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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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><tbody><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><a href="http://www.aceabio.com/" target="_blank"><img src="/img/new_window.png" /> Roche Group</a></td><td>05469830001</td><td>For xCelligence impedance unit </td></tr><tr><td>Luma plate 96 </td><td>PerkinElmer, Inc.</td><td>6005630</td><td>For measuring radioactivity in the Top Count NXT. </td></tr><tr><td>xCelligence RTCA DP Station </td><td><a href="http://www.aceabio.com/" target="_blank"><img src="/img/new_window.png" /> Roche Applied Science</a></td><td>RTCA DP</td><td>Impedance unit </td></tr><tr><td>Top Count NXT </td><td>PerkinElmer, Inc.</td><td>C990601</td><td>Scintillation &amp;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, &amp;#954;</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

Universidad de Cartagena UDEC

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

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

科研

癌症研究

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

免疫与感染

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 ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

在预处理肿瘤细胞的同种异体共培养环境中分析人 T 细胞活性

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

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 ...

<meta charset="utf8"></head><body>甲状腺癌细胞作为癌症免疫治疗的 ADCC 生物测定中的效应子

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

<meta charset="utf8"></head><body>作者聚焦:通过抗体依赖性细胞介导的细胞毒性测定优化基于抗体的癌症治疗

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

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

摘要

探索更多视频

此视频中的章节

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

定量高通量单细胞的细胞毒性T细胞的测定

基于细胞的流式细胞仪检测，以测量细胞毒活性

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

在预处理肿瘤细胞的同种异体共培养环境中分析人 T 细胞活性

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

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

使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性

使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性