We are grateful to Dr. Jos&#233; Luis Daniotti for comments. We thank the assistance to Dr. J. Arturo Pimentel and the Laboratorio Nacional de Microscop&#236;a Avanzada-UNAM for acquisition of confocal images. I.M.-D. and T.M.V.C. were supported by grants 157634 and 253596 from Consejo Nacional de Ciencia y Tecnolog&#236;a (CONACYT), Sociedad Latinoamericana de Glicobiolog&#236;a, A.C. T.M.V.-C. is recipient of a scholarship (245192) from CONACYT. We also thank the support of the Red Tem&#225;tica Glicociencia en Salud - CONACYT (253596).

与知情同意和炫酷德Investigaciòn连接DinámicaCelular-自治大学德尔斯卡德莫雷洛斯生物伦理委员会批准获得从健康男性献血者外周血。 1.分离人幼稚CD4 + T细胞活化<ol><li>收集2毫升从通过知情同意健康人供体衍生的外周血，并用2毫升无菌PBS-EDTA的稀（1×磷酸盐缓冲盐水（PBS），2毫摩尔EDTA，pH 7.4）中。关于3毫升蔗糖溶液的顶端缓慢加入稀释的血液与1.077密度如前所述8。 注意:所引起的密度差在离心分离期间微分迁移将导致红细胞完全沉淀和密度较小的单核细胞层将形成以上。加入2ml外周血将purificati后呈现约0.5×10 6幼稚CD4 + T细胞在步骤。 </li><li>在在21℃（使用转子用于与为30mm固定角度圆底管），并且没有断点250 XG离心30分钟至产生外周血单核细胞（PBMC）的梯度。离心后，将PBMC中可以清楚地观察到，为白色环。收集用移液器将PBMC和转移到无菌管中，洗涤。 </li><li>洗涤三次后，在250×g离心10分钟，PBS-EDTA溶液进行幼稚CD4 + T细胞的纯化。排序方法或几种类型的纯化试剂盒可用于该目的。优选地，使用&gt; 1×10 7个PBMC中纯化。 <ol><li>通过用磁珠阴性选择纯化幼稚CD4 + T细胞。用抗CD45RO，CD8，CD14，CD15，CD16，CD19，CD25，CD34，CD36，CD56，CD123，抗TCRγ/δ，抗HLA-DR，和CD235a（血型糖蛋白A）的抗体进行阴性选择。由至f确定的CD4 + CD45RA +细胞评估纯度的百分比低流式细胞仪。 注意:任选地，进行到PBMC中的孵育在37℃和5％的CO 2与在10ml补充的培养基的1×10 6个细胞/ ml的密度，以促进单核细胞粘附，并继续与幼稚CD4 + T细胞纯化过夜。从外周血幼稚CD4 + T细胞的有效回收很大程度上取决于净化系统。 </li></ol></li><li>通过加入每PBS的孔250微升含5微克/ ml抗CD3单克隆抗体制备的24孔细胞培养板中，并在37°C孵育至少2小时。 注:96孔板可以用来当使用从幼稚CD4 + T细胞数量较少（ 例如，2×10 4 -1×10 5）。 </li><li>除去从24孔或96孔板的抗CD3抗体的稀释和洗涤用无菌1×PBS洗涤两次板。 </li><li>用稀释纯度&gt; 95％的幼稚CD4 + T细胞（CD4 +CD45RA +）以补充有胎牛血清的3％的1×10 6个细胞/ ml，在先进的RPMI 1640培养基中的密度（或RPMI补充有血清的10％），2mM谷氨酰胺和抗生素（1 100U / ml青霉素，1 100μg/ ml链霉素）。 注意:如果神经节苷脂本地化外周血单个核细胞中要求，遵循同样的协议进行稀释和刺激，如下所述。 </li><li>加入500微升细胞稀释的抗CD3包被的孔，并在24孔板非涂布孔中（对照细胞）。备选地，加入100微升细胞稀释到96孔板的孔中。 </li><li>通过加入抗CD28抗体的1微克/毫升完成幼稚的 CD4 + T细胞在抗CD3包被的孔的刺激条件。 </li><li>保持静止的 CD4 + T细胞在非包被的孔控制在不加抗CD28抗体。孵育0静止和活化的CD4 + T细胞，以72小时的37℃的标准条件下C和5％ 的 CO 2。 </li><li>为了验证一个适当的活化静息流式细胞仪检测CD69早期活化标记（16小时激活后）或CD25晚期活化标志（48小时后激活）的表达，评估和激活，在37℃下培养幼稚CD4 + T细胞和5％的CO 2在不存在或抗CD3 / CD28抗体9,10的存在。 </li></ol> 2.休息的坚持和激活幼稚的CD4 + T细胞<ol><li>消毒通过高压和紫外线照射12毫米圆形盖玻片至少15分钟。 </li><li>地方盖玻片在无菌的24孔细胞培养板。 