这项工作得到了国家杜邦癌症 (印加) (et)、甲组织癌症 (et)、大学大学巴黎太阳 (et)、Selectimmunco (et)、Labex 免疫肿瘤学 (et)、SIRIC CARPEM (CG 等) 赠款的资助。电子是由一个基金会的研究金弧形。EV 和 CD 由 APHP (交易所 année 研究) 的奖学金资助。慢性乙肝由大学索邦巴黎太阳 (合同博士) 的奖学金资助。作者感谢布里斯托尔迈尔斯宝在这个项目上的资助。作者感谢医院 europ 乔治蓬皮杜和尼克 (Laurianne Chambolle, Elodie 米歇尔和 Gisèle Legall) 的病理学系。作者感谢 PARCC, 医院 europ 乔治蓬皮杜 (科琳 Lesaffre) 的组织学平台。

这项研究是按照《赫尔辛基宣言》进行的, 并得到当地道德委员会 (2012-05-04) 的批准。从参与人中获得了知情同意。
1. 组织材料
<ol><li>在手术当天收集碾压混凝土组织标本。在室温下处理手术标本 (病理科)。</li>
	<li>在干管中收集大约0.5 厘米 x 0.5 cm x 0.5 厘米大小的肿瘤样本。在液氮中冷冻, 贮存在-80 ° c。</li>
	<li>将样品涂在最佳切削温度复合物上。部分样品与低温在-20 ° c 入 4-6 µm 厚实的部分。让样品的空气干燥12小时, 并直接储存在-80 ° c, 以避免干燥。</li>
	<li>通过查看苏木精和曙红染色切片检查样品的质量。</li>
</ol>2.原位til 免疫荧光染色
<ol><li>
程序指南
	<ol>
<li>在室温下执行所有实验步骤。</li>
		<li>对于所有步骤, 执行稀释与三缓冲盐水 (TBS; 请参见材料表)。</li>
		<li>除主抗体孵育后, 在 TBS 中执行所有步骤的洗涤。对于后者, 使用三缓冲盐水 Tween20 (TBST, 请参阅材料表)。对于缓冲区组成和重构, 请参见补充表 1。</li>
		<li>使用湿度室的抗体孵化和染色罐洗涤。</li>
		<li>在手术过程中不要让部分干涸。</li>
	</ol>
</li>
	<li>
预处理
	<ol>
<li>将含有组织样品的幻灯片解冻, 并用纸巾小心地将试样周围的滑动干燥。用疏水屏障笔界定包含组织的反应区域 (请参见材料表)。干燥2分钟。</li>
		<li>将100% 丙酮中的样品固定5分钟, 干燥2分钟, 用 TBS 洗涤10分钟。</li>
	</ol>
</li>
	<li>
饱和和封锁
	<ol>
<li>预处理的幻灯片为10分钟, 3 滴素 0.1%, 点击和/或轻弹幻灯片, 以分发素和消除气泡, 然后治疗10分钟与3滴的生物素 0.01% (见材料表), 点击, 和/或轻弹。用 TBS 洗。</li>
		<li>执行 Fc 受体阻滞。应用100µL 5% 体积/体积的正常血清稀释在 TBS. 使用同一寄主物种的血清作为标记的二级或三级抗体 (见材料表);在这里, 驴血清被使用。孵育30分钟。</li>
		<li>在孵化期间, 简要地旋转 anti-CD8、PD-1 和 Tim-3 抗体 (材料目录)。准备在 TBS 的主要抗体混合, 如补充表 2中所述。</li>
	</ol>
</li>
	<li>
CD8、PD-1 和 Tim-3 的免疫染色
	<ol>
<li>准备主抗体混合物。有关所使用的抗体 (anti-CD8、PD-1、Tim-3 及其相应的二级抗体) 及其浓度, 请参阅材料表和补充表 2 。</li>
		<li>轻拍和/或挥出剩余的驴血清 (添加在步骤 2.3.2)。用100µL 的非标记主抗体混合在湿化室中孵育1小时的幻灯片。</li>
		<li>在孵化期间, 简要地旋转青菁5抗兔, AF488-anti-goat, 和化抗鼠抗体, 并准备在补充表 2中描述的二次抗体混合。</li>
