质的产生, 都有助于诱导调节 T 细胞 (Tregs), 同时也有效抑制 Th1 和 Th17 分化和扩张15。在过去的两年中, TolDCs 的治疗潜力已经被几个调查员报告。在这些研究中, 体外产生的 TolDCs 不仅改善了自身免疫性疾病的不同临床前模型的病理症状 16 , 而且还导致了患者免疫耐受的发展17 ,18。有趣的是, 今天 TolDCs 疗法已被认为是一种替代或辅助方法的自身免疫性疾病的几个临床试验, 包括1型糖尿病19, 类风湿关节炎20, 21, 多发性硬化症 (MS)22,23,24, 克罗恩病25。
有各种各样的协议被使用了开发 TolDCs 和几个实验室报告了方法为 TolDCs 的世代和表型特征。这些方法可用于从造血祖重现性生成 TolDCs体外, 并稳定地维护它们在致耐受性状态在体内26,27,28,29。iDCs 可以通过接触各种免疫调节药理剂或抗炎性细胞因子转化为 TolDCs。例如, 维生素 D3 是已知的一种已知的药理剂, 可以增加 IL-10 的生产和抑制 IL-12 分泌物, 从而增强其免疫抑制功能30。此外, 当 DCs 暴露于强烈的炎症刺激, 如脂多糖 (LPS), 一些药理剂, 如地塞米松 31, 雷帕霉素 32, 皮质类固醇33已显示, 以诱导TolDC 表型通过减少 CD40、CD80、CD86 和 MHCII34的 DC 表面表达式。IL-10 和 TGF β是最受研究的抗炎性细胞因子, 以诱导直流耐受35和伴随暴露于这两种细胞因子已被证明诱导致耐受性表型在dc36。
由于致耐受性 DC 是由功能特征而不是表型标记来定义的, 所以需要对 TolDCs 的细胞和功能特性进行一致的方法。此外, 必须建立一个严格和一致的协议, 以便对致耐受性 DC 表型进行一致的评价和表征, 如果我们要有效和重现性比较新的药物诱导 TolDC 表型的能力实验室。在这里, 我们提供了一个详细的协议, 分步方法分离 iDCs 与造血祖细胞的小鼠, 并随后分析的功效, 评估新的药物的能力, 以转换 iDCs 为 TolDCs, 提供了一个强大的TolDCs 的功能和表型特性体外和体内。这个描述包括一个精心的方法来表征 TolDCs 的表面配体, 细胞因子剖面和免疫抑制功能在体内。我们还提供了一个方法的例子, 探讨这些 TolDCs 的潜在治疗应用在 MS, 实验性自身免疫脑脊髓炎 (EAE) 的临床前模型。这项既定的议定书将帮助调查人员评估新的药物促进诱导 TolDCs 的能力, 并将促进扩大 TolDC 治疗发展范围的努力。

