This work was supported by NIH R21CA162259, NIH R01CA209829, NIH P20 MD006988, NIH 2R25 GM060507, a Hyundai Hope on Wheels Scholar Hope Grant, the Department of Pathology and Human Anatomy, the Department of Basic Sciences, and the Center for Health Disparities and Molecular Medicine at Loma Linda University School of Medicine and by a Grant to Promote Collaborative and Translational Research from Loma Linda University (KJP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

<html>根据洛马琳达大学的机构动物护理和使用委员会（IACUC）和机构审查委员会（IRB）批准的协议和根据所有联邦指南进行研究。 注意:PDX和人体组织应按照安全规程处理，以防止血液传播的病原体传播。 细胞因子转导的基质细胞的培养和扩增注意:每次基质传代，收获培养上清（1 mL等分试样），并储存在-80°C，以便将来通过 ELISA测定定量细胞因子水平。 <ol><li>如材料表中所述，制备R10培养基和冷冻培养基。 </li><li>产生10,11或获得对照基质（用空载体转导）和产生细胞因子（TSLP +）的基质。"&gt; 6在液氮（LN 2 ）中储存，直到使用，如所述12解冻基质细胞，并将其置于分开的T-25细胞培养瓶中的5 mL R10培养基中，培养细胞在37℃的培养箱中， CO 2 ，这是解冻后的"第1号"。 </li><li>一旦基质汇合（24-48 h），通过胰蛋白酶消化，使用1×胰蛋白酶EDTA（0.25％）并在T-150培养瓶中重新铺板。这是解冻后的"通道＃2"。 </li><li> 当基质在T-150烧瓶（3-4天后）中达到融合时，通过胰蛋白酶消化细胞。使用这种细胞悬浮液用于实验需要。 <ol><li>细胞膨胀到多个烧瓶<ol><li>将收获的细胞在三个T-150烧瓶之间分离并培养直至汇合。如果需要基质扩张，重复此步骤。在T-150烧瓶中汇合时典型的人间质（HS-27A）细胞计数为〜5×10 6 。 </li></ol></li><li>细胞torage <ol><li>将收获的细胞悬浮液转移到锥形管中，并以500×g离心5分钟，倾析上清液并将细胞重悬于冷冻培养基中。冷冻并存放LN 2〜1.5×10 6个细胞/瓶。 </li></ol></li><li>对于小鼠中的periotoneal基质注射进入步骤4.1。 </li></ol></li></ol> 2.用于监测转导基质体外细胞因子生产的ELISA测定<ol><li>购买市售的ELISA测定试剂盒以检测感兴趣的细胞因子。 </li><li>解冻早期，中期和晚期传代期收获的来自对照和细胞因子产生（TSLP +）基质的基质细胞上清液（ 图2 ）。 </li><li>使用ELISA试剂盒根据制造商的说明书确定上清液样品中的TSLP浓度。编制这些序列数据来监测细胞培养期间细胞因子产生的稳定性<html> e（ 图2A-B ）。 <ol><li>鉴定并丢弃具有减少或次优的细胞因子表达的细胞因子产生基质，以及储存中任何相应的小瓶（ 图2A-B ）。 </li><li>识别和维持表现出稳定，高水平细胞因子浓度的产生细胞因子的基质。使用这些解冻进行实验。 </li></ol></li><li>使用ELISA试剂盒在细胞培养期间常规监测基质（在解冻后早期，中期和晚期至少1次），以确保稳定的细胞因子产生，或在对照基质的情况下，不存在细胞因子产生。 </li></ol> 3.磷酸流动细胞分析法测定细胞因子产生的细胞因子活性注意:在第一次实验之前评估工程基质的上清液，以验证基因产生的细胞因子的相关生物活性用于个别实验。