我们感谢下院实验室的成员提供咨询和协助。得到美国国立卫生研究院 (R01-AI087644 M.H. 和 P30-CA008748 的支持, 纪念斯隆-凯特林癌症中心)。

1. 制备刺激性玻璃表面
<ol><li>涂层八井腔 coverglass 与生物素化聚 l-赖氨酸 (生物锁相环) 稀释1:500 在蒸馏, 去离子水 (ddH2 O)。室温下孵育30分钟 (RT)。</li>
	<li>使用 H2进行清洗。</li>
	<li>在 RT 干燥2小时。</li>
	<li>块生物锁相环涂层表面与阻塞缓冲 (HEPES 缓冲盐水 [10 毫米 HEPES pH 值 7.4, 150 毫米氯化钠], 与 2% BSA) 为30分钟在 RT。<ol>
<li>将5毫克的聚 l-赖氨酸氢溴酸盐溶于1毫升的10毫米矫正假肢4, pH 8.5。</li>
		<li>添加125μmol (0.55 毫克) 的 NHS-生物素 (从100毫克/毫升的股票在亚砜) 和检查的 pH 值的反应。如果 ph 值低于 8.5, 则添加 2 ul 4 N 氢氧化钠, 将其提高到 ph 值8.5 和9.5 之间。</li>
		<li>涡流为30分钟。</li>
		<li>淬火反应与 50 ul 100 毫米甘氨酸溶解在20毫米三 pH 值8.0。</li>
		<li>全功率旋转10分钟, 将上清液转移到新管上。 注: 生物锁相环的质量通常与以前的制剂相比, 通过将材料的两倍稀释涂敷在96井 ELISA 板上, 并用碱性磷酸酶结合链亲和素进行孵化后定量化素含量。</li>
	</ol>
</li>
	<li>删除阻止缓冲区。不要让水井干。添加链亲和素 (100 µg/毫升在阻塞缓冲区)。在4摄氏度孵育1小时。</li>
	<li>在哈佛商学院洗。填充和反转室滑井 4-5 次, 从井中删除哈佛商学院。不要让水井干。</li>
	<li>将生物素化抵押贷款公司配体和黏附分子添加到表面。 注意: 可通过将邻硝基保护组 (例如, nitrophenylethyl (photocaged) 或 nitroveratryloxycarbonyl (NVOC)) 添加到关键 lysines (mcc, K12 在卵中 K7) 的ε氨基组中, 来 MCC 和卵子。Photocaged MCC 和卵子可以从商业供应商获得。重建后的水缓冲, 他们被折叠到细菌表达的 I Ek和 H2-Kb使用已建立的方法11,12。</li>
	<li>photoactivatable MCC 或卵肽的验证<ol>
<li>Decage NVOC 保护肽通过紫外线照射与手持紫外线灯 (见材料表) 在 RT 20 分钟。</li>
		<li>脉冲 1.0 x 105 apc 与1µM nonstimulatory 肽 (阴性控制,例如Hb), 非辐照 photoactivatable 肽, 辐照的 photoactivatable 肽, 和激动肽 (正控制, 例如, MCC) 为 1 h 到隔夜在37°c。</li>
		<li>刺激 T 细胞表达5C。C7/2B4/AND TCR, 使用 CH12 或 CH27 B 细胞。为了刺激 OT1 T 细胞, 使用 RMA 或 EL4 胸腺瘤细胞。</li>
		<li>将脉冲 apc 与相同数量的 T 细胞在96井圆底板上混合, 最终体积为200µL. 孵育37°c 为 12-24 h。</li>
