Name: isolation and ex vivo culture of vδ1+cd4+γδ t cells, an extrathymic αβt-cell progenitor
Uploaded: 2015-12-07T15:00:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Isolation and Ex Vivo Culture of VÃŽÂ´1+CD4+ÃŽÂ³ÃŽÂ´ T Cells, an Extrathymic ÃŽÂ±ÃŽÂ²T-cell Progenitor at JoVE.com

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>

<ol>
	<li>Bhandoola, A., von, B. H., Petrie, H. T., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commitment+and+developmental+potential+of+extrathymic+and+intrathymic+T+cell+precursors:+plenty+to+choose+from.">Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from.</a> Immunity. 26 (6), 678-689 (2007).</li><li>Von, B. H., Melchers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Checkpoints+in+lymphocyte+development+and+autoimmune+disease.">Checkpoints in lymphocyte development and autoimmune disease.</a> Nat. Immunol. 11 (1), 14-20 (2010).</li><li>Prinz, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+earliest+steps+of+gammadelta+T+cell+development+in+the+adult+thymus.">Visualization of the earliest steps of gammadelta T cell development in the adult thymus.</a> Nat. Immunol. 7 (9), 995-1003 (2006).</li><li>Ferrero, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCRgamma+silencing+during+alphabeta+T+cell+development+depends+upon+pre-TCR-induced+proliferation.">TCRgamma silencing during alphabeta T cell development depends upon pre-TCR-induced proliferation.</a> J. Immunol. 177 (9), 6038-6043 (2006).</li><li>Krangel, M. S., Carabana, J., Abbarategui, I., Schlimgen, R., Hawwari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enforcing+order+within+a+complex+locus:+current+perspectives+on+the+control+of+V(D)J+recombination+at+the+murine+T-cell+receptor+alpha/delta+locus.">Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus.</a> Immunol. Rev. 200, 224-232 (2004).</li><li>Hawwari, A., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+rearranged+variable+gene+segments+in+directing+secondary+T+cell+receptor+alpha+recombination.">Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (3), 903-907 (2007).</li><li>Hawwari, A., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+TCR+delta+and+alpha+repertoires+by+local+and+long-distance+control+of+variable+gene+segment+chromatin+structure.">Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.</a> J. Exp. Med. 202 (4), 467-472 (2005).</li><li>Hawwari, A., Bock, C., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cell+receptor+alpha+gene+assembly+by+a+complex+hierarchy+of+germline+Jalpha+promoters.">Regulation of T cell receptor alpha gene assembly by a complex hierarchy of germline Jalpha promoters.</a> Nat. Immunol. 6 (5), 481-489 (2005).</li><li>Hager, E., Hawwari, A., Matsuda, J. L., Krangel, M. S., Gapin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+constraints+at+the+level+of+TCRalpha+rearrangement+impact+Valpha14i+NKT+cell+development.">Multiple constraints at the level of TCRalpha rearrangement impact Valpha14i NKT cell development.</a> J. Immunol. 179 (4), 2228-2234 (2007).</li><li>Linton, P. J., Dorshkind, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+lymphocyte+development+and+function.">Age-related changes in lymphocyte development and function.</a> Nat. Immunol. 5 (2), 133-139 (2004).</li><li>Sprent, J., Tough, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+life-span+and+memory.">Lymphocyte life-span and memory.</a> Science. 265 (5177), 1395-1400 (1994).</li><li>Guy-Grand, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrathymic+T+cell+lymphopoiesis:+ontogeny+and+contribution+to+gut+intraepithelial+lymphocytes+in+athymic+and+euthymic+mice.">Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice.</a> J. Exp. Med. 197 (3), 333-341 (2003).</li><li>McClory, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+stepwise+program+of+extrathymic+T+cell+development+within+the+human+tonsil.">Evidence for a stepwise program of extrathymic T cell development within the human tonsil.</a> J. Clin. Invest. 122 (4), 1403-1415 (2012).</li><li>Jbakhsh-Jones, S., Jerabek, L., Weissman, I. L., Strober, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrathymic+maturation+of+alpha+beta+T+cells+from+hemopoietic+stem+cells.">Extrathymic maturation of alpha beta T cells from hemopoietic stem cells.</a> J. Immunol. 155 (7), 3338-3344 (1995).</li><li>Garcia-Ojeda, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+development+of+committed+progenitors+in+the+bone+marrow+that+generate+functional+T+cells+in+the+absence+of+the+thymus.">Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus.</a> J. Immunol. 175 (7), 4363-4373 (2005).</li><li>Arcangeli, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrathymic+hemopoietic+progenitors+committed+to+T+cell+differentiation+in+the+adult+mouse.">Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse.</a> J. Immunol. 174 (4), 1980-1988 (2005).</li><li>Maillard, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch-dependent+T-lineage+commitment+occurs+at+extrathymic+sites+following+bone+marrow+transplantation.">Notch-dependent T-lineage commitment occurs at extrathymic sites following bone marrow transplantation.</a> Blood. 107 (9), 3511-3519 (2006).</li><li>Ziegler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Peripheral+CD4(+)+Vdelta1(+)+gammadeltaT+Cells+Can+Develop+into+alphabetaT+Cells.">Human Peripheral CD4(+) Vdelta1(+) gammadeltaT Cells Can Develop into alphabetaT Cells.</a> Front Immunol. 5, 645 (2014).</li><li>Mollet, M., Godoy-Silva, R., Berdugo, C., Chalmers, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+simulations+of+the+energy+dissipation+rate+in+a+fluorescence-activated+cell+sorter:+Implications+to+cells.">Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells.</a> Biotechnol Bioeng. 100 (2), 260-272 (2008).</li></ol>

