这项工作是支持的补助基思L.克努森的苏珊科曼为治愈基金会和NIH / NCI 9R01 CA152045和P50-CA116201。

1。短期人力抗原特异性CD8 + T细胞线7代<ol><li>独立的外周血单个核细胞（PBMC）从正常健康HLA-A2阳性献血者使用聚蔗糖帕克加的血。 </li><li>对外周血单核细胞（PBMC）2毫升/加入到6孔板中以2×10 6细胞/ ml。 </li><li>添加HLA-A2结合的HER-2/neu基因P369-377肽（氨基酸序列KIFGSLAFL的）或HLA-A2结合的流感P58-66肽（GILGFVFTL）直接到各孔中，在终浓度为10微克/毫升和地点在37℃用5％CO 2的培养箱中。 </li><li>为了帮助刺激和扩大T细胞，添加，每2天，250微升/孔重组人IL-2（库存：50单位/μl）来实现的最终浓度为50单位/毫升培养基中的新鲜培养基。 </li><li>再次刺激培养细胞与2×10 6个细胞/毫升照射的自体PBMCs 10微克/毫升的相同的肽脉冲2小时，用D在步骤1.3和添加这些细胞以1ml /孔的第一肽刺激后一周的文化。 </li><li>添加人重组IL-2和新鲜的培养基，步骤1.4中所描述的，到现有的文化，每2天。 </li><li>重新刺激细胞如上述步骤1.5一个星期后的第二个肽刺激的自体PBM​​Cs肽和照射。 </li><li>准备使用6天之后，最后再刺激的最早的细胞。 </li></ol> 2。乳腺癌细胞的制备<ol><li>放置的的xCelligence阻抗测量站（站xCelligence）的培养箱中，在37℃，5％CO 2。 </li><li>收获的人类肿瘤细胞（SKBR3，BT20，MCF7或HCC1419），75％融合时，用0.25％胰蛋白酶和离心分离（1000转，5分钟）。 </li><li>肿瘤细胞计数，使用血球和重组在RPMI肿瘤介质（补充有10％胎牛血清（FBS）和500个单位/毫升IFN-γ）的浓度为7.5×10 3肿瘤cells/100的微升。 </li><li>计划的xCelligence软件通过设置布局页面来确定井布局和设置时间表页面阻抗读数40小时内每隔5分钟。 xCELLigence系统的最初18小时内，将采取的肿瘤细胞的阻抗读数。 </li><li>加入100微升的RPMI肿瘤媒体的xCelligence E-板和执行的背景下，通过测量阻抗的细胞在没有读取。 </li><li>添加到E-板的肿瘤细胞在每以及和地方在xCelligence站的100微升。 </li><li>按下启动按钮用xCELLigence软件开始服用的肿瘤细胞，立即开始坚持到E板的阻抗读数。 </li></ol> 3。 CD8 + T细胞共培养肿瘤<ol><li>一旦肿瘤细胞附着并倍增至1.5×10 4个肿瘤细胞，每孔（近似mately 18小时后收获T细胞最初被镀），第1节轻轻地刮取和搅拌</li><li>离心机以1,000 rpm 5分钟的计数使用血球和含10％FBS的RPMI培养基中的重组他们在不同的比率，从1肿瘤细胞进行最大比40 T细胞和T细胞的比例为稀释2倍，直到达到1肿瘤细胞的到1.25 T细胞的。例如，有1.5×10 4个肿瘤细胞，每孔。为了达到1:40的比例，添加每孔6×10 5个T细胞，并稀释2倍，直到T细胞的比例为1.5×10 4个肿瘤细胞，以1.875×10 4 T细胞，每孔（1:1.25的比例）的实现。 </li><li>暂停xCelligence站和删除的E盘。 </li><li>用移液管的孔中取出介质，并添加200μl的媒体单独或孔的T细胞的合适比例。 </li><li>将E-板背面在xCelligence站，并继续xCelligence软件Pro克阻抗读数采取T细胞除了约20小时后，每5分钟。 </li><li>规范化结束时的测定结果。 </li></ol> 4。铬释放实验（CRA）7 <ol><li>按照机构辐射安全程序工作时，与51 Cr和51 Cr标记的靶细胞。始终使用适当的屏蔽，以减少辐射暴露。 </li><li>脉冲肿瘤细胞2小时，在1×10 6个肿瘤细胞/毫升，10微升/毫升的新鲜的51 Cr（0.005次/毫升）。 </li><li>肿瘤细胞洗净，在10毫升的RPMI培养基含10％FBS，离心5分钟，以1,000 rpm。将肿瘤细胞在96 -孔圆形底板中的100μl/孔，并以1×10 5个肿瘤细胞。 </li><li>添加各种比例，1肿瘤细胞开始到第40的T细胞的T细胞，并稀释2倍，直到1 1.25 T细胞的肿瘤细胞的比率来实现的。例如，有1×10 5个肿瘤细胞<html>每口井。为了达到1:40的比例，每孔加入4×10 6个T细胞，并稀释2倍，直到T细胞的比例为1×10 5个肿瘤细胞，以1.25×10 5个T细胞，每孔（1:1.25的比例）的实现。 </li><li>添加自发的和最大的肿瘤细胞控制的板块。为了计算从肿瘤细胞自发释放的51 Cr，添加六口井每片含1×10 5个肿瘤细胞在100μl100μl的RPMI培养基含10％FBS。来计算最大释放的51 Cr从肿瘤细胞中，添加六口井每片含1×10 5个肿瘤细胞，在100微升至100微升0.1％的Triton X-100的PBS中，所有细胞裂解。 </li><li>用5％的CO 2，在37℃下孵育5小时的板。 </li><li>培养后，从每个孔中取出50微升上层清液，并把它添加到亮度板。 </li><li>干板过夜，使用γ计数器测量CPM。 </li></ol>乐“&gt; 5。裂解百分比计算<ol><li>阻抗测定：取细胞指数（CI）报道，在不同时间点的阻抗阅读，使用下面的公式来确定％溶解：（CI肿瘤 - （CI肿瘤的T细胞））/（CI肿瘤） * 100。 </li><li> CRA：使用下面的公式来确定％溶解：（CPM实验 - CPM自发）/（CPM的最大 - CPM自发）* 100。 </li></ol> 6。代表结果 T细胞本身不增加阻抗在一般情况下，T细胞，不坚持最坚实的表面，并不要求遵守活化和增殖。因此，有人推测，T细胞不应该有助于阻抗用xCELLigence E-板。为了测试这一点，井装载任何SKBR3乳腺癌细胞或新鲜纯化的人CD8 + T细胞。超过40小时的过程，一个代表性的例子演示如何测量阻抗图1A中所示的评估基本上是T细胞的影响不大。相反，它可以说，T细胞可能减少背景阻抗，虽然只是轻微。尽管如此，共培养后，增加T细胞在18小时后启动阻抗测量的过程中导致的阻抗中断（ 图1B）。其他的研究显示，中断是由于在确保的T细胞的溶液的温度，就可以减少因为缩减信号处理部分相同的孵化器内部。 由T细胞介导的​​阻抗减少是剂量依赖性的。 基于阻抗的测定样品的细胞毒性T细胞的活性进行比较的实用程序需要不同浓度的抗原特异性T细胞之间的分辨能力。为了测试是否这是可能的，人乳腺癌SKBR3细胞与不同浓度的共培养从HER-2/neu的P369特异性T细胞的T细胞衍生的蒸发散。如在图2A中所示，减少阻抗依赖于T细胞的剂量。 如图2B所示，在10小时的细胞指数T细胞的范围内按照一个简单的二阶多项式。因此，xCelligence站CD8 T细胞介导的​​死亡SKBR3肿瘤细胞指数下降实时监控。 的阻抗为基础的检测中检测到抗原特异性T细胞要确定是否由HER-2/neu的阻抗减少，这是一个HER-2/neu和HLA-A2阳性的乳腺癌肿瘤细胞系，人乳腺癌SKBR3 P369特异性T细胞抗原孵育特定的，独立的孔中SKBR3与来自流感（FLU） - 特异性T细胞系的T细胞。流感特定的T细胞作为一种非特异性的控制，HLA-A2阳性，但没有任何能够识别HER-2/neu的。如图中所示的网络连接3 GURE，有FLU-特异性T细胞（P &lt;0.0001）相比，是一个显着的区别HER-2/neu基因P369特异性T细胞的溶解活性。在某些情况下，不特定的肿瘤的T细胞（例如，FLU特定或anti-CD3/anti-CD28的刺激）溶解活性（ 图3A-B）展示了一个较低的水平。 T细胞可以杀死两种方式之一，无论是通过Fas非特异性：Fas配体的相互作用或抗原 - 特别是通过释放颗粒酶和穿孔素。为了进一步证实，HER-2/neu-specific T细胞在抗原特异性的方式杀死的概念，我们将阻塞的抗Fas配体抗体。 如图3B中所示，该抗体的HER-2/neu基因P369特异性T细胞的溶解活性并没有降低。该抗体能抑制Fas和Fas配体之间的相互作用，表现出与T细胞非特异性激活淋巴球珠。这些结果表明，当肿瘤特异性T细胞与非肿瘤特异性T细胞，Fas配体的封锁测试，可能需要在最低限度，以减少非T细胞受体介导的相互作用。可选地，当使用短期培养的T细胞线，其中只有一小部分的T细胞用于体外生成的特定的抗原，MHC不匹配介导的T细胞的活化的可能性可能发生的非特异性裂解。在这种情况下，除了不相关的HLA的抗体，阻止可能被担保。 ，以确定是否P369特异性T细胞，杀伤肿瘤细胞的余依赖性，无论是抗-HLA-I类抗体或同种型对照抗体加入到共培养中的HLA类。 图3C中所示，裂解由P369-特异性T细胞的抑制夹杂物的抗体展示HLA-I类限制。最后，由于使用SKBR3的发展主要是做这个实验，这是重要的，以确定是否其他附着HLA-A2和HER-2/neu表达的肿瘤细胞P369特异性T细胞可杀死。因此，制备的T细胞的HLA-A2和HER-2/neu-expressing肿瘤细胞，人乳腺癌SKBR3，MCF-7和HCC1419细胞以1:5的比例加入。 BT20的HLA-A2阴性的肿瘤细胞系，用作为阴性对照。所示图3D，所有这些额外的肿瘤细胞，除了BT20也被杀害了所评估的阻抗。因此，该方法似乎，多个目标粘附肿瘤细胞是有用的。 基于阻抗的测定是比标准铬释放测定在检测细胞毒性T细胞的活性更加敏感铬（51铬）释放法（CRA）的措施杀伤活性长期以来一直是黄金标准来监测CD8 + T细胞应答。在该试验中，用51 Cr标记的靶细胞（如肿瘤细胞）。在大多数情况下，健康的靶细胞可以保留大部分的在检测过程中的放射性标记。被添加到CD8 + T细胞的靶细胞中，通常会在不同的浓度，和杀死靶细胞检测到培养基中释放的51 Cr的 。除了事实，即它使用危险的辐射，与CRA的两个主要问题是缺乏敏感性的测定法通常需要大量的CD8 T细胞，限制了它的实用程序。要确定阻抗为基础的检测更敏感，比HER-2/neu的CRA，P369特异性T细胞在体外和应用产生，数额不等，任E板含有未标记的肿瘤细胞或标准的聚丙烯板51 Cr标记的靶细胞。