		<li>用链亲和素-辣根过氧化物酶比色检测法, 从各井中恢复上清液, 并进行酶联免疫分析 IL-2。 注: 强烈建议通过电喷雾质谱法确认所有肽的锁定状态, 然后再复性为 MHC 复合体。</li>
	</ol>
</li>
	<li>对 MHC 进行折叠和纯化, 以尽量减少光照射。例如, 用铝箔包裹折叠反应, 用 UV 灯关闭凝胶过滤层析。 注: 发现 photoactivatablek和卵 H2-Kb在高密度下诱导紫外独立 T 细胞活化, 可能是由于功能不完全的锁定。因此, 在固定化步骤中, photoactivatable 抵押贷款公司被稀释成 10 30x 以上的 nonstimulatory 抵押贷款公司 (例如I -e k,其中包含血红蛋白64-76 肽 (Hb))。这提高了后续成像实验的信噪比。</li>
	<li>若要 photoactivate CD4+ T 单元格, 请集体使用生物素化k (3 µg/毫升)、生物素化 photoactivatableMCC -e k (0.1 µg/毫升) 的混合物, 并对 i类MHC 生物素化 k (0.5 H2-K/mL). anti-H2-KK抗体鼓励5C。C7/2B4/AND T 细胞, 表达 H2-KK, 扩散到玻璃表面而不进行激活。</li>
	<li>对于 CD8+ T 单元格, 请使用生物素化H2-D b 的混合体, 其含肽 KAVYDFATL (1 µg/毫升)、生物素化photoactivatable OVA-H2-K b (0.1 µg/毫升) 和黏附分子 ICAM-1 的胞外域 (2 µg/毫升由昆虫细胞培养产生的13)。ICAM-1 通过将整合素 LFA1 在 T 细胞表面上, 鼓励紧密的接触形成。</li>
	<li>将所有蛋白混合物应用于阻塞缓冲液中, 然后在 RT 中孵化1小时, 或至少2小时4摄氏度。 注: 此类表面的分子密度为每µm2的 8000, 以前已确定为6。鉴于 photoactivatable 抵押贷款公司代表了生物素化蛋白在表面上的~ 1/30 th, 它的密度将是 decaging之前每µm 2 的267个分子。</li>
	<li>在步骤1.6 中进行清洗, 并在哈佛商学院离开, 直到准备使用。</li>
	<li>将 20万 CD4+或 CD8+ T 单元格表示为每个井中适当的 TCR, 并允许单元格在37摄氏度内坚持15分钟。一旦细胞在表面上附着并扩散, 它们就可以 photoactivation 和成像。 注: 荧光成像探针对效应 T 细胞的逆转录病毒转导, 详细描述 elswhere6,7。钙信号也可以研究使用 untransduced T 细胞加载的钙敏感染料 Fluo-46。比例染料 Fura-2 是不推荐的, 因为它需要在紫外线范围内的激发, 这也诱导 decaging photoactivatable 抵押贷款公司。</li>
</ol>2. 图像采集
<ol><li>使用带有 UV 兼容150X 物镜的倒置 TIRF 显微镜进行图像采集。紫外线照射用户定义的区域使用一个数字隔膜系统连接到 100 W 汞灯 (HBO)。使用 400 nm 长的后视镜将这盏灯上的紫外线直接照射到样品上。</li>
	<li>使用图像分析软件进行局部 photoactivation 和时间推移采集。在大多数实验中, 分别使用 488 nm 和 561 nm 激发激光器对绿色和红色通道中的探头进行监测。激光光被定向到样品上, 使用双通带分色镜, 也传输在紫外线范围 (以使 decaging)。有关显微镜配置的其他信息, 请参阅材料表和图 6 。</li>