注意:对于具有小于1×10 5细胞培养物是优选使用滑动室有适于小孔，以防止细胞分散在盖玻片。 </li><li>涂层盖玻片（或滑动室）用200μl聚L-赖氨酸（MW 150-300千道尔顿）的0.1％（重量/体积）溶液，并孵育在r 5分钟OOM温度（RT），取出，晾干，在室温至少1小时。 </li><li>仔细混合并收集来自24孔板的休息和激活幼稚的 CD4 + T细胞。计数细胞，并在必要时用新鲜补充的培养基调整至1×10 5个细胞转移到聚-L-赖氨酸涂覆的盖玻片。孵育所述细胞在37℃下至少6小时和5％的CO 2。 </li></ol> 3.收集和休息的固定和激活幼稚的CD4 + T细胞<ol><li>小心地从培养的休息和激活幼稚的 CD4 + T细胞的培养基。 </li><li>添加RT无菌PBS500μl的慢。 注意:从井溶液仔细添加和去除防止细胞的分离从盖玻片。 </li><li>弃去PBS，并添加另外500微升PBS彻底清除培养基。 </li><li>加入500微升过滤4％多聚甲醛（FILT修复细胞配给移除可与显微镜分析干扰），并在RT（优选的）或在4℃过夜温育30分钟微粒。 注意:多聚甲醛是一种有毒的挥发性化合物。它被称为是一个人类致癌物。用适当的安全协议，请谨慎使用。 </li><li>除去固定剂，并在室温用PBS洗涤至少两次。 </li><li>继续进行下一步骤，或保持板孔用500μl的PBS在4℃下数天，直至染色。 注:在此过程中不育有助于避免微生物的生长。 </li></ol> 4.染色，安装和可视化激光共聚焦显微镜<ol><li>从孔中取出PBS中。加入500微升冷封闭缓冲液（1×PBS，0.5％的标准级牛血清白蛋白，1％胎牛血清pH 7.4）中。孵育在冰上20分钟。 </li><li>小心地取出封闭液，添加的主要抗体（抗神经节苷脂GD3克隆R24，GD2克隆14G2a）PE标记的抗CD25和APC缀合的抗TCR和同种型在封闭缓冲液以2.5微克/毫升稀释的控制。 </li><li>在冰上孵育2小时或过夜，在4℃。 注:缓慢轨道搅动是优选的。 </li><li>小心取出抗体和缓慢加入500微升的PBS。重复两次。 </li><li>添加二次抗体在封闭缓冲液以0.1微克/毫升稀释（为R24抗GD3，的Alexa Fluor为14G2a抗GD2 488缀合的或的Alexa Fluor 647缀合的抗IgG2a的FITC缀合的抗IgG3的）。 </li><li>孵育在黑暗冰1小时无晃动。 </li><li>在PBS中洗涤三次如4.4所述）。保持有足够的PBS的孔中，以避免样品脱水。 </li><li>添加赫斯特333258染色在PBS中稀释至0.1微克/毫升。 </li><li>孵育15分钟，在室温和删除。在PBS中洗涤三次。 </li><li>放置在纸巾上的幻灯片，并添加20微升安装溶液（在PBS中的50％的甘油）或商业安装溶液s到避免褪色和样品淬火。 </li><li>小心地拿起用细镊子盖玻片，并将其放置在安装的解决方案。确保在细胞附着在盖玻片的侧面与所述溶液接触。密封指甲油盖玻片并用荧光或共聚焦显微镜分析。 </li></ol> 5.隔离的RNA <ol><li>制备抗CD3抗体（5微克/毫升）包被的96孔板上。孵育4×10 4个幼稚CD4 + T细胞/孔在100μl补充培养基与抗CD28抗体（1μg/ ml的）来完成的刺激。在37℃和5％CO 2的0至72小时。 </li><li>不同的激活后次后放置细胞成离心管中。 </li><li>在250 XG添加的PBS 500微升，离心在RT 5分钟。 </li><li>除去上清液，加入1ml Trizol试剂。 </li><li>在室温下孵育5分钟，并保持样品在-8076℃持续至少24小时。在需要时与RNA分离进行。 注意:在-80℃下离开样品至少24小时提高RNA的产率。此外，利用手的手套是必不可少由RNA酶，以避免RNA降解。 </li><li>除霜温育的样品在37℃水浴中2分钟。 </li><li>加入200μl氯仿，用手30秒（以避免RNA侵略性混合涡旋）剧烈震荡。孵育5分钟，在室温。 </li><li>在4℃下12000 xg离心离心15分钟。 </li><li>回收水相到一个新的离心管中。 </li><li>加入500微升异丙醇并倒转试管三次。 </li><li>孵育在室温10分钟，离心10分钟以12,000 xg离心在4℃下。 </li><li>丢弃由倒置上清。 </li><li>以12,000 xg离心加入1ml冰冷的75％乙醇，离心10分钟，在4℃。 </li><li>完全弃去上清液并通过在离开管打开的盖干燥RNA沉淀。 不E:在这一点上的粒料不是清晰可见。仍要继续。 </li><li>重悬沉淀于20μl的分子级的水。通过测定使用的NanoDrop 260 nm和280nm处的吸收计算浓度和核酸纯度。 </li><li>通过使用任何传统的逆转录酶试剂盒，并按照manufacturer&#39;s协议认真着手合成cDNA。 注意:从4×10 4个幼稚的 CD4 + T细胞中获得约0.5-2微克的RNA。 cDNA可从100ng的RNA的被原料合成，并无需低RNA输入试剂盒在实时PCR反应中使用。 </li></ol>