一旦工程基因被验证，进一步的测试是不必要的，除非引入了用新载体产生的基质。 <ol><li>购买MUTZ5和/或MHH-CALL4白血病细胞系（TSLP反应性）， 14种重组人细胞因子，以及批准用于磷酸流式细胞术检测以检测合适的下游磷酸化靶标的抗体。 </li><li>从对照和表达细胞因子的基质中解冻收获的上清液。 </li><li>在96孔组织培养板中以0.2×10 6个细胞浓度将R20培养基（参见表格 ）中的白血病细胞系平板化。每个条件平板3孔以允许一式三份测定。在37℃孵育2小时。这一次允许在先前培养条件下发生的任何磷酸化的损失。 注意:每个细胞系12个孔为步骤3.4所示的每个实验条件提供一式三份测定。 </li> &lt;在培养2小时后，将细胞从每孔转移到4mL管中，并在4℃下以500×g离心5分钟。倾倒上清液。 <ol><li>对于以下实验条件中的每一个，将细胞重新悬浮在200μL中（一式三份进行测定）:1）仅阴性对照培养基，2）在饱和浓度下（TSLP为15ng）的重组细胞因子的阳性对照培养基/ mL 等 ），3）控制来自对照基质的基质上清液，以及4）来自细胞因子产生基质的细胞因子 - 基质上清液。 </li></ol></li><li>在特定商业磷酸化测定指定的持续时间内孵育细胞（ 例如 ，对于TSUP，MUTZ5或MHH-CALL4中的磷酸化STAT5为30分钟）。 </li><li>在4℃下以500xg离心5分钟并倾析上清液。 </li><li>按照制造商的方案进行磷酸流式细胞仪染色，并收集流式细胞术数据。 15 </li><li><strong&gt;分析流式细胞仪数据。 15 <ol><li>基于正向（FSC，表示细胞大小）和侧（SSC，表示细胞粒度）光散射的完整细胞上的门。 </li><li>确定每个样品中完整细胞的中值荧光强度（MFI）。比较从基质细胞上清液的一式三份值获得的平均MFI与阳性和阴性介质对照的平均MFI。 </li></ol></li></ol> 4.将细胞因子转导的基质注射入小鼠注意:培养HS-27A基质至少3次解冻前通道，然后将其注入小鼠，以确保健康细胞和充分的细胞因子产生。 <ol><li> 基质的制备 <ol><li>将收获的基质细胞悬浮液转移到锥形管中，并将等分试样10μL用于血细胞计数器;使用台盼蓝获得活细胞计数。 16离心剩下的ce以500×g悬浮5分钟。 </li><li>从造粒的细胞吸出上清液，并轻轻地将细胞重悬于必要体积的无菌磷酸盐缓冲盐水（PBS）中，用于小鼠注射（取决于注射的小鼠数目）。 注意小鼠注射的典型基质细胞浓度:在200μLPBS中每只小鼠0.5×10 6 -5×10 6个细胞。 </li><li>将基质细胞悬浮液保持在4°C（或冰上），直到注射前。确保细胞悬浮液至少在室温（RT）（〜25°C）下注射小鼠。 </li></ol></li><li> 基质细胞注射 <ol><li>消毒生物安全罩，组装注射材料，然后将鼠笼放在里面。 </li><li>在将200μL拉伸成结核菌素注射器之前，通过反转轻轻混合细胞悬浮液。 </li><li>使用标准腹膜内注射技术， 17约束小鼠并将200μL细胞悬浮液施用于腹膜腔。 Machholz 等人先前已经详细说明了这些细节。 18 </li></ol></li></ol> 5.小鼠外源人细胞因子的系列血液收集和血浆监测<ol><li> 通过尾巴剪取血液收集 <ol><li>消毒生物安全罩，组装鼠标垫，剪刀等工具/用品。 </li><li>将鼠标放在限制器中并固定管，以尽量减少鼠标移动。 </li><li>用异丙醇消毒尾巴和手术剪刀。在尾部涂上局部麻醉霜。 </li><li>使用非常锋利的外科剪刀剪掉约0.5 - 1毫米的小鼠尾巴。使用肝素化毛细管收集大约80μL的外周血。 注意:如果采用这种方法六次以上采集血液mes，尾部去除应保守相对于尾部的去除量。随着剪刀向上延伸，直径增加，出血更快，愈合时间更长。 </li></ol></li><li> 等离子体分离 <ol><li>使用灯泡注射器（将其从毛细管中排出）转移到标记的K 2 EDTA大容量管中;反转20次以防止凝血。 注意:通常来自尾夹的血液凝固很快。然而，如果出血持续时间超过约2分钟，请使用浸液式铅笔/粉末来辅助凝血。 </li><li>根据制造商的说明离心血液采集管，并等离子体储存（-20°C）直到准备进行ELISA测定。 </li><li>如果需要来自小鼠血液的小鼠 - 人细胞嵌合数据，则如步骤6.3.1所述处理剩余的颗粒。否则，丢弃血细胞沉淀。 </li></ol></li><li> 微量修饰ELISA小鼠血浆的umes 注意:制造商的ELISA程序从推荐的100μL样品体积修改为40μL体积，以适应从小鼠收集的微量体积。不可能以这些有限的体积一式三份运行体内小鼠血浆样品。在实验结束时，通过心脏穿刺收集大约500μL的死后血液。在100μL安乐死血浆中评估的细胞因子浓度用于验证每只小鼠的40μL 体内数据。 <ol><li>解冻冷冻的小鼠血浆样品。 </li><li>连续稀释ELISA标准品，并以40μl/孔将其平板化一式三份。 </li><li>向ELISA板中加入每只小鼠血浆样品40μL。 注意:如果小鼠样品小于40μL，则使用ELISA缓冲液将体积高达40μL。记录此稀释液体积，并使用它来计算每个稀释样品的稀释因子。什么时候分析ELISA数据将稀释的样品浓度乘以样品的稀释因子，以确定原始样品中的实际细胞因子浓度。 </li><li>根据制造商的说明完成ELISA测定。 </li></ol></li></ol> 6.将造血细胞移植入小鼠<ol><li> 亚致死辐射条件移植骨髓 注意:洛马琳达大学（LLU）使用Co-60辐射源。将动物放置在距离辐射源80cm处的20×20cm场中并且在距离限制器顶部具有0.5cm的Lexan层的饼形限制器中。计算暴露时间，以达到225 cGy剂量，根据最近的校准源（目前为2-3分钟）。 <ol><li>次级照射小鼠（全身照射剂量为免疫缺陷小鼠最大225cGy）。 </li><li>移植造血细胞〜24小时后通过&lt;静脉注射静脉注射。 </li></ol></li><li> 静脉内造血细胞移植 <ol><li>准备人体细胞悬浮液移植体积200μL无菌PBS每只小鼠:CD34 +造血干细胞（HSC）:1×10 5至5×10 5细胞/小鼠和白血病细胞系/患者样本:1×10 6至每只小鼠5×10 6个细胞。 </li><li>将细胞保持在4°C（冰上运输）直至移植前。移植前确保细胞至少在室温下。 </li><li>消毒生物安全罩，准备移植注射罩。 </li><li>将鼠标置于加温笼中至少5分钟以允许尾静脉扩张。 </li><li>轻轻混匀细胞悬浮液，并将200μL加入结核菌素注射器。安全地将针放在一边将其保持无菌的地方。 </li><li>将鼠标放在约束中，用异丙醇对尾巴进行消毒/ LI&gt; <li>使用标准静脉注射技术， 17将人细胞悬液注入小鼠尾静脉。 注意:尾静脉注射可能需要几次尝试，特别是对于新手动物处理器。 Kovacscics和Raper的JoVE视频19提供了这种动物技术的详细演示。 </li><li>监测鼠标恢复约5分钟。记录注射期间的任何不良事件（ 例如溢出的细胞，主要出血，过度的尾部创伤）。 </li></ol></li><li> 分析小鼠外周血中的人细胞嵌合 <ol><li>在血浆分离后获得剩余在血管中的血球细胞（步骤5.2.3）。 </li><li>对于红血细胞（RBC）裂解，将剩余的血球重新悬浮在等体积的PBS中，等离子体移除（以取代血浆体积）并充分混合（涡流/移液管）。 </li><li>将每个再悬浮的血液样品转移到标记的4 mL管中然后以1:9的比例向每个管中加入RBC裂解缓冲液（900μLRBC裂解缓冲液用于100μL重悬浮的血液）。在室温下孵育5分钟。离心机（5分钟，500×g）和倾析。 </li><li>进行第二次RBC裂解缓冲液孵育（室温5分钟）。离心和倾析如前。 </li><li>洗涤〜1 mL PBS中的剩余细胞。离心和倾析如前。 </li><li>将沉淀重悬于适合于标准流式细胞术染色的PBS体积（根据制造商和个别实验室方案而异）。 