	<li>安装包含 T 单元格的会议厅幻灯片后, 根据需要调整设置以获得 TIRF 或荧光照明。在 "实时" 模式下, 选择表示感兴趣的荧光探针的单元格字段。使用软件控制建立单个细胞下 photoactivation 的微米尺度区域。</li>
	<li>开始时间推移获取。通常, 获取 80 timepoints, 每个时间点之间间隔为5秒。在双色实验的情况下, 这给连续 488 nm 和 563 nm 曝光留下了足够的时间。</li>
	<li>10 timepoints 后, 通过打开数字隔膜快门 1-1.5 s, photoactivate 选定区域。</li>
	<li>完成时间后, 选择一个新的单元格字段并重复该过程。</li>
</ol>3. 数据分析
注: 固定化配体的局部 photoactivation 创建一个定义良好的固定刺激区域, 可用于定量分析信号响应和骨架重塑事件。分析协议通常涉及在辐照区域内量化荧光强度, 或者使用辐照区域作为距离测量的位置端点 (e. g. 用于评估中心体的偏振度到辐照区域)。下面介绍两种分析协议。各种交互式图像分析程序可用于进行强度和距离测量, 然后可以输出以进行额外的处理和分析。
<ol><li>
荧光强度
	<ol>
<li>确定单元格外部的背景区域 (fib) 的荧光强度 (fi)。这将用于背景更正。<ol>
<li>在单元格外绘制一个微米大小的正方形区域并制作一个面具。要确定荧光强度, 请单击分析 |掩码统计信息。选择平均值荧光强度并导出值。 注意: 过程详细信息 (例如3.1.1.1) 引用 Slidebook 软件 (请参阅材料表)。使用其他软件包 (e. g) 实现此协议将稍有不同。</li>
		</ol>
</li>
		<li>确定每个时间点的光活化作用区域内的 FI。<ol>
<li>选择掩码以突出显示光活化作用的区域。要确定荧光强度, 请单击分析 |掩码统计信息。选择平均值荧光强度并导出值。</li>
		</ol>
</li>
		<li>从区域内的实测 fi 值中减去背景 fi。然后, 通过在 photoactivation 前9帧的平均、背景校正 fi 划分, 使校正后的 fi 测量正常化。ΔF = (fib)/平均 (fi1-9)-fib))。 注: 图ΔF 作为时间的函数。</li>
	</ol>
</li>
	<li>
距离
	<ol>
<li>获取光活化作用区域中心的 x 和 y 坐标。<ol>
<li>要确定光活化作用区域中心的 x 和 y 坐标, 请选择掩码以突出显示光活化作用的区域。突出显示 photoactivation 区域后, 选择分析 |面具统计 |区域中心并导出值。</li>
		</ol>
</li>
		<li>确定每个时间点感兴趣的荧光探针的 x 和 y 坐标。这通常是通过手动或自动粒子跟踪实现的。<ol>
<li>要确定感兴趣的荧光探针的 x 和 y 坐标, 请选择手动粒子跟踪, 然后单击感兴趣的荧光探针。跟踪所有时间点后, 选择分析 |面具统计 |区域中心并导出值。</li>
		</ol>
</li>
		<li>使用等式计算每个时间点的荧光蛋白与光活化作用区域中心的距离: 距离 = √ (x2-x1)2 + (y2 1)2),其中 x2和 y2是感兴趣的荧光蛋白的坐标, x1和 y1是光活化作用区域中心的坐标。</li>
		<li>将距离图作为时间的函数。</li>
	</ol>
</li>
</ol>

