Christian Welker is funded by a grant provided by the J&#252;rgen-Manchot-Stiftung.

伦理声明：所有的程序进行，根据赫尔辛基宣言，并批准了临床伦理委员会，在图宾根大学（项目38 / 2009B02和470 / 2013B02）。 1.分离外周血单个核细胞（PBMC的） <ol><li>取50-100毫升来自健康志愿者通过静脉穿刺使用50毫升注射器含有1000 IU的肝素硫酸盐和稀释血液1:2用PBS（pH值= 7.2）。 </li><li>仔细层35毫升的血：15毫升血液中分离溶液的PBS溶液如Biocoll（D = 1.077克/毫升）的50毫升锥形管中。离心15分钟，在800×g离心在RT。 注：制动不能使用。 </li><li>收集淋巴细胞层用吸管的细胞，并在至少50毫升的PBS洗细胞两次（离心12分钟;在400×g离心）。离心用吸管后，取出上清液。 </li><li>裂解通过暂停和incub剩下的红细胞和使用本淋巴细胞馏分1-5毫升为2-4分钟低渗缓冲溶液。 注：曝光和裂解缓冲液的体积的持续时间取决于所使用的裂解缓冲液和对残留在淋巴细胞部分的红细胞数量。 </li><li>稀释细胞用PBS（1:10）和离心机在250×g离心12分钟，随后重悬，并在300×g离心10毫升PBS洗涤细胞沉淀一次12分钟。 </li><li>重悬在PBS的淋巴细胞和使用台盼蓝溶液细胞计数的Neubauer计数室。 注意：一般，&gt; 1×10 8淋巴细胞从100 ml的接收新鲜抽取的外周血。 </li></ol> 2.隔离Vδ1性T细胞<ol><li>取&lt;1×10 6个分离的淋巴细胞用于初始FACS确定Vδ1CD4 T细胞的频率。 注：此T-细胞实体的人口尺寸可以有很大的不同个体，并根据其免疫状态之间。 Typic同盟，约1％的T细胞是Vδ1+和大约1-5％Vδ1+细胞是CD4 +。 <ol><li>稀释细胞用1ml FACS缓冲液，它由含2％FCS和5mM EDTA的PBS中。离心在660×g离心2分钟，倒出上清液。约100微升FACS缓冲液的回流体积足以用于随后的染色。简要地涡旋，并培育的细胞与5微升的Fc封闭试剂5分钟在RT的FACS染色的增加特异性。 </li><li>直接向细胞中添加人单克隆抗体不除去的Fc封闭试剂。确保以染色细胞抗Vδ1（1:50），CD3（1:50），CD4（1：200），CD8（1：200）和TCRαβ（一点25）。制备同种型对照与对应的免疫球蛋白同种型。在黑暗中孵育细胞与抗体一起进行20分钟，在4℃。 </li><li>在1毫升流式细胞仪缓冲洗涤细胞（离心2分钟，在660 XG），弃去supernatant并着手FACS收购剩余的缓冲区容量。 </li></ol></li><li>造粒的细胞（未在FACS分析中使用）离心12分钟;在300克; 8°C。除去上清液和重悬细胞pelletin每1×10 7个细胞加入60μlMACS缓冲液。添加每1×10 7个细胞20微升的Fc封闭试剂孵育5分钟，在4℃。 </li><li>添加10微升FITC缀合的抗人Vδ1每1×10 7个细胞抗体直接细胞中。在黑暗中孵育12分钟，在4℃。 </li><li>洗用7-14 ml的MACS缓冲液中的细胞（离心12分钟;在300克; 8℃）。完全弃上清，悬浮细胞每7×10单元80微升MACS缓冲。 </li><li>取1-2×10 5细胞并稀释它们在100μlFACS缓冲液用于验证通过FACS分析的标记。都需要这个步骤没有进一步的添加剂。确保有一个suffi在亮度Vδ1cient差异-和Vδ1+细胞 。如果不是这种情况下，重复步骤2.3）和2.4）。 </li><li>每1×10 7个细胞加入20微升抗FITC微珠到剩余的细胞搅匀，孵育在黑暗中15分钟，在4℃。此后，洗涤用至少10 ml的MACS缓冲液中的细胞（离心12分钟，在300×g离心8℃）。 </li><li>在离心分离，达到平衡的预冷却的磁列放置在用500μlMACS缓冲液中的磁铁。 </li><li>将细胞重悬于500μl的MACS缓冲液（用于细胞数高于1×10 8，扩大这个体积相应地），并小心地应用该细胞到柱上。收集流过的含Vδ1 -细胞。 </li><li>用500μlMACS缓冲液洗柱三次，在同一管再次收集流过。