我们感谢E. Massicotte和J.主的过程中流式细胞仪和细胞分选实验，专家技术援助。此外，我们要感谢的红外对抗诊所的工作人员和所有捐助者提供血液样本。外周血样本来自谁签署知情同意书，根据赫尔辛基下批准德研究所15-9诊所RECHERCHES蒙特利尔（IRCM）的研究伦理审查委员会的研究方案宣言健康成年供体。下列试剂是通过美国国立卫生研究院艾滋病试剂计划，艾滋病，NIAID，NIH司获得的：从道格拉斯里奇曼博士的MT4细胞;与人类重组IL-2莫里斯·盖特利博士，霍夫曼 - 罗氏公司科恩博士是加拿大首席科学家在人类反转录病毒学的收件人。这项工作是由CIHR（CIHR 111226）科恩博士和来自全宗德RECHERCHE魁北克 - 桑特（FRQ-S）艾滋病网络BEGO博士和Cohen博士支持拨款。

一般注意事项 它来保持培养中的细胞，没有任何抗生素，因为即使控制细菌感染可以由PBMC的感测是非常重要的。细菌等组件的感测会诱发一个背景干扰素的产生，不能从由特定的HIV-1感测触发的区分。此外，所有的细胞都必须定期地测试用于支原体污染是否存在。 始终使用无内毒素的解决方案，只要有可能。 在共培养中使用的不同的细胞的流式细胞术表征（PBMC中，的pDC，MT4和CD4 + T细胞）是一个可选的步骤，但强烈建议，因为它可以提供所需的给定的实验的正确解释的有价值的信息。 1.准备感染的靶细胞<ol><li>维持人体的 CD4 + T细胞系MT4在RPMI 1640 MEDI一个补充有10％胎牛血清（FBS），从现在起称为RPMI-1640完全培养基中。 </li><li>隔离原代CD4 + T细胞 。 <ol><li>通过密度梯度离心分离从人外周血单核细胞。 </li><li>隔离用CD4 + T细胞的阴性选择的试剂盒，按照生产商的建议的 CD4 + T淋巴细胞。 </li><li>激活的CD4 + T淋巴细胞用PHA-L（5微克/毫升）48小时，然后维持细胞在RPMI-1640完全培养基中补充有重组IL-2（100U / ml）中。原发性感染T细胞后5天的隔离。 </li></ol></li><li>感染靶细胞（MT4 T细胞系或异源的原代CD4 + T细胞 ）的。 <ol><li>前两天共培养，感染的T细胞与HI​​V-1病毒。在这里描述的特定示例pNL4.3-GFP_ires_Nef野生型（WT）或pNL4.3-GFP_ires_Nef增量VPU（dVpu）病毒被使用。这些病毒株代表GRE恩荧光蛋白（GFP）-marked仅会有不同的表达VPU能力等基因感染性分子克隆。 </li><li>使用感染（MOI病毒）不同的多重选择的文化有类似感染水平的联合培养。共培养的天，确定的GFP +细胞通过流式细胞百分比术作为感染的标志物。使用仅培养了一系列20-50％感染细胞用于共培养物。 </li></ol></li></ol>任选的步骤：执行流式细胞染色在这一点上，以评估感兴趣分子/配体，例如BST2，和CD4的表面表达。对于细胞表面染色，悬浮0.5×10 6 T细胞在100微升洗涤液，加入1微升抗CD4_PerCP /经Cy5.5和1微升抗BST2_A660的。孵育管45分钟，在4℃，用流式细胞仪洗涤溶液洗涤之前。如果研究的目的是评估VPU介导BST2拮抗作用，进行HIV-1颗粒在这个时候释放测定先前所描述的15。 2.隔离感测单元（整体或外周血单个核细胞富集的pDC）的富集注意：当的PBMC用于传感，开始采血后半小时内分离和及时进行它。立即用于共培养物或浓缩的pDC使用的PBMC。 <ol><li>通过密度梯度离心分离从人外周血单核细胞。 任选的步骤：执行多参数流式细胞术染色，以确认新分离的PBMC包含不同细胞谱系在生理比率。对于细胞表面染色，重悬在100微升的Fc阻断溶液10 6 PBMC中，并孵育15分钟，在4℃。封闭后，加入1微升抗CD3_Pacific蓝色（T细胞），1微升抗CD14-PE_Texas红（对骨髓细胞），4微升抗BDCA2_APC和1微升抗ILT7_PE的（为的pDCs）的。孵化管中在4℃下45分钟，用流式细胞仪洗涤溶液洗涤之前。 </li><li>丰富使用PDC的阴性选择试剂盒，按照制造商的建议的pDCs。建议在工作速度快，保持细胞的冷，并用预冷的解决方案。这将防止在细胞表面上和非特异性细胞标记的抗体的封盖，以及确保最大的pDC活性。 <ol><li>执行新鲜分离的pDC的多参数流式细胞仪分析，以证实纯度，富集的百分比，以及了pdc-特定表面标志物的相对量（如ILT7和BDCA2）。对于细胞表面染色，重悬在100微升的Fc封闭液10 4的pDCs，并孵育15分钟，在4℃。封闭后，加入1微升抗CD3_Pacific蓝，1微升抗CD14_PE_Texas红，4微升抗BDCA2_APC和1微升抗ILT7_PE的。孵育管45分钟，在4℃，用洗涤流术洗涤液（PBS，5前毫摩尔EDTA，5％FBS）中。最低40％的pDC纯度建议（如果起始材料具有0.4％的pDC，这将相当于100倍富集）。 </li></ol></li></ol> 3.共培养感染的CD4 + T细胞和感测单元（整体或外周血单个核细胞富集的pDCs）的<ol><li>混合220微升的PBMC（稀释为850000个细胞/ ml）或220微升的pDCs的（在一个浓度范围为10万至50万个细胞/ ml）与30μl的感染的T细胞（稀释在10 6个细胞/ ml），每孔中一个U型底96孔板。 <ol><li>作为对照，板外周血单个核细胞或PDC和T细胞的孤独。调整好，以250微升的RPMI 1640完全培养基的体积。 </li><li>评估细胞反应的TLR7和TLR9途径的已知激动剂。为此，板220微升的PBMC（稀释为850000个细胞/ ml），每孔或200微升的pDCs的（在一个浓度范围为10万至50万个细胞/ ml）在U型底96孔板中并添加TLR7激动剂（伊米quimod，最终浓度：2.5微克/毫升）或TLR9激动剂（ODN 2216的CpG-A中，最终浓度：5微米）。孵育细胞，在37℃和5％CO 2的18至22小时，并以类似于下面描述的共培养物的方式处理它们。 </li></ol></li><li>孵育共培养物在37℃和5％CO 2的18至22小时，然后将它们传输到V型底96孔板中。离心5分钟，在400×g下。 <ol><li>转移上清液至平底96孔板中。上清液可以储存在-80℃或立即用于I型干扰素的检测。 </li><li>悬浮共培养细胞在2％多聚甲醛，并使用流式细胞仪分析它们。由于在规模和造粒的差异，MT4细胞是从基于其侧散射（SS）与前向散射（FS）型材外周血单个核细胞或pDC细胞容易辨别。如果初级T细胞被用作靶感染的细胞中，这些可以与任何细胞追踪染料染色，如CSFE之前，共培养。 </li></ol></li><li>使用HEK-蓝的IFN-α/β报道细胞的生物活性的I型干扰素的测量。 注：由HEK293细胞与人类STAT2和IRF9基因稳定转染生成这些细胞获得充分活跃I型IFN信号通路。它们还包含IFN-α/β诱导性ISG54启动子控制之下的分泌型碱性磷酸酶（SEAP）的报道基因。这些细胞与I型干扰素的刺激激活JAK / STAT / ISGF3通路，诱导产生SEAP的。 <ol><li>保持的HEK-蓝IFN-α/β细胞在DMEM补充有10％FBS中。板他们以每孔50,000个细胞的一个平底96孔板的密度，在180微升的最终体积。 </li><li>加入20微升共培养物上清液至每个孔一式两份。每块板还需要一套内部标准控件（人干扰素α，从100 U /终浓度毫升至2500单位/毫升）。孵育所述板在37℃和5％CO 2的18-22小时。 </li><li>确定碱性磷酸酶活性用孔定量蓝溶液，将其从粉红色变成紫色/蓝色在酶的存在下的水平。准备量化蓝以下制造商的建议。添加180微升的该溶液到平底96孔板的每个孔中。到每个孔中也加入20微升的诱导的HEK-蓝IFN-α/β细胞的上清液，并孵育所述板在37℃培养箱，直到颜色在标准的IFN的对照孔的发展。 </li><li>评价SEAP的使用分光光度计水平620-655纳米和确定I型干扰素的浓度通过外推法从干扰素标准曲线的线性部分。 </li></ol></li></ol>

