这项工作是由项目资助＃1010395（BQ与CP）和＃525431（CR）和项目资助＃455395（CP）来自澳大利亚，澳大利亚的中心为肝炎和HIV病毒学意向书的国家健康和医学研究委员会的支持2012赠款（CR和RJJ）和戈登和格莱特牧夫座基金会（BQ和CR）的资助。我们要感谢HARPREET Vohra保持和迈克尔Devoy其出色的保养JCSMR流式细胞实验室，澳大利亚癌症研究基金会生物分子资源设施，JCSMR，澳大利亚国立大学，用于多肽合成，和大卫博伊尔博士，CSIRO动物健康实验室，澳大利亚Geelong提供家长HIV疫苗的股票。

注:此协议下使用小鼠据澳大利亚国立大学动物实验伦理委员会的小鼠的指导方针和处理颈椎脱位处死。 1，染料和肽制剂<ol><li>染料的制备 注:染料被预稀释的不同浓度，以允许细胞在离散荧光强度标记。 CFSE是用在通过3.5倍连续稀释制成浓度7，和CTV和CPD用于在6个不同的浓度，通过3.7倍连续稀释制成（ 见表2-4）。股票的浓度在表2-4中列出的染料将被用来在表2-4中列出的最终染料浓度来标记靶细胞。染料在暴露于水溶液中反应。因此，重要的是小瓶平衡至室温开口，以减少染料的接触缩合之前。 <ol><li> CFSE 注意:CFSE被购买作为二醋酸盐琥珀酰亚胺酯（CFDA，SE;分子量557.47），通常在25毫克/瓶。 <ol><li>悬浮CFSE 25毫克在4.48毫升DMSO中，得到10mM的储备溶液。 </li><li>连续稀释CFSE加入35微升10mM的CFSE库存到87.5微升的DMSO中，并用稀释的贮存液重复此给每个染料的浓度在表3中 。 </li></ol></li><li> CTV 注意:CTV在9小瓶，每一个都可以与10微升的DMSO中复溶，以产生染料的10mM的溶液包通常购买。 <ol><li>重组，集中CTV的所有9小瓶90微升的DMSO给一个10毫米的解决方案。 </li><li>连续稀释CTV加入11微升10mM的CTV库存到29.7微升的DMSO中，并用稀释的贮存液重复此给每个染料浓度在表2中 。 </li></ol></li><li>持续专业发展 注:CPD（MW 792.6）是典型的LY购买的0.5毫克/瓶。 <ol><li> 63毫升DMSO中得到一个10毫米重悬浮液0.5毫克的CPD。 </li><li>连续稀释CPD:加入11微升10毫CPD股票为29.7微升DMSO中，用稀释的原液重复此给每个染料浓度见表4。 注意:上述的染料股可以存储在-20℃下数个月，无功能显著损失可以解冻和再冷冻数次。 </li></ol></li></ol></li><li>肽制剂 注意:MHC I类结合肽通常用于脉冲的靶细胞在6个不同的浓度，用10倍稀释液从1μM的（ 表5）的起始浓度进行。 MHC II类结合肽通常用于脉冲的靶细胞在6个不同浓度，使用3倍稀释度从400μM（ 表5）的起始浓度进行。每个肽是由在PBS这样的临屋区的2倍终浓度吨每肽浓度为250微升的最小容积。肽的股票可以事先向FTA建设来制备并储存于-20℃下无功能显著损失。 <ol><li>准备股票的MHC I类结合肽</li><li>准备每个肽抗原表位的最高浓度为2微米的储备液（2×1微米） </li><li>连续稀释的MHC I类结合肽:加入44.4微升2微米肽原液为400微升PBS。重复此用稀原液，得到每种肽的浓度在表5中 。 </li><li>准备股票的MHC-II结合肽</li><li>准备每个表位肽的浓度最高为800微米的储备液（2×400微米）。 </li><li>连续稀释的MHC-II结合肽:加入150μl的800微米肽溶液为300微升PBS。重复此用稀原液，得到每种肽的浓度在表5中</st荣&gt;。 </li></ol></li></ol> 2。FTA准备注意:下面的步骤概述包括细胞6种不同浓度（ 即 42细胞团。）反复6次，以产生252可辨别的细胞簇脉冲具有7种不同的抗原表位肽的自贸区的建设。