需要注意的是水库必须将缓冲到列前空。 </li><li>从磁性装置取出柱，并将其放置入15ml锥形管中。很快就被牢牢地推动活塞入列冲洗用1毫升MACS缓冲柱。收集管中的洗脱液。 注意：为了获得&gt; 98％的纯度，通常需要重复步骤2.7-2.9）与第二列。 </li><li>验证细胞的纯度通过FACS分析18具有相同的抗体面板之前（见2.1.2），并计数细胞。 </li></ol> 3.隔离Vδ1CD4+ T细胞<ol><li>重悬25微升的CD4珠用涡旋为&gt; 30秒。通过将围成一个磁铁1分钟在1.5ml反应管洗在1ml MACS缓冲液珠（如所描绘由生产的最低量）。小心弃去上清液用小规模的吸移管（200微升），重悬珠子在MACS缓冲原始卷。 </li><li>造粒Vδ1+细胞（离心12分钟;在300×g离心），并吸出上清，重悬所有Vδ1+细胞在500μlMACS缓冲液并将其添加到含有珠的管中。混合大力孵育20分钟，在4℃的恒定倾斜（例如，在旋转体）。 </li><li>放置含有细胞中的磁铁，2分钟的管。确保有在盖子没有残余。 注：CD4 +细胞将已绑定磁珠并连接到面对磁体的管的一侧。 </li><li>用一个小规模的吸管Vδ1+细胞-仔细打开盖子，同时保持该管的磁装置内，并收集包含CD4上清液。将CD4 - Vδ1+细胞到一个单独的管中，并把该管插入磁体再次以避免可能来自人口剩余的CD4细胞或珠。 <ol><li>一，在5毫升MACS缓冲细胞（300 XG离心12分钟） -洗CD4第二重悬它们在每100微升媒体1×10 5个细胞的浓度。的CD4 -细胞可以在下面（4·2）给出的条件下可以容易地培养。 </li></ol></li><li>放置管与磁场以外的CD4 +细胞的目标，重悬细胞在500μlMACS缓冲液，并把它们放回磁性装置。重复步骤3.3-3.5两次，以获得更高的纯度。通过移液除去上清液</li><li>重悬的CD4 +细胞在100μl培养基（RPMI 1640,10％FCS，1％L-谷氨酰胺，1％青霉素/链霉素），加入珠分离溶液的10微升。在室温下孵育45分钟，恒定的倾斜（例如，在一个旋转器）。 </li><li>放置细胞的磁铁1分钟。小心收集使用小型吸管含珠-自由Vδ1+ CD4 +细胞的上清液。 </li><li>磁铁之外，悬浮用100微升培养基和重复珠步骤3.6和3.7倍，以获得CD4 +细胞的更高的细胞数。 </li><li>造粒的细胞（离心，在300×g离心，12分钟），并通过移液完全丢弃上清液。悬浮细胞在新鲜，预热媒体和计数。检查Vδ1+ CD4 +细胞通过FACS分析在步骤2.1.1-2.1.3中描绘的纯度。 </li></ol> 4.单细胞克隆有限稀释<ol><li>隔离来自同种异体供体的外周血单核细胞（PBMC），为在1.1-1.6所示。 </li><li>用γ射线照射2.5×10 7个同种异体PBMC中与80戈瑞在25毫升培养基中。 </li><li>添加IL-2（200U / ml）的IL-7（20毫微克/毫升）和PHA（2微克/毫升），以照射饲养细胞和分发它们在96孔U字形板，5×10 4个饲养细胞以每孔50微升。 </li><li>稀释Vδ1+ CD4 +细胞以每50μl0.3个细胞的浓度。吸取德50微升的细胞溶液以每孔窝藏照射细胞和细胞因子。 注：因此，细胞因子稀释至100U / ml的IL-2，10纳克/毫升的IL-7和1微克/毫升PHA的最终浓度。孵育96孔板在培养箱在37℃，5％CO 2的潮湿气氛中。 <ol><li>可选，培育剩余的净化Vδ1+ CD4 +细胞批量培养相同的培养条件的克隆下。 </li></ol></li><li>供给细胞每3-4天用细胞因子和新鲜的培养基。对于这一点，在孔中加入50μl新鲜培养基通过移液培养基的交换一半。添加的细胞因子的2倍浓度，以每补充。加入每孔每隔一轮饲养的5×10 4照射的饲养层细胞。 </li><li>利用显微镜，观察第一克隆成为3-4周后可见的，因为它们代谢，从而改变培养基和形态菌落的颜色。 注：对于皮草疗法培养，悬浮克隆井并将它们传送到单独的96孔板中。如2.1.1-2.1.3指示通过FACS分析细胞。克隆在这些条件下保持可能最终从Vδ1改变它们的TCR对αβTCR。此转分化的时间点，从克隆的克隆而异。 </li></ol>