的测量，阻抗或自由铬，测定在5个小时后，T细胞此外，一个代表性的例子， 图4示出的阻抗测定法优于铬释放。 批内变异阻抗为基础的检测通常是在20％以下。 批内的变化进行了检查，在此检测中，因为更广泛的使用，将在很大程度上取决于它的可重复而变化（ 图5）。独立生成两个P369特异性T细胞系，间隔30天，并一式三份的增加，在一个范围内的肿瘤细胞在T细胞的比率。 图5中的曲线图的插图示出的线在大致相当于在SKBR3细胞溶解。变异系数（％）在每个细胞的比率计算，并绘制。如在图5中示出，1:40和1:5比率％的变异系数（CV）为15％以下。然而，在1:2.5和1:1.25的CV高或不可预知的。由于广泛的生物学变异，它是不可能测量批间变异的主要的T细胞系。 <img src="/files/ftp_upload/3683/3683fig1.jpg" alt="图1"/> 图1。阻抗需要的另外的T细胞进行归一。 A组：表明，肿瘤细胞（红色线）和T细胞（蓝线）是负责的阻抗。阻抗描超过40小时为肿瘤或T细胞。从第二个实验中获得相似的结果。请注意，在约20个小时的信号扰动代表点的另外的T细胞不含有肿瘤细胞的孔，B组：示出阻抗测量的另外的T细胞对肿瘤细胞的瞬时中断。这需要归一化的另外的T细胞后，在某个时间点（参见图中标记点的归一化）。痕迹是由重复的数据点。肿瘤细胞本身的跟踪显示为红色。其他的痕迹代表肿瘤细胞共同培养与HER-2/neu基因P369特异性T细胞（蓝色：1.25 T细胞/肿瘤细胞，格林比：40个T细胞/肿瘤细胞）的比例。每个跟踪通过的持续时间的培养物收集每5分钟重复数据点计算。结果是代表3个独立的实验。 <a href="http://www.jove.com/files/ftp_upload/3683/3683fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/3683/3683fig2.jpg" alt="图2"/> 图2。减少由T细胞介导的阻抗是剂量依赖性的。 A组：显示的是单独或与不同浓度的肿瘤特异性T细胞的肿瘤细胞培养人乳腺癌SKBR3阻抗的痕迹。通过每5分钟收集培养的持续时间的一式三份的数据点计算出每个跟踪。结果有代表性的3个独立的实验B组：显示的是的细胞指数跨越所有的T细胞/肿瘤细胞浓度在10小时。而虚线表示的人肿瘤细胞的细胞指数为一个。每个符号是三次重​​复测定的平均值（±标准差）。结果是代表3个独立的实验。 <a href="http://www.jove.com/files/ftp_upload/3683/3683fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/3683/3683fig3.jpg" alt="图3"/> 图3。基于阻抗的测定法检测抗原特异性细胞毒性T细胞应答。面板A显示HER-2/neu的P369-特异性T细胞（红色）或共培养后的FLU-特异性T细胞（蓝色）5，10，和15小时的SKBR3细胞的裂解水平。每个数据点的平均（±SEM）一式三份测量。结果代表3个独立的实验面板B显示了人乳腺癌SKBR3 P369特异性T细胞或者非特异性激活的T细胞的裂解，证明该抗原特异性裂解是不是由于Fas的Fas配体的相互作用。黑条代表共同培养载有Fas配体抗体。每个长条的平均值（±标准差），在5小时后，加入T细胞一式三份的裂解。结果有代表性的3个独立的实验。 面板C显示了10小时后，T细胞SKBR3此外P369特异性T细胞的裂解是HLA-I类依赖。无论是HLA-I类阻塞或同型对照共同培养过程中加入。每个酒吧是在一式三份的平均（±SEM）裂解。实验重复2次，具有相似的结果。 图D显示了几个HLA-A2 +，HER-2/neu-expressing肿瘤细胞系P369特异性T细胞的T细胞加入10小时后的％裂解。 BT20，HLA-A2阴性乳腺癌细胞系作为阴性对照使用。从三次测量计算每个点代表一个独特的实验。 <a href="http://www.jove.com/files/ftp_upload/3683/3683fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img srC =“所有/ files/ftp_upload/3683/3683fig4.jpg”ALT =“图4”/&gt; 图4。的阻抗为基础的分析是比铬释放法检测肿瘤抗原特异性T细胞的检测更敏感。显示的是人乳腺癌SKBR3％溶解在不同浓度的P369-特异性T细胞，肿瘤细胞和T细胞后，在5小时内计量混合使用基于阻抗的测定法和铬释放测定。中各符号是三次重​​复的平均值（±标准差）。结果是代表3个独立的实验。 <img src="/files/ftp_upload/3683/3683fig5.jpg" alt="图5"/> 图5。 ％批内的变异系数的基于阻抗的T细胞的细胞毒性测定。显示的是平均值（±SD）％批内的变异系数在一个范围内的浓度的抗原特异性T细胞在人乳腺癌SKBR3溶解。每个符号是重复三次的平均值。图中显示的数字是t他的意思是％的CV。 插入面板显示的平均（±SEM，N = 3％）裂解SKBR3，P369-377特异性T细胞在不同浓度的。示两个独立产生的P369特异性T细胞系，使用两个单独的供者的T细胞的。

