D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

在这项研究中，成年（6-8周）雌性C57BL / 6小鼠。小鼠根据根特大学动物饲养的指导原则安置和饲养。所有动物的程序获得批准的机构动物护理和伦理委员会。 1.准备单核从小鼠脾细胞（跨国公司）的<ol><li>通过颈脱位牺牲鼠标。 </li><li>把鼠标放在解剖板背下来，并修复后和前肢与引脚。 </li><li>消毒皮肤表面，用70％（体积/体积）乙醇/ H 2 O </li><li>切割沿腹部中线到胸部皮肤和使用无菌手术器械暴露脾脏。 </li><li>修整周围脂肪组织后，将脾脏成24孔板含1ml 1×磷酸盐缓冲盐水（PBS）在室温下。 </li><li>用无菌手术刀解剖脾脏和部分转移到70微米的尼龙过滤器放置在50毫升的浴缸即</li><li>采用2毫升注射器活塞，轻轻捣烂脾片通过细胞过滤和洗涤用5毫升1X PBS。 </li><li>加入3 ml的菲可帕克的至15ml管中并覆盖与5 ml的细胞悬浮液的密度梯度介质。 </li><li>离心在400×g离心20分钟，在RT下无制动。 </li><li>相间转移到新鲜15ml试管，加入10毫升冷的1×PBS和离心机在300×g离心10分钟，在4℃。 </li><li>重悬在2ml的1×PBS含有0.5％牛血清白蛋白（BSA）和1mM乙二胺四乙酸（EDTA）（FACS缓冲液）中的细胞。转移10微升细胞悬浮液到一个小管，混合，用10微升0.4％的</li></ol> 2.富集，检测和鼠标iNKT细胞的分离纯化<ol><li>富集的小鼠脾脏CD5positive（CD5 +）淋巴细胞。 <ol><li> （可选的）染色10 5个细胞与FITC缀合的抗CD5（4微升1点半稀释液/ 10 6个细胞）30分钟，在4℃下在黑暗中，洗净用200μlFACS缓冲液重悬在500μlFACS缓冲液。准备的4&#39;，6-二脒基-2-苯基吲哚（DAPI）在1×PBS中20μM的工作溶液中，加入1微升其至10 5个细胞在FACS缓冲液，并在室温下孵育5分钟。获得10 4个预富集细胞上的流式细胞仪。 <ol><li>使用FCS-H和FCS-W，电栅极上的FSC-W 罗细胞以排除细胞双峰和聚集物（ 图1A）。然后，电子门出DAPI +（死）了FSC-W 罗群（ 图1B）内的细胞，并使用SSC-A和FCS-A按大小（ 图1C）电子选择淋巴细胞群。 </li><li>使用淋巴细胞门控内的细胞，画出SSC-A与CD5和如图1D电子栅极CD5 +淋巴细胞。获取的流式细胞仪的预富集的样品的剩余部分。 </l我&gt; </ol></li><li>调整细胞计数，以每90微升10 7个细胞在FACS缓冲液，并添加10微升抗CD5微珠。 </li><li>孵育4ºC15分钟，用4ml FACS缓冲液重悬在500μl冷FACS缓冲液洗一次。富集CD5 +使用磁性细胞分离（MACS）淋巴细胞根据制造商的建议列。 </li><li>可选）染色10 5个细胞与FITC缀合的抗CD5（4微升1:30稀释/ 10 6个细胞）和DAPI作为2.1.1。收购10 4富集淋巴细胞的流式细胞仪，并验证电子门2.1.1创建的。选择CD5 +淋巴细胞中富集的细胞部分。获得10 5个细胞的流式细胞仪分析的比例存在于富集馏分CD5 +淋巴细胞如图1E - 小时。 </li></ol></li><li>检测和小鼠的iNKT CE隔离LLS用流式细胞仪。 <ol><li>重悬富集CD5 +淋巴细胞在约10 6个细胞每20微升在含有抗CD16 / CD32的FACS缓冲液（4微升1:50稀释/ 10 6个细胞）和PE缀合的αGalCer的浓度/ CD1d的四聚体（1微克/ 10 6个细胞）。 </li><li>下室温温育30分钟，在黑暗中。 </li><li>稀释小鼠抗体（mAbs）的抗CD3εV500（4微升1:20稀释/ 10 6个细胞），抗-CD19 E450（4微升1:50稀释/ 10 6个细胞）和抗CD8αE450（4微升1:50稀释/ 10 6个细胞）在FACS缓冲液中，添加到CD5 +淋巴细胞和在黑暗中温育30分钟，在4℃。 </li><li>通过在300×g离心FACS缓冲液离心10分钟，在4℃下重悬在4毫升，以10 7个细胞/ ml的终浓度清洗细胞用1ml的FACS缓冲液中。 </li><li>通过30微米的细胞滤网过滤细胞，然后将细胞在冰上。 </li><li>校准流式细胞仪对细胞根据制造商的建议14,15排序。 注意：流式细胞仪的细胞分选的校准需要培训，并且可以由有经验的操作者，或受过训练的技术人员进行。 </li><li>加入10微升的DAPI每10 6个细胞20μM的工作溶液，并在室温下孵育5分钟。获得大约10 4富集CD5 +淋巴细胞上的流式细胞仪。上FSC-W 罗细胞电子门，以排除细胞双峰和聚集物（ 图2A）。然后，电子门出DAPI + CD8 + CD19 +细胞 （转储通道）的FSC-W 罗群（ 图2B）内，并使用SSC-A和FCS-A作为2.1.1.1中描述的电子选择淋巴细胞（ 图2C） 。 <ol><li>剧情αGalCer/ CD1d的四聚体由CD3ε和电子门αGalCer/ CD1d的如图2D四聚体+CD3ε+ iNKT细胞。获得不超过2×10 5个CD5 +富集的淋巴细胞，以确定百分比αGalCer/ CD1d的四聚体+CD3ε+ iNKT细胞存在于富集细胞部分。 </li></ol></li><li>利用在2.2.7中创建的电子门。使用70微米的喷嘴处的&#39;1.5&#39;的流速，FACS排序αGalCer/ CD1d的四聚体+CD3ε+ iNKT细胞上的流式细胞仪，在70磅。收集在含有1ml 100％FCS一FACS管排序iNKT细胞。接着用10ml RMPI 1640培养基和离心将细胞在300×g离心10分钟，在4℃下冲洗从FACS管侧排序iNKT细胞。 </li><li>重悬排序iNKT细胞在1ml的RPMI 1640培养基中，并获得40微升其上的流式细胞仪评估排序iNKT细胞的纯度。选择FSC-W LO DAPI -淋巴细胞参考译文]在2.2.7所述。 （ 图2E - G）和电子栅极αGalCer/ 如图2H的CD1d四聚体+CD3ε+ iNKT细胞。 </li></ol></li></ol> 3.文化和扩展鼠标iNKT细胞 0天<ol><li>外套平底96孔板中，每孔纯化的抗CD3ε（3微克/毫升）处理1小时，在室温。用200μl的1×PBS和一次用200μl的完全RPMI 1640培养基（10％体积/体积的FCS，100U / ml青霉素 - 链霉素，5.5μMβ巯基乙醇）洗涤两次。 </li><li>计数使用0.4％血球作为可行排序iNKT细胞（步骤2.2.8）的数量在1.11，离心机在300×g离心10分钟，在4℃，重悬在5×10 5个细胞/在完全RPMI 1640培养基毫升含有重组鼠IL-2（10毫微克/毫升），重组鼠IL-12（1纳克/毫升）和可溶性抗小鼠CD28（5微克/毫升）。薄板S在抗CD3ε涂层钢板orted iNKT细胞在10 5个细胞/孔，并在 CO 2培养箱中在37℃下进行2天。 </li></ol>第2天<ol start="3"><li> 在 CO 2培养箱转移细胞到一个新的未涂覆的平底96孔板中并培养静止的条件下将细胞在完全RPMI 1640培养基中，在37℃下进行2天。 </li></ol>第4天<ol start="4"><li>通过温和移液移走从培养板上的细胞，将细胞转移到一个新的15ml试管。离心机在300×g离心10分钟，在4℃下，悬浮在5×10 5个细胞/在完全RPMI 1640培养基毫升含重组鼠IL-7（5毫微克/毫升），和板10 5细胞/孔在将平在 CO 2培养箱底的96孔板4天，在37℃。 </li></ol> 8天<ol start="5"><li>收集细胞的新管，离心300×g于4℃下用含有可溶性抗CD28（5微克/毫升）10分钟，并重新悬浮于5×10 5细胞/ ml，在完全RPMI 1640培养基。 </li><li>外套平底96孔板中，每孔纯化的抗CD3ε（3微克/毫升）洗如3.1。板10 5个细胞/孔抗CD3ε包被的板并在37℃孵育在 CO 2培养箱直到11天。 </li></ol>第11天 - 18 <ol start="7"><li>重复步骤3.4至3.6。 </li></ol> 19-20日<ol start="8"><li>转移细胞到一个新的15ml试管中，离心以300×g离心，重悬在5×10 5细胞/ ml，在完全RPMI 1640培养基。 </li><li>板的细胞在10 5个细胞/孔在平底96孔板中，并允许细胞休息2天，在37℃在一个实验使用之前CO 2培养箱。 </li></ol> 4，细胞因子生产用鼠标iNKT细胞使用的CD1d开发平台电子商务结合的分析<ol><li>外套平底96孔板用100μl重组鼠的CD1d的（在1×PBS中10微克/ ml）的每孔，孵育1小时，在37℃。 </li><li>用200μl的1×PBS洗涤四次，在1×PBS（封闭溶液），每孔加入200微升2％FCS的孵育2小时，在37℃。 </li><li>除去封闭溶液，每孔添加200微升αGalCer（5毫微克/微升），孵育所述板在37℃下16小时。 [对于制备αGalCer溶液，加1085μ1×PBS中至55微克αGalCer的溶于含96毫克/毫升蔗糖，10毫克/毫升的脱氧胆酸钠和5毫克/毫升吐温20的车辆。 </li><li>温育后，弃去αGalCer溶液和洗两次以及用200μl1×PBS中，随后通过加入200μl的完全RPMI 1640培养基。 </li><li>收集在体外扩增的 iNKT细胞在第21天，离心将细胞在300×g离心10分钟，在4℃下重悬在完成RPMI 1640培养基以0.5×10 6细胞/ ml的浓度。 </li><li>加入200μl每孔的细胞的iNKT再悬浮并在 CO 2培养箱中孵育72小时，在37℃。 </li><li>通过根据制造商的协议酶联免疫吸附试验（ELISA）收集感兴趣的上清液并测量细胞因子。 </li></ol>