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这里所描述的协议测检测感染了GFP标记的HIV-1病毒仅会有不同的表达VPU能力，在我们最近的一份报告18中描述的T细胞。但是，这些方法可以很容易地进行修改，以研究传感未标记病毒以及缺乏其它病毒基因可能潜在调节先天传感病毒。如果使用不表达GFP的病毒，感染的靶细胞的百分比应通过共同培养，如p24的（衣壳）细胞内染色和流式细胞术或通过p24的ELISA现有的另一种手段来确定。此外，这些协议可以使用不同的靶T细胞在内的各种可用的细胞系和原代细胞。 时相比，用于研究HIV-1的感测方法早在本手稿中描述的方法具有许多优点。首先它已经建立由多个基团的无细胞的HIV-1 P文章是I型干扰素2,13,14不良诱导。此外，早期的研究依赖于较旧的IFNα基于ELISA的方法来量化的I型干扰素的产生量。这种检测技术的局限性是，大多数老IFNαELISA试剂盒仅测量一种类型的IFNα，而忽略了其他类型的IFNα和IFNβ的。使用所描述的报道细胞系的克服了这一限制作为人类I型IFN的所有生物活性形式的浓度可以一次进行测定。该技术是高度敏感的，比新产生的IFN EL​​ISA试剂盒和用于许多捐助者可以很容易地检测出大量的I型干扰素的成本更低。然而，该协议可以很容易地适于与其他的I型干扰素的读出的方法，如ELISA中使用。 关键步骤：重要的是，所有的细胞成分（包括靶和效应）的质量进行严格控制。潜在的污染，即使当asymptomatic，需要被监视和控制。正如我们前面所提到的，抗生素控制无症状细菌污染，以及潜在的其他细胞内病原体如支原体，可以产生类似的免疫应答的那些后HIV-1感染，即生产的I型干扰素的观察。此外，所有的试剂需要自由LPS或其他潜在的内毒素。同样，监测PBMC和的pDCs也是很重要的，因为人类样本通常可以通过底层持续感染，这可能会影响正常传感打底。我们建议总是包括建议的阴性对照，例如没有靶细胞，以及与模拟感染的细胞共培养。受感染的细胞的存活力也是正确的pDC传感自I型干扰素不产生以下共培养细胞凋亡2,14重要。 该技术的一个重要的限制是需要新鲜分离的原代细胞。这一程序是不使用要么以前冻结或保存在培养外周血单个核细胞或pDC的测试。的PBMC的分离是劳动密集型的，并且这些细胞具有非常有限的寿命。此外，虽然与人血液的工作应当用于防止血源性病原体标准协议。经常有变异性的几个来源与工作相关联的涉及新鲜分离的人细胞。其中是实施以分离这些细胞的不同的程序，和从供体到供体中遇到的遗传变异性。而这是不可能的，以避免供体间变异性，使用了建议的阳性对照，如测量响应于TLR7和TLR9的途径已知激动剂，将用于这些实验的一个重要的内部控制。我们观察到，一个强大的响应于TLR9途径的激动剂可以与健康的pDC响应相关联，而一个响应TLR7通路所需的抗HIV-1 3的适当的反应。 2，通常不需要从外周血单个核细胞富集的pDC的。然而，如果实验设计需要时（例如在上下文在需要的特定的pDC表面标志物耗竭）负选择是优选超过正选择，作为细胞选择过程后保持自由的抗体。孤立的pDC进行文化迅速凋亡。对于需要这些细胞的实验是在延长的时间周期中培养，培养基可以补充有IL-3。这种细胞因子诱导的pDC细胞增殖并抑制其凋亡16。然而，IL-3的使用需要被严格控制，因为它可能导致的pDC成熟或分化。 这里所描述的方法结合，以重新创建先天传感期间所涉及的初始步骤的目标HIV-1感染细胞的新鲜分离的PBMC或pDC细胞。这种方法无疑是有益的EXPLOR与HIV感染有关ê早期先天免疫活动。虽然相关协议的目的是测量I型干扰素，他们可以很容易地进行修改，以衡量在识别受感染细胞的pDC细胞释放的其它生物活性分子。这些可包括：III型干扰素（IFN-λ），促炎细胞因子（如TNF-α和IL-6）和趋化因子CXCL10，CCL4，CCL5和17。