<table><tbody><tr><td>CDDO-DFPA (RTA-408)</td><td>Reata Pharmaceuticals</td><td>in house synthesis</td><td>Cell culture</td></tr><tr><td>Mouse GM-CSF</td><td>Peprotech Inc.</td><td>315-03</td><td>BMDC differentiation</td></tr><tr><td>Mouse IL-4</td><td>Peprotech Inc.</td><td>214-14</td><td>BMDC differentiation</td></tr><tr><td>Lipopolysaccharides (LPS)</td><td>Sigma Aldrich Inc.</td><td>L2880</td><td>Cell culture</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma Aldrich Inc.</td><td>516732</td><td>Cell culture</td></tr><tr><td>Pertussis toxin (PTX)</td><td>R&amp;amp;D systems</td><td>3097</td><td>EAE induction</td></tr><tr><td>MOG (35&amp;ndash;55) peptide</td><td>21stCentury Biochemicals</td><td>in house synthesis</td><td>EAE induction</td></tr><tr><td>Trypan blue</td><td>Gibco, Life Technologies</td><td>15250-061</td><td>Cell culture</td></tr><tr><td>RPMI-1640 plus L-glutamine</td><td>ThermoFisher Scientific</td><td>11875-093</td><td>Cell culture</td></tr><tr><td>Non-essential amino acid (100X)</td><td>ThermoFisher Scientific</td><td>11140050</td><td>Cell culture</td></tr><tr><td>HEPES</td><td>ThermoFisher Scientific</td><td>15630080</td><td>Cell culture</td></tr><tr><td>penicillin/streptomycin</td><td>ThermoFisher Scientific</td><td>15140122</td><td>Cell culture</td></tr><tr><td>40 &amp;#956;m cell strainer</td><td>Corning</td><td>352340</td><td>Cell isolation</td></tr><tr><td>PE-conjugated CD80</td><td>BD Biosciences</td><td>557227</td><td>Flow cytometry</td></tr><tr><td>PE-conjugated CD86</td><td>BD Biosciences</td><td>555665</td><td>Flow cytometry</td></tr><tr><td>PE-conjugated PD-L1</td><td>BioLegend</td><td>124307</td><td>Flow cytometry</td></tr><tr><td>APC-conjugated MHCII</td><td>Miltenyi Biotec Inc.</td><td>130-112-388</td><td>Flow cytometry</td></tr><tr><td>APC-conjugated CD11c</td><td>BD Biosciences</td><td>340544</td><td>Flow cytometry</td></tr><tr><td>Isotype matched PE</td><td>Miltenyi Biotec Inc.</td><td>130-091-835</td><td>Flow cytometry</td></tr><tr><td>Isotype matched APC</td><td>Miltenyi Biotec Inc.</td><td>130-091-836</td><td>Flow cytometry</td></tr><tr><td>CFSE</td><td>BioLegend</td><td>423801</td><td>T cell proliferation assay</td></tr><tr><td>Pan dendritic cell isolation kit</td><td>Miltenyi Biotec Inc.</td><td>130-100-875</td><td>T cell proliferation assay</td></tr><tr><td>FcR Blocking Reagent</td><td>Miltenyi Biotec Inc.</td><td>130-100-875</td><td>T cell proliferation assay</td></tr><tr><td>Pan Dendritic Cell Biotin-Antibody Cocktail</td><td>Miltenyi Biotec Inc.</td><td>130-100-875</td><td>T cell proliferation assay</td></tr><tr><td>Anti-Biotin MicroBeads</td><td>Miltenyi Biotec Inc.</td><td>130-100-875</td><td>T cell proliferation assay</td></tr><tr><td>CD4+ T cell isolation kit</td><td>Miltenyi Biotec Inc.</td><td>130-104-454</td><td>T cell proliferation assay</td></tr><tr><td>CD4+ T cell Biotin-Antibody Cocktail</td><td>Miltenyi Biotec Inc.</td><td>130-104-454</td><td>T cell proliferation assay</td></tr><tr><td>Anti-Biotin MicroBeads</td><td>Miltenyi Biotec Inc.</td><td>130-104-454</td><td>T cell proliferation assay</td></tr><tr><td>ACK lysing buffer</td><td>ThermoFisher Scientific</td><td>A1049201</td><td>BMDC differentiation</td></tr><tr><td>1 ml syringe</td><td>BD Biosciences</td><td>309626</td><td>T cell proliferation assay</td></tr><tr><td>3 ml syringe</td><td>BD Biosciences</td><td>309588</td><td>BMDC differentiation</td></tr><tr><td>25G needle</td><td>BD Biosciences</td><td>309626</td><td>T cell proliferation assay</td></tr><tr><td>23G needle</td><td>BD Biosciences</td><td>309588</td><td>BMDC differentiation</td></tr><tr><td>BSA</td><td>Sigma Aldrich Inc.</td><td>A2058</td><td>T cell proliferation assay</td></tr><tr><td>EDTA</td><td>ThermoFisher Scientific</td><td>15575020</td><td>T cell proliferation assay</td></tr><tr><td>LS Column</td><td>Miltenyi Biotec Inc.</td><td>130-042-401</td><td>T cell proliferation assay</td></tr><tr><td>Pre-Separation Filter</td><td>Miltenyi Biotec Inc.</td><td>130-095-823</td><td>T cell proliferation assay</td></tr><tr><td>collagenase D</td><td>Sigma Aldrich Inc.</td><td>11088858001</td><td>T cell proliferation assay</td></tr><tr><td>HBSS</td><td>ThermoFisher Scientific</td><td>14025076</td><td>T cell proliferation assay</td></tr><tr><td>ovalbumin (OVA) peptide 323&amp;ndash;329</td><td>Sigma Aldrich Inc.</td><td>O1641</td><td>T cell proliferation assay</td></tr><tr><td>Mouse IFN-&amp;gamma; TaqMan probe</td><td>ThermoFisher Scientific</td><td>Mm01168134_m1</td><td>qRT-PCR</td></tr><tr><td>Mouse IL-12a TaqMan probe</td><td>ThermoFisher Scientific</td><td>Mm00434165</td><td>qRT-PCR</td></tr><tr><td>Mouse IL-12 p70 DuoSet ELISA</td><td>R&amp;amp;D systems</td><td>DY419-05</td><td>ELISA</td></tr><tr><td>Mouse EDN-1 ELISA</td><td>RayBiotech</td><td>ELM-EDN1-1</td><td>ELISA</td></tr><tr><td>TNF-&amp;alpha; TaqMan probe</td><td>ThermoFisher Scientific</td><td>Mm00443258</td><td>qRT-PCR</td></tr><tr><td>Mouse TNF-&amp;alpha; Quantikine ELISA Kit</td><td>R&amp;amp;D systems</td><td>MTA00B</td><td>ELISA</td></tr><tr><td>IL-6 TaqMan probe</td><td>ThermoFisher Scientific</td><td>Mm00446190</td><td>qRT-PCR</td></tr><tr><td>Mouse IL-6 Quantikine ELISA Kit</td><td>R&amp;amp;D systems</td><td>M6000B</td><td>ELISA</td></tr><tr><td>IL-23a TaqMan probe</td><td>ThermoFisher Scientific</td><td>Mm01160011</td><td>qRT-PCR</td></tr><tr><td>Mouse IL-23 DuoSet ELISA</td><td>R&amp;amp;D systems</td><td>DY1887-05</td><td>ELISA</td></tr><tr><td>IL-4 TaqMan probe</td><td>ThermoFisher Scientific</td><td>Mm99999154_m1</td><td>qRT-PCR</td></tr><tr><td>IL-10 TaqMan probe</td><td>ThermoFisher Scientific</td><td>Mm01288386_m1</td><td>qRT-PCR</td></tr><tr><td>TGF-&amp;beta; TaqMan probe</td><td>ThermoFisher Scientific</td><td>Mm01178820_m1</td><td>qRT-PCR</td></tr><tr><td>Anti-Heme Oxygenase 1 antibody</td><td>Abcam</td><td>ab13248</td><td>Western blotting</td></tr><tr><td>Anti-&amp;beta;-actin antibody</td><td>Abcam</td><td>ab8226</td><td>Western blotting</td></tr><tr><td>CFX96 Touch Real-Time PCR Detection System</td><td>Bio-Rad Inc.</td><td></td><td>qRT-PCR</td></tr><tr><td>BD FACSCalibur Cell Analyzer</td><td>BD Biosciences</td><td></td><td>Flow cytometry</td></tr></tbody></table>