在每个重复，没有脉冲与任何表位（无）的细胞群，包括作为对照。它是有利于自由贸易协定具有CD45同种异型差异从宿主动物，以允许从受体细胞的鉴别由抗体标记，在分析的时间。否则自由贸易协定可以带有其它染料如PKH-26用于此目的（在步骤2.6中所述）。通常情况下，自由贸易协定的准备过程，从开始进行时一口气完成。 <ol><li>标签42，将10毫升的锥形底的塑料管1-42（如表1中的例子）。 </li><li>细胞的制备<ol><li>分离脾和/或鲤鱼门公园hnodes从小鼠。通过一个70微米的糖化制备自组织单细胞悬液细孔筛用5毫升注射器柱塞，并用血细胞计数器计数细胞。 </li><li>重悬细胞于高达200×10 6个细胞/ 11.5毫升罗切斯特公园纪念研究所1640（RPMI，或等同物）含有5％胎牛血清（FCS）的毫升。注:使用一个缓冲高胺含量为染料的毒性最小化到细胞3是很重要的。 </li></ol></li><li> CTV标签 注:细胞最初标记​​为6的浓度CTV的。 <ol><li>彻底颠倒离心管几次重悬细胞。加1.9毫升细胞悬液至6 10 ml离心管标记为37-42的，小心不要弄湿管的上半部分。 </li><li>与CTV标记细胞，取出管帽和水平打下管。 </li><li>加入83微升PBS，以在管上方的非接液部分，并把它添加17微升股票CTV的（见表乐2的量原液被分配给该管）。注意:非润湿的管是很重要的，以防止与染料溶液的细胞悬浮液的运动和细胞溶液的过早混合。 </li><li>盖上管和涡旋快速彻底混合细胞悬液，用染料。 </li><li>重复步骤2.3.2-2.3.4为CTV在表2中说明的指定管中的储备溶液。 </li><li>孵育细胞至少5分钟在室温（20℃）。 </li></ol></li><li> CFSE标记<ol><li>视标记后，加入5 ml RPMI的含5％FCS，每管和由涡旋令其充分悬浮细胞。 <ol><li>从管37传送1 ml的细胞悬液，以管31，25，19，13，07和1。 </li><li>从管38传送1 ml的细胞悬液，以管32，26，20，14，08和2。 </li><li>从管39传送1 ml的细胞悬液至离心管，33，27，21，15，09和3中。 </li><li>从管40传送1 ml细胞的悬浮液至管34，28，22，16，10，和4。 </li><li>从管41传送1 ml的细胞悬液的试管35，29，23，17，11，和5。 </li><li>从管42传送1 ml的细胞悬液，以管36，30，24，18，12，和6 注意:小心不要在步骤2.4.1.1-2.4.1.6湿管的上半部分。 </li></ol></li><li>为了用CFSE标记细胞，取出管帽和水平打下管。 </li><li>加103毫升的PBS中，以在所述管上方的非润湿的部分，并把它添加7毫升库存CFSE（ 参见表3的量原液被分配给该管）。 </li><li>盖上管和涡旋快速彻底混合细胞悬液，用染料。 </li><li>做在表3中说明的指定管为原液的CFSE的步骤2.4.2-2.4.4。 </li><li>孵育细胞至少5分钟在室温（20℃）。 </li><li>洗涤细胞:稀释细胞悬液，用9ml 20℃RPMI含5％FCS，沉淀通过离心分离，在300×g离心10分钟，在20℃，通过抽吸去除上清液用移液管。 </li></ol></li><li>肽脉冲和细胞清洗 注意:在细胞已被标记为CTV和CFSE，它们是脉冲与MHC I类和/或类MHC-II分子结合肽（在1.2的方法制备）。一个细胞群，也不得用肽（ 例如 ，无在表1中）的脉冲，并用作阴性对照的T细胞应答的计算。 <ol><li>肽脉冲<ol><li>将细胞再悬浮于250微升的RPMI含5％FCS的总体积。注意:通常将50μl细胞悬浮液保持在上清液在步骤2.4.7的端部的抽吸之后，因此添加200μl的培养基对细胞沉淀。 </li><li>加入250μl的预先制备的肽库（如表5中），以适当地指定管（如表1），并河畔e来包括PBS的对照管单独加入而不肽作为一个零控制。 </li><li>混合的细胞悬浮液用旋涡。注意:每种肽的表位，每个肽浓度被分配给单个管和关键是此记录清楚的基础上，细胞在该管的预期荧光，由于该群集的荧光签名将限定该肽（如表1例如）。 </li><li>孵育的细胞，在37℃下反应1小时。 </li></ol></li><li>细胞洗涤<ol><li>加入5 ml冰冷的含5％FCS的细胞悬浮液和悬浮细胞通过反相管（4℃）的RPMI。仔细底图细胞悬液用3毫升冰冷（4℃）FCS的。 </li><li>通过离心在4℃下沉淀细胞，在300×g离心10分钟使用慢加速和制动，以确保FCS的接口和细胞悬浮液的溶液被维持。 </li><li>小心吸过的RPMI然后用移液管在FCS，而使洗涤细胞沉淀不受干扰。注意:使用一个FCS底图洗细胞，有助于确保尽可能多的肽溶液被从细胞尽可能地除去，从而限制细胞群体暴露于多种游离肽时，细胞被汇集。 </li><li>再次洗涤细胞:重悬细胞沉淀物在含5％FCS加入10ml 4℃RPMI中。通过离心在4℃下沉淀细胞，在300×g离心10分钟倒出上清液。 </li><li>池中的所有细胞群汇集成使用含5％FCS6毫升4℃RPMI吸管单管。沉淀池的细胞通过离心分离，在300×g离心10分钟，在4℃下抽吸去除上清液用移液管。 </li></ol></li></ol></li><li> CPD细胞标记 注意:此时6内重复测定可通过标签肽脉冲的细胞会产生6种不同浓度的持续专业发展。 <ol><li>添加11.4毫升20℃含5％FCS的汇集细胞沉淀重悬彻底用移液管的RPMI。 </li><li>加1.9毫升细胞悬液至6，10 ml离心管标记为AF，小心不要弄湿管的上半部分。 </li><li>与CPD标记细胞，取出管帽和水平打下管。 </li><li>加入92微升PBS，以在管上方的非接液部分，并为此增加股票持续专业发展（ 见表4原液分配给每个管的数量）。 </li><li>盖上管和涡旋快速彻底混合细胞悬液，用染料。 </li><li> 。千万步骤2.6.3-2.6.5的持续专业发展的在表4中所述的指定管股票的解决方案注:与CFSE和CTV，防贪处标示浓度不能精确线性相关标记的细胞所产生的荧光强度，所以用来获得几个标记种群的等距荧光峰的标记浓度一直凭经验确定（ 见表4）。 </li><li>孵育细胞至少5分钟在室温（20℃）。 </li><li>用含5％FCS 20℃的RPMI重悬细胞至10 ml:洗涤细胞两次。通过离心分离，在20℃下沉淀的细胞在300×g离心10分钟抽吸去除上清液用移液管。重复。 </li><li>池的所有单元汇集成使用含5％FCS的用移液管8毫升4℃RPMI单个管。沉淀池的细胞通过离心分离，在300×g离心10分钟，在4℃下，吸离上清液用移液管。 </li></ol></li><li> （可选）PKH-26细胞标记 注意:如果自由贸易协定不能与细胞表达CD45的一个异型差异来承载小鼠构建，它们可以被标记为与PKH-26，以允许从受体细胞的鉴别。 <ol><li>加2.9毫升20℃PBS中汇集的细胞沉淀并用移液管重悬浮彻底。 </li><li>加入细胞悬液，以无Ñ​​润湿10毫升管，小心不要弄湿管的上半部分。 </li><li>取下管盖和水平打下管。 </li><li>加入58微升稀释液C（在PKH-26染料套件），以在管上方的非接液部分，并把它添加42微升的1毫米的股票PKH-26。 </li><li>盖上管，彻底涡旋混匀细胞溶液与染料。 </li><li>孵育细胞至少10分钟，在室温（20℃）。 </li><li>用含5％FCS 20℃的RPMI重悬细胞至10 ml:洗涤细胞两次。通过离心分离，在20℃下沉淀的细胞在300×g离心10分钟抽吸去除上清液用移液管。重复。 </li></ol></li><li>自由贸易协定注入到宿主动物 注意:为了测量在体内的T细胞应答，自由贸易协定被注入到具有一个活跃的免疫反应，并留在原地长达24小时的动物。