支持由NIH R01 CA121258，R01 CA125244，R01 CA154256，NIH / NCATS UCLA CTSI授权号UL1TR000124，USAMRMC W81XWH-08-1-0734，和琼·S·霍姆斯纪念研究基金的一部分。 MJH和GCO接到琼S.福尔摩斯纪念博士后奖学金，在加州大学洛杉矶分校的支持。 CSM的装置是由创新科学方法获得：www.creative-sci.com。慢病毒载体是从加州大学洛杉矶分校矢量的核心，这是支持CURE / P30 DK041301收到。

1.将准备<ol><li>将细胞沉淀歧管滑入杀菌袋和密封用高压灭菌胶带。面对滑板袋的纸侧，以避免在井中塑料沉积物的特氟隆涂层的一面。 </li><li>高压釜袋15分钟，在121℃。 </li><li>在无菌的150×15毫米的无菌培养皿中取出的幻灯片从生物安全柜和地点内杀菌袋。多达四个幻灯片每盘可以容纳。广场旁边的幻灯片35×10毫米的培养皿。加入2-3毫升无菌H 2澳提供加湿。 </li><li>预涂载玻片用聚-D-赖氨酸，在100微克/毫升用足够体积，以覆盖整个井。 1小时，在室温之后，吸出溶液，并通过移液的1×PBS在孔的表面冲洗井两次的表面上。 </li><li> （可选）添加纤维连接蛋白或其他ECM组件，以确保更强的肿瘤细胞的粘附。添加纤维连接蛋白在5微克/毫升足够量塔吨的井不会时间在RT在短时间内干燥。 <ol><li> 1小时后，吸剩的解决方案，并通过上下吹打与1X PBS洗两次澡。 </li></ol></li><li>保持PBS的井，直到肿瘤准备电镀。使用滑动的同一天。 </li><li>收获的所需粘附的肿瘤细胞类型的汇合烧瓶中。在适当缓冲的生长培养基，例如计数通过光学显微镜和重悬使用台盼蓝染料排斥的活细胞以5×10 6个细胞/ ml，Dulbecco氏最小基本培养基（DMEM）中，用10％胎牛血清（FBS），L-谷氨酰胺和丙酮酸钠。 </li><li>如果粘附细胞群的荧光成像是期望的，以一个重要的荧光染料或细胞增殖的染料由以下由制造商提供的协议标签的粘附细胞群。 </li><li>顺利吸取10微升完全培养基到滑动照顾的每个孔中，以避免在WEL产生气泡LS，因为这些可以预防均匀肿瘤细胞的粘附。 </li><li>添加2.5-5.0×10 4细胞（5-10微升的细胞悬浮液），以在各孔中（ 图1A）的介质。调整取决于肿瘤细胞类型和生长的天所需的数目的最佳数目。较大的肿瘤细胞或快速成长的细胞可能需要较少的细胞开始。通过加入培养基和吸管上下，以确保均匀分布的细胞带来良好体积多达40微升。 </li><li>毕竟井种子，使肿瘤细胞附着在RT，然后将盖体上的150×15毫米培养皿中并小心地将皿在37℃/ 5％CO 2的加湿培养箱中在至少24小时。 </li><li>每日更换培养基。小心吸取从单层废介质的一部分，并替换为新鲜的完全培养基。 注意：由于蒸发，更容积将需要比取出要添加。井将准备测定时为偶数，confluent层细胞的存在。这通常是初始播种后1-3天。 </li><li>如步骤1.4中所述并继续到步骤2，如果单层需要的话，或以下面的步骤1.13从单层提取ECM蛋白用PBS洗单层轻轻一次。 </li><li>使0.5％的Triton-X（V / V）在完全培养基中的溶液，并添加30微升至每个孔中。让在引擎盖单层消化2分钟，然后用完全培养基通过移液如上所述洗涤两次。 注：细胞外基质层可以是通过光学显微镜（ 图2）可见。使用幻灯片马上，或保持在完全培养基中的加湿的CO 2培养箱中培养1-2天。不要让水井干涸。 </li></ol> 2.沉淀非附着性T细胞上的滑动<ol><li>如果所述非粘附细胞群的荧光成像是期望的，标记的非粘附细胞群与重要的荧光染料，如CFSE，细胞追踪红CMPTX或eFluor670按照预先测定的由制造商提供的至少1小时的协议。