20使用验证流式细胞术的免疫分型抗体鉴定小鼠细胞（小鼠CD45 +）和人类细胞（人类CD45 +）6,21 以及对感兴趣的细胞系特异性的其它人白细胞标志物。 注意:建议从每个小鼠样品取小体积（5μL-20μL）以创建"合并样品";用于未染色，活力和同种型对照样品。对于每个实验条件，最佳做法有未染色和同种型对照。 </li></ol></li></ol> 7. 体内细胞因子活性的功能评估<ol><li> 小鼠安乐死和组织收获 <ol><li>在指定的实验时间点通过 CO 2窒息来安乐死小鼠。 </li><li> 通过心脏穿刺获取血液并收集血浆，如步骤5.2.1-5.2.2所示。 注意:在其他组织之前收集血液，因为它在死亡后立即开始凝结。 </li><li>按步骤5.2.2处理小鼠血液，等分并储存血浆。 <ol><li>随后，根据制造商的方案， 通过 ELISA来评估在安乐死中获得的血浆中的外源性细胞因子浓度。评估并比较来自连续体内的小鼠血浆中的外源性细胞因子浓度采血和安乐死血浆样品（见5.3节）。 </li></ol></li><li>将PBS添加到剩余的血液样品中以取代等离子体体积和过程，如步骤6.3.1和6.3.2所述。立即进行流式细胞术检测或将细胞重新悬浮于冷冻介质中以保留LN 2 。 </li></ol></li><li>从PDX小鼠收获骨髓（BM）和脾脏，并如前所述制备单细胞悬液。 22 </li><li>使用3％乙酸与亚甲蓝（裂解细胞膜和RBCs离开活细胞的完整核），以1:1的稀释度和使用血细胞计数器计数细胞获得每个PDX小鼠组织样品的细胞计数。列出每只动物的骨髓和脾脏样品的总细胞计数。 </li><li>离心机（500×g，15分钟）细胞悬浮液并倒出上清液。立即进行骨髓和/或脾细胞的流式细胞术测定，或重悬于冷冻培养基中，sub&gt; 2存储。 注意:每只小鼠冷冻10个BM等分试样（用于未来的生化测定）和〜5个脾脏等分试样（用于未来的PDX移植）。 </li><li> 免疫分型鉴定体内功能的影响 <ol><li>为感染目标细胞因子和第二MM同种型对照抗体（ 图5和材料表）制备免疫表型造血细胞群的流式细胞计数抗体的一种主混合物（MM）。 </li><li>在步骤7.4中制备的解冻PDX组织等分试样（37℃珠浴）。 </li><li>通过将解冻的细胞转移到锥形管中并加入至少3倍体积的PBS来洗涤解冻的细胞。离心（500×g，5分钟）并倒出上清液。 </li><li>重复洗涤步骤（7.5.3）。 </li><li>通过从每个PDX样品中获取〜10μL-20μL细胞，用于未染色的对照，活力对照和同种型对照（参见步骤6.4.6注释），创建合并的等分试样。污染所有样品，除了您使用可固定的活力染料（死细胞标记）进行染色控制，根据制造商的方案孵育和洗涤。 </li><li>根据标准流式细胞术染色方案，用表型-MM染色每个PDX细胞样品;用同种型MM相似地染色合并的同种型对照样品。孵育所有样品，用PBS洗涤，并通过重悬浮于1％多聚甲醛中固定样品。 </li><li>收集流式细胞术数据并使用适合感兴趣的细胞群体的门控策略分析数据（ 图5 ）。典型的门控如下: <ol><li>通过绘制排除碎片的FSC和SSC门来定义完整的单元格。 </li><li>通过选择对死细胞标记为阴性的细胞（这是"总活细胞"）来识别活细胞群体。 </li><li>在"总活细胞"中使用子门来定义具有所需免疫表型的细胞多数（参见图5）。 </li><li>获得来自流式细胞术分析的总活细胞计数中子集细胞群的频率。 6，21 </li></ol></li><li>通过将B细胞亚群的频率乘以总活细胞（步骤7.5.6.4中获得）乘以步骤7.3中获得的每组织的总活细胞计数（％B细胞亚群x"）来计算每个组织中的靶细胞数目血细胞计数器细胞计数"; 见表1 ） </li><li>比较对照PDX小鼠和表达细胞因子（TSLP +）的PDX小鼠之间的靶细胞群的细胞计数，以确定外源人细胞因子的体内功能效应（ 表1 ）。 </li></ol></li></ol>