至关重要的是，该自由贸易协定也被注入到一个幼稚的动物作为阴性对照。<ol><li>计数和重悬的细胞以每毫升2.5×10 8个细胞于PBS中。注入200微升细胞静脉注射到宿主小鼠​​，其中包括一个天真的动物作为对照。 </li></ol></li></ol> 3，流式细胞仪<ol><li>自由贸易协定后注射收获血液或脾（或其他感兴趣的组织）从主机小鼠18-24小时。 </li><li>通过一个70微米的糖化制备单细胞悬液，从组织细孔筛用5毫升注射器的柱塞。 </li><li>重悬的细胞在含有0.1％BSA的PBS中达到65×10 6个细胞/ ml。 </li><li>免除细胞悬液100μL分装成一个微量滴定板抗体标记的水井。 </li><li>标记细胞荧光标记的抗体和荧光探针的可行性与频谱兼容荧光CFSE，CTV和CPD。注:抗体对B细胞标记，如B220和活化标记物，如CD69是必不可少的，如果使用FT测量B细胞活化一种测量T H细胞反应2。包括抗体CD45.1和/或CD45.2如果自由贸易协定和主机的小鼠有CD45异型差异。 </li><li>加入100μl的抗体/活力染料2倍原液（PBS中含0.1％BSA）对细胞的100微升等分拌匀，在冰上孵育（4℃）30分钟。 </li><li>离心300×g离心5分钟，在4℃下沉积的细胞，取出上清液:洗涤细胞。通过离心重悬细胞于200μl的PBS中含有0.1％BSA，沉积物的细胞在300×g离心5分钟，在4℃，并除去上清液。 </li><li>将细胞再悬浮在400微升PBS中含有0.1％BSA，过滤器单元通过一个70微米的筛网，并通过流式细胞术在流式细胞仪能够检测有关的荧光染料共轭物和分析的总体积。收集到3×10 6细胞的活动，以解决每个FTA细胞群，并获得足够的细胞进行统计学卡尔分析。注意:确保所有典型的控件（如单染对照组）进行流式细胞就业。通常情况下，CFSE需要激励来自蓝色激光源（通常为488 nm）和检测与集中在520 nm的带通滤波器;视需要激励从紫激光源（通常在405 nm）和检测与集中在450 nm的带通滤波器;和CPD需要激励从红色激光光源（通常为633纳米或640纳米）和检测与集中在670 nm的带通滤波器。 </li></ol> 4，数据分析<ol><li>分析流式细胞仪用标准的流式细胞术软件数据（见代表性的结果为门控策略中使用的类型的一个例子）。 </li><li>为％特异性杀伤，计算细胞的脉冲与MHC I类结合肽和FTA簇的未肽脉冲（"无"），每个FTA细胞簇的数目，并使用以下公式来计算％比杀害。 ％比杀害 <img alt="公式" fo:content-width="2in" src="/files/ftp_upload/51627/formula.jpg" width="200" /> 注意:在上述公式中"底漆"，是指从目标被认为具有针对靶肽的免疫应答的动物，"幼稚"是指从幼稚的动物对象，"肽"是指脉冲与肽和"无"目标是指未用任何肽冲激的目标。 </li><li>对B细胞活化的T H活性的量度，计算对脉冲与在"底漆"的动物的MHC-II结合肽并从该相减的GMFI FTA B细胞的CD69抗体荧光的几何平均荧光强度（GMFI）在相应的自贸区B细胞在"天真"的动物给的T H活性的测量值CD69的表达。 </li><li>从在步骤4.2和4.3，使用生成的统计信息数学的电子表格软件如的GraphPad棱镜计算其他定量和定性参数，如根据曲线和EC 50区（在深入介绍如何将这些计算的AUC执行可以在这里找到 http://graphpad.com/guides/prism/6/statistics/index.htm?stat_area_under_the_curve.htm，以及EC 50:http://www.graphpad.com/guides/prism/6/curve-fitting/index。 HTM？reg_the_ec50.htm）。 </li></ol>