替代地，操纵细胞以表达由病毒载体编码的荧光蛋白质。 </li><li>如上面1.2中描述的高压釜的细胞沉淀歧管在高压釜中袋。 </li><li>删除包含从孵化器和地方生物安全柜Teflon涂层幻灯片加湿室。 </li><li>移液器向上洗井用1×PBS和向下，然后加入45微升完全培养基中含有至少10％的血清。 </li><li>从消毒信封移除歧管，小心地将在过孔中，直到“钩”在歧管的端部接触的滑动（ 图1B）的底部。 </li><li>确保该培养基是在每一个信道的可见。如果没有看到，起飞歧管，并添加更多的媒体到对应得很好，然后更换并再次检查。 </li><li>每个陈重复步骤2.5歧管的NEL，或尽可能多的水井所需。 </li><li>计数非粘附细胞，并重新悬浮于不低于1.0-2.0×10 5个/微升。制定1微升的细胞并缓慢吸取他们进入通道（ 图1C）。做到这一点的每口井所需。 </li><li>离开，整个电池装载装置中， 即 ，歧管和滑动，生物罩内20-30分钟以使细胞在通道沉降。 </li><li>通过触摸两端并轻轻提起它垂直向上从滑动，以便不打扰灶性接种于孔中（ 图1D）的中心的细胞移除歧管。 </li><li>检查各孔，以确保非粘附细胞已经成​​功地接种在井停留在歧管通道的圆周内的中心。非粘附细胞将理想地稍微粘附在单层或ECM和对方，因此轻轻移动滑动周围不会导致细胞PELLE吨驱散。不要挤的幻灯片。 </li></ol> 3.荧光显微镜<ol><li>通过适当的实验设备，进行拍摄沉淀被确认之后。理想情况下，使用配有CO 2的湿度室中的显微镜。要采取更大的面积的图像在低功率， 即 ，4X或10X，以允许多个图像，使得井的整体可以看出来。 </li><li>获得使用图像捕捉软件的数字图像。执行明和/或多通道荧光成像。 </li><li>如果需要的话，设置了一个时间推移在给定区域，以记录不同频道的图像，或保持在滑动加湿，并在37℃/ 5％CO 2培养箱并手动取出到图像在所需的时间点（ 图1E＆F，建议在较长的时间点）。 </li></ol> 4.分析和显示<ol><li>使用图像编辑软件，合并轻，FLuorescent图​​像转化成1层，使多种细胞类型和标记物可以看出，在相同的图像。拖放图像文件为每个单独的颜色到程序中，出现提示时，选择“添加图层”选项来创建具有多个图层的图像文件。 <ol><li>在更高的放大倍率，拿，对齐和缝合跨越以及图像组合在一起数字。通过观察两个图像之间的保守的区域，并覆盖在另一个的顶部的一个图像，直到产生一个完整的画面对准的文件。 </li></ol></li><li>使用采集软件来确定距离单个细胞迁移收购过程中添加微米酒吧的图像。 </li><li>以确定在不同时间的细胞的迁移，使表面强度荧光地图与图像软件， 即 ImageJ的，量化荧光T细胞聚集或分布在时间。 <ol><li>要生成表面图，采取步骤4.1和图层窗口下产生分层图像，取消除对应​​所需的通道之外的所有图层。例如，如果一个面积希望可视EMD表达，只有“绿色”对应于用于该标记的过滤层应该是可见的。 </li><li>拼合图像并将其保存在识别的文件格式（ 例如 ，.png格式），并加载图像到程序中。要生成曲面图，改变图像类型为8位，根据需要减少背景更改亮度/对比度，并选择“分析/曲面图”选项。通过检查“阴影”，“绘制轴”，“一个多边形每行&#39;和&#39;平滑”复选框中生成图5中的图像。 </li><li>可替代地，采取利用从焦点细胞存半径或直径的测量，在时间零，并比较所述的细胞在最外点在不同的时间。特氟隆孔的直径为6.0毫米。 </li></ol></li></ol>

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The authors declare they have no competing financial interests.

