Name: phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice
Uploaded: 2016-06-22T15:00:00.000+00:00
Duration: 7 min 17 s
Description: Watch this Scientific Journal Video about Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice at JoVE.com

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A3A01020610 to HJP) and a grant from the Korean Health Technology R&amp;D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (HI15C0493 to SJH).

在这项研究中，将小鼠保持在延世大学的延世实验动物研究中心的一个特定的无病原体的设施。所有的动物实验均按照利用在延世大学延世实验动物研究中心的国际动物护理和使用委员会批准协议的韩国食品和药物管理局的指导方针进行。 1.溶液的制备<ol><li>通过在RPMI稀释牛胎儿血清（FBS），以2％和青霉素 - 链霉素1％制备2％RPMI培养基</li><li>准备完整的RPMI媒体。以RPMI培养基添加10％FBS的，青霉素 - 链霉素1％L-谷氨酰胺的1％，和50μM2-巯基乙醇。 </li><li>制备荧光激活细胞分选（FACS）缓冲液。与FBS的2％这样做，补充磷酸盐缓冲盐水（PBS）。 </li><li>准备T细胞的隔离缓冲。这样做，补充的PBS与FBS的2％和2mM ethylenediaminetetraacetic酸。 </li></ol> 2.脾淋巴细胞的分离<ol><li>如先前所述19从幼稚或LCMV CL13感染的小鼠中取出脾脏，并将其放置至60mm含有10ml 2％RPMI培养基的×15毫米的培养皿中。 注意:在此研究中的实验中，5至6周龄C57B1L / 6J雌性小鼠接受LCMV CL13的2×10 6噬斑形成单位（pfu）通过经由尾静脉静脉内注射。在牺牲16天感染后（16天PI）25只。分析锗匹配的幼稚小鼠在同一天。 </li><li>放置细胞过滤到50ml管中并用2ml的2％RPMI培养基的冲洗。广场上的70微米的细胞过滤健脾和使用注射器的活塞来转动。冲洗细胞过滤用2ml 2％RPMI培养基的并从50ml管中移除。 </li><li>填满用2％RPMI培养基和离心管中，在300×g离心在4℃下5分钟。弃上清，悬浮在1ml的ACK裂解缓冲液的细胞沉淀。孵育在室温下（RT）将样品5分钟。 注:ACK裂解缓冲液的上面指出的量是一个脾脏。相应地扩大该卷汇集脾样本。 </li><li>填满用2％RPMI培养基和离心管中，在300×g离心在4℃下10分钟。弃去上清液，并在完全RPMI培养基一个1×10 7个细胞/ ml的密度重悬细胞。 注:对于活细胞计数，染色细胞台盼蓝和计数只活了不使用血球染色的细胞。 </li></ol> 3.脾常规T（T CONV）细胞和T reg细胞的表型注意: 调节性T细胞的分离之前，检查通过用各种抗体染色细胞并通过流式细胞仪分析它们从幼稚或感染的小鼠分离脾淋巴细胞的表型。 <ol><li>转移50微升细胞（5×10 5细胞）的总脾淋巴细胞到一个新的U型底96孔板的每个孔中，并添加每孔150μl的流式细胞仪缓冲液中。离心机在300×g离心在4℃下2分钟。 </li><li>弃去上清液。搅动细胞沉淀并每孔添加200μl的FACS缓冲液中。离心机在4℃（2次）300 xg离心2分钟。 </li><li>制备用于染色的细胞表面标志物的抗体混合物中，每孔50微升FACS缓冲液中具有以下的抗体和试剂。抗CD4紫色染料，抗CD25绿色染料，抗PD-1紫色染料，抗CD8 PerCP-经Cy5.5染料和细胞生存力检测试剂的近红外（IR）荧光活性染料。 注意:要进一步分析活化的T reg细胞的表型，抗CD103 23,25可以加入用于细胞表面标记物染色。 </li><li>悬浮用50μl每孔抗体混合物的细胞沉淀。孵育在4℃下在黑暗中20分钟。用洗涤细胞两次FACS缓冲液在300×g离心2分钟离心在4℃下。 </li><li>最后洗涤步骤后，倒出上清液，并用100微升固定缓冲液根据制造商的方案制备定为在黑暗中20分钟后的细胞于4℃。 </li><li>与透洗涤缓冲液（根据制造商的方案制备）通过离心洗涤细胞两次，在300×g离心2分钟，在4℃。 </li><li>制备用于细胞内的Foxp3染色的抗体溶液中，并重新悬浮用50μl抗Foxp3的抗体溶液中的细胞沉淀。 注意:要进一步分析ŤCONV和T reg细胞，可以在这个步骤中加入抗CTLA的表型。 </li><li>孵育在黑暗中20分钟，在4℃并重复步骤3.6。最后洗涤步骤后，重悬在200μlFACS缓冲液中的细胞沉淀，并用流式细胞仪25检查的细胞的表型。 </li><li>门活细胞普惠化。分析ŧCONV细胞LCMV CL13感染过程中的表型，检查Foxp3的频 ​​率- PD-1 +细胞CD4 +和CD8 + T细胞中。 注意:基于实验结果，Foxp3的百分比- PD-1 +为50％以上，在CD4 +和CD8 + T细胞群在含有LCMV CL13 16天圆周率。 <ol><li>分析调节性T细胞的频率和表型检验的Foxp3 +或FOXP3 + PD-1 +细胞的CD4 + T细胞中的频率。 注意:基于实验结果，Foxp3的+或FOXP3 + PD-1 +细胞的百分比在CD4 + T细胞群体的20％以上，分 ​​别。 </li></ol></li></ol> 4.隔离CD4 + CD25 + T reg细胞的注:所有试剂的量如下所示是一个开始1×10 7总脾细胞数。 <ol><li>准备细胞用天真和慢性LCMV感染小鼠第2节。通过加入10ml的T细胞分离缓冲液洗细胞。离心机在300×g离心在4℃下10分钟。完全弃上清，重悬细胞沉淀在40微升缓冲。 </li><li>对于使用磁性细胞分离系统CD4 + T细胞富集，加入10微升生物素抗体混合物拌匀。孵育在4℃下10分钟。 </li><li>添加缓冲液30微升，20微升抗生物素微珠用于非CD4 + T细胞的标记和10μlCD25-PE抗体对CD25 +细胞的荧光标记。拌匀，孵育在黑暗中15分钟。 </li><li>加2ml缓冲液中，通过40微米的细胞过滤的细胞传递到一个新的50ml管中，以除去细胞碎片。离心300 xg离心洗细胞10分钟，在4℃。完全弃去上清液并以高达1.25×10 8个细胞的密度重新悬浮于500μl缓冲液细胞沉淀。 </li><li>应用该细胞在柱上，并收集穿过所述列中的未标记细胞。通过在4℃下加入2ml缓冲液和离心机在300×g离心10分钟，洗柱。完全弃上清，悬浮隔离的CD4 + T细胞的90微升缓冲。 </li><li>对于CD25 +细胞的磁贴标签，加10微升抗PE微珠，拌匀，并在4℃孵育在黑暗中15分钟。 </li><li>加入2毫升缓冲液中，并在4℃以300 xg离心离心洗细胞10分钟。完全弃去上清液，并在高达1×10 8个细胞的密度重新悬浮于500μl缓冲液中的细胞沉淀。 </li><li>以丰富的CD4 + CD25 + T reg细胞，应用细胞上的阳性选择列，洗柱通过加入2ml缓冲的n个。重复洗涤三次。 </li><li>当列是最后的洗涤步骤后空，加入1毫升缓冲液上柱，并冲洗出磁性标记的CD4 + CD25 +使用柱塞细胞。 </li><li>重复步骤4.8-4.9，以提高分离的CD4 + CD25 + T reg细胞的纯度。离心300 xg离心洗涤用FACS缓冲液将细胞10分钟，在4℃。弃去上清液，并在完全RPMI培养基上2×10 6个细胞/ ml的浓度重悬分离的CD4 + CD25 + T reg细胞。 </li><li>要检查孤立的CD4 + CD25 + T reg细胞的纯度，使用2×10 5细胞从总的孤立的CD4 + CD25 + T reg细胞。细胞染色用含有抗CD4 FITC和细胞生存力检测近红外荧光活性染料试剂50微升FACS缓冲液。 注:CD25已经被隔离在与PE标记。 </li><li>孵育在4℃下在黑暗中20分钟。离心300 xg离心洗涤用FACS缓冲液将细胞2分钟，在4℃。洗涤后，悬浮于100μlFACS缓冲液中的细胞沉淀并通过流式细胞术检查纯度。 </li><li>门活细胞群。分析调节性T细胞的纯度，确认CD4 + CD25 +细胞的百分比。 注意:基于实验结果，CD4 + CD25 +细胞的纯化的细胞之间的比例是在约80％以上。 </li></ol> 5.隔离CD8 + T细胞和CD8 + T细胞的标记注:所有的试剂的体积如下所示是用于1×10 7个总脾细胞的起始细胞数。 <ol><li>由于使用天真的小鼠在第2节准备的细胞。通过加入10ml T细胞分离的buff洗涤细胞呃。离心机在300×g离心在4℃下10分钟。完全弃去上清液，并且重悬细胞沉淀在40微升缓冲液中。 </li><li>对于使用磁性细胞分离系统CD8 + T细胞的分离，加入10微升非CD8 + T细胞标记生物素抗体混合物拌匀。孵育在4℃下5分钟。 </li><li>添加缓冲液30微升和20微升抗生物素微珠。拌匀，并在4℃孵育10分钟。 </li><li>应用该细胞在柱上，并收集穿过所述列中的未标记细胞。通过加入缓冲2毫升三次洗柱。 </li><li>离心机在300×g离心在4℃下10分钟。完全弃上清，悬浮隔离的CD8 + T细胞在2毫升缓冲。 </li><li>要检查分离细胞的纯度，准备50微升的FACS缓冲抗体溶液。其中重悬孤立的CD8 + T CEL的2×10 5个细胞LS在50μl抗体溶液。 