作者感谢南特大学医院动物设施 (犹特人) 的工作人员畜牧和护理, 南特大学的细胞和 tissular 成像核心设施 (MicroPICell) 的成像, 和从南特的细胞术设施 (Cytocell)他们的专家技术援助。这项工作由 INSERM、CNRS、大学 de 南特、国立杜肿瘤研究所 (INCa#PLBio2014-155)、国家民族反癌症 (AO 区域间 2017) 和欧洲联盟时代网 Transcan2 (Immunoglio) 资助。这支队伍由研究所 Medicale (DEQ20170839118) 资助。这项工作是在 LabEX 监察员办公室和 IHU Cesti 方案的范围内实现的, 该项目分别由艾文莉和 ANR-11-LABX-0016-01 的国家研究机构 Investissements d ' ANR-10-IBHU-005 支助。IHU-Cesti 项目也支持南特都市和支付 la 卢瓦尔地区。作者感谢 Chirine Rafia 为改正手稿提供帮助。

下列涉及动物的程序是根据机构指南 (协议 #00186) 执行的. 02; 意大利 (法国) 的区域道德委员会人类 PBMCs 从知情的健康捐献者的血液中分离出来 (景区法国法国南特)。所有步骤都是在无菌条件下进行的。
1. 细胞毒性效应 T 淋巴细胞的非特异扩张
<ol><li>准备并照射35的馈线细胞。为刺激 2 x 105 -4 x 105个效应细胞, 准备 10 x 106 PBMCs 和 1 x 106 BLCLs 从三个不同, 健康捐赠者。</li>
	<li>并用重悬15毫升 RPMI 中的饲养细胞和效应细胞, 辅以10% 热灭活 FCS、2毫米 l-谷氨酰胺、青霉素 (100 毫升/ml)、链霉素 (0.1 µg/毫升) 和300毫升/ml 重组 IL-2。</li>
	<li>在最终浓度为1µg/毫升, 仔细混合, 并分配150µL 的细胞悬浮每井在96井 U 底板。</li>
	<li>孵育在37°c 和与 5% CO2在一个湿润的大气。</li>
	<li>每日检查板材直到大细胞团簇形成在养殖井 (~ 天 7)。</li>
	<li>在新鲜培养基中将细胞转化为 1 x 106细胞/毫升的培养瓶。</li>
	<li>通过计数 (每周 2x) 确定细胞总数, 并在新鲜培养基中保持 1 x 106细胞/毫升。 注意: 效应免疫细胞应准备好治疗管理的休息状态 (通常3周后, 最初的放大刺激)。在体内注射前应检查这些效应细胞的纯度和反应性 (如体外测定)。</li>
</ol>2. 手术前效应细胞的制备
<ol><li>在检查效应细胞计数后, 通过离心 (300 x g 5 分钟) 收集50毫升管中的效应细胞。 注: 为弥补任何损失, 准备过量的细胞 (例如, 50 x 106)。</li>
	<li>小心取出上清液, 在15毫升的无菌 PBS 和离心机中并用重悬细胞, 在 300 x g 5 分钟进行洗涤。</li>
	<li>仔细和彻底清除上清, 然后并用重悬细胞颗粒在1毫升的无菌 PBS。</li>
	<li>将悬浮细胞转移到一个1.5 毫升的微细中, 以 300 x g的离心为5分钟。</li>
	<li>小心, 完全清除上清吹打缓慢。</li>
	<li>并用重悬细胞颗粒在8µL 不育 PBS 每只老鼠。 注意: 这是一个关键步骤。</li>
	<li>使用微测量细胞悬浮液的体积。如果需要, 添加无菌 pbs (20 x 106细胞在每剂量的 PBS 的15µL), 并仔细混合。</li>
	<li>使用微检查, 每个鼠标的最终音量介于15和 20 ul 之间 (势在必行 < 20 µL)。</li>
	<li>保持细胞在冰上, 直到立体定向注射。 注: 超过3小时 timepoints 未测试。</li>
</ol>3. 立体定向注射
<ol><li>
设备安装
	<ol>
<li>根据制造商的指示, 组装和校准小型动物立体定向框架, 以确保颅内注射的准确性 (如注射器大小、所需体积和注射率)。 注: 建议采用慢速输液 (即2-3 µL/分钟)。</li>
		<li>在微生物安全柜 (MSC) 下安装材料, 以维持器械的不育, 保护小鼠不受感染。 注: 在37摄氏度的水浴中放置 "等温集团"。这个系统限制了小鼠在手术中的体温过低。加热垫, 这是必要的后程序护理, 必须使用在连续的温度监测。</li>