肿瘤细胞中的单个细胞悬浮液移液到特氟隆掩模滑动的孔中。使细胞附着，然后在湿润的5％CO 2，37℃培养箱（ 图1A），形成单分子膜。从单层衍生既定单层或ECM蛋白可收获这些测定（ 图1B）。标记有重要的荧光染料或转导的载体编码的EMD效应T淋巴细胞接种到使用CSM阱的中央。然后非粘附效应T淋巴细胞的迁移上的单层或ECM蛋白随时间的能力进行了评估和量化的光?...

径向单层细胞迁移测定法最初开发到载玻片上涂覆有细胞外基质（ECM）蛋白5-7或具有单个ECM组分，如纤连蛋白或层粘连蛋白1,2-测量粘附的肿瘤细胞1-4的浸润特性。该技术涉及接种肿瘤细胞的单细胞悬浮液在使用不锈钢细胞沉降支管（CSM）的孔的中心。沉淀后，将肿瘤细胞将附着在井和在初始细胞群随时间的直径的改变的底物用于建立横向运动的速率。径向单层细胞迁移测定法提供了一种视觉优势所采用的transwell板，以测定细胞的体外迁徙功能等现有的方法;这些测定法是非利于成像8。同时，它也是在选择时间点所提供的自由量很大■当迁移评估，对时间点的数量没有限制的研究员能沉淀后，选择图像。 因为迁移的能力是对非贴壁细胞的一个重要的功能，特别是在免疫治疗或者它们可以用作递送载体为病毒载体领域，我们适于使用CSM来评价非粘附细胞的迁移类型对肿瘤细胞单层，除了ECM蛋白。的显微镜可视化上存活的肿瘤细胞单层非粘附细胞的迁移，在复杂的ECM从肿瘤，或单个ECM组分分离的额外的好处，使该测定的通用性。雇用孔涂有单一胞外蛋白分析不准确地反映ECM组织基质或肿瘤的细胞将通过迁移体内 。 这里，我们使用同种异体细胞毒性T淋巴细胞（alloCTL），敏感地认识到主要histocomp使用单向混合淋巴细胞肿瘤细胞反应（MLTR）或混合淋巴细胞反应（MLR）9，作为我们的代表性非粘附细胞类型atibility复合体（MHC）蛋白。我们测试了人和鼠源的细胞。当迁移测定肿瘤单层，采用的肿瘤细胞或者部分相关指标，显示一些对用于敏感的效应，还是完全相关的目标，拥有全套MHC分子的细胞群发现了同样的MHC蛋白质的效应已被致敏对。在一些实验中，我们使用了荧光CellTracker红色CMPTX或细胞增殖染料eFluor 670效应细胞和靶细胞之间进行区分。我们还使用转用病毒载体编码荧光蛋白作为可视化细胞的另一种方式。对于某些分析，我们转了alloCTL与逆转录病毒的复制载体（RRV）编码翡翠绿（EMD）荧光蛋白10,11;对于超视距器，肿瘤细胞被转导的慢病毒载体编码mStrawberry。 所述alloCTL被通过歧管的一个通道进入任一肿瘤细胞单层或ECM从肿瘤细胞单层收获的中心接种。粘附和非粘附细胞相互作用显现由光和/或通过在一段时间的荧光显微镜。中断在肿瘤细胞单层在低功率，或肿瘤细胞具有片段化的细胞核在高功率是细胞损伤的由裂解和细胞凋亡的指标，分别。我们创建数字表面强度荧光地图显示非粘附荧光T细胞在单层培养的迁移。我们还注意到毒性集群的形成叠加的非贴壁alloCTL之后主义及其对贴壁胶质瘤细胞单层。同时，观察RRV-EMD从alloCTL到胶质瘤单层水平传导。

