Name: t cells capture bacteria by transinfection from dendritic cells
Uploaded: 2016-01-13T13:00:00.000+00:00
Duration: 11 min 39 s
Description: Watch this Scientific Journal Video about T Cells Capture Bacteria by Transinfection from Dendritic Cells at JoVE.com

This work was supported by grants BFU2011-29450, BFU2008-04342/BMC from the Spanish Ministry of Science and Innovation and PIES201020I046 from Consejo Superior de Investigaciones Cient&#236;ficas (CSIC).

注:实验过程批准了委员会的马德里自治大学的研究伦理和动物福利与健康的马德里自治大学负责人的监督下，按照西班牙和欧洲的准则进行的。小鼠饲养在无特定病原（SPF）的住房，他们用二氧化碳培训并合格的人员（CO 2）吸入法实施安乐死。 1.鼠标骨髓来源的DC分化和感染注意: 图1总结了该第一步骤。所有过程应进行在从该点上的引擎盖，仅使用无菌培养基，仪器，枪头和培养皿。 <ol><li>小鼠骨髓来源的DC分化<ol><li>解剖胫骨和股骨从一个C57 / BL6小鼠22和转移到的塑料培养皿用10ml RPMI培养基。 </l我&gt; <li>互砍骨骺关闭，用无菌剪刀和揭露骨髓，其中有一个特色鲜明的红色。 </li><li>注入的骨的内部与上使用无菌的Petri无菌注射器10 ml的磷酸盐缓冲盐水的（PBS）中含有0.5％牛血清白蛋白（BSA）和5mM乙二胺四乙酸（EDTA）（PBS / BSA / EDTA缓冲液）菜。 </li><li>收集细胞悬浮液，并离心在一个管中于600×g离心在冷冻离心机（4℃）。 </li><li>将细胞重悬于1毫升铵-氯化物钾（ACK）裂解缓冲液（0.15M的氯化铵 ，10毫KHCO 3和0.1mM EDTA）中，为了消除红血细胞温育30秒，室温。 </li><li>加入10 mL的RPMI 1640培养基中含10％胎牛血清（FBS），并过滤通过细胞过滤网（70微米）离心以相同的速度（600×g离心），因为团块通过滤波消除之前去除骨碎片和细胞团块。 </li><li>细胞计数的d调整至5×10 5个细胞/毫升的RPMI 10％FBS（含50μM的β-巯基乙醇，1mM丙酮酸钠），并添加20纳克/毫升的重组鼠粒细胞-巨噬细胞集落刺激因子（RM-GM-CSF）作为终浓度。 </li><li>添加该悬浮液到无菌的，微生物学品质15厘米培养皿，并培养在 CO 2培养箱中（37℃，5％的CO 2）。 </li><li>每三天，降速两个非贴壁细胞和分离的细胞用5mM EDTA的PBS重悬在细胞密度为5×10 5个细胞/ ml，用含有20纳克/毫升RM-GM-CSF的新鲜培养基中。 </li></ol></li><li>的DC和抗原负载成熟<ol><li>在9天，重悬细胞在培养基含有20纳克/毫升RM-GM-CSF的5×10 5细胞/密度毫升。然后加入20纳克/毫升脂多糖（LPS），以允许高的MHC-II表达的细胞上，并培育在非组织培养处理过的无菌的15厘米的培养皿24小时。 </li><li>一前一后对LPS刺激的一天，染色一些DC细胞的抗鼠的CD11c，MHCII和Gr-1通过流式细胞仪检查的DC分化。流式细胞DC的分析的一个例子示于图3A和3B。 <ol><li>孵育树突（5×10 5个细胞/样品）用抗小鼠CD16 / CD32的单克隆抗体的终浓度在50μlPBS / BSA / EDTA每个样品缓冲液2.5微克/毫升。之后，加入50微升的抗体的抗MHCII（IA / IE）的CD11c和Gr-1在1偶联有不同荧光:100,1:200和1:500分别在PBS / BSA / EDTA缓冲液。 </li><li>最后，洗净树突用PBS / BSA / EDTA缓冲液中，并根据制造商的协议通过流式细胞术分析。 注意:如果所述细胞是分化良好，细胞准备用于抗原负载。 </li></ol></li><li>洗涤的DCs用RPMI 10％FBS（无抗生素）中，用10微克/毫升OTII OVAp（OVA 323-339孵育它们; ISQAVHAAHAEINEAGR）上，在37ºC在1ml每5×10 6个树突状最小1小时RMPI 10％FBS培养基的塑料管。作为阴性对照，留下的DC而不OVAp孵化。 </li></ol></li><li> DC的感染<ol><li> 在 CO 2培养箱中感染的DC在感染复数为10的细菌（每直流10细菌）1小时（MOI）（37℃，5％CO 2）。 </li><li>洗涤直流细胞3×PBS中和1x在RMPI 10％FBS和离心机在450×g离心以洗掉大部分胞外细菌。最后，重悬细胞RMPI 10％FBS的培养基（无抗生素），在20×10 6个细胞/ ml。 </li></ol></li></ol>从OTII转基因小鼠2.隔离CD4 + T淋巴细胞注意: 图1总结了该第二步骤。