	</ol>
</li>
	<li>
手术前动物制剂
	<ol>
<li>麻醉小鼠腹腔注射氯胺酮 (10 毫克/毫升) 和甲苯噻嗪 (0.1 毫克/毫升) 在10µL/克体重的鼠标。</li>
		<li>执行脚趾捏试验, 以确保完全麻醉和镇痛的动物。 注意: 任何运动都是不深镇痛的征兆, 如果出现这种情况, 在重复手术前需要几分钟的时间。</li>
		<li>一旦鼠标被正确麻醉, 使用剪刀从手术部位 (两个耳朵之间, 由鼻子) 去除皮毛。</li>
	</ol>
</li>
	<li>
手术前细胞准备
	<ol>
<li>在每次注射前用吸管 (几次) 仔细并用重悬细胞, 以防止细胞结块。</li>
		<li>仔细绘制所需的细胞悬浮量 (15-20 µL) 到22克微量泵, 以避免气泡的渴望。 注意: 这个单元格加载步骤进入微量泵是很重要的, 以尽量减少差异的注入量。在程序之间重新加载每个单独注入的单元格, 以防止细胞聚集, 并确保队列中的效应细胞管理甚至数量。</li>
		<li>然后, 把注射器放进适应的注射器泵中。</li>
	</ol>
</li>
	<li>
程序护理
	<ol>
<li>使用聚维酮碘5% 溶液浸泡的拭子对手术部位进行消毒, 至少3x。</li>
		<li>在鼠标的眼睛里放一个润滑的眼膏, 防止角膜的任何干燥。</li>
		<li>将麻醉的鼠标放在立体定向的框架上, 在一个温暖的等温块上, 覆盖着无菌塑料包装, 以维持在手术中的老鼠的体温, 并限制死亡率。 注: 鼠标的鼻子和牙齿应适当地放置在牙杆上方, 以确保在手术过程中有足够的呼吸流量。</li>
		<li>一旦鼠标定位在牙杆上方, 将耳棒牢牢地拧紧在鼠标的耳朵下, 以固定头部。 注意: 小心不要破坏耳膜或损害呼吸。</li>
		<li>用无菌剪刀在颅骨的上半部, 从前到后, 使头骨显露出 1-2 厘米中线矢状皮肤切口。</li>
		<li>确定矢状和冠状缝线 (Bregma) 的交叉点, 作为注射前立体定向定位的地标 (如图 1所示)。</li>
		<li>把微量泵放在这一点上。</li>
		<li>移动微量泵2毫米右侧面和0.5 毫米前的 Bregma。</li>
		<li>使用 microdrill, 在预定坐标处用无菌钻头在头骨上做一个小孔。小心保持肤浅, 以避免任何外伤的大脑。 注: 在本议定书中, 免疫细胞注射在一个已建立的肿瘤 (肿瘤细胞注射后一周)。皮肤应重新打开 (疤痕) 和注射是执行在同一坐标用于肿瘤细胞植入 (孔通常仍然存在2周后注射)。在脑实质13、14中为注射肿瘤和效应细胞选择了坐标。</li>
	</ol>
</li>
	<li>
免疫效应细胞的注射
	<ol>
<li>小心地将微量泵插入钻孔中, 缓慢移动, 将针3毫米向下的硬脑膜, 然后向后0.5 毫米到最后深度2.5 毫米之前注入效应细胞。</li>
		<li>在 2-3 µL/分钟内运行效应细胞注射, 并在注射时间内监测小鼠。</li>
		<li>一旦注射完成, 取出针仅1毫米, 并保持微量泵在一个额外的分钟, 然后慢慢地完全撤回微量泵, 以防止任何渗漏从输液现场。 注: 在将动物从立体定向装置移走后, 立即清洁注射设备进行实验。</li>
	</ol>
</li>
	<li>
小鼠术后护理及随访
	<ol>
<li>将动物从立体定向的框架中取出, 立即在切口部位应用聚维酮碘5% 溶液, 用适当的手术缝合闭合皮肤。</li>
		<li>在伤口上直接应用2% 利多卡因凝胶, 用皮下注射治疗丁丙诺啡0.15 µg/克, 术后镇痛。</li>
		<li>将被麻醉的老鼠放回它的笼子上面的加热垫设置为37°c, 以保持适当的老鼠体温和避免任何体温过低。 注: 不需要单独的住房。</li>
		<li>监视鼠标直到它完全从麻醉中恢复, 并将其转移到一个住房室。 注意: 到目前为止, 该协议得到很好的支持, 因为很少发生意外并发症 (< 5% 的注射小鼠)。</li>
		<li>每日监测小鼠和弄死, 当任何下降的健康迹象被观察 (例如, 驼背的姿势, 减少流动性, 虚脱, 或重大体重下降 [≥ 15%])。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