		<li>在 TBST 中清洗幻灯片5分钟。</li>
		<li>在湿化室中孵育30分钟的二次抗体的100µL 的幻灯片。</li>
		<li>如补充表 2所述, 准备含有 Cy3-streptavidin 的三级抗体混合物。</li>
		<li>在 TBS 中冲洗幻灯片10分钟, 烘干幻灯片。</li>
		<li>孵育的幻灯片与100µL 的第三抗体混合物30分钟。</li>
		<li>在 TBS 中冲洗幻灯片10分钟, 烘干幻灯片。</li>
	</ol>
</li>
	<li>
单元安装
	<ol>
<li>安装在 4 ", 6-Diamidino-2-吲, 盐酸 (DAPI)-包含安装介质 (1.5 µg/毫升 DAPI) 与片兼容荧光显微镜 (见材料表) 的幻灯片。 注意: 作为对每项实验的阴性对照, 一张与同种匹配的抗体的滑动与相应的抗体浓度相同。对于这种染色, 我们使用了小鼠 IgG1, 兔 igg, 和山羊 igg 阴性控制抗体 (参见材料表)。作为一个积极的控制, 我们使用人类增生扁桃体, 这是已知的是积极的测试标记, 每一个实验。结果中描述了一个典型的染色 (图 1)。CD8、PD-1 和 Tim-3 的单染色应单独进行 (每张幻灯片染色一次), 并进行相同的前处理和无 DAPI。在同样的实验条件下, 没有任何抗体和只有 DAPI 安装介质的滑动也应进行。一张幻灯片应使用相同的实验条件, 没有任何抗体和没有 DAPI 成像。</li>
	</ol>
</li>
</ol>3. 荧光分析和自动细胞计数
<ol><li>图像采集与自动显微镜
	<ol>
<li>创建一个协议。<ol>
<li>打开自动显微镜软件和荧光照明灯。</li>
			<li>在菜单栏中, 单击 "文件"、"创建协议"、"选择 DAPI"、"FITC" 和 "TRITC"。</li>
			<li>单击 "下一步"、"组织部分", 然后单击 "下一步"、"协议名称"。选择一个名称, 例如, "CD8-PD-1-Tim-3", 单击 "下一步" 和 "保存"。</li>
			<li>手动将幻灯片放在舞台上。</li>
			<li>在菜单栏中, 单击 "安装"、"设置"。在新窗口中单击 "设置主页 Z"。</li>
			<li>使用正控制滑块调整曝光时间即CD8、PD-1 和 Tim-3 三重染色的 DAPI 样本。</li>
			<li>在控制栏中单击 "设置曝光"。</li>
			<li>调整每个滤镜的曝光时间, 直到获得足够但不饱和的信号。</li>
			<li>对于单色成像, 请执行以下操作:</li>
			<li>选择购置和在 ' 自动对焦 ' 选择 DAPI 过滤器。</li>
			<li>在 "扫描区域限制" 中, 将目标上移到幻灯片的左上角。单击 "标记"。在右下角做同样的事情。 注: 此步骤 delimitates 4X 低功耗成像 (LP 成像) 的区域;注意将标记放置好, 以便围绕系列的所有示例。</li>
			<li>对于大功率 (HP) 成像, 请执行以下操作:</li>
			<li>在 "自动对焦" 下, 选择 "DAPI"。</li>
			<li>在 "获取" 波段下, 选择 "DAPI"、"FITC"、"Cy3" 和 "Cy5"。单击 "确定"。在菜单栏上单击 "文件", "保存协议"。</li>
		</ol>
</li>
		<li>扫描幻灯片。<ol>
<li>通过单击菜单栏中的 "文件"、"加载协议" 来加载协议。单击 "开始"。</li>
			<li>输入 "实验室 ID" (用于图像存储的文件夹位置)。输入幻灯片 ID 以标识幻灯片。单击 "下一步"。</li>
			<li>单击 "单色成像" (在明亮的光场下扫描整个幻灯片, 并使用4x 目标获取序列图像)。 注意: 这将生成幻灯片的灰度概览。</li>