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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=Design+and+evaluation+of+multi-gene,+multi-clade+HIV-1+MVA+vaccines.">Design and evaluation of multi-gene, multi-clade HIV-1 MVA vaccines.</a> Vaccine. 27, 5885-5895 (2009).</li><li>Wild, J., 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=Preclinical+evaluation+of+the+immunogenicity+of+C-type+HIV-1-based+DNA+and+NYVAC+vaccines+in+the+Balb/C+mouse+model.">Preclinical evaluation of the immunogenicity of C-type HIV-1-based DNA and NYVAC vaccines in the Balb/C mouse model.</a> Viral Immunol. 22, 309-319 (2009).</li><li>Takeshita, T., 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=Molecular+analysis+of+the+same+HIV+peptide+functionally+binding+to+both+a+class+I+and+a+class+II+MHC+molecule.">Molecular analysis of the same HIV peptide functionally binding to both a class I and a class II MHC molecule.</a> J Immunol. 154, 1973-1986 (1995).</li><li>Krutzik, P. O., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+cell+barcoding+in+flow+cytometry+allows+high-throughput+drug+screening+and+signaling+profiling.">Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.</a> Nature Methods. 3, 361-368 (2006).</li><li>Gilbert, D. F., Wilson, J. C., Nink, V., Lynch, J. W., Osborne, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+labeling+of+viable+cells+for+high-throughput+analysis+of+glycine+receptor+function+using+flow+cytometry.">Multiplexed labeling of viable cells for high-throughput analysis of glycine receptor function using flow cytometry.</a> Cytometry A. 75, 440-449 (2009).</li><li>Quah, B. J., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+carboxyfluorescein+diacetate+succinimidyl+ester+(CFSE)+to+monitor+lymphocyte+proliferation.">The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor lymphocyte proliferation.</a> J Vis Exp. , (2010).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nat Protoc. 2, 2049-2056 (2007).</li><li>Durward, M., Harms, J., Splitter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+specific+killing+assay+using+CFSE+labeled+target+cells.">Antigen specific killing assay using CFSE labeled target cells.</a> J Vis Exp. , (2010).</li><li>Hermans, I. F., 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=The+VITAL+assay:+a+versatile+fluorometric+technique+for+assessing+CTL-+and+NKT-mediated+cytotoxicity+against+multiple+targets+in+vitro+and+in+vivo.">The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo.</a> J Immunol Methods. 285, 25-40 (2004).</li><li>Busch, D. H., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+affinity+maturation+by+selective+expansion+during+infection.">T cell affinity maturation by selective expansion during infection.</a> J Exp Med. 189, 701-710 (1999).</li><li>Savage, P. A., Boniface, J. J., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+kinetic+basis+for+T+cell+receptor+repertoire+selection+during+an+immune+response.">A kinetic basis for T cell receptor repertoire selection during an immune response.</a> Immunity. 10, 485-492 (1999).</li><li>Wang, X. L., Altman, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caveats+in+the+design+of+MHC+class+I+tetramer/antigen-specific+T+lymphocytes+dissociation+assays.">Caveats in the design of MHC class I tetramer/antigen-specific T lymphocytes dissociation assays.</a> J Immunol Methods. 280, 25-35 (2003).</li><li>von Essen, M. R., Kongsbak, M., Geisler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+behind+functional+avidity+maturation+in+T+cells.">Mechanisms behind functional avidity maturation in T cells.</a> Clin Dev Immunol. , (2012).</li><li>Prussin, C., Metcalfe, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracytoplasmic+cytokine+using+flow+cytometry+and+directly+conjugated+anti-cytokine+antibodies.">Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies.</a> J Immunol Methods. 188, 117-128 (1995).</li><li>Foster, B., Prussin, C., Liu, F., Whitmire, J. K., Whitton, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracellular+cytokines+by+flow+cytometry.">Detection of intracellular cytokines by flow cytometry.</a> Curr Protoc Immunol. , (2007).</li><li>Czerkinsky, C. C., Nilsson, L. A., Nygren, H., Ouchterlony, O., Tarkowski, 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+solid-phase+enzyme-linked+immunospot+(ELISPOT)+assay+for+enumeration+of+specific+antibody-secreting+cells.">A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells.</a> J Immunol Methods. 65, 109-121 (1983).</li><li>Klinman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISPOT+assay+to+detect+cytokine-secreting+murine+and+human+cells.">ELISPOT assay to detect cytokine-secreting murine and human cells.</a> Curr Protoc Immunol. , (2008).</li><li>Yerly, 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=Increased+cytotoxic+T-lymphocyte+epitope+variant+cross-recognition+and+functional+avidity+are+associated+with+hepatitis+C+virus+clearance.">Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance.</a> J Virol. 82, 3147-3153 (2008).</li></ol>