I型干扰素生成的pDC表示防御病毒感染的第一道防线，并且因此作为先天免疫和适应性免疫1之间的重要环节。而不含细胞的HIV-1的颗粒是通过先天免疫系统检测到不良的HIV-1感染的 CD4 + T细胞通过的pDC 2有效地检测到。所述感测机构，需要在PDC上，然后接着病毒捕获和内由PDC被感染的T细胞和CD4分子的表面上的病毒包膜糖蛋白（gp120的）之间的初始接触。由TLR7后续识别的转移的HIV-1 RNA的触发MYD88 / IRF7途径的活化，并最终导致I型IFN的产生3。重要的是，第二感测途径中存在的pDCs，这是由TLR9介导的，由病毒糖蛋白，gp120的相互作用的抑制，与PDC特异性表面标记BDCA2 4。 5另一个的pDC-比表面抑制性受体调制。 BST2被以高水平表达的pDC在和一些癌细胞的表面上，但在相对较低的水平，其他类型的细胞，如B细胞，巨噬细胞和T细胞。重要的是，BST2可以通过I型干扰素在一些转化细胞系的，以及在人类和小鼠起源6的原代细胞培养诱导。除了 ​​其免疫调节功能，BST2最近发现抑制HIV-1的释放和其它包膜病毒颗粒由交联新生病毒颗粒到感染细胞表面 7。在HIV-1的情况下，该病毒编码辅助蛋白U（VPU）被证明作为BST2拮抗剂。据信，VPU下调BST2来自HIV-1感染的细胞的细胞表面上，它的系绳的部位荷兰国际集团的活性，其结果增强了病毒释放和传播7，虽然这一概念已被质疑8,9。在没有VPU，大量的完全成形的和成熟的子代的HIV-1颗粒被保留在细胞表面。无论这些拴颗粒可能代表免疫检测有效的目标仍然是一个悬而未决的问题。此外，目前还不清楚VPU是否能够通过调节表面BST2水平dysregulate I型IFN的产生从pDC细胞。 旨在评估HIV-1的感测早期研究用无细胞HIV-1的颗粒，其一般被认为是I型干扰素3,10-12的差诱导剂进行。鉴于最近的研究显示，PBMC和pDC的有效检测HIV感染的CD4 + T淋巴细胞2,13,14，我们在这里介绍一个简单的方法来衡量在识别HIV-1感染细胞的体外pDC的触发先天的反应。