			<li>点击 "查找标本"。选择低分辨率扫描的组织区域 (4x): 按住 Ctrl 键并使用光标单击字段以选择或取消选中字段 (图 2A)。</li>
			<li>单击 "LP 成像" (4x 成像), 获取每个字段的荧光红蓝绿色图像。</li>
			<li>对于 HP 字段选择, 按住 "Ctrl" 键并使用光标单击字段以选择或取消选中与将在高分辨率 (20x) 扫描的组织区域相对应的字段。选择5字段 (图 2B)。</li>
			<li>单击 "HP 成像" (20x 成像), 获取每个字段的多光谱图像 (图 2C)。</li>
			<li>单击 "数据存储" 以将图像存储在 LabID 开始处选定的文件夹中。 注意: 该过程在图 2中进行了总结和说明。对于每个步骤 (组织检测, 字段选择), 一个算法可以增加和培训的用户对整个自动化扫描过程。</li>
		</ol>
</li>
	</ol></li>
	<li>用耦合分析软件进行荧光图像分析和自动计数
	<ol>
<li>构建频谱库。<ol>
<li>打开图像分析软件。</li>
			<li>在左侧面板中, 打开 "生成库"。在 "加载图像" 下, 单击 "浏览" 并选择一个单声道彩色图像 (例如, CD8/Cyanine 5)。</li>
			<li>选择荧光, (例如, 菁 5)。单击 "提取"。单击 "保存" 以存储在库中。单击 "保存"。</li>
			<li>重复相同的过程, PD-1, Tim-3, 和 DAPI 单染色幻灯片, 以整合的频谱, 每荧光的兴趣。 注意: 构建频谱库集成了用于特定组织 (这里, 肾脏) 的每个荧光的频谱, 但是已经存在的 "合成" 库可以使用。由于自体荧光光谱是严格的相对于组织, 它是重要的是执行一个自动萤光和光谱库的每个项目。</li>
		</ol>
</li>
		<li>创建新项目。<ol>
<li>从菜单栏中, 单击 "文件"、"新建项目"。在 "查找功能" 选项卡中, 选择 "单元格分割"。在 "分" 选项卡中, 选择 "分"。单击 "创建"。</li>
			<li>将代表图像集成到项目中。<ol>
<li>在 "文件" 选项卡上, 单击 "打开图像"。选择10到30代表性的整个系列图像。添加非着色幻灯片以删除荧光。在右侧面板: 选择库源。选择荧光并选择对应于以前构建的频谱库的。</li>
			</ol>
</li>
			<li>要去除组织的自发荧光, 请单击 "AF 按钮", 然后选择空白幻灯片上的自发荧光区域。</li>
			<li>图像处理和复合图像生成。<ol>
<li>单击 "准备全部" 以集成荧光库和自发荧光光谱来生成复合图像 (图 3)。单击 "眼睛" 图标打开 "视图编辑器" 面板, 然后选择显示的数据: 选择 CD8、PD-1、Tim-3 和 DAPI 的标记。去掉荧光。按照软件提供的步骤操作。</li>
			</ol>
</li>
			<li>分割单元格。<ol>
<li>要分割细胞, 在 ' 隔间 ', 选择 "细胞核" 和 "膜"。在 "细胞核" 标签中, 设置 DAPI 为核 counterstain。</li>
				<li>在 "最大和最小大小 (px)" 选项卡中分别输入 "40" 和 "176" 作为最小和最大大小。对于 "最小" 信号, 设置 "0.13"。在 "拆分" 选项卡中, 设置 "2.60"。在 "细胞核" 标签中, 设置 "0.81"。选择 "使用膜信号帮助分割"。</li>
				<li>单击屏幕底部的 "分段单元格"。检查细胞的细胞核和细胞膜的分割, 如果不符合细胞的 DAPI 和细胞膜荧光, 则重试不同的参数。 注: 该软件识别的细胞与核 DAPI 染色, 并根据细胞大小。根据项目调整单元格参数 (大小、拆分、像素)。</li>
			</ol>
</li>
