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>

<ol>
	<li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., 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=Fluorescent+target+array+killing+assay:+A+multiplex+cytotoxic+T-cell+assay+to+measure+detailed+T-cell+antigen+specificity+and+avidity+in+vivo.">Fluorescent target array killing assay: A multiplex cytotoxic T-cell assay to measure detailed T-cell antigen specificity and avidity in vivo.</a> Cytometry A. 81, 679-690 (2012).</li><li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., 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=Fluorescent+target+array+T+helper+assay:+a+multiplex+flow+cytometry+assay+to+measure+antigen-specific+CD4++T+cell-mediated+B+cell+help+in+vivo.">Fluorescent target array T helper assay: a multiplex flow cytometry assay to measure antigen-specific CD4+ T cell-mediated B cell help in vivo.</a> J Immunol Methods. 387, 181-190 (2013).</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=New+and+improved+methods+for+measuring+lymphocyte+proliferation+in+vitro+and+in+vivo+using+CFSE-like+fluorescent+dyes.">New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.</a> J Immunol Methods. 379, 1-14 (2012).</li><li>Mata, M., Paterson, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1+T+cell+responses+to+HIV-1+Gag+protein+delivered+by+a+Listeria+monocytogenes+vaccine+are+similar+to+those+induced+by+endogenous+listerial+antigens.">Th1 T cell responses to HIV-1 Gag protein delivered by a Listeria monocytogenes vaccine are similar to those induced by endogenous listerial antigens.</a> J Immunol. 163, 1449-1456 (1999).</li><li>Earl, P. 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。

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		<col />
		<col />
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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">RPMI</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">R8758</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fetal calf serum</td>
			<td style="width:71px;">Serana</td>
			<td style="width:119px;">FCS-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CTV</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C34557</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CFDA, SE</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C1157</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CPD</td>
			<td style="width:71px;">eBioscience</td>
			<td style="width:119px;">65-0840-90</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">anti-B220 PerCp-Cy5.5</td>
			<td style="width:71px;">eBioscience</td>
			<td style="width:119px;">110730</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">anti-CD69 brilliant violet 605</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">104529</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">PKH-26</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">EQUIPMENT:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Material Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments (optional)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Vortex</td>
			<td style="width:71px;">Scientific Industries Inc&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Flow cytometer (Fortessa or equivalent with blue, red and violet laser source)</td>
			<td style="width:71px;">BD Bioscience</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></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 ...

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Immunology and Infection

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

14.3K 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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...