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The authors have nothing to disclose.

研究一种稀缺（T-）细胞实体的表型，生物学和功能，即Vδ1+ CD4 + T细胞中，我们使用两个标记：Vδ1和CD4其正磁性细胞分离。 Vδ1是孤儿受体，而CD4表达于T辅助细胞，在单核细胞和树突状细胞一个较低的水平，并在对造血祖细胞非常低的水平。 对于细胞高纯度的浓缩和选择的技术包括荧光激活细胞分选（FACS），分隔细胞转化为基于荧光标记亚群的人口的技术。细胞用?...

在脊椎动物中，该构造中的细胞和免疫的体液部分适应性免疫起着抵御病原体是重要的作用。大范围的抗原的识别通过hyperpolymorphic T细胞和B细胞受体（TCR / BCR），这对于T细胞被假定为主要产生在胸腺1介导的。于此，造血干细胞（HSC），来源于骨髓，种子胸腺和分化沿良好定义阶段最后到所有T细胞谱系引起。胸腺播种祖细胞CD4 -和CD8 -从而构成不成熟，双阴性（DN）胸腺细胞部分。胸腺产生信号，然后诱使他们的后裔承诺，并分化成任何αβ或γδT细胞。在DN2 / 3胸腺功能性重排的TCR-γ和TCR-δ链基因的表达导致对γδTCR络合物，其中驱动细胞增殖的ð促进分化成γδT细胞2,3。与此相反，一官能的TCR-β链的重排，可以配对preTα构建preTCR PT，诱导DN3胸腺细胞的TCR的γ链的转录沉默和其过渡到的CD4 + CD8 +双阳性胸腺细胞4 。在这个阶段中，TCR-α链的重组发生，删除的TCR-δ基因座的TCR-α基因座内伫立，因而废除了生产γδTCR在这些细胞中不可撤销地5-9。重新排列αβTCRs随后选择其结合自MHC弱（阳性选择）的能力，这可能不超过一定的阈值，以避免自身免疫（阴性选择）。根据它们的结合MHC I类或II的容量，所选择的αβT细胞发育成的单阳性的 CD4 +或CD8 + T细胞，其出口胸腺作为幼稚T细胞。 然而，胸腺退化的生活导致幼稚T细胞的指数降低输出，几乎灭绝后青春期早期10开始。然而，T细胞池的大小保持整个生命，这只能在部分地由T细胞后胸腺稳态扩散和长寿命的免疫存储器 11中的扩散进行说明恒定。因此，胸腺外T细胞发育必须发生。最近的研究已经获得了大量的景点，其特征αβT细胞前体，其中，在胸腺外的网站，就产生了功能αβT细胞12-17。然而，关于胸腺外αβT细胞前体独立于胸腺分化成αβT细胞详细知识是作为局部的，我们有他们采取从而在路径上的背景。 我们最近发现Vδ1+小T细胞的实体</suP&gt; CD4 +γδT细胞作为胸腺外αβT细胞prognitor 18，它从健康的人供体的外周血中分离时，可以分化为αβT细胞有轻度炎症的环境。有趣的是，违反后胸腺T细胞的稳态增殖，Vδ1CD4 +细胞转分化产生新的T细胞受体，从而扩大了剧目的多样性，从 ​​而使潜在的新的抗原可以被识别，并且可以防止新收购的病原体的宿主。这增加了T细胞的可塑性，并增加了对胸腺外T细胞发育一个至今没有受到重视的新途径。 从淋巴细胞来源定量隔离，单细胞克隆的产生及其有效扩张是必不可少的，目标是鉴定那些触发此αβT细胞precursor`s胸腺外发育标记物和分子。