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The authors have nothing to disclose.

所述协议可以用于本地化神经节苷脂或蛋白质中的 CD4 + T细胞或其它免疫细胞（ 例如 ，PMBCs， 图5）由少数细胞起始的细胞悬浮液。因为T细胞和非粘附性的小尺寸的，收购的信息差或低质量的荧光显微图像的结果，如果该细胞不正确地附着。 该协议具有良好质量的共聚焦显微镜分析相结合是对使用技术如薄层色谱法检测神经节苷脂的重要?...

的CD4 + T细胞协调通过其效应子功能由抗原呈递细胞1活化后的免疫应答。被激活期间调制的细胞机制的研究允许洞察免疫功能的一基本过程。然而，因为它们在血液周2代表细胞的非常小的群体幼稚CD4 + T细胞的研究可能是复杂的。 通过荧光显微镜几个报告已经研究参与CD4 + T细胞活化的不同分子，主要关联至质膜3蛋白的定位。神经节苷脂含唾液酸鞘糖脂，虽然他们已经在他们丰富，其他细胞等免疫细胞的神经细胞被广泛研究，也表达了与生物学相关职能4,5神经节苷脂。我们以前报道THA人幼稚CD4 + T细胞的活化有所述α2,8唾液酸转移酶ST8Sia 1（GD3合酶）和GM2 / GD2合酶的上调在t，诱导GD2的显著表面neoexpression和GD3神经节苷脂6的上调。在免疫细胞GD3，GD2和其他神经节苷脂的进一步研究是必要的，以补充免疫功能的蛋白质为基础的局部视图。 通常，神经节苷脂表达的研究是基于技术如薄层色谱法（TLC）7，但该技术不允许神经节苷脂的空间定位在质膜或在亚细胞区室，限制生物分析。 在这项工作中，我们描述了在人幼稚CD4 + T细胞和PBMC中抗CD3 /抗CD28活化后GD3和GD2神经节苷脂的抗体介导的识别和定位的协议。有了这个协议，我就也有可能以分析神经节苷脂的基因和分子表达在低数量在悬浮细胞，以获得高质量的图像6，考虑淋巴细胞的小尺寸（9微米）。