<table border="0" cellpadding="0" cellspacing="0" width="652">
	<colgroup>
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	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:203px;">MT4 cells</td>
			<td style="width:87px;">NIH AIDS Reagent Program</td>
			<td style="width:103px;">120</td>
			<td style="width:260px;">This reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: MT-4 from Dr. Douglas Richman.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK-Blu IFN-&#945;/&#946; reporter cells </td>
			<td style="width:87px;">Invivogen</td>
			<td style="width:103px;">HKB-IFNAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">RPMI</td>
			<td style="width:87px;">Wisent</td>
			<td style="width:103px;">350-000-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM</td>
			<td style="width:87px;">Wisent</td>
			<td style="width:103px;">319-005-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">FBS</td>
			<td style="width:87px;">Wisent</td>
			<td style="width:103px;">080-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Ficoll-P&#226;que Plus</td>
			<td style="width:87px;">Myltenyi</td>
			<td style="width:103px;">17-1440-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CD4+ T cell isolation kit II</td>
			<td style="width:87px;">Myltenyi</td>
			<td style="width:103px;">130-091-155</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Diamond Plasmacytoid Dendritic Cell Isolation Kit II </td>
			<td style="width:87px;">Myltenyi</td>
			<td style="width:103px;">130-097-240</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">PHA-L</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">O2769</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:203px;">Human rIL-2</td>
			<td style="width:87px;">NIH AIDS Reagent Program</td>
			<td style="width:103px;">136</td>
			<td style="width:260px;">This reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Human rIL-2 from Dr. Maurice Gately, Hoffmann - La Roche Inc.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Imiquimod</td>
			<td style="width:87px;">Invivogen</td>
			<td style="width:103px;">TLRL-IMQS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">ODN 2216 CpG-A</td>
			<td style="width:87px;">Hycult Biotec</td>
			<td style="width:103px;">HC4037</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:203px;">Human IFN&#945;</td>
			<td style="width:87px;">PBL Interferon source</td>
			<td style="width:103px;">11100-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QUANTI-Blue </td>
			<td style="width:87px;">Cedarlane</td>
			<td style="width:103px;">REP-QB2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;"> </td>
			<td style="width:87px;"> </td>
			<td style="width:103px;"> </td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">Flow cytometry (Antibodies/reagents)</td>
			<td style="width:87px;"> </td>
			<td style="width:103px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Fc blocking solution (composition below):</td>
			<td style="width:87px;"> </td>
			<td style="width:103px;"> </td>
			<td style="width:260px;">Note: make in washing solution (PBS, 5mM EDTA, 5% FBS)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">10% Goat serum</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">G 9023</td>
			<td style="width:260px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">10% Rabbit serum</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">R 9133</td>
			<td style="width:260px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">10% Mouse serum</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">M 5909</td>
			<td style="width:260px;"> </td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">2.5 mg/ml human IgG</td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">I 4506</td>
			<td style="width:260px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-CD3_Pacific Blue</td>
			<td style="width:87px;">Biolegend</td>
			<td style="width:103px;">300417</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">anti-CD14_PE-TxRed</td>
			<td style="width:87px;">Life Technologie</td>
			<td style="width:103px;">MHCD1417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-BDCA2_APC</td>
			<td style="width:87px;">Myltenyi</td>
			<td style="width:103px;">130-090-905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-ILT7_PE</td>
			<td style="width:87px;">Biolegend</td>
			<td style="width:103px;">326408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-CD4_PerCP/Cy5.5</td>
			<td style="width:87px;">Biolegend</td>
			<td style="width:103px;">317427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">anti-BST2_Alexa 660</td>
			<td style="width:87px;">eBioscience</td>
			<td style="width:103px;">50-3179</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:203px;">Paraformaldehyde </td>
			<td style="width:87px;">Sigma-Aldrich</td>
			<td style="width:103px;">P 6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Cell strainer</td>
			<td style="width:87px;">BD Falcon </td>
			<td style="width:103px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;"> </td>
			<td style="width:87px;"> </td>
			<td style="width:103px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Equipment</td>
			<td style="width:87px;"> </td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cyan ADP instrument </td>
			<td style="width:87px;">Beckman Coulter</td>
			<td style="width:103px;">CY20030</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing the innate sensing of hiv-1 infected cd4+ t cells by plasmacytoid dendritic cells using an ex vivo co-culture system.