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The authors have nothing to disclose.

该手稿描述了一种用于工程化PDX来表达外源人细胞因子的简单，快速且相对成本有效的方法。这里描述的策略是基于每周腹膜内注射转染以表达人细胞因子TSLP的基质细胞系。在进行本文描述的方法之前，产生用于表达高水平的感兴趣细胞因子（TSLP）和类似工程化的对照基质的基质。在本文提出的方案中，基质在培养物中扩增，并筛选随时间提供稳定，高水平细胞因子产生的能力（或在对照基质?...

患者来源的异种移植物（PDX）是一种强大的体内模型，用于研究"天然"哺乳动物环境中正常和恶性造血细胞的产生。最常见的是，通过将人细胞注射或移植到免疫缺陷小鼠中来产生PDX。使用正常人造血干细胞的PDX生产允许对正常人血液和免疫细胞发育的体内研究。由白血病或其他癌细胞产生的PDX可以研究致癌机制，并在人群中存在的遗传景观和突变范围的背景下鉴定有效的治疗方法。 1因此，PDX是目前翻译生物医学研究的黄金标准，用于识别有效的治疗方法，也是理解癌症进展机制的重要工具。 PDX模型是帮助研究健康差异疾病的重要工具<html>遗传病变或任何疾病，其中患者遗传景观的变化可以显着地促成肿瘤形成和治疗结果。 小鼠 - 人PDX模型是可能的，因为许多小鼠细胞因子在它们在小鼠内部时激活人类细胞的细胞因子受体时充分模拟了它们的人类类似物。例如，白细胞介素-7（IL-7）为人B细胞发育提供了关键信号。 2在这种情况下，小鼠IL-7与人IL-7具有足够的同源性，小鼠细胞因子刺激人B细胞前体中的信号通路。 2，3，4然而，胸腺基质淋巴细胞生成素（TSLP），其中其他细胞因子（IL-3，粒细胞 - 巨噬细胞 - 巨噬细胞集落刺激因子（GM-CSF），干细胞因子（SCF） ，对于正常和恶性人造血细胞的生产来说重要的是，当小鼠和人类细胞因子显示出低同源性时，小鼠细胞因子不会在人细胞上激活它们各自的受体，为了克服这个障碍，已经使用了许多策略在PDX小鼠中设计人类细胞因子的表达，包括注射重组人细胞因子，流体动力注射DNA，慢病毒表达，转基因表达和敲入基因置换7本报告介绍了一种通过基质介导的方法制备人类细胞因子细胞因子递送（ 图1 ）。 在本文所示的方法中，PDX小鼠被工程化以表达人细胞因子，TSLP，或用作细胞因子阴性对照。表达TSLP的PDX通过每周腹膜内注射已被转导以表达高水平人TSLP的基质细胞来实现。细胞因子阴性PDX"对照"小鼠被类似地设计;虽然控制基质用控制载体转导。该方法在注射TSLP +基质的PDX小鼠中达到人TSLP的正常生理水平。在接受细胞因子阴性基质的PDX小鼠中未观察到可检测的TSLP。我们选择人类基质细胞系HS-27A作为我们的研究，因为它在培养中强壮地生长，并且显示非常低水平的细胞因子产生，其不支持共培养物中分离的祖细胞的增殖。 8对于人TSLP表达，使用来自先前描述的骨架9的先进一代自身灭活慢病毒载体转导基质，并且包括cPPT / cts元件和土拨鼠肝炎后转录调节元件（WPRE）以增加转基因表达。在延伸因子-1的控制下将人TSLP基因构建到该载体中（EF-1）α启动子，以实现强壮，组成型和长期表达。 这种人细胞因子增强的PDX模型的工程包括4个主要步骤。首先，转导的基质在体外扩增 ，并通过酶联免疫吸附测定（ELISA）来评估稳定的高水平细胞因子产生。第二，使用磷酸流式细胞术验证转导的基质细胞产生的人细胞因子的活性（和来自对照基质的细胞因子活性的缺乏）。已知对感兴趣的细胞因子（本例中为TSLP）有反应的细胞系与基质细胞上清液一起温育，并测定细胞因子诱导的磷酸化。第三，将小鼠注射转导的人类基质，然后每周一次通过ELISA评估人细胞因子水平的小鼠血浆。第四，移植人类造血细胞，并在已知靶标上评估人细胞因子的体内功能效应（ <em&gt;例如细胞群体）。 <img alt="图1" src="/files/ftp_upload/55384/55384fig1.jpg" /> 图1:用于在小鼠中产生外源人细胞因子的PDX模型。 （ 1A ）设计实验并获得转导的人类基质细胞（ 1B ）获得人类细胞（造血干细胞，白血病细胞等 ）产生PDX（患者来源的异种移植物）小鼠。 （ 2A ）根据实验时间表将注射工程基质和（ 2B ）将人类细胞移植入免疫缺陷小鼠。 （ 3A-B ）通过ELISA监测基质上清液和小鼠血浆中的细胞因子浓度。 （ 4 ）收获人体细胞并评估PDX中存在的人细胞因子的体内功能效应。 <a href="http://ecsource.jove.com/files/ftp_upload/55384/55384fig1large.jpg" target="_blank">请点击这里</a>以查看该图的较大版本。 与其他在PDX小鼠中递送/产生人细胞因子的方法相比， 通过基质细胞递送人细胞因子提供了优点和缺点。与注射重组人细胞因子相比，基质介导的递送通常较便宜（基质细胞培养的成本低于重组细胞因子的成本）和较少的劳动密集型（每周一次注射，而每周多次注射）。短暂的细胞因子半衰期的问题也被缓解，因为基质不断产生外源细胞因子。 通过流体动力注射DNA递送细胞因子可能比通过基质递送更便宜。然而，它类似地是短暂的，并且可能需要比基质介导递送所需的简单的每周腹膜内注射更多的技术技能。小鼠中的慢病毒基因表达可以提供较少的转录细胞因子递送的方法;然而，在我们手中，生理TSLP水平未达到。另外，这种方法是劳动密集型的，需要连续生产慢病毒载体。转基因或敲入小鼠提供稳定的细胞因子的长期表达，并且可以被设计用于组织特异性表达，这可以是一个优点。另一方面，人类细胞因子基因在PDX小鼠所需的免疫缺陷小鼠背景上的转基因表达在模型的价值建立之前需要大量的资源投入。此外，转基因模型通常不允许改变细胞因子起始的时间或体内细胞因子产生的水平的选择。这些可以通过简单地改变引发基质细胞注射的时间点或注射的基质细胞的剂量来实现基质介导的递送。 基质细胞介导的细胞因子转运在这里详细描述的用于开发PDX以评估TSLP在正常人B细胞发育4,6和高风险B细胞急性淋巴细胞白血病中的作用。该方法提供了替代细胞因子递送方法，用于产生与TSLP以外的人细胞因子相似的模型。该模型还可用于产生初步数据，可以帮助确定细胞因子转基因或细胞因子敲入PDX模型的价值是否值得大量的时间和金钱投资。