			<li>细胞表型。<ol>
<li>在 "表型" 标签中, 单击 "添加"。创建细胞表型分类: CD8 (蓝点)/CD8-pd-1 (红点)/CD8-pd-1-Tim-3 (绿点)/其他 (黑点) (图 4)。</li>
				<li>选择每个类别中5多个单元格的示例。单击 "训练分类器"。 注意: 这个软件生成了一个统计分类算法。</li>
				<li>点击 "表型全部" 获得每个细胞的表型。 注: 该软件给出了一个具有置信区间 (CI) 精度的细胞表型。</li>
				<li>通过训练软件改进算法, 直到眼睛和自动计数之间的差异是一致的 (错误 < 5%)。选择对感兴趣的单元格可以接受的 CI。 注: 我们选择在 55% CI 的基础上进行培训, 可以接受。</li>
			</ol>
</li>
			<li>保存算法和项目。</li>
			<li>在 "文件" 下选择 "项目"。命名它并单击 "保存"。</li>
		</ol>
</li>
		<li>对系列进行批量分析。<ol>
<li>选择 "批次分析"。选择项目。添加要分析的图像。为数据选择一个存储文件夹。单击 "运行"。检查复合图像的质量。验证该系列的所有图像的分。 注: 耦合图像分析软件集成了所有的细胞隔间 (膜/细胞质/核) 信号。分步骤是至关重要的, 尽管算法的训练可以是一个耗时的步骤。每个图像的所有数据都在一个 .txt 文件中。一个细胞的所有数据 (特别是它的表型给与它的 CI) 在一条线。对于每张幻灯片, 图像获取和后续计数都在5字段上执行。</li>
		</ol>
</li>
	</ol></li>
	<li>原始数据的综合化和自动细胞计数的世代
	<ol>
<li>用统计程序计算数据。<ol>
<li>计算所有的数据从5图像对应的5字段分析为1患者。使用统计平台, 有经验丰富的统计学家、数据管理人员或数据科学家进行分析。建立一个脚本, 自动计数的细胞取决于他们的表型, 选择 CI > 55%。</li>
			<li>将该系列的所有 .txt 文件放在同一个文件夹中。</li>
		</ol>
</li>
		<li>提取数据。<ol>
<li>将所有 .txt 文件放在一个文件夹中。运行在步骤3.3.1 中构建的自动脚本。打开由 R 脚本生成的输出. csv 文件。另存为. xl 格式。输出为每个患者收集每个表型的单元格数 (即、单独的 CD8、CD8-PD-1、CD8-PD-1-Tim-3)。</li>
			<li>计算每个字段每个患者的平均单元格数: 按字段个数 (即, 5) 划分单元格数, 以规范化每个字段每个患者的单元格数。</li>
			<li>计算总 CD8+ t 单元格中的 PD-1+单元格的百分比, 在总 CD8 + t 单元格中 PD-1 + Tim-3+单元格。有关此步骤的摘要, 请参见图 5 。 注意: R 语言 (或其他编程软件的选择) 是需要正确的创建和执行命令, 所以有经验的用户或统计学家/数据科学家是必不可少的。R 程序可以计算同一病人的数据, 但一个病人的 5 .txt 文件的名称应该有一个相同的开始, 对应于一个独特的病人标识符。</li>
		</ol>
</li>
	</ol></li>
	<li>统计分析
	<ol>
<li>使用统计软件对结果进行统计分析。</li>
		<li>在统计学家的帮助下进行适当的统计分析。<ol>
<li>使用非参数魏氏的签名秩测试, 比较 PD-1 在两个细胞表型之间 (图 6)。协和组织学特征与皮尔逊的 chi 平方检验相关联。</li>
			<li>使用卡普兰-梅尔方法估计的进展自由生存, 和 Cox 回归模型, 以估计协影响的时间-事件的结果, 如整体生存和无病生存。</li>
			<li>请考虑p-值小于0.05。</li>
		</ol>
</li>
	</ol></li>
</ol>