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The authors have no competing interests to disclose.

在当前协议关键步骤包括分离和CD5 +淋巴细胞（第1和2）的随后的富集，FACS分选（第3节）和iNKT细胞的初始接种（第4节）。第1节执行的步骤，切记要仔细层中的脾细胞悬浮液密度梯度介质，使得不同的细胞相间的离心后产生的。随后的富集CD5 +淋巴细胞通过磁性细胞分离（第2部分）最大程度地减少染色所需的试剂和时间和FACS分类iNKT细胞（第3节），这有助于增加的iNKT细胞产量后?...

鼠不变的自然杀伤T（iNKT细胞）细胞是由CD1d的表达皮质胸腺细胞1,2在胸腺选择的先天T淋巴细胞的独特群体。 iNKT细胞表达由一个不变的Vα14-Jα18TCR链配对任Vβ8，Vβ7或Vβ2TCR的3，其能够识别内源性以及外国脂质抗原的CD1d的上下文中的T细胞受体（TCR）。例如，鼠iNKT细胞识别并通过内源性脂质抗原称为isoglobotrihexosylceramide（iGb3）4，以及α-半乳糖神经酰胺（αGalCer）5,6-，糖脂自海绵中分离被激活。 TCR依赖性iNKT细胞的活化促进适应性免疫应答的引发，并作为其结果，iNKT细胞已被证明是功能上参与的范围病状包括风湿性疾病7和癌症的改善或发展&lt;SUP&gt; 8。目前，合成的iNKT细胞的配体构成有希望的新的疫苗佐剂可以是能够调节的一些免疫病理状况。 先前已经证明，iNKT细胞可以在体外从以下然而小鼠组织分离来产生;许多研究中采用的使用主要抗原呈递细胞（APC）和/或细胞系9，Vα14TCR转基因（Tg）为10只小鼠，或胸腺瘤为的iNKT细胞衍生的杂交瘤11,12的产生。此外，大量的老鼠，大批量试剂如αGalCer加载CD1d的二聚体，和漫长的文化时代使一些出版协议不太道德和经济上的吸引力9,13。 在本报告中，我们描述了用于分离和在 iNKT细胞的小鼠脾脏体外扩增的方法适于。更具体地，协议描述了一种方法富集从小鼠脾脏这减少了所需的用于FACS细胞分选小鼠，试剂和时间，并提出了在体外扩展排序脾iNKT细胞的优化方法iNKT细胞。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>eBiosciences</td>
			<td>14-8021</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-12</td>
			<td>eBiosciences</td>
			<td>39-8122-65</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-7</td>
			<td>eBiosciences</td>
			<td>14-8071</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3e (145-2C11)</td>
			<td>eBiosciences</td>
			<td>16-0032-86</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD28 (37.51)</td>
			<td>eBiosciences</td>
			<td>16-0281-85</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD19 (eBio1D3)</td>
			<td>eBiosciences</td>
			<td>48-0193-82</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD8&#945; (53-6.7)</td>
			<td>eBiosciences</td>
			<td>48-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-mouse CD5 (53-7.3)</td>
			<td>eBiosciences</td>
			<td>11-0051-81</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-conjugated anti-mouse CD3&#949; (500A2)</td>
			<td>BD biosciences</td>
			<td>560771</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD5 (Ly-1) microbeads</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-049-301</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD16/32 (2.4G2)</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% (wt/vol)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-056</td>
			<td>[Ca2+/Mg2+ - free]</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00 AF</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate F-bottom</td>
			<td>Greiner-bio one</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner-bio one</td>
			<td>665-180</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Greiner-bio one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>227-261</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>Greiner-bio one</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>30 &#181;m filter</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacketed CO2 incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Kit</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

刺激后迅速分泌细胞因子的能力是不变的天然杀伤T（的iNKT）细胞谱系的功能特性。因此iNKT细胞的特征是一种天生的T细胞群体能够激活和转向适应性免疫应答。的改进的技术用于小鼠的iNKT细胞的培养和扩大发展能促进的iNKT细胞生物学中的体外和体内模型系统研究。这里，我们描述了分离和扩增的鼠脾iNKT细胞的优化过程。 从C57BL / 6小鼠脾脏被除去，解剖和紧张，将所得细胞悬浮液被层叠在密度梯度介质。离心后，脾单核细胞（MNCs）被收集和CD5阳性（CD5 +）淋巴细胞富集使用磁珠。是在CD5 +分数在iNKT细胞随后用^5;神经酰胺加载的CD1d四聚体和通过荧光激活细胞分选（FACS）纯化。 FACS分选iNKT细胞然后最初在体外培养的小鼠重组IL-7存在下被扩展之前使用的重组鼠细胞因子和板结合的T细胞受体（TCR）的刺激的组合。使用这种技术，大约可以在18-20天的培养，之后可用于功能测定法在体外 ，或在小鼠体内转移实验，他们会产生10 8 iNKT细胞。

使用密度梯度脾的单核细胞的分离需要大约1小时，并消除了使用以裂解红血细胞（红细胞）所需的试剂的。使用这种方法和碎屑的脏器应变被去除期间产生得到一种高收率的活细胞。通常，iNKT细胞的脾淋巴细胞池内的频率总T淋巴细胞的1至5％的范围内然而，这可以根据所使用的动物的年龄，性别和健康状况而变化。大约10 6 iNKT细胞可以从3只小鼠的合并脾和产量最高iNKT细胞来获取从6-8周?...

Watch this Scientific Journal Video about An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen at JoVE.com

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

an-optimized-method-for-isolating-expanding-invariant-natural-killer

Research

JoVE Journal

Immunology and Infection

11.9K 次观看.  Ghent University Hospital. Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents. 

视频: An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

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

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

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

T细胞受体在小鼠T细胞的逆转录病毒转导

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

科研

免疫与感染

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

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

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

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

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

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...