<table><tbody><tr><td>&lt;strong&gt;Wet lab reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>T-25 Nunc Cell Culture Treated EasYFlasks, 7 mL capacity,&amp;nbsp; 25cm&amp;sup2; culture area</td><td>Thermo Scientific / Fisher</td><td>156367</td><td></td></tr><tr><td>T-75 Falcon Tissue Culture Treated Flask, 250 mL capacity, 75 cm&amp;sup2; culture area &amp;amp; vented cap</td><td>Corning Inc. / Fisher</td><td>353136</td><td></td></tr><tr><td>T-150 Falcon Tissue Culture Treated Flask, 600 mL capacity, 150 cm&amp;sup2; culture area &amp;amp; vented cap</td><td>Corning Inc. / Fisher</td><td>355001</td><td></td></tr><tr><td>10 mL serological pipettes</td><td>Falcon</td><td>357551</td><td></td></tr><tr><td>15 mL&amp;nbsp; polypropylene conical tubes</td><td>Fisher</td><td>05-539-12</td><td></td></tr><tr><td>5 mL round-bottom polystyrene tubes</td><td>Falcon</td><td>352054</td><td></td></tr><tr><td>50 mL polypropylene conical tubes</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>2.0 mL cryogenic vials, externally threaded</td><td>Corning Inc. / Fisher</td><td>4230659</td><td></td></tr><tr><td>1 mL pipette tips</td><td>Fisher</td><td>2707509</td><td></td></tr><tr><td>200 &amp;mu;L pipette tips&amp;nbsp;</td><td>Denville Scientific</td><td>P3020-CPS</td><td></td></tr><tr><td>10 &amp;mu;L pipette tips</td><td>Denville Scientific</td><td>P-1096-CP</td><td></td></tr><tr><td>Sterile Disposable Filter Units with SFCA Membrane, Nalgene Rapid-Flow</td><td>Fisher</td><td>09-740-28D</td><td></td></tr><tr><td>2 NH&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&amp;nbsp;&lt;/sub&gt;</td><td>BioLegend</td><td>423001</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline (PBS) without calicium and magnesium, 1&amp;times;</td><td>Corning cellgro/ Mediatech</td><td>21-031-CV</td><td></td></tr><tr><td>Human TSLP ELISA Max Deluxe Set</td><td>BioLegend</td><td>434204</td><td></td></tr><tr><td>3% Acetic Acid with Methylene Blue</td><td>Stemcell Technologies</td><td>7060</td><td></td></tr><tr><td>Trypan Blue&amp;nbsp;</td><td>Corning</td><td>25-900-Cl</td><td></td></tr><tr><td>&quot;Trypsin&quot; 1x 0.25% Trypsin 2.21 mM EDTA in HBSS</td><td>ThermoFisher</td><td>25-053</td><td></td></tr><tr><td>TWEEN 20 BioXtra</td><td>Sigma-Aldrich</td><td>P-7949</td><td></td></tr><tr><td>&lt;strong&gt;&amp;ldquo;R10&amp;rdquo; cell culture medium, % of total volume (makes 565 mL)&lt;/strong&gt;</td><td>-</td><td>&lt;strong&gt;-&lt;/strong&gt;</td><td>&lt;strong&gt;Lab Recipe&lt;/strong&gt;</td></tr><tr><td>RPMI, 88.5% (500 mL)&amp;nbsp;</td><td>Mediatech</td><td>10-040-CV&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>FBS, 9.9% (56 mL)&amp;nbsp;</td><td>Mediatech</td><td>35-011-CV</td><td></td></tr><tr><td>L-Glutamine, 0.99% (5.6 mL)&amp;nbsp;</td><td>Mediatech</td><td>25-005-Cl</td><td></td></tr><tr><td>Penicillin-Streptomycin, 0.50% (2.8 mL)&amp;nbsp;</td><td>Mediatech</td><td>30-002-Cl</td><td></td></tr><tr><td>2-Mercaptoethanol, 0.10% (560 &amp;micro;L)&amp;nbsp;</td><td>MP</td><td>190242</td><td></td></tr><tr><td>&lt;strong&gt;&amp;ldquo;R20&amp;rdquo; cell culture medium, % of total volume (makes 195 mL)&lt;/strong&gt;</td><td>-</td><td>-</td><td>&lt;strong&gt;Lab Recipe&lt;/strong&gt;</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; RPMI, 76.84% (150&amp;nbsp; mL)</td><td>see above</td><td>see above</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; FBS, 20.49% (40 mL)</td><td>see above</td><td>see above</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 2mM L-glutamine, 1.02% (2 mL)</td><td>see above</td><td>see above</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1 mM Na pyruvate, 1.02% (2 mL )</td><td>see above</td><td>see above</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Penicillin-Streptomycin,&amp;nbsp; 0.51% (1 mL)</td><td>see above</td><td>see above</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 2-Mercaptoethanol, 0.10% (0.1 M),&amp;nbsp; 0.10% (200 &amp;micro;L)</td><td>see above</td><td>see above</td><td></td></tr><tr><td>&lt;strong&gt;&amp;ldquo;Freezing medium&amp;rdquo;, % of total