HIV-1的固有感测需要感染的CD4 + T细胞与浆细胞样树突细胞（pDC细胞）直接接触。为了研究这个过程中，此处所描述的协议使用新鲜分离的人外周血单核细胞（PBMC）或浆细胞样树突细胞（pDC细胞）以感测在任一T细胞系（MT4）或异源的原代CD4 + T细胞感染。为了确保正确的感测，至关重要的是，将PBMC采血和被感染的T细胞的所使用的最佳百分比后立即分离。此外，多参数流式细胞染色可用于确认PBMC样品含有不同细胞谱系在生理比率。一些控制也可包括评估生存能力和的pDCs的功能。这些包括，特定表面标志物的存在下，评估细胞反应Toll样受体激动剂已知（TLR）的途径，和确认缺乏斯波的taneous I型干扰素（IFN）的生产。在这个系统中，新鲜分离的PBMC或pDC细胞共培养与HIV-1感染的细胞在96孔板为18-22小时。从这些共培养物的上清液，然后用于通过监测ISGF3通路在HEK-蓝IFN-α/β细胞的活化，以确定生物活性的I型IFN的水平。事先和在共培养条件下，靶细胞可以经过流式细胞仪分析，以确定多个参数，包括感染细胞的杀伤差动感染细胞的百分比，特定表面标志物的水平，和。虽然这些协议最初开发到后续的I型干扰素的产生，它们可能被用来研究来自pDC细胞释放其他imuno-调节分子，并进一步了解理事HIV-1先天敏感的分子机制。