作者宣称，他们有没有竞争的财务权益。

的FTA为基础的检测的优点在于，它们允许从流动的单一宿主动物术&gt; 250可行的和全功能的靶细胞群的判别。这提供了复杂性的一个级别一直未能前体内流式细胞仪为基础的分析。这是在上面所示的2动物实验，其中反应7不同的病毒表位，6的浓度可在6次重复同时在一个单一的动物允许将用于体内的T细胞应答所确定的参数如AUC和EC 50监视高亮。 除了 ​​?...

T细胞发挥的适应性免疫应答的核心作用，并且通常针对操作在免疫治疗。通过分泌细胞因子，调节免疫力的许多方面，也可以直接帮助B细胞制造抗体外来抗原的CD4 +效应T细胞反应。 CD8 +细胞毒性T细胞（CTL）还可以以外来抗原通过分泌细胞因子以及打在直接杀伤细胞中表达外来抗原核心作用作出响应。发起这些T细胞的效应子功能的基本相互作用涉及T细胞受体（TCR）与细胞表面上显示的MHC分子外来肽的相互作用。 CD4 + T细胞识别的抗原呈递细胞和CD8 + T细胞的MHC-II类分子的肽显示的认识上是通常显示在微生物感染细胞的MHC-I类分子的肽显示。 为了以评估的作用的T细胞在免疫反应中发挥，其效应子功能是通过可靠和灵敏的技术来测量它是必不可少的。 T细胞反应评估常用的方法包括; MHC class-I/II/peptide四聚体反应;细胞因子的产生通过ELISPOT和细胞内细胞因子染色;并通过51 Cr释放测定杀伤能力。这些测定法，但是，通常是进行先体外后体内与体外刺激，或提供有限的洞察T细胞的功能。理想的情况下，测量T细胞应答时，将所发生的，与无处理的T细胞，从而避免通过体外刺激，可能会发生变化的功能参数是有利的，以评估它们的原位 ， 在体内 。一些最常用的体内 T细胞功能测定法中使用是基于测量的CTL介导的杀伤脉冲与I类MHC结合肽的靶细胞，所列举的体内通过他们的检测通过与重要的染料如CFSE荧光标记。虽然这些类型的测定可以监视的CTL介导的杀伤的目标时，它们发生在体内 ，他们以前有一个相对有限的能力来评估杀灭的多个目标呈现不同浓度和不同类型的肽表位，这是必需的，以允许定性参数，例如作为功​​能性抗体亲抗原性和特异性表位变体的交叉反应性进行评估。这些检测也没有提供对CD4 + T细胞介导的反应的任何信息。 为了克服许多与用于评估的T细胞反应当前方法的局限，我们最近开发了一种基于荧光的目标阵列（自由贸易协定），它同时允许T细胞反应的监测对&gt; 250靶细胞在一个动物由多重检测流式细胞仪1，2。自由贸易协定是由淋巴细胞标记的SEV像CFSE，CTV和CPD活体染料允许产生独特的荧光&gt; 250细胞团ERAL浓度和组合。由于这些细胞保持活力和功能齐全的，它们可以被注射到动物，让监控它们的相互作用与效应性T细胞在体内3。例如，在FTA细胞簇可与MHC I类结合肽进行脉冲，让抗原特异性的CTL介导的杀死靶细胞1的评估。另外，在FTA细胞团，也可与MHC II类结合肽脉冲，使抗原特异性T辅助细胞（T H）通过评估活化（通过激活标记物如CD69，CD44和/或评估活动的评估CD62L的自由贸易协定中的B细胞轴承同源肽2）。