<ol>
	<li>Zhang, N., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8(+)+T+cells:+foot+soldiers+of+the+immune+system.">CD8(+) T cells: foot soldiers of the immune system.</a> Immunity. 35, 161-168 (2011).</li><li>Garboczi, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+complex+between+human+T-cell+receptor,+viral+peptide+and+HLA-A2.">Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.</a> Nature. 384, 134-141 (1996).</li><li>Knutson, K. L., dela Rosa, C., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+analysis+of+T-cell+immunity.">Laboratory analysis of T-cell immunity.</a> Front Biosci. 11, 1932-1944 (2006).</li><li>Andersen, M. H., Schrama, D., Thor Straten, P., Becker, 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=.">.</a> Cytotoxic T cells. J. Invest. Dermatol. 126, 32-41 (2006).</li><li>Weren, A., Bonnekoh, B., Schraven, B., Gollnick, H., Ambach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometric+assay+focusing+on+perforin+release+mechanisms+of+cytotoxic+T+lymphocytes.">A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes.</a> J. Immunol. Methods. 289, 17-26 (2004).</li><li>Aktas, E., Kucuksezer, U. C., Bilgic, S., Erten, G., Deniz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+CD107a+expression+and+cytotoxic+activity.">Relationship between CD107a expression and cytotoxic activity.</a> Cell Immunol. 254, 149-154 (2009).</li><li>Knutson, K. L., Schiffman, K., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+a+HER-2/neu+helper+peptide+vaccine+generates+HER-+2/neu+CD8+T-cell+immunity+in+cancer+patients.">Immunization with a HER-2/neu helper peptide vaccine generates HER- 2/neu CD8 T-cell immunity in cancer patients.</a> J. Clin. Invest. 107, 477-484 (2001).</li><li>Asiedu, M. K., Ingle, J. N., Behrens, M. D., Radisky, D. C., Knutson, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta/TNF(alpha)-mediated+epithelial-mesenchymal+transition+generates+breast+cancer+stem+cells+with+a+claudin-low+phenotype.">TGFbeta/TNF(alpha)-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype.</a> Cancer Res. 71, 4707-4719 (2011).</li><li>Strijbosch, L. W., Buurman, W. A., Does, R. J., Zinken, P. H., Groenewegen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+dilution+assays.+Experimental+design+and+statistical+analysis.">Limiting dilution assays. Experimental design and statistical analysis.</a> J. Immunol. Methods. 97, 133-140 (1987).</li><li>Gallimore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+exhaustion+of+lymphocytic+choriomeningitis+virus-specific+cytotoxic+T+lymphocytes+visualized+using+soluble+tetrameric+major+histocompatibility+complex+class+I-peptide+complexes.">Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.</a> J. Exp. Med. 187, 1383-1393 (1998).</li></ol>