在图1中所描述的共培养系统允许HIV-1的固有感测的对照研究。由于主电池的可变性质，重要的是，髓样细胞和T细胞（CD14分别+和CD3 +）的正常比率观察到，如在图 2A所示的例子中此外，只有一个（与非活化的T细胞更高FS和更高水平的CD3）活化T细胞的数目低是所希望的，在新鲜分离的PBMC培养物。的PBMC与不同细胞类型之间的不正常的比率往往无法安装在体外 ，有可能适当的先天免疫反应，由于已激活的表型作为正在进行的感染的结果。最重要的是，需要的pDC的相对百分比，如图2B进行评估。虽然这个百分比可以变化从0.2％至1.2％，异常检测的表型，也观察到与两个奥妙该范围MES。使用阴性选择的pDC的富集往往产生50-95％的pDC纯度，如在两个例子中，从图2B（92％和72％）看出。的原型实例为整体实验示于图3和4。在这个例子中，MT4细胞用pNL4.3-GFP_ires_Nef WT或增量VPU达到30％的感染在共培养（图3A）的时间。如先前所描述，仅与WT病毒的感染导致显著下调表面BST2（图3B）。由于它们在尺寸和粒度的差异，MT4细胞可从外周血单个核细胞通过流式细胞术（图4A）容易辨别。共培养温育后，与已知的TLR7或TLR9激动剂和PBMC中的接触与HIV-1感染的细胞释放显著量的I型干扰素（图4B）的接触只的PBMC。在外周血单个核细胞中未检测到IFN独自一人，在外周血单个核细胞合铜ltured与模拟感染的MT4细胞中，或在单独感染的MT4 细胞 （图4B）。在这里介绍的实例中，HIV-1感染的MT4细胞的PBMC的固有感测被发现在VPU的存在下显著降低。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/51207/51207fig1highres.jpg" src="/files/ftp_upload/51207/51207fig1.jpg" width="500px" /> 图1：实验设计的示意图 SUP：上清<a href="https://www.jove.com/files/ftp_upload/51207/51207fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/51207/51207fig2highres.jpg" src="/files/ftp_upload/51207/51207fig2.jpg" width="500px" /> 图2：新鲜分离的PBMC和ENR表型特征iched的pDC。（A）的骨髓细胞，并在PBMC中从健康供体分离的观察T细胞的普通分布（骨髓细胞的百分比应之间在10-20％之间和T细胞55-65％的范围内）。使用识别表面CD14（髓系标志物）和CD3（T细胞标志物）荧光标记的抗体外周血单个核细胞样本进行染色。 （B）的pDCs的表型特征。之间细胞的PBMC中的总数（在实施例0.99％示出）的0.2-1.2％之前富集的pDC表示。这些细胞可以基于特定的标记，如BDCA2和ILT7，存在于细胞表面的表达来鉴定。负选择可以用来显著丰富的pDC的数量，并消除潜在的有害污染物，如CD14 +髓样细胞（浓缩达到92％和72％，在两个例子示出<a href="https://www.jove.com/files/ftp_upload/51207/51207fig2highres.jpg" target="_blank">）。Cli的</a>CK这里查看大图。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/51207/51207fig3highres.jpg" src="/files/ftp_upload/51207/51207fig3.jpg" width="500px" /> 图3：感染细胞的感染pNL4.3-GFP_ires_Nef WT或dVpu的MT4文化中的HIV-1感染靶细胞MT4表征（A）的百分比由GFP阳性细胞的百分比来确定。表面染色后，在感染的细胞（B）的BST2细胞表面表达进行评价。灰色实心直方图代表染色的免疫前兔血清（未染色的对照）假感染细胞;剩余的直方图代表染色的细胞用抗BST2多克隆兔血清。直方图用实线表示控制GFP负（负）细胞;而直方图用虚线对应于受感染的GFP阳性（正）的细胞。平均感orescence强度（MFI）值是表示各样品。流式细胞仪，如图2所述进行分析。 <a href="https://www.jove.com/files/ftp_upload/51207/51207fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/51207/51207fig4highres.jpg" src="/files/ftp_upload/51207/51207fig4.jpg" width="500px" /> 图4：新鲜分离的PBMC和感染的MT4 T细胞共培养流式细胞PBMC和MT4 T细胞之间的观察到的单独的MT4 T细胞的FS / SS型材，单独的PBMC，或共培养物的（A）的比较。由于在大小和粒度的差异，MT4 T​​细胞可以是从在使用流式细胞仪共培养的PBMC容易辨别。 （B）中的I型干扰素的PBMC的刺激TLR后释放的量的代表性例子7或TLR9激动剂（前），或在共培养与模拟或感染pNL4.3-GFP_ires_Nef WT或dVpu指定的MT4细胞。如图OD原始数据650值及其相应的converseion到I型干扰素的浓度以U表示/毫升。 <a href="https://www.jove.com/files/ftp_upload/51207/51207fig4highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System. at JoVE.com

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

不像无细胞的HIV-1颗粒，感染的CD4 + T细胞是由浆细胞样树突细胞（pDC细胞）有效地感测。这个手稿描述了一种方法，其中外周血单核细胞（PBMC）或分离的pDC是共培养的HIV-1感染的T细胞的pDC评估先天感测的评估，I型干扰素的释放。

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

assessing-innate-sensing-hiv-1-infected-cd4-t-cells-plasmacytoid

Research

JoVE Journal

Immunology and Infection

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

8.6K 次观看.  Institut de Recherches Cliniques de Montréal (IRCM). 不像无细胞的HIV-1颗粒，感染的CD4 + T细胞是由浆细胞样树突细胞（pDC细胞）有效地感测。这个手稿描述了一种方法，其中外周血单核细胞（PBMC）或分离的pDC是共培养的HIV-1感染的T细胞的pDC评估先天感测的评估，I型干扰素的释放。 

视频: Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

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 ...

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 ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

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 ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...