经选择的本机或工程免疫效应细胞的移植, 是有效治疗肿瘤的一种有希望的方法, 如浸润性脑癌, 注意限制活性对非转化细胞15, 16,17,18。然而, 包括大脑在内的中枢神经系统具有特殊的免疫状态, 特别是由于血脑屏障的存在以及缺乏典型的淋巴引流系统19、20。...

肾小球 (IV. 型星形细胞瘤) 是成人最常见和最有攻击性的原发性脑癌。尽管有积极的治疗, 结合手术和射频化疗, 肾小球仍然与一个非常糟糕的预后 (中位存活率14.6 月和2年死亡率 > 73%)1。这一证据表明, 在过去十年2中, 没有有效的治疗进展得到证实。在设计更有效的治疗策略3,4,5, 免疫疗法6的候选者中, 目前正在探索追踪和消除高度侵入性和无线电/抗化疗肿瘤细胞, 怀疑他们的关键贡献迅速和致命的肿瘤复发7。对免疫疗法的各种潜在免疫指标进行了鉴定和建议, 涉及天然或改良的αβ或υδ T 淋巴细胞, 如肾小球特异肿瘤抗原或应力诱导分子8,9, 10。管理选定的肾小球活性免疫细胞效应器的可能性代表了一个独特的机会, 提供高数量的效应淋巴细胞直接进入残余恶性肿瘤的部位。为支持这一策略, 我们最近发现, 基于 immunodeficient 小鼠的同种异体肾小球移植的模型忠实地重述了肾小球肿瘤的发展9,11。此外, 这些模型被用来证明过继转输转移异体人 vυ9vδ2t 淋巴细胞的强抗癌功效。
本协议描述了在同种异体 T 淋巴细胞的移植基础上实现脑肿瘤立体定向免疫疗法的关键实验步骤。本文说明: (i) 治疗同种异体免疫效应 T 淋巴细胞 (如人 vυ9vδ2t 淋巴细胞) 的扩增;(ii) 制备注射用的这些效应 T 淋巴细胞;(iii) 大脑内立体定向管理的程序, 靠近肿瘤。本文还介绍了立体定向注射后这些细胞效应的行为。
这里提出的治疗方法是基于每剂量 20 x 106效应细胞为每个脑肿瘤-轴承 immunodeficient 鼠注射。一个初始的体外扩张步骤是需要产生大量的免疫细胞。因此, 非特异细胞扩张是使用 phytohemagglutinin (PHA L) 和辐照异基因饲养细胞: 外周血单个核细胞 (PBMCs) 的健康捐献者和 eb 病毒 (eb) 转换的 B-永生细胞线 (BLCLs), 从 PBMCs 的体外感染, 从狨 B95-8 细胞系的 eb 病毒的培养上清液中提取, 在1µg/毫升环孢素 a 的存在。
从体外测定9中比较和选择了肾小球活性效应免疫细胞。这些效应细胞的激活和放大使用标准协议, 根据其性质 (例如, 人类 Vγ9Vδ2 T 淋巴细胞9或人抗疱疹病毒αβ T 淋巴细胞12) 与最低纯度 > 80%, 例行由细胞分析检查。下面详述的细胞扩张程序适用于各种人类淋巴细胞子集。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PBMCs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:232px;">from 3 different healthy donors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">BLCLs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td>from 3 different donors</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">Roswell Park Memorial Institute medium (RPMI)</td>
			<td style="width:136px;">Gibco</td>
			<td>31870-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">FCS</td>
			<td style="width:136px;">Dutscher</td>
			<td style="width:119px;">S1810-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">L-glutamine</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">penicillin/streptomycin</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">IL-2</td>
			<td style="width:136px;">Novartis</td>
			<td style="width:119px;">proleukin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PHA-L</td>
			<td style="width:136px;">Sigma</td>
			<td>L4144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Stereotaxic frame</td>
			<td>Stoelting Co.</td>
			<td align="right">51600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Mouse adaptator for stereotaxic frame&#160;&#160;</td>
			<td>Stoelting Co.</td>
			<td align="right">51624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microsyringe pump injector&#160;</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">UMP3-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoFil Syringe</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">NF34BV-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">NSG mice</td>
			<td style="width:136px;">Charles River</td>
			<td style="width:119px;">NSGSSFE07S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Ketamine</td>
			<td style="width:136px;">Merial</td>
			<td style="width:119px;">Imalg&#232;ne 1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylazine</td>
			<td style="width:136px;">Bayer</td>
			<td style="width:119px;">Rompur 2%</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Scissors</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">201758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Forceps</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">501215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">OmniDrill 115/230V</td>
			<td style="width:136px;">WPI</td>
			<td align="right">503598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Vicryl 4-0</td>
			<td style="width:136px;">Ethicon</td>
			<td>VCP397H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylocaine</td>
			<td style="width:136px;">Astrazeneca</td>
			<td>3634461</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts

胶质瘤多形性 (肾小球) 是成人中最常见和最有攻击性的原发性脑癌, 通常与预后较差有关, 在过去十年中提出了缺乏有效的治疗方法。在设计新的治疗策略的有希望的候选者中, 细胞免疫疗法已被用来消除高度侵袭性和化疗放射抗拒肿瘤细胞, 可能涉及迅速和致命复发的癌症。因此, 在肿瘤附近的同种异体肾小球-反应性免疫细胞效应剂 (如人 vυ9vδ2 T 淋巴细胞) 的管理, 将是一个独特的机会, 以提供高效和高度集中的治疗药物直接进入脑恶性肿瘤现场。在这里, 我们提出了一项协议, 以制备和立体定向管理的异基因人淋巴细胞在 immunodeficient 小鼠携带原位人原发性脑肿瘤。本研究为这些细胞免疫疗法的可行性和抗肿瘤效果提供了一个临床前的概念证明, 它们依赖于 intrabrain 肿瘤床内的异基因人淋巴细胞的立体定向注射。

这项研究描述了在肿瘤携带的小鼠脑内进行细胞免疫效应细胞转移的策略, 其基础是直接在瘤床内进行的立体定向注射。
为了尽量减少与大注射量相关的脑损伤的风险, 需要将效应细胞悬浮液集中 (20 x 106细胞在 PBS 的 15-20 µL 中)。为了检查这个细胞浓度步骤是否可能影响效应细胞的生存能力, 这些细胞是根据所描述的协...

Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

在这里, 我们提出了一项协议, 以制备和立体定向管理的异基因人淋巴细胞在 immunodeficient 小鼠携带原位人原发性脑肿瘤。本研究为 intrabrain 细胞免疫疗法的可行性和抗肿瘤疗效提供了概念验证。

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

stereotactic-adoptive-transfer-cytotoxic-immune-cells-murine-models

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  CRCINA. 在这里, 我们提出了一项协议, 以制备和立体定向管理的异基因人淋巴细胞在 immunodeficient 小鼠携带原位人原发性脑肿瘤。本研究为 intrabrain 细胞免疫疗法的可行性和抗肿瘤疗效提供了概念验证。

视频: Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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

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

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

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

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

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

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

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

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

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

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

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

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

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

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

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

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

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

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