生产和自由地进入这个视频文章是由罗氏诊断。

CD8 + T细胞是重要的适应性免疫反应，可以直接杀死感染的细胞或恶性细胞4。虽然有几种方法可以测量抗原特异性CD8 + T细胞（如酶联免疫斑点法，四聚体，流式细胞术）的数目，通常是有帮助的知道是否这些细胞功能的细胞毒性，3。最知名的检测评估细胞毒性功能的CRA，这被认为是黄金标准检测4。 CRA的主要限制是（1）的放射性的要求，（2）缺乏敏感性，（3）缺乏的机理信息，（4）要求大量的细胞，及（5）组费力。因此，在过去的十年中，一直在采取新的策略，以尽量减少这些限制的兴趣。较新的方法，包括那些措施蛋白酶释放4，BLT酯酶activity5，表面表达CD107 6。本手稿表明，基于阻抗的测定，罗氏xCELLigence系统上执行的，也可能是有用的作为替代的CRA。适应能力的阻抗为基础的方法在于观察T细胞，从而不紧紧地附着表面，似乎并没有产生多大的影响，阻抗，从而减少与测量的T细胞的直接干预任何关注。使用阻抗测定系统所需的劳动力预计将显着少，主要是由于消除监管关注使用的放射性。阻抗为基础的系统的唯一显着的缺点是50,000元至60,000元的支出是必需的购买xCelligence单元。然而，的xCelligence单元消除了需要的辐射计数器，更为通用，可用于评估细胞反应的T细胞的细胞毒作用8除了药物和迁移。 的观察，在肿瘤细胞的阻抗的减少是剂量依赖性的， 图2中所示，一个合理的陡坡（LDAS）限制稀释测定来估计细胞毒性细胞的频率为9，该方法的可用于一个简单的二次二次函数表明。与基于阻抗的方法的一个优点是，T细胞和肿瘤细胞的数目是小于的CRA，LDA的潜在的更可行的。例如，在CRA，按1:40的比例，为1×10 5个肿瘤细胞，每孔镀敷，需要4×10 6总T细胞，每孔。然而，阻抗为基础的检测中，只有1.5×10 4个肿瘤细胞，每孔都需要，消耗只有6×10 5个T细胞，每孔。因此，基于阻抗的测定，使用85％的T细胞和肿瘤细胞。这种减少表明，基于阻抗的系统可以适用于在人体临床试验的免疫监视，其中t他血量平可能会受到限制。另外，而CRA被限制在一个单一的时间点，基于阻抗的检测收集连续的实时数据，从而使用户能够以最少的附加劳动和细胞上的相同的实验比较多的溶解时间点。频率计算可能被结合与其他策略，不测量，如四聚体分析的功能，建立功能的细胞毒性T细胞的比例，以总抗原特异性CD8 + T细胞10。阻抗为基础的方法最吸引人的特点之一是，不像大多数其他实验，细胞毒性，可实时监控，有可能使它更容易使用抑制剂或抗体细胞相互作用研究。另一种方法是，该方法可能是合适的为抗蚀标签的肿瘤细胞。最后，由于非杀死肿瘤细胞是未标记的，并保持无菌，还收获和T细胞的抗性细胞的研究是possib文件中。 总之，尽管这两个系统提供了特异性T细胞的肿瘤的杀伤中的数据，基于阻抗的测定是比CRA更敏感，提供了更灵活的平台。