淋巴结应当被用来代替脾脏分离CD4 + T细胞，因为外周血CD4 +淋巴细胞在淋巴结秒（〜50％）比在脾（〜25％），因此纯化会更有效放大。 <ol><li>让单细胞悬液淋巴结<ol><li>删除腹股沟，腋窝，上臂，颈和肠系膜淋巴结23 OTII转基因小鼠，并转移到一个塑料菜10 RPMI 10％FBS培养基中。 </li><li>在使用两个磨砂显微镜载玻片无菌罩研磨淋巴结。放置在一个显微镜载玻片的磨砂侧淋巴结，擦第二张幻灯片，直到机关已接地的磨砂面。 </li><li>洗单细胞悬浮液在PBS / BSA / EDTA缓冲液。 </li><li>过滤该细胞虽然细胞过滤网（70微米）以除去结缔组织和洗涤用PBS / BSA / EDTA缓冲液。 </li><li>将细胞重悬于1毫升ACK裂解缓冲液和为了消除红血细胞下室温温育1分钟。 </li></ol></li><li> CD4 + T细胞的分离<ol><li>如步骤2.1.3和计数再次清洗细胞重悬在它们在PBS / BSA / EDTA缓冲液100×10 6个细胞/ ml。 30分钟250在冰上购买负选择CD4 + T细胞的:添加生物素化的抗CD8，IgM抗体，B220，CD19，MHC II类（Ⅰ抗体），细胞CD11b，CD11c的和DX5 1。 </li><li>在PBS / BSA / EDTA缓冲液中洗涤细胞，并培育的细胞（100×10 6个细胞/ ml）与链亲和素微珠在推荐在生产商的方案，在冰上15分钟的浓度。 </li><li>在PBS / BSA / EDTA缓冲液中洗涤细胞，并筛选它们虽然之前的 CD4 + T细胞的分离细胞过滤网（30微米）。 </li><li>通过使用磁性细胞分离机根据生产商的方案负向选择隔离的CD4 + T细胞。 </li><li>计数细胞，并调整到4×10 6个细胞/毫升的RPMI 10％FBS的培养基（无抗生素）。 </li><li>修复并保持在冰上分离的CD4 + T细胞 （3×10 5个细胞），以检查纯度，流动流式细胞仪所描述的24。的CD4 + T细胞的纯化的实例示于图3C。 </li></ol></li></ol> 3. T细胞Transinfection测量庆大霉素保护测定注意: 图2总结了协议的这个第三步骤。 <ol><li> T细胞Transinfection <ol><li>孵育分离的CD4 + T细胞与受感染DC的细胞（1:1）（所述的DC感染1小时并洗涤以消除游离的细菌）处理30分钟，以允许在 CO 2培养箱中（37℃，免疫突触的形成，5％ 的 CO 2）。 </li><li>添加0.5分离CD4 + T细胞（2×10 6个细胞）和0.1感染的DC（2×10 6个细胞）上的24孔培养板的毫升毫升。作为阴性对照，直接CD4 + T细胞的0.5 ml的感染在10细菌感染复数为30分钟。 </li><li>包括使用从T细胞分离感染的DC额外的控制聚碳酸酯的transwell屏障（有3微米孔径的），阻碍的DC-T物理接触。加入0.1毫升的transwell内的DC和0.5ml CD4 + T细胞的入井（24孔板）的下室中。 </li><li>最后，完成与RMPI中，直到为0.6毫升每口井，使等量的所有样本。 </li><li>免疫突触形成30分钟后，添加庆大霉素100微克/毫升与在 CO 2培养箱中收集细胞前孵育1小时（37℃，5％CO 2）。 </li></ol></li><li>重隔离的 CD4 + T细胞的<ol><li>收集非粘附细胞，重悬它们在PBS / BSA / EDTA缓冲液。大多数的DC仍然附着于塑料培养板。轻轻涡管，以确保细胞分解。保持500微升细胞上清液作为对照，表明庆大霉素一直运作良好。 </li><li>从包含DC和T细胞一起在步骤2.2样品再分离CD4 + T细胞虽然大多数的DC的应安装在该板上。置于冰上对照样品。仅考虑小于2％的污染实验。的transinfection后CD4 + T细胞纯化的一例示于图3D。 </li></ol></li><li>裂解性T细胞和种子它们对细菌培养基琼脂板。 <ol><li>用PBS洗两次CD4 + T细胞。 </li><li>计数细胞，并重悬它们以2×10 6个细胞/ PBS中毫升。 </li><li>加入500微升0.1％的Triton X-100的在500μl的T细胞以裂解它们以1×10 6个细胞/ ml，释放细胞内细菌出T细胞。 </li><li>使裂解的细胞的3串行十倍稀释和种子将50μl每种稀释液对细菌培养基琼脂平板上。分割板分成4部分和种子裂解的细胞的不同稀释度的每一个部分。代表性的结果示于图4。 <ol><li>作为对照，还对板在琼脂上的缀合物的储存细胞上清液，以确保庆大霉素运作良好。另外板块感染的DC作为额外的控制。万一低的DC有污染发生（&lt;2％），减去对应于低直流污染菌落形成单位（CFU的）。 </li></ol></li></ol></li></ol> 4.定量流式细胞术检测T细胞Transinfection 注意: 图2总结了这一步骤。 <ol><li> T细胞Transinfection <ol><li>感染的BMDCs如在步骤1.3，但使用表达GFP的细菌在这种情况下。 </li><li>染色CD4 + T细胞（分离自淋巴结在步骤2.2）用细胞追踪氯甲基氨基香豆素（CMAC）30分钟，在37ºC根据制造商的协议。 </li><li>用RMPI 10％FBS（无抗生素）洗两次标记的CD4 + T细胞。 </li><li>孵育感染的DC（后1小时培养的细菌）与标记的CD4 +T细胞30分钟，以允许免疫突触的形成，在37ºC包括所有说明步骤3.1.1控制。 </li></ol></li><li>感染的CD4 + T细胞染色<ol><li>直流-T的免疫突触的形成在30分钟后，收集细胞和在PBS / BSA / EDTA清洗。 </li><li>之后洗涤两次用PBS / BSA / EDTA缓冲液，分离凝集细胞轻轻涡旋管中。 </li><li>固定的样品在0.2ml / PBS含3％PFA中15分钟，在室温的管中。然后把它们洗干净的PBS。 </li><li>孵育抗小鼠CD16 / CD32单克隆抗体（2.5微克/毫升）的PBS / BSA / EDTA为50μl/样品15分钟在冰上阻断树突状细胞的Fc受体。 </li><li>在50μl/ PBS / BSA / EDTA的样品添加兔抗细菌抗体（10μg/ ml的作为最终浓度）处理30分钟在冰上染色细胞外细菌。 </li><li>用PBS / BSA / EDTA洗涤细胞并孵育抗小鼠的CD11c抗体偶联有藻红蛋白（PE）和一个本身抗兔condary抗体偶联有其它荧光像的Alexa-647氟在1:PBS / BSA / EDTA对在冰上30分钟，200在100μl/样品。 </li><li>洗涤细胞并通过流式细胞仪分析样品。不要忘记包括使用非荧光的细菌作为阴性对照和样品标记有在实验中使用，以补偿样品和调整仪设置25的流中的每个荧光染料的只是一个transinfected T细胞的样品。代表性的分析结果列于图5。 注意:如果细菌不表达GFP，以区分细胞内外的细菌，标记胞菌，修复和透化的样品用Triton X-100，在0.5％在PBS中5分钟。随后染色细菌总数（内+外）用不同的荧光染料。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentamicin+antibacterial+activity+in+the+presence+of+human+polymorphonuclear+leukocytes.">Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes.</a> Antimicrobial Agents and Chemotherapy. 16 (6), 743-749 (1979).</li><li>Zhang, X., Goncalves, R., Mosser, D. M., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+14,+The+isolation+and+characterization+of+murine+macrophages.">Chapter 14, The isolation and characterization of murine macrophages.</a> Current protocols in immunology. , Unit 14.1 (2008).</li><li>Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+th17+differentiation+of+na&#239;ve+CD4+T+lymphocytes.">Isolation and th17 differentiation of na&#239;ve CD4 T lymphocytes.</a> Journal of Visualized Experiments: JoVE. (79), e50765 (2013).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> Journal of Visualized Experiments: JoVE. (41), (2010).</li><li>Foucar, K., Chen, I. M., Crago, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+operation+of+a+flow+cytometric+immunophenotyping+laboratory.">Organization and operation of a flow cytometric immunophenotyping laboratory.</a> Seminars in diagnostic pathology. 6 (1), 13-36 (1989).</li><li>Itano, A. A., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+to+naive+CD4+T+cells+in+the+lymph+node.">Antigen presentation to naive CD4 T cells in the lymph node.</a> Nature Immunology. 4 (8), 733-739 (2003).</li></ol>