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。细胞用荧...

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><tbody><tr><td>Biocoll Solution</td><td>Biochrom</td><td>L 6113</td><td>lymphocyte separating solution</td></tr><tr><td>Lysing Buffer</td><td>BD BioSciences</td><td>555899</td><td>lysis of erythrocytes&amp;nbsp;</td></tr><tr><td>Phosphate-buffered Saline</td><td>Sigma Aldrich</td><td>D8537&amp;nbsp;</td><td></td></tr><tr><td>MACS buffer</td><td>Miltenyi Biotec</td><td>130-091-222</td><td>supplement with BSA and pre-cool before use</td></tr><tr><td>BSA</td><td>Miltenyi Biotec</td><td>130-091-376</td><td>not mandatorily from this supplier</td></tr><tr><td>anti-human Vd1 FITC (clone:&amp;nbsp; TS8.2)</td><td>Thermo Scientific</td><td>TCR2730</td><td>not mandatorily from this supplier</td></tr><tr><td>anti-human CD3 PerCP (clone: SK7)</td><td>BD BioSciences</td><td>345766</td><td>not mandatorily from this supplier or this flurochrome</td></tr><tr><td>anti-human TCRab PE (clone: T10B9.1A-31)</td><td>BD BioSciences</td><td>555548</td><td>not mandatorily from this supplier or this flurochrome</td></tr><tr><td>anti-human CD4 VioBlue (clone: M-T466)&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-097-333</td><td>not mandatorily from this supplier or this flurochrome</td></tr><tr><td>anti-human CD8 APC-H7 (clone: SK1)</td><td>BD BioSciences</td><td>641400</td><td>not mandatorily from this supplier or this flurochrome</td></tr><tr><td>Anti-FITC MultiSort Kit</td><td>Miltenyi Biotec</td><td>130-058-701</td><td>yields better results than anti-FITC MicroBeads</td></tr><tr><td>MS columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td>pre-cool before use</td></tr><tr><td>MiniMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>CD4 Positive Isolation Kit</td><td>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 VÃŽÂ´1+CD4+ÃŽÂ³ÃŽÂ´ T Cells, an Extrathymic ÃŽÂ±ÃŽÂ²T-cell Progenitor at JoVE.com

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#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 Vδ1+CD4+γδ T Cells, an Extrathymic αβT-cell Progenitor

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

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 ...

免疫与感染

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&#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 ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

来自分离人产品的γ-Delta（γδ）T细胞的扩增和富集

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

癌症研究

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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 ...

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). 
The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).
The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

通过使用来自辐照的Buffy外套的饲养细胞的T淋巴细胞的分离和培养来研究主动脉瓣钙化

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...

<meta charset="utf8"></head><body>用于检测 T 细胞中 Ca2+ 微结构域的高分辨率成像技术

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

<meta charset="utf8"></head><body>作者聚焦:揭示治疗干预的适应性免疫反应的初始激活

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

原代 T 细胞中初始 Ca2+ 微结构域的成像

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

分离和

摘要

探索更多视频

此视频中的章节

胸腺表征祖定居在小鼠胚胎使用

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

隔离和幼稚的CD4 T细胞的Th17细胞分化

来自分离人产品的γ-Delta（γδ）T细胞的扩增和富集

鼠标朴素CD4

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

通过使用来自辐照的Buffy外套的饲养细胞的T淋巴细胞的分离和培养来研究主动脉瓣钙化

原代 T 细胞中初始 Ca²⁺ 微结构域的成像

原代 T 细胞中初始 Ca²⁺ 微结构域的成像

原代 T 细胞中初始 Ca²⁺ 微结构域的成像