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ficoll-Paque Plus </td>
 <td>GE Healthcare </td>
 <td>17-1440-03 </td>
 <td>Used to isolate PBMCs from blood </td>
 </tr>
 <tr>
 <td>RPMI 1640 </td>
 <td>Cellgro </td>
 <td>10-040-CV </td>
 <td>Cell culture media </td>
 </tr>
 <tr>
 <td>FBS </td>
 <td>Denville Scientific </td>
 <td>FB5001-H </td>
 <td>Cell culture media </td>
 </tr>
 <tr>
 <td>Human IL-2 </td>
 <td>eBioscience </td>
 <td>14-8029-81 </td>
 <td>For stimulating growth of T cells </td>
 </tr>
 <tr>
 <td>Human Interferon gamma </td>
 <td>Cell Sciences </td>
 <td>CR2026 </td>
 <td>For stimulating MHC expression in tumor cells </td>
 </tr>
 <tr>
 <td>HER-2/neu p369-377 peptide </td>
 <td>Mayo Clinic peptide synthesis core </td>
 <td>amino acid sequence KIFGSLAFL </td>
 <td>Binds to HLA-A2 </td>
 </tr>
 <tr>
 <td>Influenza p58-66 peptide </td>
 <td>Mayo Clinic peptide synthesis core </td>
 <td>amino acid sequence GILGFVFTL </td>
 <td>Binds to HLA-A2 </td>
 </tr>
 <tr>
 <td>Human CD3/CD28 Dynabeads </td>
 <td>Invitrogen </td>
 <td>111-61D </td>
 <td>For non-specific T cell activation and expansion </td>
 </tr>
 <tr>
 <td>Chromium-51 </td>
 <td>MP Biomedicals </td>
 <td>62015 </td>
 <td>CRA labeling reagent </td>
 </tr>
 <tr>
 <td>E-plate </td>
 <td>Roche Applied Science </td>
 <td>05469830001 </td>
 <td>For xCelligence impedance unit </td>
 </tr>
 <tr>
 <td>Luma plate 96 </td>
 <td>Perkin Elmer </td>
 <td>6005630 </td>
 <td>For measuring radioactivity in the Top Count NXT. </td>
 </tr>
 <tr>
 <td>xCelligence RTCA DP Station </td>
 <td>Roche Applied Science </td>
 <td>RTCA DP </td>
 <td>Impedance unit </td>
 </tr>
 <tr>
 <td>Top Count NXT </td>
 <td>Perkin Elmer </td>
 <td>C990601 </td>
 <td>Scintillation &amp; Luminescence Counter </td>
 </tr>
 <tr>
 <td>Fas Ligand antibody</td>
 <td>BD Biosciences</td>
 <td>556372</td>
 <td>For blocking Fas-Fas ligand interactions</td>
 </tr>
 <tr>
 <td>HLA-A2 BB7.2 antibody</td>
 <td>BD Biosciences</td>
 <td>551230</td>
 <td>For blocking HLA class I</td>
 </tr>
 <tr>
 <td>Mouse IgG2b, &kappa;</td>
 <td>BD Biosciences</td>
 <td>555740</td>
 <td>Isotype control for HLA class I</td>
 </tr>
 <tr>
 <td>SKBR3 tumor line</td>
 <td>ATCC</td>
 <td>HTB-30</td>
 <td>Adenocarcinoma</td>
 </tr>
 <tr>
 <td>MCF7 tumor line</td>
 <td>ATCC</td>
 <td>HTB-22</td>
 <td>Adenocarcinoma</td>
 </tr>
 <tr>
 <td>HCC1419 tumor line</td>
 <td>ATCC</td>
 <td>CRL-2326</td>
 <td>Primary ductal carcinoma</td>
 </tr>
 <tr>
 <td>BT20 tumor line</td>
 <td>ATCC</td>
 <td>HTB-19</td>
 <td>Carcinoma</td>
 </tr>
 </tbody>
</table>