The authors have nothing to disclose.

T细胞或T淋巴细胞是一种类型的白细胞中发挥在细胞介导的免疫中发挥中心作用，并属于适应性免疫反应26。 T细胞是难治被感染在体外 ，但一些报道表明，它们可以感染体内 14,15。 APC和T细胞的过程中免疫突触的紧密接触作为交换生物材料 13，包括某些病毒如HIV 11的平台。据最近表明，违背了教条，T细胞，适应性免疫细胞的范例，也能够有效地捕获细菌通过transinfection从感染的DC 11,16。在这里我们展示他的详细方案，以量化的T细胞细菌transinfection 在体外感染的BMDCs。 T细胞细菌transinfection是由抗原识别16增强。我们发现，通过使用CD4 + T细胞分离自OTII转基因小鼠识别特定OVAp，DC培养上清装载OVAp和感染不同的细菌（这里的数据是由肠道沙门氏菌介绍）小鼠。 CD4 + T细胞从OTII小鼠分离孵育与受感染的DC 30分钟，以允许免疫突触的形成。这里给出的数据对应于T细胞可以从发达国家在这短短的时间段捕获细菌。我们知道，T细胞再暴露于感染的DC导致更大的细菌捕获（未示出），但由于T细胞也破坏了具有摄取细菌非常快，在较长的DC / T细胞时的接触发生的细菌transinfection定量证明是困难。庆大霉素存活测定法是一种非常灵敏的方法来定量T细胞transinfection因为它能够检测一个单侵染细菌21。孵化与庆大霉素之后，刚刚杀死细菌外，T细胞分离并溶解到种子上的细菌培养基琼脂PL阿泰。 T细胞的形成缀合物与树突后隔离是在协议中的一个关键步骤。仅与污染​​物的小于2％的实验，应考虑在内。 DC的污染可以通过流式细胞仪和相应的低直流污染的CFU应减去进行测量。可替代地，CD4 + T细胞的纯度可以通过使用细胞分选得到改善。 另一种方法来定量T细胞transinfection是通过流式细胞术，使用表达GFP的细菌和与相容的荧光团标记的胞外细菌。这种方法的一个限制是，细菌有以表达绿色荧光蛋白是足够明亮的反对自体荧光背景检测感染的细胞。另外，细菌感染可能与树突状细胞跟踪器之前被染色。另一种方法是染色细胞外的细菌，透化的T细胞，然后标记所有细菌（细胞内+胞外）用不同的荧光染料。 关于这个协议的未来发展方向，我们正在试图建立一个协议，以量化T细胞捕获较长的论述后细菌感染的DC，使用细菌饰有磁性粒子的数量。 T细胞捕获细菌将保留磁性粒子，因此它应该很容易通过使用磁铁孤立他们。

