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 border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">FITC Rat Anti-Mouse CD4</td>
			<td style="width:67px;">RM4-5</td>
			<td style="width:173px;">BD Biosciences</td>
			<td style="width:104px;">553047</td>
			<td style="width:249px;">Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytofix/Cytoperm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-Bottom Tissue Culture Plates</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation buffer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td align="right">554655</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC Rat Anti-Mouse CD25</td>
			<td>7D4</td>
			<td>BD Biosciences</td>
			<td>553072</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 70mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td>Use this strainer for grinding the whole spleen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 40mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td>Use this strainer for filtering the cells before column enrichment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 421 Anti-mouse CD279 (PD-1)</td>
			<td>29F.1A12</td>
			<td>BioLegend</td>
			<td>135217</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 605 Anti-Mouse CD4</td>
			<td>RM4-5</td>
			<td>Biolegend</td>
			<td align="right">100547</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC Anti-Mouse/Rat Foxp3&#160;</td>
			<td>FJK-16s</td>
			<td>eBioscience</td>
			<td>17-5773</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td></td>
			<td>eBioscience</td>
			<td>00-5223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP-Cyanine5.5 Anti-Mouse CD8a</td>
			<td>53-6.7</td>
			<td>eBiosicence</td>
			<td>45-0081</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-gamma Platinum ELISA</td>
			<td></td>
			<td>eBiosicence</td>
			<td>BMS606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X)</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK Lysing Buffer</td>
			<td></td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine, 200mM solution</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin, 10,000U/mL</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td></td>
			<td>Life technologies</td>
			<td>L-34975</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 500X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8a+ T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4+CD25+ Regulatory T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-091-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LD columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-901</td>
			<td>Use this column for Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LS columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-401</td>
			<td>Use this column for CD8+ T cell and Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA, 0.5M (pH 8.0)</td>
			<td></td>
			<td>Promega</td>
			<td>V4231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td></td>
			<td>Sigma Life Science</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30919.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Violet Cell Proliferation Kit</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Canto II flowcytometer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer*</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowjo</td>
			<td></td>
			<td>TreeStar</td>
			<td></td>
			<td>Flow cytometry software&#8224;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematocytomer</td>
			<td></td>
			<td>Marienfeld superior</td>
			<td></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

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

科研

免疫与感染

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

神经干/祖细胞在小鼠EAE的慢性全身性注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

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

从外周血人类同种异体抗原特异性T细胞的生成

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

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

生物膜启动对病毒感染的细胞测定细菌和真菌

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

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

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

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

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

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