determining optimal cytotoxic activity of human her2neu specific cd8 t cells by comparing the cr51 release assay to the xcelligence system

细胞毒性CD8 + T细胞构成的子群的T细胞，能够诱导感染或恶性的宿主细胞的死亡。这些细胞表达一个专门的受体，称为T细胞受体（TCR），它可以识别一个特定的抗原肽结合HLA类分子2。接合感染的细胞或肿瘤细胞通过HLA I类分子生产裂解的分子，如颗粒酶和穿孔在目标细胞死亡所导致的结果。虽然它是有用的，以确定频率的抗原特异性CD8 + T细胞，使用如酶联免疫斑点法或流式细胞仪检测，也是有帮助的，以确定强度的CD8 + T细胞的反应，使用细胞毒性试验3。最知名的检测评估抗肿瘤细胞毒作用的是铬释放含量（CRA），这被认为是一个标准的测定4。 CRA有一些限制，包括细胞暴露γ辐射，升再现性，并且要求大量的细胞应答。在过去的十年中，一直有兴趣在采取新的战略来克服这些限制。较新的方法，包括那些措施蛋白酶释放4，BLT酯酶活性和表面表达CD107 6。的阻抗为基础的检测，使用罗氏xCELLigence系统，检查在本纸，其潜在的作为替代的CRA。电流阻抗或反对时发生黏附肿瘤细胞结合电极板。肿瘤细胞分离后杀戮和降低电阻抗由xCELLigence系统可测。的观察能力，以适应基于阻抗的方法来评估细胞介导的​​杀戮在于，T细胞不能坚持大多数表面紧密，不会出现阻抗太大的影响，从而减少直接干扰T细胞的任何关注同测量。结果表明，基于阻抗的方法能够检测具有更高的灵敏度相对于标准CRA的抗原特异性细胞毒性CD8 + T细胞的水平变化。基于这些结果，阻抗为基础的方法可能是很好的替代品，评级机构或其他方法，旨在测量细胞毒性CD8 + T细胞功能。

Watch this Scientific Journal Video about Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System at JoVE.com

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

铬释放法检测细胞毒性T细胞的活性，常见的检测，有几个限制。阻抗为基础的方法，在本文章中使用抗原特异性CD8 + T细胞和人乳腺癌肿瘤细胞系，SKBR3，检验检测细胞的杀伤能力。

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

determining-optimal-cytotoxic-activity-human-her2neu-specific-cd8-t

Research

JoVE Journal

Immunology and Infection

24.9K 次观看.  College of Medicine, Mayo Clinic. 铬释放法检测细胞毒性T细胞的活性，常见的检测，有几个限制。阻抗为基础的方法，在本文章中使用抗原特异性CD8 + T细胞和人乳腺癌肿瘤细胞系，SKBR3，检验检测细胞的杀伤能力。

视频: Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

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

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

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

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

科研

免疫与感染

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

猴免疫缺陷病毒特异性CD8分析<sup&gt; +</sup&gt; T细胞猕猴由肽MHC-I四聚体染色

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...