当病原体感染其宿主，通常的先天和适应性免疫反应所必需的细菌清除的活化。先天性免疫是防御的第一道防线，防止大多数感染。先天免疫区分以精确的方式元件被间微生物大类保守的（病原体相关分子模式）1。先天免疫的机制包括物理障碍如皮肤，化学品的障碍（抗菌肽，溶菌酶）和先天的白细胞，包括吞噬细胞（巨噬细胞，中性粒细胞和树突状细胞），肥大细胞，嗜酸性粒细胞，嗜碱性粒细胞，和天然杀伤细胞2。这些细胞识别和消灭病原体，无论是通过接触或通过细胞吞噬功能，其中包括病原体吞噬和杀害攻击他们。该系统不允许终身防守，而相比之下，适应性免疫，从而赋予针对p免疫记忆athogens。适应性免疫系统是防御的第二行，并且能够识别和反应以多种微生物和非微生物物质3的特定抗原。适应性免疫系统的主要组成部分是淋巴细胞，它包括B和T细胞。 B细胞参与了体液应答，分泌抗病原体或外源蛋白质。然而，T细胞代表了细胞介导的免疫力，调节细胞因子分泌或杀死病原体感染的细胞4的免疫应答。 抗原呈递细胞（APC），包括树突状细胞或巨噬细胞，先天免疫系统的组分，可以识别吞噬病原体和过程细菌成分成抗原，其由主要组织相容性复合物（MHC）5-7呈现在细胞表面上。后的APC已吞噬的病原体，它们通常迁移到引流淋巴结，在那里它们以T相互作用细胞。 T淋巴细胞可以通过它们的T细胞受体识别特异性的肽-MHC复合物。免疫突触（IS）发生在负载抗原的APC和淋巴细胞之间的界面中的抗原呈递8,9。有些细菌可以生存的吞噬功能，并在装甲运兵车系统传播。这种观点认为，受感染的APC作为细菌的水库或"特洛伊木马"，以促进细菌的蔓延10。装甲运兵车和采取的是在形成过程中发生淋巴细胞之间的亲密接触也可作为一个平台，交换膜，遗传物质和外来体的一部分，可以被劫持用于某些病毒感染的T细胞;这个过程被称为transinfection 11-13 一些病原性细菌（ 李斯特菌， 沙门氏菌和志贺氏菌 ）能够侵入T淋巴细胞在体内和修改其行为14-16。我们有最近描述了T淋巴细胞也能抗原呈递16的过程中捕获的细菌通过从以前感染树突细胞（DC）transinfection。 T细胞细菌捕获由transinfection极其比直接感染更有效（1,000-4,000x）。 T细胞捕获病原体和非病原菌显示比transinfection是由T细胞驱动的过程。引人注目的是，transinfected T（TIT）细胞迅速杀死细菌捕获和这样做的效率比专业的吞噬细胞16。这些结果，这打破免疫学的教条，表明适应性免疫的细胞可以执行是假想排他性的先天免疫的功能。此外，我们发现，乳头细胞分泌大量的促炎细胞因子和体内的细菌感染保护。 这里，我们提出用于研究细菌transinfection过程中的不同协议小鼠模型。该模型是基于使用CD4 + T细胞的转基因小鼠OTII，其中承担特异性的TCR的OVA（OVAp）的肽323-339中的I-17抗体的相互作用是特异性地与细菌感染的骨的上下文marrow-衍生的DC（DC培养上清）18,19装有OVAp，形成稳定的免疫突触。 T细胞transinfection可以可视化，并使用荧光显微镜进行跟踪。此外，流式细胞仪可用于通过取由细菌表达绿色荧光蛋白（GFP）16,20发出的荧光的优点检测感染的细胞。此外，T细胞transinfection可以通过更灵敏的方法，庆大霉素存活测定法，允许大量事件的测量来定量。庆大霉素是一种不能渗透的真核细胞的抗生素。因此，使用这种抗生素可以胞内细菌的分化幸存抗生素除了FROM外那些被打死21。