volume&lt;/strong&gt;</td><td>-</td><td>&lt;strong&gt;-&lt;/strong&gt;</td><td>&lt;strong&gt;Lab Recipe&lt;/strong&gt;</td></tr><tr><td>&amp;ldquo;R10&amp;rdquo; medium, 45%</td><td>see &quot;R10&quot; recipe</td><td>-</td><td></td></tr><tr><td>FBS, 20%</td><td>see above</td><td>35-011-CV</td><td></td></tr><tr><td>DMSO, 10%</td><td>Corning</td><td>25-950-CQC</td><td></td></tr><tr><td>D(-)(+)-Trehalose dihydrate, 5%</td><td>Fisher Scientific</td><td>BP2687-100</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catolog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Biologics&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&quot;HS-27&quot; HS-27A human stroma line</td><td>ATCC</td><td>CRL-2496</td><td></td></tr><tr><td>&quot;CALL-4&quot; MHH-CALL-4 cell line, human B cell precursor leukemia</td><td>DSMZ</td><td>ACC 337</td><td></td></tr><tr><td>&amp;ldquo;NSG mice&amp;rdquo; NOD.Cg-Prkdc&lt;sup&gt;scid&lt;/sup&gt; Il2rg&lt;sup&gt;tm1Wjl&lt;/sup&gt;/SzJ, male or female, age 6 &amp;ndash; 8 weeks</td><td>JAX Mice&amp;nbsp;</td><td># 005557</td><td></td></tr><tr><td>MUTZ-5 cell line, human B cell precursor leukemia</td><td>DSMZ</td><td>ACC 490</td><td></td></tr><tr><td>Recombinant Human TSLP protein</td><td>R&amp;amp;D Systems</td><td>1398-TS-010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catolog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Flow cytomery antibodies (clone)*&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse CD45 FITC (30F11)</td><td>Miltenyi Biotec</td><td>130-102-778</td><td></td></tr><tr><td>CD127 PE (MB15-18C9) (alternate name is IL-7R&amp;alpha;)</td><td>Miltenyi Biotec</td><td>130-098-094</td><td></td></tr><tr><td>CD34 APC (8G12)</td><td>BD Biosciences</td><td>340667</td><td></td></tr><tr><td>CD45 PE-Cy7&amp;nbsp; (HI30)</td><td>eBioscience</td><td>25-0459</td><td></td></tr><tr><td>&quot;FVD&quot; Fixable viability dye&amp;nbsp; eFluor 780&amp;nbsp;</td><td>eBioscience</td><td>65-0865</td><td></td></tr><tr><td>Ig &amp;kappa; light chain FITC&amp;nbsp; (G20-193)</td><td>BD Pharmingen</td><td>555791</td><td></td></tr><tr><td>Ig &amp;lambda; light chain FITC (JDC-12)</td><td>BD Pharmingen</td><td>561379</td><td></td></tr><tr><td>IgD PE (IA6-2)</td><td>BioLegend</td><td>348203</td><td></td></tr><tr><td>IgM PE-Cy5 (G20-127)</td><td>BD Biosciences</td><td>551079</td><td></td></tr><tr><td>pSTAT5 PE,&amp;nbsp; mouse anti-STAT5 (pY694)</td><td>BD PhosphoFlow</td><td>612567</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catolog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other materials &amp;amp; equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Animal Implantable Nano Transponder with Canula</td><td>Trovan&amp;nbsp;</td><td>ID-100B(1.25)</td><td></td></tr><tr><td>EMLA lidocaine anesthetic cream (obtain by presciption through animal care facility)</td><td>perscription</td><td>perscription</td><td></td></tr><tr><td>BD &amp;frac12; cc LO-DOSE U-100 Insulin Syringe 28G&amp;frac12;&amp;nbsp;</td><td>BD</td><td>#329461</td><td></td></tr><tr><td>BD Microtainer Tubes with K&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>BD&amp;nbsp;</td><td>#365974</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Alegra X-15R&amp;nbsp;</td><td></td></tr><tr><td>FisherBrand Capillary Tubes (Heparinized)</td><td>Fisher Scientific</td><td>22-260-950</td><td></td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>02-671-6</td><td></td></tr><tr><td>MACSQuant Analyzer 10</td><td>Miltenyi Biotec</td><td>130-096-343</td><td></td></tr><tr><td>Mouse Pie Cage</td><td>Braintree Scientific, Inc.</td><td>MPC-1</td><td></td></tr><tr><td>Mouse Tail Illuminator Restrainer</td><td>Braintree Scientific, Inc.</td><td>MTI STD</td><td></td></tr><tr><td>Pistol Grip Implanter</td><td>Trovan&amp;nbsp;</td><td>IM-300(1.25)</td><td></td></tr><tr><td>&amp;micro;Quant&amp;nbsp;</td><td>Bio-Tek Instruments Inc.</td><td>MQX200</td><td></td></tr><tr><td>FlowJo flow cytometry analysis software</td><td>FlowJo, LLC</td><td>Version 10</td><td></td></tr><tr><td>*All antibodies are anti-human unless otherwise stated.</td><td></td><td></td><td></td></tr></tbody></table>