因为超过250个目标可以同时检测，也可以对测量脉冲具有n个多靶细胞簇的CTL和T H的反应umerous肽在不同浓度及加入许多重复的。因此，自由贸易区法提供的体内 T细胞效应反应评估了前所未有的水平。 在这里，我们详细描述了自贸区的建设和展示如何将它们应用到评估在体内的 T细胞反应。该过程描述通过使用三个重要的染料，包括6重复脉冲与MHC I类和II-结合肽42细胞团组成的252分辨细胞簇一个自贸区的建设。 42细胞簇标记出现在10ml锥形底管，它是有帮助的，如表1中所示来放置这些在一个试管架上。该方法可用于更小的数字可辨别的集群根据需要通过降低标记的量来调节各染料进行1。 我们强调检测的实用程序，通过展示它如何衡量RESPONS通过重组痘病毒接种在一个小队列小鼠的多个表位产生的ES。这显示了自由贸易协定法可用于通过，测量累计反应和功能分别亲和力，曲线下面积（AUC）评估和产生半数最大反应（EC 50）所需的有效肽浓度测量的使用面积。

<table><tbody><tr><td>RPMI</td><td>Sigma</td><td>R8758</td><td></td></tr><tr><td>Fetal calf serum</td><td>Serana</td><td>FCS-500</td><td></td></tr><tr><td>CTV</td><td>Invitrogen</td><td>C34557</td><td></td></tr><tr><td>CFDA, SE</td><td>Invitrogen</td><td>C1157</td><td></td></tr><tr><td>CPD</td><td>eBioscience</td><td>65-0840-90</td><td></td></tr><tr><td>anti-B220 PerCp-Cy5.5</td><td>eBioscience</td><td>110730</td><td></td></tr><tr><td>anti-CD69 brilliant violet 605</td><td>Biolegend</td><td>104529</td><td></td></tr><tr><td>PKH-26</td><td>Sigma</td><td>PKH26GL-1KT</td><td></td></tr><tr><td>Vortex</td><td>Scientific Industries Inc</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Flow cytometer (Fortessa or equivalent with blue, red and violet laser source)</td><td>BD Bioscience</td><td></td><td></td></tr></tbody></table>