<table><tbody><tr><td>RPMI</td><td>Fisher Scientific</td><td>SH3025501</td><td></td></tr><tr><td>r-GMCSF</td><td>Peprotech</td><td>315-03</td><td></td></tr><tr><td>LPS</td><td>SIGMA</td><td>L2630-10mg</td><td></td></tr><tr><td>Na Pyruvate</td><td>Thermo Scientific</td><td>SH3023901</td><td></td></tr><tr><td>2-ME</td><td>Gibco</td><td>31350-010</td><td></td></tr><tr><td>OVAp OTII (323&amp;ndash;339)</td><td>GenScript</td><td></td><td></td></tr><tr><td>Cell Strainer 70uM</td><td>BD</td><td>352350</td><td></td></tr><tr><td>&amp;nbsp;30 uM Syringe Filcons Sterile</td><td>BD</td><td>340598</td><td></td></tr><tr><td>AutoMacs Classic</td><td>Miltenyi Biotec</td><td>130-088-887</td><td></td></tr><tr><td>Gentamicin</td><td>Normon</td><td>624601.6</td><td></td></tr><tr><td>Transwell</td><td>Costar</td><td>3415</td><td></td></tr><tr><td>LB</td><td>Pronadisa</td><td>1231</td><td></td></tr><tr><td>Agar</td><td>Pronadisa</td><td>1800</td><td></td></tr><tr><td>Paraformaldehyde 16%</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td></td><td></td><td></td></tr><tr><td>CD8 biot</td><td>BD Biosciences</td><td>553029</td><td></td></tr><tr><td>IgM Biot</td><td>ImmunoStep</td><td>Clone RMM-1</td><td></td></tr><tr><td>B220 Biot</td><td>BD Biosciences</td><td>553086</td><td></td></tr><tr><td>CD19 biot</td><td>BD Biosciences</td><td>553784</td><td></td></tr><tr><td>MHC-II Biot (I-A/I-E)</td><td>BD Biosciences</td><td>553622</td><td></td></tr><tr><td>CD11b biot</td><td>Immunostep</td><td>11BB-01mg</td><td></td></tr><tr><td>CD11c biot</td><td>Immunostep</td><td>11CB3-01mg</td><td></td></tr><tr><td>DX5 biot</td><td>BD Biosciences</td><td>553856</td><td></td></tr><tr><td>Gr-1 biot</td><td>BD Biosciences</td><td>553125</td><td></td></tr><tr><td>CD16/CD32</td><td>ImmunoStep</td><td>M16PU-05MG</td><td></td></tr><tr><td>anti Salmonella</td><td>ABD Serotec</td><td>8209-4006</td><td></td></tr><tr><td>CD11cPE</td><td>BD Biosciences</td><td>553802</td><td></td></tr><tr><td>CD4-APC</td><td>Tonbo Biosciences</td><td>20-0041-U100</td><td></td></tr><tr><td>Gr-1 APC</td><td>BD Biosciences</td><td>553129</td><td></td></tr><tr><td>MHC-II (I-A/I-E) FITC</td><td>BD Biosciences</td><td>553623</td><td></td></tr><tr><td>Alexa-Fluor 647 Goat Anti-Rabbit IgG (H+L) Antibody, highly cross-adsorbed</td><td>Invitrogen</td><td>A-21245</td><td></td></tr><tr><td>CMAC (7-amino-4-chloromethylcoumarin)</td><td>Life technologies</td><td>C2110</td><td></td></tr><tr><td>BSA</td><td>SIGMA</td><td>A7030-100G</td><td></td></tr><tr><td>Streptavidin MicroBeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td></td></tr><tr><td>BD FACSCanto II</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

t cells capture bacteria by transinfection from dendritic cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