注:要准备抗体溶液，加入抗CD8 FITC和细胞活力检测试剂（近红外荧光活性染料）到FACS缓冲。 </li><li>孵育在4℃下在黑暗中20分钟。离心300 xg离心洗涤用FACS缓冲液将细胞2分钟，在4℃。洗涤后，悬浮于100μlFACS缓冲液中的细胞沉淀，并通过流式细胞术检查细胞的纯度。 </li><li>门活细胞群。要检查的 CD8 + T细胞的纯度，确认CD8 + T细胞的百分比。 注意:基于实验结果，CD8 +细胞的纯化的细胞之间的百分比超过约90％。 </li><li>添加的PBS分离的CD8 + T细胞。离心机在300×g离心在4℃下10分钟。弃去上清液。悬浮分离CD8 + T细胞在PBS中1×10 7细胞/ ml的浓度。 </li><li>标注的CD8 + T CE用于体外抑制测定法LLS，稀释细胞增殖跟踪紫色染料在PBS中在RT以获得为5μM的浓度。 注:在该研究中使用的细胞增殖跟踪紫色染料的近似激发和发射峰405和450处，分别。 </li><li>在一个15毫升管混合细胞增殖跟踪紫色染料（5微米）和细胞悬浮液（1×10 7个细胞/ ml的CD8 + T细胞的）的井等体积，并在20分钟孵育在37℃。涡管每隔10分钟。 </li><li>填补了冷完全RPMI媒体管，并离开管在室温下10分钟。离心在300×g离心在RT 10分钟。完全弃去上清液，并以2×10 6个细胞/ ml用预温热的完全RPMI培养基的浓度重悬细胞。孵育所述细胞在室温下15分钟。 </li></ol> 6.设置在体外抑制测定中使用的CD4 + CD25 + T &lt;子&gt;章和CD8 + T细胞<ol><li>为了制备抗CD3 / CD28包被的珠子，磁珠适当体积转移到15毫升管（2.5微升/ 1×10 5个细胞）。添加的PBS等体积并混合。离心在300 xg离心洗净2分钟，在4℃，弃上清。稀释在完全培养基的磁性珠（50μl/孔）。 </li><li>等分试样50的CD4 + CD25 + T reg细胞的微升每U型底96孔板的孔（1×10 5细胞/孔）。添加CD8 + T细胞作为应答T（T RESP）细胞每孔（1×10 5个细胞/孔）的50微升。加入50μl的稀释抗CD3 / CD28包被的珠子进入每孔。 注意:在此步骤中，标签和设置对照孔如下:无抗CD3 / CD28包被的珠子"未刺激的 CD8 + T仅细胞"; "只有CD8 + T细胞"用抗CD3 / CD28包被的珠子;只有"CD8 + T细胞"用抗CD3 / CD28包被的珠子;"。 调节性T细胞仅"用抗CD3 / CD28包被的珠子调节性T细胞可以通过完整的媒体和共培养和T RESP细胞在T的不同比例稀释RESP细胞: 调节性T细胞（1:0.25-1:1）。 </li><li>加入50微升或介质适当的音量到所有的井200微升总体积。覆盖箔的板，并在37℃下在CO 2培养箱中孵育72小时。 </li></ol> 7. CD8分析+ T细胞的增殖和细胞因子的产量从CD8 + T细胞<ol><li>用于细胞因子产生的分析，在培养3天后，每个分离上清液以及到另一个板并执行酶联免疫吸附测定（ELISA）。 注:上清液可被等分并储存在-70℃。在这个实验中，使用抗小鼠IFN-γ抗体包被的板，以检测IFN-γ的生产符合ING制造商的规程。来确定IFN-γ的生产上的单细胞水平增殖的CD8 + T细胞，能够进行细胞内细胞因子染色。 </li><li>从每个孔中分离上清液后，冲洗含有用FACS缓冲液和离心将细胞板在300×g离心2分钟，在4℃（3次）。 </li><li>洗涤后，弃上清。悬浮用50μl抗体混合物的对增殖的 CD8 + T细胞的染色细胞沉淀。孵育在4℃下在黑暗中20分钟。 注:要准备抗体混合，添加抗CD4 FITC，抗CD8 PerCP-经Cy5.5和细胞活力检测试剂（近红外荧光活性染料）到FACS缓冲。 注:对各种标记物如CD44或CD69的抗体可以与其他抗体结合，以确认CD8 + T细胞的活化。请记住，CD8 + T细胞已被标记的细胞增殖跟踪v在步骤5.10 iolet染料。 </li><li>在4℃以300 xg离心离心洗涤两次2分钟。最后洗涤步骤后，弃去上清液，并且用100μl的固定缓冲液中固定在黑暗中20分钟后的细胞于4℃。 </li><li>在4℃以300 xg离心离心洗涤两次2分钟。悬浮细胞用200μlFACS缓冲液中，并测量细胞增殖跟踪紫色色素标记CD8 + T细胞的通过流式细胞术的扩散。 <ol><li>栅极活细胞中的 CD8 + T细胞群。根据细胞增殖跟踪紫色染料，并根据下列等式的 CD8 + T细胞稀释液的稀释测量分割和不可分割的细胞的百分比。 ％抑制= [（增殖的CD8 + T细胞在不存在调节性T细胞的％ -增殖的CD8 + T细胞的百分比在调节性T细胞的存在）/（增殖的CD8 + T细胞的百分比缺乏调节性T细胞的）]×100此外，通过使用流式细胞仪软件25进行数据分析。 </li></ol></li></ol>