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Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.</a> Science. 348 (6230), 124-128 (2015).</li><li>Roussel, H., 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=Composite+biomarkers+defined+by+multiparametric+immunofluorescence+analysis+identify+ALK-positive+adenocarcinoma+as+a+potential+target+for+immunotherapy.">Composite biomarkers defined by multiparametric immunofluorescence analysis identify ALK-positive adenocarcinoma as a potential target for immunotherapy.</a> OncoImmunology. 6 (4), e1286437 (2017).</li><li>Pages, F., Granier, C., Kirilovsky, A., Elsissy, C., Tartour, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarqueurs+pr&#233;dictifs+de+r&#233;ponse+aux+traitements+bloquant+les+voies+de+costimulation+inhibitrices.">Biomarqueurs pr&#233;dictifs de r&#233;ponse aux traitements bloquant les voies de costimulation inhibitrices.</a> Bull Cancer. 103, S151-S159 (2016).</li><li>Tumeh, P. C., 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=PD-1+blockade+induces+responses+by+inhibiting+adaptive+immune+resistance.">PD-1 blockade induces responses by inhibiting adaptive immune resistance.</a> Nature. 515 (7528), 568-571 (2014).</li><li>Fourcade, 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=Upregulation+of+Tim-3+and+PD-1+expression+is+associated+with+tumor+antigen-specific+CD8++T+cell+dysfunction+in+melanoma+patients.">Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients.</a> J Exp Med. 207 (10), 2175-2186 (2010).</li><li>Koyama, S., 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=Adaptive+resistance+to+therapeutic+PD-1+blockade+is+associated+with+upregulation+of+alternative+immune+checkpoints.">Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.</a> Nat Commun. 7, 10501 (2016).</li><li>Wherry, E. J., Kurachi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+insights+into+T+cell+exhaustion.">Molecular and cellular insights into T cell exhaustion.</a> Nat Rev Immunol. 15 (8), 486-499 (2015).</li><li>Cai, C., 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=Tim-3+expression+represents+dysfunctional+tumor+infiltrating+T+cells+in+renal+cell+carcinoma.">Tim-3 expression represents dysfunctional tumor infiltrating T cells in renal cell carcinoma.</a> World J Urol. 34 (4), 561-567 (2016).</li><li>Sakuishi, K., 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=Targeting+Tim-3+and+PD-1+pathways+to+reverse+T+cell+exhaustion+and+restore+anti-tumor+immunity.">Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity.</a> J Exp Med. 207 (10), 2187-2194 (2010).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+a+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</li><li>Bethmann, D., Feng, Z., Fox, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprofiling+as+a+predictor+of+patient's+response+to+cancer+therapy-promises+and+challenges.">Immunoprofiling as a predictor of patient's response to cancer therapy-promises and challenges.</a> Curr Opin Immunol. 45, 60-72 (2017).</li><li>Badoual, C., 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=PD-1-expressing+tumor-infiltrating+T+cells+are+a+favorable+prognostic+biomarker+in+HPV-associated+head+and+neck+cancer.">PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer.</a> Cancer Res. 73 (1), 128-138 (2013).</li><li>Nizard, 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=Induction+of+resident+memory+T+cells+enhances+the+efficacy+of+cancer+vaccine.">Induction of resident memory T cells enhances the efficacy of cancer vaccine.</a> Nat Commun. 8, 15221 (2017).</li><li>Nghiem, P. T., 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=PD-1+Blockade+with+Pembrolizumab+in+Advanced+Merkel-Cell+Carcinoma.">PD-1 Blockade with Pembrolizumab in Advanced Merkel-Cell Carcinoma.</a> N Engl J Med. 374 (26), 2542-2552 (2016).</li><li>Roussel, H., 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=Composite+biomarkers+defined+by+multiparametric+immunofluorescence+analysis+identify+ALK-positive+adenocarcinoma+as+a+potential+target+for+immunotherapy.">Composite biomarkers defined by multiparametric immunofluorescence analysis identify ALK-positive adenocarcinoma as a potential target for immunotherapy.</a> Oncoimmunology. (4), (2017).</li><li>Woods, K., 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=Mismatch+in+epitope+specificities+between+IFNgamma+inflamed+and+uninflamed+conditions+leads+to+escape+from+T+lymphocyte+killing+in+melanoma.">Mismatch in epitope specificities between IFNgamma inflamed and uninflamed conditions leads to escape from T lymphocyte killing in melanoma.</a> J Immunother Cancer. 4, 10 (2016).</li><li>Granier, C., 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=Tim-3+Expression+on+Tumor-Infiltrating+PD-1+CD8++T+Cells+Correlates+with+Poor+Clinical+Outcome+in+Renal+Cell+Carcinoma.">Tim-3 Expression on Tumor-Infiltrating PD-1+CD8+ T Cells Correlates with Poor Clinical Outcome in Renal Cell Carcinoma.</a> Cancer Res. 77 (5), 1075-1082 (2017).</li><li>Feng, Z., 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=Multispectral+Imaging+of+T+and+B+Cells+in+Murine+Spleen+and+Tumor.">Multispectral Imaging of T and B Cells in Murine Spleen and Tumor.</a> J Immunol. 196 (9), 3943-3950 (2016).</li><li>Feng, Z., 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=Multispectral+imaging+of+formalin-fixed+tissue+predicts+ability+to+generate+tumor-infiltrating+lymphocytes+from+melanoma.">Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.</a> J Immunother Cancer. 3, 47 (2015).</li><li>Fridman, W. 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所有作者没有利益冲突声明。