本文我们描述了如何从感染的骨髓衍生-DC和如何通过两种不同的方法来测量细菌transinfection执行小鼠T细胞细菌transinfection:流式细胞仪和庆大霉素存活测定法图1总结的步骤，以获得细胞。通过骨髓细胞与GM-CSF连续9天的孵化产生的DC。随后，区议会熟化与LPS以增加它的膜MHCII与OVA（OVAp）的特定的肽加载​​它们以后。作为对照，有些区议会没装OVAp。二者的DC（OVAp加载的和非加载）感染在感染复数为10的细菌（MOI）。另一方面，CD4 + T细胞是从OTII的转基因小鼠，其识别特定OVAp的淋巴结中分离;图2示出了执行量化和T细胞transinfection包括合适的阴性对照的步骤。作为descri睡前，有些区议会没装OVAp。在这种情况下，存在的DCs和T细胞，但不是免疫突触的形成之间的相互作用由于缺乏OVAp的。一个附加的控制包括，与聚碳酸酯的transwell屏障（有3微米孔径的），阻碍的DC-T物理接触分离DC和T细胞。域控制器都包含在里面的透孔;因此它们不能够穿过该孔径。另外一个不利的控制也纳入，直接T细胞的感染，直接在10细菌MOI培养的T细胞。 T细胞transinfection可以量化由两种方法:流式细胞仪或庆大霉素存活测定法。为了通过流量测量仪，DCS应已预先用细菌-GFP和CD4 + T细胞感染的染色用细胞追踪器（T细胞-蓝）。后缀合物的形成，细胞应染色抗体的DC（CD11c的-PE）。胞外细菌也应以quantif染色ÿ感染的CD4 + T细胞通过流式细胞术以后的百分比。为了测量transinfection庆大霉素存活测定法，细胞与庆大霉素1小时杀死胞外细菌，则T细胞被再分离到种子上琼脂平板上。小数连续稀释制成种子50微升每种稀释液上琼脂平板和便利的CFU的数量。 如图3，DC分化已被通过流式细胞术（图3A和3B）检查。议会应表达的CD11c和MHCII（图3A），而不是的Gr-1，这是嗜中性粒细胞的标志物。如示于图3B中，一些嗜中性粒细胞（的的Gr-1 +，MHC 3.27％ - ）被呈现在培养，但培养的DCs与嗜中性白细胞污染的5％以上，不应使用。 CD4 + T细胞的纯度应在分离后从LY进行检查英里节点（图3C），并从树突分离后缀合物的形成（图3D）。考虑到实验后缀合物形成的污染物小于2％。 图4显示了使用沙门氏菌 Ťtransinfection实验的一个例子。 如图4A所示，板被分为四个部分的种子在每一个部分。 沙门氏菌的连续稀释都生长在LB琼脂平板和菌落形成单位进行计数。 如图4A所示 ，沙门氏菌，从感染的DC捕获由T细胞和此过程明显增强用OVAp识别（上图4A和图4B的左侧板）。然而，当有一个物理屏障阻碍DC和T细胞之间的接触，基本上没有transinfection，如在操作的一个直接T的情况下<html>细胞感染（上图4A和图4B的右板）。 如图5，T细胞transinfection可以通过流动进行定量仪利用细菌表达明亮的GFP。 DC和T细胞可以按大小和复杂性在向前和侧向散射图中有区别，但不同的标记物可以用来很好区分。 的 CD4 + T细胞用细胞追踪器（CMAC）和DC用针对CD11c的抗体。胞外细菌用针对沙门氏菌和第二抗体抗兔兔抗体缀合的Alexa Fluor 647因此，受感染的 CD4 + T细胞的百分比可以量化，因为它们被标以CMAC（未用表面CD11c-PE）和表达绿色荧光蛋白（细菌-GFP），但他们并没有用针对外菌（的Alexa Fluor 647）的抗体。 ntent"FO:保持together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/52976/52976fig1.jpg" /> 直流发生和 CD4 + T 细胞的分离 的图1流程图 。在该图的左手侧，则处理区分骨髓细胞的树突状细胞（DC）是详细。解剖股骨和胫骨后，骨髓细胞进行提取，从骨骼内部。随后，红血细胞（RBC）裂解和骨髓细胞是用重组鼠粒细胞 - 巨噬细胞集落刺激因子（GM-CSF）的9天培养。一旦被分化良好，树突一起保温脂多糖（LPS），以增加主要组织相容性复合物II（MHCII）的表达。然后，树突加载与特定卵白蛋白的肽（OVAp）和感染的感染复数为10的细菌（MOI）。它们中的一些不装载OVAp作为对照。在图的右手侧，如何隔离CD4 + T细胞从OTII转基因小鼠被表示。淋巴结（逻辑节点）是除去，地面，从而获得单细胞悬浮液。最后，CD4 + T细胞是通过使用磁性细胞分离机的负筛选分离得到。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52976/52976fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52976/52976fig2.jpg" /> 图2流程图的T细胞transinfection过程。感染（细菌在绿色描绘）和OVAp加载-DCS（红细胞）孵育CD4 + T细胞（蓝色细胞），以允许免疫突触的形成。感染的DC（但不装载OVAp）也一起孵育的T细胞，这使得这两个单元之间，但没有免疫突触的形成的相互作用。一个额外的控制包括，分离CD4 + </SUP&gt; T细胞和树突状与聚碳酸酯的transwell屏障（+ TW），这阻碍的DC-T细胞接触。直接T细胞的感染也纳入为阴性对照。最后，T细胞transinfection可以通过两种方法进行定量，通过流式细胞术（图的左侧），这表明T细胞捕获的细菌的数量，并通过庆大霉素存活测定法（图右侧的）一个非常敏感的方法（可以检测到一个感染细菌），表明细菌感染率。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52976/52976fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52976/52976fig3.jpg" /> 图3.流式细胞仪骨髓-DC和分离的CD4 + T细胞的分析 。代表性点阵图骨髓-DC的由分析流式细胞仪。 （A）中的DCs表达MHCII和的CD11c（85.8％;右上象限）（B）中的DCs表达MHCII但不是的Gr-1（85％;右下象限）。但是，有细胞中表达的Gr-1，但不MHCII细胞（左上部分）的3.27％。这些细胞轻微污染的中性粒细胞。 （C和D）占CD4 + T细胞从淋巴结（C）中分离出纯度直方图，并结合形成以树突状细胞（D）后， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52976/52976fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52976/52976fig4.jpg" /> 图4. T细胞transinfection庆大霉素存活试验量化。 LB琼脂平板分成4部分相当于Decima的裂解CD4 + T细胞的升连续稀释。 （A）的菌落形成沙门氏菌为单位在LB琼脂平板生长相应于T细胞transinfection在存在（+）或不存在（ - ）OVAp的感染的DC的表面上，并在条件允许的DC / T细胞接触（ - TW），或在一个物理屏障阻碍这种接触（+ TW）的存在。还示出对应于直接的T细胞感染（阴性对照）的空盘。曾几何时，DC / T细胞接触受到阻碍（​​+ TW）几乎没有transinfection。 （B）中从几个实验示出的细菌通过T细胞感染的DC中捕获的速率量化的菌落形成单位（CFU的）。直接T细胞的感染和条件阻碍DC / T细胞接触产生很少或几乎没有级别的T细胞的感染。当被允许的DC / T细胞的接触，有T细胞transinfection，并且它是由抗原识别（+ OVAp）增强。柱条代表的3 I的平均ndependent实验。误差棒表示标准差。显著差异是由***表示，对应P &lt;0.0001。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52976/52976fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52976/52976fig5.jpg" /> 图5. T细胞transinfection定量流式细胞仪。代表散点图显示树突状细胞和T细胞的结合物transinfection过程之后。 CD4 + T细胞是小而圆的前向和侧向散射（FCS - SSC）斑点印迹显示，并为了很好地分化，它们进行染色用细胞追踪器（CMAC）。区议会表达的CD11c凡是不注CMAC，并于T CD4 + T细胞形状不规则较大。含有胞内细菌-GFP但阴性extracel感染的CD4 + T细胞细胞性细菌（6.19％），显示在右下象限。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52976/52976fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about T Cells Capture Bacteria by Transinfection from Dendritic Cells at JoVE.com