the use of fluorescent target arrays for assessment of t cell responses in vivo

监测T细胞应答在体内的功能是对我们的免疫应答的理解的发展和免疫疗法的设计很重要。在这里，我们描述了使用的荧光目标阵列（FTA）技术，它利用活体染料，如羧基荧光素琥珀酰亚胺酯（CFSE），紫激光激发的染料（CellTrace紫罗兰:CTV）和红色激光激发的染料（细胞增殖染料eFluor 670:CPD ）以组合性标签小鼠淋巴细胞为&gt; 250分辨荧光细胞簇。这些自由贸易协定内细胞团可以是脉冲与主要组织相容性复合体（MHC）I类和MHC II类结合肽，从而作为靶细胞对CD8分别+和CD4 + T细胞。这些FTA细胞保持存活，功能齐全的，并且因此可以被施用到小鼠体内，以允许的CD8 + T细胞介导的杀伤FTA靶细胞和CD4 + T细胞预谋HEL的评估FTA B细胞的靶细胞的对实时体内通过流式细胞术。因为&gt; 250靶细胞可以一次进行评估，该技术允许对多个抗原表位的T细胞应答的若干浓度和在多次重复的监控。因此，该技术可以在两个定量（ 如累积响应的幅度）和定性（ 例如 ，功能性抗体亲抗原性和响应的表位的交叉反应性）水平测量T细胞应答。本文中，我们描述了如何将这些自由贸易协定的构造，并给予他们如何可以应用于评估诱导的重组痘病毒疫苗的T细胞反应的例子。

由于使用了FTA测定的一个例子，一个BALB / c小鼠进行免疫接种的重组牛痘病毒（VV）的表达HIV-I表位（VV-HIV）和响应的HIV-CTL表位，加格，加格MUT，的env和聚合物，该VV CTL表位F2L和F2L MUT和HIV-T H细胞表位，加格钍（如2所述）通过使用252参数FTA测定法（ 图1B）进行评估。加格（GAG MUT）和F2L（F2L MUT）的表位变异并不表示在VV-HIV载体，因此对这些表位的反应是反射性的表位变?...

Watch this Scientific Journal Video about The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo at JoVE.com

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

监测体内详细的T细胞应答的能力，对于我们的免疫应答的理解的发展非常重要。在这里，我们描述了评估同时采用流式细胞仪&gt; 250参数体内 T细胞检测使用荧光目标阵列（自由贸易协定）的。

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

the-use-fluorescent-target-arrays-for-assessment-t-cell-responses

Research

JoVE Journal

Immunology and Infection

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

14.4K 次观看.  Australian National University. 监测体内详细的T细胞应答的能力，对于我们的免疫应答的理解的发展非常重要。在这里，我们描述了评估同时采用流式细胞仪> 250参数体内 T细胞检测使用荧光目标阵列（自由贸易协定）的。 

视频: The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

科研

癌症研究

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

免疫与感染

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 ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

生物工程

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

Photoactivatable 激动剂对 T 细胞活化的时空控制

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

荧光目标阵列的T细胞反应评价中的应用

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Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

定量高通量单细胞的细胞毒性T细胞的测定

T细胞实时成像实时信号复合物的形成

测量TCR-的pMHC结合

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

成像的CD4 T细胞间质迁移发炎真皮

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

Photoactivatable 激动剂对 T 细胞活化的时空控制

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

荧光目标阵列的T细胞反应评价中的应用

摘要

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Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

定量高通量单细胞的细胞毒性T细胞的测定

T细胞实时成像实时信号复合物的形成

测量TCR-的pMHC结合

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

成像的CD4 T细胞间质迁移发炎真皮

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

Photoactivatable 激动剂对 T 细胞活化的时空控制

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术