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biocoll Solution</td>
			<td style="width:93px;">Biochrom</td>
			<td style="width:119px;">L 6113</td>
			<td style="width:361px;">lymphocyte separating solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysing Buffer</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555899</td>
			<td>lysis of erythrocytes&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered Saline</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MACS buffer</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>supplement with BSA and pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human Vd1 FITC (clone:&#160; TS8.2)</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td>TCR2730</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD3 PerCP (clone: SK7)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>345766</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human TCRab PE (clone: T10B9.1A-31)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555548</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD4 VioBlue (clone: M-T466)&#160;</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-097-333</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD8 APC-H7 (clone: SK1)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>641400</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Anti-FITC MultiSort Kit</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-058-701</td>
			<td>yields better results than anti-FITC MicroBeads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MS columns</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td>pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MiniMACS Separator</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CD4 Positive Isolation Kit</td>
			<td style="width:93px;">life technologies</td>
			<td>11331D</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and ex vivo culture of v&#948;1+cd4+&#947;&#948; t cells, an extrathymic &#945;&#946;t-cell progenitor

胸腺，主机关αβT细胞和适应性免疫系统在脊椎动物的骨干的产生，长期以来被认为是αβT细胞的唯一来源。然而，胸腺衰退开始在生活中导致幼稚αβT细胞显着减少输出到外围月初。不过，甚至百岁老人可以增强免疫力对新收购的病原体。最近的研究表明，胸腺外αβT细胞发育，但我们途径的理解是可以补偿功能胸腺损失仍残留。 γδT细胞是构成该组织的主要T细胞亚群先天淋巴细胞。最近，我们通过展示一个冲高迄今赏识优秀功能于γδT细胞亚群的CD4的稀缺实体+Vδ1+γδT细胞能分化为αβT细胞在炎症条件。在这里，我们provide的协议这个祖细胞从外周血中分离和随后的培养。 Vδ1细胞是正从使用磁珠健康人供体的PBMC中富集，随后其中我们的目标CD4 +细胞与另一磁性标记技术的稀缺馏分的第二步骤。第二标记的磁力超过第一磁标签中的一个，并且因此允许感兴趣的人口的高效，定量和特定正隔离。然后我们引入所需克隆并有效地扩大了的细胞和用于识别所产生的克隆通过FACS分析的技术和培养条件。因此，我们提供了净化，文化和体外扩增的CD4 +Vδ1+γδT细胞的详细的协议。这方面的知识是前提，是涉及到这个αβT细胞progenitor`s生物学研究，并为那些谁的目标是IDEntify参与其转分化的分子触发器。

图1描述了不同的阶段和Vδ1T细胞的分离自外周血的结果。 图1A示出 Vδ1+细胞的典型分布的 CD3 +淋巴细胞，以及Vδ1+群体的共受体的表达。在该供体，Vδ1+细胞 （红色）的频率为总淋巴细胞计数的2.3％和Vδ1+淋巴细胞的CD4表达（绿色）为2.6％。总而言之，目标人群隔离代表总淋巴细胞在这个供体的0.06％。 图1B示出了一个...

Watch this Scientific Journal Video about Isolation and Ex Vivo Culture of VÎ´1+CD4+Î³Î´ T Cells, an Extrathymic Î±Î²T-cell Progenitor at JoVE.com

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

isolation-ex-vivo-culture-v1-cd4-t-cells-an-extrathymic-t-cell

Research

JoVE Journal

Immunology and Infection

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

9.2K 次观看.  Children's University Hospital Tübingen. Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral Vδ1+CD4+ T cells that is, as we showed recently, an extrathymic αβ T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

视频: Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

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

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

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

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

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

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

从肾脏双重否定αβ性T细胞的分离

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

该<em&gt;离体</em&gt;文化与模式识别小鼠肠道组织体受体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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 ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...