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Organ. 105, 59-72 (2014).</li><li>Grusch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporally+precise+activation+of+engineered+receptor+tyrosine+kinases+by+light.">Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.</a> EMBO J. 33, 1713-1726 (2014).</li><li>Yi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+repositioning+in+T+cells+is+biphasic+and+driven+by+microtubule+end-on+capture-shrinkage.">Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage.</a> J. Cell Biol. 202, 779-792 (2013).</li></ol>

作者没有什么可透露的。

近年来, 光已成为 spatiotemporally 控制激活细胞过程的绝佳工具。开发了各种方法, 各有关联的优缺点。这里描述的系统, 这是基于 decaging 的固定化, 胞外配体, 是非常适合分析快速, 亚细胞, 极化信号响应。这种方法已被应用于检测 T 细胞的形成, 如上文所述。此外, 其他受体的笼型配体已被设计, 使我们能够应用同样的策略来评估自然杀伤细胞中的抑制信号和转录因子 NF-nf-κb 的核易位, 以回应像受体...

t 淋巴细胞 (t 细胞) 通过识别细胞表面 MHC 的抗原肽, 在细胞免疫中起重要作用。抗原识别是由 TCR 介导的, 它驱动了幼稚 T 细胞的分化, 促进了溶细胞的传递, 并推动了效应人群的交际反应。TCR 的参与也诱发了细胞结构的戏剧性变化。在几分钟内, T 细胞 gloms 到抗原呈现细胞 (APC) 的一侧, 形成一个极化接口, 称为免疫突触 (是)1,2。增强 t 细胞效应反应通过使细胞因子的定向释放, 或者, 在细胞毒性 T 淋巴细胞 (CTLs), 裂解蛋白, 破坏 APC。
TCR 参与抵押贷款公司诱导了多个下游适配器分子的快速磷酸化, 包括连接器的活化 T 细胞 (LAT), 这最终促进强健的重塑突触细胞骨架2。皮质丝状肌动蛋白 (F 肌动蛋白) 驱动 T 细胞传播在 APC 表面, 然后解决成环状结构的特点是 F 肌动蛋白积累在外围和耗尽从中心。F-肌动蛋白环形成紧密耦合到重新定位微管组织中心 (MTOC, 也称为中心体在 T 细胞) 到一个位置只是在界面的中心。这两种事件都发生在初始抗原识别的几分钟内, 并建立了随后激活事件和效应响应发生的体系结构上下文。
研究的是形成, 各种实验室已经开发的方法, 其中 APC 被一个玻璃表面取代, 其中包括固定化 TCR 配体或支持脂双层, 本身包含配体3,4。t 细胞形成类似的接触在这些表面上, 可以通过全内反射荧光显微镜 (TIRF) 或共聚焦显微成像, 使高分辨率研究早期 T 细胞活化和形成。
虽然这些方法已经允许充分组装的优秀可视化是, 许多信号跟随 TCR: 抵押贷款公司结扎发生在几秒钟之内, 复杂化努力确定事件序列在 TCR 活化以后准确地.为了规避此问题, 开发了一种 photoactivation 方法, 其中 photoactivatable 抵押贷款公司用于实现 TCR激活5、6、7的时空控制.在这一系统中, T 细胞附着在含有 photoactivatable 抵押贷款公司的玻璃表面上, 而非刺激 TCR 直到紫外线 (UV) 光照射。在 t 细胞下的微米尺寸区域的紫外线照射会使 photocage 产生一个可由 t 细胞识别的刺激区。随后的信号事件和骨架重塑, 然后监测使用基因编码荧光记者。两个 photoactivatable 版本的抗原肽, 蛾细胞色素 c88-103 (MCC) 和卵清蛋白257-264 (卵), 这是在第二类 mhc I-Ek和类 I mhc H2-Kb分别提出, 已已开发 (图 1)。这样, 就可以对 CD4k中特定的+ T 单元格进行分析 (表示5C。C7、2B4 或 TCRs) 和 CD8 OVA-H2-Kb (表示 OT1 TCR) 的+ T 单元格。
在过去的十年中, TCR photoactivation 和成像方法已被用来建立早期 TCR 信号步骤的精确动力学, 并确定的分子路径控制极化骨架重塑5,6,7,8,9,10. 例如, 这项化验有助于确定中心体转向 APC 的方向是由甘油中心的脂质第二信使的局部梯度介导的。预计这种方法将继续对要求高分辨率的 T 细胞功能成像分析的应用有价值。