<table border="0" cellpadding="0" cellspacing="0" width="920">
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			<td height="22" style="height:22px;width:312px;">Petri Dish, 150mm</td>
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			<td height="22" style="height:22px;width:312px;">20 &#956;L Pipetman</td>
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			<td style="width:239px;">F123600</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L Pipetman</td>
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			<td style="width:239px;">F123601</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L pipette tips</td>
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			<td style="width:239px;">89003-060</td>
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			<td style="width:239px;">25-054-CL</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:312px;">TrypLE Express (optional)</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:239px;">12604</td>
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			<td height="22" style="height:22px;width:312px;">Tumor cell culture media (e.g. DMEM)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">10-013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">AIM-V serum-free media</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">12055-083</td>
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			<td height="22" style="height:22px;width:312px;">Fetal Bovine Serum</td>
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			<td style="width:203px;"></td>
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			<td style="width:203px;">Millipore</td>
			<td style="width:239px;">A-003-E</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fibronectin</td>
			<td style="width:203px;">BD</td>
			<td style="width:239px;">354008</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">31/2 x 51/4 Autoclave Pouches</td>
			<td style="width:203px;">Crosstex</td>
			<td style="width:239px;">SCXS2</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypan Blue</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-900-CI</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Cell Proliferation eFluor 670</td>
			<td style="width:203px;">eBioscience</td>
			<td style="width:239px;">65-0840-85</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">ImageJ</td>
			<td style="width:203px;">NIH</td>
			<td style="width:239px;"></td>
			<td></td>
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radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive t lymphocytes

我们报告的径向单层细胞迁移测定法的一个新颖的适应，首次报道以测量粘附的肿瘤细胞的细胞外基质蛋白的径向迁移，用于测量荧光标记，非粘附人或鼠效应免疫细胞的运动性。这种技术采用一个不锈钢歧管和10以及特氟隆滑动灶性沉积非贴壁的T细胞变成制备与任一汇合的肿瘤细胞单层或细胞外基质蛋白的孔中。光和/或多信道荧光显微镜被用于随时间跟踪效应细胞的移动和行为。荧光染料和/或荧光的转基因编码用于差异标记的细胞类型进行成像的病毒载体。这种方法是从类似型的体外测定该跟踪水平或垂直的迁移/侵袭利用滑动室，琼脂或Transwell小板明显。该法允许详细的影像数据，以BË与特定的荧光标记区分不同类型的细胞收集;即使特定的细胞亚群（ 即 ，转导/ nontransduced）可以被监控。使用对应于所述转移的小区类型特定的荧光通道中产生的表面强度的荧光曲线。这允许在特定时间更好的可视化的非粘附免疫细胞迁移的。因此能够收集的其他效应细胞的功能，如细胞毒性或从效应器病毒载体的转移证据到靶细胞，以及。因此，该方法允许研究人员用显微镜记录差异标记的，非粘附的细胞 - 细胞间的相互作用与各种类型的贴壁细胞。这样的信息可以是在生物-操纵或激活免疫细胞类型，其中的功能性的视觉证据期望与肿瘤靶细胞将其用于癌症治疗前的评估特别相关。

对于荧光蛋白编码病毒载体可以除了，或代替，荧光染料可以使用。病毒转导应提前运动检测来进行。既粘附和非粘附的细胞类型可以是差异标记。的协议转导将取决于载体的使用的类型。在这里，我们转导在图3，图5和图6与RRV-EMD至少两天的alloCTL提前使用协议测定的描述12。我们还使用的慢病毒载体，CMV-草莓-IRES-FLUC2，转导的肿瘤细胞以表达mStrawberry红色荧光蛋?...

Watch this Scientific Journal Video about Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes at JoVE.com

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

径向移动性和逆转录病毒复制的载体转导，非粘附Alloresponsive T细胞的杀伤功能

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

radial-mobility-cytotoxic-function-retroviral-replicating-vector

Research

JoVE Journal

Immunology and Infection

7.7K 次观看.  UCLA David Geffen School of Medicine. We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality. 

视频: Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

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 ...

科研

免疫与感染

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...