修改和故障排除:
组织质量是一个重要的参数;它可以很容易地通过苏木精和曙红着色检查。
该技术的一个优点是可能的多路复用染色, 但为了避免荧光溢出, 它建议选择的发射波长的三角洲 10 nm 最小。在这里, 因为我们有4染色包括 DAPI, 我们选择展开波长 (DAPI: 460 nm, AF488: 519 nm, 菁 3: 570 nm, 菁 5: 670 nm)。通过使用软件计算混合发射信号中的荧光?...

在过去的几年中, 免疫治疗已经成为一种非常有前途的疗法, 许多类型的癌症, 包括 RCC。特别是, 基于抑制像 PD-1 和 CTLA-4 等抑制检查站的免疫疗法已被报告为临床有效的1,2,3,4,5,6. 抗 CTLA-4、PD-1 或程序死亡配体 1 (PD-L1) 的单克隆抗体已在几种癌症中获得批准, 并导致20% 以上患者的长期持续临床反应7。然而, 并非所有的病人都是应答者, 治疗的费用很高, 这些治疗是有毒的, 导致潜在的严重的自身免疫性副作用。因此, 当前的挑战是确定那些新的免疫的预测标记。据报道, 肿瘤的突变率, PD-L1 的表达, 或 CD8 细胞浸润的水平, 与临床反应相关.然而, 该协会仍然过于薄弱, 建议在临床实践中使用这些临床生物标志物, 除了在非小细胞肺癌 (NSCLC) 患者的 Pembrolizumab 前 PD-L1 的同伴试验,8 ,9,1011,12。结果表明, PD-1、Tim-3、Lag-3 和 CTLA-4 等多种抑制受体的共同表达, 诱导细胞衰竭表型和抗性治疗13,14,15。由于外周血不是肿瘤微环境的代表, 因此, 分析细胞的表型特征原位是很有意义的。PD-1 和 Tim-3 共表达 T 细胞已知是功能受损的细胞在几个上下文13,16,17。在这项研究中, 评估了两个抑制受体 PD-1 和 Tim-3 在 CD8 的 T 细胞上的共同表达对预后的影响.
到目前为止, 研究多标记物在肿瘤浸润淋巴细胞 (til) 中的协同表达主要是通过流式细胞仪分析进行的, 因此有必要对新鲜肿瘤进行研究, 从而排除回顾性分析。与常规的原位染色, 一次只能进行一次染色, 而与表达标记的细胞类型的表征是不可能的。例如, PD-L1 是由肿瘤微环境的许多细胞类型表达的, 使得传统的免疫组织化学分析难以定义 PD-L1 细胞的表达与相关研究的相关性更大。在这项工作中, 我们开发了一个创新的原位多免疫荧光法与计算机计数, 以关联 PD-1 和 Tim-3 的共同表达的肿瘤浸润 CD8+ T 细胞与临床结果在 RCC。这项技术有几个优点, 包括有可能在一个单一的细胞水平和多个标记同时使用多光谱相机, 可以捕获限制的时间间隔 > 10 nm 通过液晶滤镜18。此外, 该过程是自动的, 使互操作性和缩短分析相比, 手动技术19。在癌症领域, 几项研究报告有说服力的多染色免疫分子如 PD-1, PD-L1, 和 CD8 在默克尔-细胞癌, 肺癌, 头颈癌20,21,22,23. 通过用户的培训 (分步骤), 可以实现自动单元计数。荧光是在不同的细胞室 (细胞核, 细胞质, 和膜) 测量。
在大量的碾压混凝土中, 不同的肿瘤浸润 CD8+ T 细胞表达 PD-1 和/或 Tim-3, 结果与临床重力评分和生存参数相关。在细胞分辨率下, 也可以利用细胞内的平均荧光强度来分析细胞膜的荧光强度。据我们所知, 这是第一次使用这种基于多光谱成像的计数技术报告预后结果的研究。