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S.-J.H. has a patent and receives patent royalties related to the PD-1 pathway. The other authors have no financial conflicts of interest.

虽然在小鼠和人类只存在调节性T细胞的数量少，必须了解它们的功能，因为它们在调节免疫反应和维持免疫耐受了至关重要的作用是重要的。 T的数目和抑制功能的慢性病毒感染15-20以及癌症进展13,14期间reg细胞增加。这大概是由于持续抗原刺激。为了评估调节性T细胞的抗原下的持久性和疾病的发展发挥作用，其抑制活性需要测量。 ?...

调节性T（ 调节性T细胞）表达叉头框P3（Foxp3的），作为其发育和功能1的转录因子。此外， 调节性T细胞表达的各种其它分子如CD25 2，淋巴细胞激活基因3（LAG-3）3，糖皮质激素诱导的肿瘤坏死因子受体4，和细胞毒性T淋巴细胞相关蛋白4（CTLA-4）5在其表面上或细胞内区域。在慢性感染多种病原体如病毒6,7，细菌8,9和寄生虫10-12，或在癌症发展13,14的过程中，T reg细胞变得分化成活化细胞，显示增强抑制功能靶向效应CD4 +和CD8 + T细胞。若干篇论文表明，扩展和活化的T reg细胞向受损CD8 + T细胞respons朋友逆转录病毒（FV）感染15-17时即FV诱导的T reg细胞抑制IFN-γ或颗粒酶B的表达和CD8 + T细胞15-17的细胞毒活性。此外，在单纯疱疹病毒感染模型，据报道CD4 + CD25 + 调节性T细胞的耗竭导致病毒特异性CD8 + T细胞和由免疫病理学CD4 + T细胞18-20的浸润严重的组织损伤的扩大。 与淋巴细胞脉络丛脑膜炎病毒的克隆13菌株慢性感染的小鼠（LCMV CL13）21-24已被广泛用于慢性病毒感染过程中表征效应T细胞（T EFF）和T reg细胞的表型和功能。期间持续LCMV感染，病毒特异性-T EFF细胞逐渐失去其效应器功能，并成为耗尽T（ŤEXH）细胞。另一方面，Treg细胞增强其抑制病毒特异性T细胞反应能力25。在T EFF细胞的功能能力的降低可以通过几个因素，如对T EFF细胞抑制性受体的上调，抗原呈递细胞的功能改变，生产免疫调节细胞因子，和增加的频率或增强调节性T的功能进行说明细胞26。间参与T细胞抑制因子，细胞程序性死亡蛋白-1（PD-1）-expressingŤEXH细胞和T reg细胞已被广泛认为是抗原持久性和抑制性环境的特点。最近，有人报告说， 调节性T细胞的PD-1途径和消融的封锁导致增强的T细胞功能和LCMV慢性感染27期间降低病毒载量。此外， 调节性T细胞的小鼠慢性感染过程中与LCMV 23,25激活</sUp&gt;和他们的抑制功能得到加强25。 PD-1的高表达对T reg细胞以及为T EXH细胞，和PD-1被T reg细胞表达的水平与他们的抑制功能的强度，以抑制T细胞增殖25相关。 这里，我们描述一个比较来自感染LCMV CL13和从幼稚小鼠分离静止T reg细胞的小鼠中分离的活化T reg细胞的特性的方法。此外，我们解释一系列过程分离活化的T reg细胞，并考察其体外的表型，以及衡量其体外抑制活性。

<table><tbody><tr><td>FITC Rat Anti-Mouse CD4</td><td>RM4-5</td><td>BD Biosciences</td><td>553047</td></tr><tr><td>Cytofix/Cytoperm</td><td></td><td>BD Biosciences</td><td>554714</td></tr><tr><td>U-Bottom Tissue Culture Plates</td><td></td><td>BD Biosciences</td><td>353077</td></tr><tr><td>Fixation buffer</td><td></td><td>BD Biosciences</td><td>554655</td></tr><tr><td>FITC Rat Anti-Mouse CD25</td><td>7D4</td><td>BD Biosciences</td><td>553072</td></tr><tr><td>Cell strainer, 70 mm</td><td></td><td>BD Biosciences</td><td>352350</td></tr><tr><td>Cell strainer, 40 mm</td><td></td><td>BD Biosciences</td><td>352340</td></tr><tr><td>Brilliant Violet 421 Anti-mouse CD279 (PD-1)</td><td>29F.1A12</td><td>BioLegend</td><td>135217</td></tr><tr><td>Brilliant