development and functional characterization of murine tolerogenic dendritic cells

免疫系统的运作方式是在协调对外来抗原的反应和保持对自抗原的无反应状态以及从共生生物体中提取的抗原之间保持紧密的平衡。这种免疫稳态的破坏会导致慢性炎症和自身免疫的发展。树突状细胞 (DCs) 是一种专业的抗原呈现细胞的先天免疫系统参与激活幼稚 T 细胞启动免疫应答外来抗原。然而, DCs 也可以被区分为 TolDCs, 以维持和促进 T 细胞耐受性和抑制效应细胞, 促进发展的任何一种自身免疫或慢性炎症条件。最近在我们理解 TolDCs 的进展表明, 直流耐受可以通过调节它们的分化条件来实现。这种现象已经导致了巨大的增长, 发展 TolDC 疗法的许多免疫障碍造成的免疫耐受性的突破。对前体免疫性小鼠模型的成功研究进一步证实了 TolDCs 治疗自身免疫性疾病的免疫治疗效用。今天, TolDCs 已成为临床上一个有希望的免疫治疗工具, 以恢复免疫耐受的各种免疫障碍的目标致病性自身免疫反应, 同时保持保护免疫完好。虽然多个实验室提出了一系列的策略来诱导 TolDCs, 但在表征细胞和功能表型方面没有一致性。该协议为骨髓源 DCs 的大量开发提供了一步一步的指导, 一种独特的方法, 用于将其与合成萜 2-氰基-312-dioxooleana-19-奠 28-伊斯兰会议组织区分为 TolDCs。酸-difluoro-丙酰胺 (CDDO-DFPA), 以及用于确认其表型的技术, 包括分析 TolDCs 的基本分子特征。最后, 通过在多发性硬化的临床前模型中测试其免疫抑制反应体外和体内, 我们展示了一种评估 TolDC 功能的方法。

BMDCs 的分化与选择:
骨髓祖细胞在全 RPMI 培养基中培养, 在 GM-CSF 和 IL-4 的存在下, 可分化为 iDCs 7 天 (图 1A)。1天, 细胞体积小, 呈球形形态。在3天更换新鲜培养基之前, 使用 PBS 进行洗涤有助于细胞形成簇群, 也增加了 CD11c+细胞的数量。在4天, BMDCs 扩大了规模, 并开始了集群的形成。黏?...

Watch this Scientific Journal Video about Development and Functional Characterization of Murine Tolerogenic Dendritic Cells at JoVE.com

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

在这里, 我们提出了一个协议, 开发和表征致耐受性树突状细胞 (TolDCs), 并评估其免疫治疗效用。

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

development-functional-characterization-murine-tolerogenic-dendritic

Research

JoVE Journal

Medicine

10.6K 次观看.  Case Western Reserve University. 在这里, 我们提出了一个协议, 开发和表征致耐受性树突状细胞 (TolDCs), 并评估其免疫治疗效用。

视频: Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

科研

生物学

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

代骨髓衍生的双光子成像使用的小鼠树突状细胞

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

免疫与感染

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate na&#239;ve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Here we provide a protocol for screening potential transcription factors involved in the development of dendritic cell (DC) using lentiviral transduction of shRNA to obtain stable knockdown cell lines for in vitro DC differentiation.

短发夹RNA介导的基因敲低在造血干和祖细胞系体外发育的树突状细胞发育研究

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

一种简单有效的方法，用于测试从人 CD14+ 单核细胞生成致耐受性树突状细胞的免疫调节剂

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

Source: Sim, W. J. et al., <a href="https://app.jove.com/v/54128">Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes.</a>&#160;J. Vis. Exp.&#160;(2016).
This video demonstrates the generation of immature, mature, and tolerogenic dendritic cells, or DCs, from human monocytes. The incubation of monocytes with growth factors and cytokines leads to the production of immature DCs. In response to immune regulatory molecules, immature DCs differentiate into tolerogenic and mature DCs.