expression of exogenous cytokine in patient-derived xenografts via injection with a cytokine-transduced stromal cell line

通过将人细胞移植到免疫缺陷小鼠中来产生患者来源的异种移植（PDX）小鼠。这些模型是研究正常和恶性造血机制的重要工具，是确定许多恶性肿瘤的有效化学疗法的黄金标准。 PDX模型是可能的，因为许多小鼠细胞因子也对人细胞起作用。然而，对于所有细胞因子来说，情况并非如此，包括许多对于研究人类细胞中正常和恶性造血作用至关重要的细胞因子。设计小鼠产生人细胞因子（转基因和敲入模型）的技术在模型的有用性已被证明之前需要大量费用。其他技术是劳动密集型（注射重组细胞因子或慢病毒），在某些情况下需要高水平的技术专长（流体动力学注射DNA）。本报告描述了一种产生具有外源性人类细胞的PDX小鼠的简单方法（TSLP，胸腺基质淋巴细胞生成素） 通过每周腹膜内注射已被转导过表达该细胞因子的基质。使用这种方法提供了实现小鼠循环中人细胞因子生理水平的连续细胞因子产生的体内来源。人细胞因子的血浆水平可以基于注射的基质细胞的数量而变化，并且细胞因子产生可以在实验的任何点开始。该方法还包括类似地产生的细胞因子阴性对照小鼠，但是通过腹膜内注射用对照载体转导的基质。我们以前已经表明，与对照PDX相比，从表达TSLP的PDX收获的白血病细胞表现出更像原始患者样品的基因表达模式。通过这种方法产生的细胞因子产生和细胞因子阴性的PDX小鼠一起提供了一个我们已经成功地用于研究的模型系统TSLP在正常和恶性造血中的作用。

模型的概述如图1所示 。一旦产生了细胞因子产生（TSLP +基质）和对照基质，它们在培养物中扩增。在将基质注入小鼠之前，通过ELISA评估基质细胞上清液以验证细胞因子产生（ 图2 ），并使用磷酸流式细胞术（方案＃3）来验证由基质产生的细胞因子的活性。当细胞传代时，上清液从融合的基质细胞培养物（对照基质和TSLP +基质）中收集。 ELISA用?...

Watch this Scientific Journal Video about Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line at JoVE.com

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

这里描述了通过每周腹膜内注射细胞因子转导的基质细胞系，在患者来源的异种移植物（PDX）小鼠中产生外源性细胞因子的方法。该方法扩大了PDX的效用，并提供了许多PDX模型中瞬时或持续的外源细胞因子递送的选择。

通过细胞因子转导的基质细胞系注射外源性细胞因子在患者来源的异种移植物中的表达

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...

expression-exogenous-cytokine-patient-derived-xenografts-via

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.3K 次观看.  Loma Linda University. 这里描述了通过每周腹膜内注射细胞因子转导的基质细胞系，在患者来源的异种移植物（PDX）小鼠中产生外源性细胞因子的方法。该方法扩大了PDX的效用，并提供了许多PDX模型中瞬时或持续的外源细胞因子递送的选择。 

视频: Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

前肿瘤反应性T细胞的体外扩增Bryostatin 1/Ionomycin手段和共同的γ链细胞因子配方

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

科研

免疫与感染

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

合成肺腺癌细胞原位移植研究 pd-l1 表达

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

癌症研究

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

用于转化免疫肿瘤学(I-O)研究的人类外周血单核细胞(PBMC)的人工化异种移植模型

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

在异种性鼠模型中的移植-无源疾病的诱导和评分,以及使用数字PCR对小鼠组织中人类T细胞的定量

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

异种移植皮肤模型在体内操纵人类免疫反应

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry

Source: Coats, J. S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55384">Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line.</a> J. Vis. Exp. (123) (2017).
This video demonstrates the evaluation of stromal cytokine-induced intracellular phosphorylation of proteins in leukemia cells. These intracellular phosphorylated proteins are quantified using the phospho-flow cytometry technique.

使用磷酸流式细胞术评估基质细胞因子诱导的细胞内蛋白磷酸化

1. Phospho-flow Cytometry Assays to Evaluate the Activity of Cytokine Produced by Stroma
NOTE: Assess the supernatant from the engineered stroma before ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Ex Vivo T Cell Stimulation in the Pancreatic Tumor: A Method to Increase Cytokine Production in T Cells

Source: Spear, S., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/60622/" target="_blank">Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity</a>. J. Vis. Exp.(2019).
This article describes the technique for ex vivo T cell stimulation to increase the intracellular concentration of cytokines such as interferon-gamma or IFN-gamma and tumor necrosis factor-alpha or TNF-alpha in the T cell. These cytokines&#160; mediate an effective anti-tumor response and can have a therapeutic role in the tumor.&#160;

胰腺肿瘤中的离体 T 细胞刺激:一种增加 T 细胞中细胞因子产生的方法

Source: Spear, S., et al. Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity. J. Vis. ...