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><tbody><tr><td>Teflon-masked microscope slides</td><td>Creative Scientific Methods</td><td>CSM002</td><td></td></tr><tr><td>Cell Sedimentation Manifold</td><td>Creative Scientific Methods</td><td>CSM001</td><td></td></tr><tr><td>Petri Dish, 150mm</td><td>Corning</td><td>430597</td><td></td></tr><tr><td>Petri Dish, 35mm</td><td>Corning</td><td>430588</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Mediatech</td><td>21-040</td><td></td></tr><tr><td>20 &amp;mu;l Pipetman</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>200 &amp;mu;l Pipetman</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>200 &amp;mu;l pipette tips</td><td>VWR</td><td>89003-060</td><td></td></tr><tr><td>Distilled, deionized water (sterile)</td><td>Mediatech</td><td>25-055</td><td></td></tr><tr><td>Trypsin</td><td>Mediatech</td><td>25-054-CL</td><td></td></tr><tr><td>Celltracker Red CMPTX</td><td>Invitrogen</td><td>C34552</td><td></td></tr><tr><td>TrypLE Express (optional)</td><td>Gibco</td><td>12604</td><td></td></tr><tr><td>Tumor cell culture media (&lt;em&gt;e.g.&lt;/em&gt; DMEM)</td><td>Mediatech</td><td>10-013</td><td></td></tr><tr><td>AIM-V serum-free media</td><td>Invitrogen</td><td>12055-083</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Omega Scientific</td><td>FB-02</td><td></td></tr><tr><td>Inverted microscope</td><td></td><td></td><td></td></tr><tr><td>SPOT Advanced</td><td>Diagnostic Instruments</td><td></td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Millipore</td><td>A-003-E</td><td></td></tr><tr><td>Fibronectin</td><td>BD</td><td>354008</td><td></td></tr><tr><td>3&lt;sup&gt;1/2 &lt;/sup&gt;x 5&lt;sup&gt;1/4 &lt;/sup&gt;Autoclave Pouches</td><td>Crosstex</td><td>SCXS2</td><td></td></tr><tr><td>Trypan Blue</td><td>Mediatech</td><td>25-900-CI</td><td></td></tr><tr><td>Cell Proliferation eFluor 670</td><td>eBioscience</td><td>65-0840-85</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr></tbody></table>

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

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

瞬时转染293T细胞复制缺陷型逆转录病毒生产

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

生物学

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 ...

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 ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

简化的单细胞 TCR 分离和逆转录病毒载体的生成, 用于体外和体内表达人类 TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

CXCR4在Murine B-1a细胞上的逆转录病毒过度表达和靶向B-1a细胞向骨髓和IgM生产转移的采用转移

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

破译急性 LCMV 感染中的早期滤泡辅助性 T 细胞定型术

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

作者聚焦:阐明急性 LCMV 挑战中 TFH 细胞分化的途径

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...