<table border="0" cellpadding="0" cellspacing="0" width="1152">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Nunc Lab-Tek Chambered Coverglass</td>
			<td style="width:211px;">Thermofischer Scientific</td>
			<td align="right" style="width:119px;">155361</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
			<td>Will need to make Biotinylated Poly-L-Lysine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-Link NHS-Biotin</td>
			<td>Thermofischer Scientific</td>
			<td align="right">20217</td>
			<td>Will need to make Biotinylated Poly-L-Lysine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin</td>
			<td style="width:211px;">Thermofischer Scientific</td>
			<td align="right">434301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BirA-500: BirA biotin-protein ligase standard reaction kit</td>
			<td>Avidity</td>
			<td>BirA500</td>
			<td>Will be used to biotinylate proteins</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated Hb I-EK&#160;</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td>For protein folding, see reference 6. For biotinylation, use BirA kit</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated NPE-MCC I-EK&#160;</td>
			<td>Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom NPE-MCC (H-ANERADLIAYL-K(Nvoc)-QATK-OH) can be purchased from Anaspec</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated &#945;H2-Kk antibody</td>
			<td style="width:211px;">BD Biosciences</td>
			<td align="right">553591</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated NPE-OVA H2-Kb</td>
			<td style="width:211px;">Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom NPE-OVA (H-SIINFE-K(Nvoc)-L-OH) can be purchased from Anaspec</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated KAVY H2-Db&#160;</td>
			<td style="width:211px;">Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom synthesized protein (KAVYDFATL) can be purchased from Anaspec</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated ICAM1&#160;</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td>For protein folding, see reference in protocol. For biotinylation, use BirA kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Hand held UV lamp</td>
			<td style="width:211px;">UVP</td>
			<td style="width:119px;">UVGL-25</td>
			<td>Lamp is held &#60; 1 cm from the sample.&#160; 30 s of 365 light is sufficient for detectable decaging, 20 min for quantitative decaging.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Olympus IX-81 OMAC TIRF system.</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Additional information about the imaging system can be found in Figure 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Mosaic digital diaphragm</td>
			<td style="width:211px;">Andor</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">Slidebook software</td>
			<td style="width:211px;">Intelligent Imaging Innovations</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spatial and temporal control of t cell activation using a photoactivatable agonist

T 淋巴细胞参与快速, 极化信号, 发生在几分钟后 TCR 激活。这诱发了免疫突触的形成, 一种定型的细胞间结, 调节 T 细胞活化和定向靶效应反应。为了有效地研究这些过程, 需要一种适合捕获快速、极化响应的成像方法。该协议描述了这样一个系统, 它是基于 photoactivatable 肽-主要的组织相容性复合物 (抵押贷款公司), 它是不刺激的, 直到它暴露在紫外线。该试剂的靶 decaging 在 videomicroscopy 实验中, 通过全内反射 (TIRF) 成像, 实现对 TCR 活化和对后续细胞反应的高分辨率监测的精确时空控制。这种方法也符合遗传和药理的摄动策略。这允许组装明确定义的分子通路, 将 TCR 信号与免疫突触的极化骨架结构的形成联系起来。

photoactivation 和成像方法允许观察和简便量化快速, 极化信号响应。为了说明它的能力, 这里转载的是一个实验, 研究了 TCR 诱导的中心体的时空关系。5C。C7 T 细胞爆炸是 retrovirally 转基因与两个荧光记者: 一项由蛋白激酶 C-θ链接到与 GFP (C1-GFP) 和 RFP 蛋白的串联 C1 域的一台检测生物传感器, 以监测中心体。然后将 T 细胞连接到包含 photoactivatable coverglassk的腔内。C1-GFP ...

Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Photoactivatable 激动剂对 T 细胞活化的时空控制

该协议描述了一种基于成像的方法, 激活 t 淋巴细胞使用 photoactivatable 肽 MHC, 使精确的时空控制 t 细胞活化。

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

spatial-temporal-control-t-cell-activation-using-photoactivatable

Research

JoVE Journal

Immunology and Infection

6.1K 次观看.  Memorial Sloan-Kettering Cancer Center. 该协议描述了一种基于成像的方法, 激活 t 淋巴细胞使用 photoactivatable 肽 MHC, 使精确的时空控制 t 细胞活化。

视频: Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

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

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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 ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号:对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...