<table>
	<tbody>
		<tr>
			<td>Vectra 3 Automated Quantitative Pathology Imaging&#160;</td>
			<td>Perkin Elmer</td>
			<td>CLS142338</td>
			<td></td>
		</tr>
		<tr>
			<td>inForm cell analysis 2.1.</td>
			<td>Perkin Elmer</td>
			<td>CLS135781</td>
			<td></td>
		</tr>
		<tr>
			<td>R software</td>
			<td>https://www.r-project.org</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dakopen delimiting pen</td>
			<td>Dako</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Buffer Salin TBS Tablets</td>
			<td>Takara, Bio Inc.</td>
			<td>TAKT9141Z</td>
			<td>pH7.6 100 tablet</td>
		</tr>
		<tr>
			<td>Tris Buffer Salin Tween 20 TBS(+Tween20)</td>
			<td>Takara, Bio Inc.</td>
			<td>TAKT9142Z</td>
			<td>pH 7.6 100 tablets</td>
		</tr>
		<tr>
			<td>Biotin blocking system</td>
			<td>Dako</td>
			<td>X0590</td>
			<td>Avidin 0.1% and Biotin 0.01%</td>
		</tr>
		<tr>
			<td>normal donkey serum</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-001</td>
			<td>5% vol./vol. concentration</td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-aldrich</td>
			<td>F6057</td>
			<td>1.5 &#181;g/mL concentration&#160;</td>
		</tr>
		<tr>
			<td>Knittel glass coverslip</td>
			<td>Knittel&#160;Gl&#228;ser,</td>
			<td>100039</td>
			<td>24x60 mm 100 cover slips</td>
		</tr>
		<tr>
			<td>Rabbit anti-CD8 Clone P17-V</td>
			<td>novus</td>
			<td>NBP1-79055</td>
			<td>use at 4&#181;g/mL</td>
		</tr>
		<tr>
			<td>Mouse anti-PD-1 Clone NAT</td>
			<td>abcam</td>
			<td>ab52587</td>
			<td>use at 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-Tim-3</td>
			<td>R&#38;D</td>
			<td>AF2365</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Rabbit anti-PD-L1 Clone SP142</td>
			<td>Roche</td>
			<td>7309457001</td>
			<td>use at 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Mouse AF647 labeled pan- Keratin Clone C11&#160;</td>
			<td>Cell Signalling</td>
			<td>4528</td>
			<td>use at 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-human gal9&#160;</td>
			<td>R&#38;D</td>
			<td>AF2045</td>
			<td>use at 0.3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cyan 5 conjugated donkey anti-rabbit&#160;</td>
			<td>Jackson Immunoresearch</td>
			<td>711-175-152</td>
			<td>use at 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Biotinylated F(ab&#8217;2) donkey anti-mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>715-066-150</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor488 conjugated donkey anti-goat IgG</td>
			<td>abcam</td>
			<td>ab150133</td>
			<td>use at 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cy3 labeled streptavidin&#160;</td>
			<td>Amersham</td>
			<td>PA43001</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>negative control mouse IgG1</td>
			<td>Dako</td>
			<td>X0931</td>
			<td>use at 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IgG from goat serum</td>
			<td>Sigma-aldrich</td>
			<td>I5256</td>
			<td>use at 3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>IgG from rabbit serum</td>
			<td>Sigma-aldrich</td>
			<td>I5006</td>
			<td>use at 4&#181;g/mL</td>
		</tr>
	</tbody>
</table>