<table border="0" cellpadding="0" cellspacing="0" width="1052">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:481px;">Advanced RPMI 1640&#160;</td>
			<td style="width:241px;">Thermo Fisher Scientific</td>
			<td style="width:137px;">12633-012</td>
			<td style="width:193px;">Suplemented with 3% FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD3 antibody</td>
			<td>eBioscience</td>
			<td>16-0037-81</td>
			<td>OKT3 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human CD28 antibody</td>
			<td>ebioscience</td>
			<td>16-0288-81</td>
			<td>CD28.6 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD3&#160; antibody</td>
			<td>Abcam</td>
			<td>ab11779</td>
			<td>R24 clone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti human GD2 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53831</td>
			<td>14G2a clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:481px;">FITC-conjugated anti-mouse IgG3 
			antibody</td>
			<td>Abcam</td>
			<td>ab97259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647 conjugated antimouse</td>
			<td>Thermo Fisher Scieni&#161;tific</td>
			<td>A-21241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 333258</td>
			<td>Sigma-Aldrich</td>
			<td>861405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Ficoll Paque-Plus</td>
			<td>GE</td>
			<td>17-1440-02&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Naive CD4+ T Cell Isolation Kit II, human</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Sorting&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-tek chamber slide system</td>
			<td>Sigma-Aldrich</td>
			<td>C7182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus FV 1000 Laser Confocal Microscope (Olympus)&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Upright BX61WI and IX81&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GD3 synthase</td>
			<td>5&#180;-GAGCGTTCAGGAAACAAATGG- 3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GD3 synthase</td>
			<td>5&#180;-CCTGTGGGAAGAGAGAGTAAG-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forward sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-CAACACAGCAGACACAGTCC-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse sequence primer GM2/GD2 synthase</td>
			<td>5&#180;-GTGGCAATCGTGACTAGAGC-3&#180;</td>
			<td></td>
			<td>Ref. 7</td>
		</tr>
	</tbody>
</table>

preparation of cd4+ t cells for analysis of gd3 and gd2 ganglioside membrane expression by microscopy

在悬浮液中和对幼稚CD4 + T细胞的活化本文描述的方法及其在盖玻片共聚焦显微镜分析依从允许参与CD4 + T细胞活化神经节苷脂的空间定位和可视化，即补体表达分析实验，如流式细胞仪，西印迹或实时PCR。神经节苷脂表达通过流式细胞仪，并通过显微镜它们的细胞定位的量化可以通过使用抗神经节苷脂抗体以高亲和力和特异性来获得。然而，在悬浮细胞的适当处理涉及培养板的处理，以促进对荧光或共聚焦显微镜获得所需的必要的粘附性。在这项工作中，我们描述了幼稚的 CD4 + T细胞活化期间确定GD3和GD2神经节苷脂表达并与TCR共定位的协议。此外，真正的-T使用&lt;40,000个细胞IME PCR实验为GD3和GM2 / GD2合酶基因的测定中描述，这表明基因分析实验可以低数量的细胞，无需额外的低输入的RNA试剂盒来进行。

在这个手稿中描述的协议使得培养良好的质量和附着静息和活化的人幼稚CD4 + T细胞（ 图1）。活化的CD4 + T细胞显示出相比于静止状态（ 图1A）的特征的增殖曲线（ 图1B）。的CD25迟活化标志是评价在通过共聚焦显微镜（ 图1C）观察到72小时的有效激活是有用的。的CD69标记当前通过流式细胞仪或显微镜用作?...

Watch this Scientific Journal Video about Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy at JoVE.com

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

我们描述了在显微镜使用，以确定在静止神经节苷脂和激活的人幼稚CD4 + T细胞的细胞膜表达和定位一个标准抗体染色方案。还使用&lt;40,000个细胞，不需要额外的低输入RNA试剂描述的实时PCR实验。

CD4的制备<sup&gt; +</sup&gt; T细胞通过显微镜分析GD3和GD2神经节苷脂膜表达的

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

preparation-cd4-t-cells-for-analysis-gd3-gd2-ganglioside-membrane

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

8.4K 次观看.  Universidad Autónoma del Estado de Morelos. 我们描述了在显微镜使用，以确定在静止神经节苷脂和激活的人幼稚CD4 + T细胞的细胞膜表达和定位一个标准抗体染色方案。还使用

视频: Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

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

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

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

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

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

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

成像的CD4 T细胞间质迁移发炎真皮

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

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

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.

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 ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

CD4的制备

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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

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

隔离和幼稚的CD4 T细胞的Th17细胞分化

评估的HIV-1感染的CD4的先天传感

诱导小鼠肠道炎症由效应CD4的过继转移

成像的CD4 T细胞间质迁移发炎真皮

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

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

猕猴免疫缺陷病毒感染 CD4⁺ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

CD4的制备

摘要

探索更多视频

此视频中的章节

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

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

隔离和幼稚的CD4 T细胞的Th17细胞分化

评估的HIV-1感染的CD4的先天传感

诱导小鼠肠道炎症由效应CD4的过继转移

成像的CD4 T细胞间质迁移发炎真皮

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

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

猕猴免疫缺陷病毒感染 CD4+ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

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

猕猴免疫缺陷病毒感染 CD4⁺ T 细胞单细胞定量的 mRNA 和表面蛋白的表达