Violet 605 Anti-Mouse CD4</td><td>RM4-5</td><td>Biolegend</td><td>100547</td></tr><tr><td>APC Anti-Mouse/Rat Foxp3&amp;nbsp;</td><td>FJK-16s</td><td>eBioscience</td><td>17-5773</td></tr><tr><td>Foxp3/Transcription Factor Staining Buffer Set</td><td></td><td>eBioscience</td><td>00-5223</td></tr><tr><td>PerCP-Cyanine5.5 Anti-Mouse CD8a</td><td>53-6.7</td><td>eBiosicence</td><td>45-0081</td></tr><tr><td>Mouse IFN-gamma Platinum ELISA</td><td></td><td>eBiosicence</td><td>BMS606</td></tr><tr><td>RPMI 1640</td><td></td><td>GE Life Sciences</td><td>SH30027</td></tr><tr><td>PBS (1x)</td><td></td><td>GE Life Sciences</td><td>SH30256</td></tr><tr><td>ACK Lysing Buffer</td><td></td><td>Gibco</td><td>A10492-01</td></tr><tr><td>L-Glutamine, 200 mM solution</td><td></td><td>Gibco&amp;nbsp;</td><td>25030</td></tr><tr><td>Penicillin-Streptomycin, 10,000 U/ml</td><td></td><td>Gibco&amp;nbsp;</td><td>10378-016</td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td><td></td><td>Life technologies</td><td>L-34975</td></tr><tr><td>CD8a&lt;sup&gt;+&lt;/sup&gt; T Cell Isolation Kit, mouse</td><td></td><td>Miltenyibiotec</td><td>130-104-075</td></tr><tr><td>CD4&lt;sup&gt;+&lt;/sup&gt; CD25&lt;sup&gt;+&lt;/sup&gt; Regulatory T Cell Isolation Kit, mouse</td><td></td><td>Miltenyibiotec</td><td>130-091-041</td></tr><tr><td>MACS Separation Columns, LD columns</td><td></td><td>Miltenyibiotec</td><td>130-042-901</td></tr><tr><td>MACS Separation Columns, LS columns</td><td></td><td>Miltenyibiotec</td><td>130-042-401</td></tr><tr><td>EDTA, 0.5 M (pH 8.0)</td><td></td><td>Promega</td><td>V4231</td></tr><tr><td>2-Mercaptoethanol</td><td></td><td>Sigma Life Science</td><td>M7522</td></tr><tr><td>Fetal Bovine Serum</td><td></td><td>Thermo Fisher Scientific</td><td>SH30919.03</td></tr><tr><td>CellTrace Violet Cell Proliferation Kit</td><td></td><td>Thermo Fisher Scientific</td><td>C34557</td></tr><tr><td>BD Canto II flowcytometer</td><td></td><td>BD Biosciences</td><td></td></tr><tr><td>Flowjo</td><td></td><td>TreeStar</td><td></td></tr><tr><td>Hematocytomer</td><td></td><td>Marienfeld superior</td><td></td></tr></tbody></table>

phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice

调节性T（ 调节性T细胞），其表达的Foxp3作为转录因子，是CD4 + T细胞的亚群。 调节性T细胞通过调节免疫应答发挥免疫耐受和内环境稳定的维护至关重要的作用。 调节性T细胞的主要作用是抑制效应T（T EFF）细胞的增殖和产生细胞因子如IFN-γ，TNF-α和IL-2的。它已被证明持久病原体感染和癌症发展过程中调节性T细胞的抑制T EFF细胞的功能能力得到增强。为了澄清调节性T细胞静息或发炎条件下的功能，已经制订了各种使用鼠标或人类T reg细胞在体外抑制测定的。这项研究的主要目的是建立一个比较表型和休息之间的抑制功能和差异激活调节性T方法细胞。以分离活化的T reg细胞，小鼠感染的淋巴细胞脉络丛脑膜炎病毒（LCMV）克隆13（CL13），LCMV的慢性菌株。与静息从幼稚小鼠分离的T reg细胞比从LCMV CL13感染小鼠的脾分离的T reg细胞表现出两个活化表型和增强的抑制活性。在这里，我们描述了体外的表型分析，从静止T reg细胞区分活化的T reg细胞的基本协议。此外，我们描述了完全活化的T reg细胞的抑制活性的测量的协议。

我们用2×10 6 PFU LCMV CL13的静脉注射为其生成与持续性病毒感染小鼠。调查在调节性T细胞和慢性病毒感染在t CONV细胞的表型的变化，从幼稚和被感染的小鼠获得脾淋巴细胞用各种抗体染色并通过流式细胞术进行分析。 的 CD4 + T CONV（ 图1A，上图）和Foxp3 - - CD8 + T CONV（ 图1B，...

Watch this Scientific Journal Video about Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice at JoVE.com

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

phenotypic-functional-analysis-activated-regulatory-t-cells-isolated

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.7K 次观看.  Yonsei University. Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from naïve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

视频: Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

科研

免疫与感染

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

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

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

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

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

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

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

人类免疫缺陷小鼠模型中的慢性、急性和再激活HIV感染

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

破译急性 LCMV 感染中的早期滤泡辅助性 T 细胞定型术

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

作者聚焦:阐明急性 LCMV 挑战中 TFH 细胞分化的途径

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

摘要

探索更多视频

此视频中的章节

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

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

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

细菌病原体在细胞和有机水平宿主反应探针中的应用

人类免疫缺陷小鼠模型中的慢性、急性和再激活HIV感染

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

摘要

探索更多视频

此视频中的章节

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

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

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

细菌病原体在细胞和有机水平宿主反应探针中的应用

人类免疫缺陷小鼠模型中的慢性、急性和再激活HIV感染

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

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

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化