multiplexed immunofluorescence analysis and quantification of intratumoral pd-1+ tim-3+ cd8+ t cells

免疫细胞是肿瘤微环境的重要组成部分, 在癌变的各个阶段影响肿瘤的生长和进化。值得注意的是, 现在已经证实, 人体肿瘤的免疫渗透可以与预后和治疗反应相关联。对肿瘤微环境中免疫浸润的分析已成为患者分类和治疗反应的主要挑战。
抑制受体的共同表达, 如程序细胞死亡蛋白 1 (PD1; 也称为 CD279), 细胞毒 t 淋巴细胞相关蛋白 4 (CTLA-4), t 细胞免疫球蛋白和含 Protein-3 (Tim-3, 也称为 CD366), 淋巴细胞活化基因 3 (Lag-3, 也称为 CD223), 是 T 细胞衰竭的标志。我们开发了一个多的原位免疫荧光染色, 以确定和量化的细胞水平, 这些抑制受体的共同表达。在回顾性肾细胞癌 (RCC) 冰冻组织中, 应用荧光多光谱成像技术与图像分析软件结合, 发现 PD-1 和 Tim-3 对肿瘤浸润 CD8+ T 细胞的联合表达与碾压混凝土的预后差相关。根据我们的知识, 这是第一个研究表明, 这种自动复用的原位技术可能有一些临床相关性。

使用上述的一般协议, 我们的目的是量化肿瘤 CD8+ T 细胞联合表达的抑制受体 PD-1 和 Tim-3 在冰冻组织的患者与临床结果25。
CD8/PD-1/Tim-3 染色的优化:
为了避免不同抗体之间的交叉反应, 选择了不同种类的抗体 (小鼠、兔和山羊, 见材料表</st...

Watch this Scientific Journal Video about Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells at JoVE.com

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

肿瘤 PD-1 的多路免疫荧光分析及量化研究+ Tim-3+ CD8+ T 单元格

研究肿瘤微环境可以确定预后或预测的标志物的临床反应免疫治疗。本文介绍了一种基于原位荧光多光谱成像的创新方法, 对 CD8+ T 细胞的各种亚群进行自动分析和计数。这种可重现和可靠的技术适用于大型队列分析。

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it ...

multiplexed-immunofluorescence-analysis-quantification-intratumoral

Research

JoVE Journal

Cancer Research

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

14.4K 次观看.  Université Paris Descartes. 研究肿瘤微环境可以确定预后或预测的标志物的临床反应免疫治疗。本文介绍了一种基于原位荧光多光谱成像的创新方法, 对 CD8+ T 细胞的各种亚群进行自动分析和计数。这种可重现和可靠的技术适用于大型队列分析。

视频: Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

科研

癌症研究

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

细胞毒性细胞因子诱导的杀伤性T细胞的分离和扩张，用于癌症治疗

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

来自分离人产品的γ-Delta（γδ）T细胞的扩增和富集

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...