T Cells Capture Bacteria by Transinfection from Dendritic Cells

这里的协议，提出通过该期间抗原呈递发生经由transinfection从预感染树突状细胞（DC）的CD4 + T细胞以测量细菌捕获。我们说明如何执行必要的步骤:原代细胞中，DC，DC / T细胞共轭形成感染和细菌的T细胞转染的测量隔离。

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

t-cells-capture-bacteria-by-transinfection-from-dendritic-cells

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

11.8K 次观看.  Spanish National Biotechnology Centre (CNB-CSIC). 这里的协议，提出通过该期间抗原呈递发生经由transinfection从预感染树突状细胞（DC）的CD4 + T细胞以测量细菌捕获。我们说明如何执行必要的步骤:原代细胞中，DC，DC / T细胞共轭形成感染和细菌的T细胞转染的测量隔离。 

视频: T Cells Capture Bacteria by Transinfection from Dendritic Cells

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

科研

免疫与感染

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

四环素调节细胞株产生高滴度的慢病毒载体，专门针对树突状细胞

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.

We outline a 9-day protocol for the transduction and expansion of rhesus macaque peripheral blood mononuclear cells which yields cells with excellent co-expression of the genes of interest in sufficient number for infusion studies of cell efficacy.

在保持中央记忆表型的九天内，原T细胞的转导与扩展

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting ...

生物工程

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

性T细胞Transinfection从树突状细胞捕捉细菌

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