这项工作是由美国胃肠病协会（AGA）研究学者奖和克罗恩和美国（CCFA）职业发展奖（以ENS），美国国立卫生研究院NIDDK F30 DK089692（以ECS）结肠炎基金会，以及北卡罗莱纳州中心大学的胃肠生物学支持与疾病格兰特P30 DK34987（组织学核心）。在UNC流式细胞仪核心设施是由NCI中心的核心支持格兰特（P30CA016086）的UNC莱恩伯格综合癌症中心支持的一部分。我们感谢卢克B.博斯特从兽医北卡罗莱纳州立大学对他的帮助与组织病理学分析和免疫组化。

注：确保所有的动物协议通过并符合机构动物护理和使用委员会（IACUC）的规定和国家研究委员会的指南实验动物的护理和使用的批准。供体小鼠可以是男性或女性，但受体小鼠应该是雄性。如果女性收件人使用，供体小鼠必须是女性5。使用常规的，非无菌床上用品和非酸化水中维持菌落，因为这些可能会影响肠道菌群，并且因此，小鼠5,6的结肠炎的表型。 1.实验准备<ol><li>用冰冷的媒体和缓冲区。保持整个实验的细胞在冰上。 </li><li>执行使用无菌技术的实验在无菌生物危害罩。 </li></ol> 2.分离脾T细胞的<ol><li>安乐死捐助鼠标/鼠标在CO 2室其次是宫颈dislocatioñ。喷腹部用70％乙醇。 </li><li>做水平切口在腹部和剥离皮肤暴露腹膜。持有腹膜远离内脏的镊子，并在左腹部腹膜切口暴露和切除脾脏。 </li><li>放置在10ml完全培养基的脾脏在培养皿。删除和脾丢​​弃多余的组织。 </li><li>使用2消毒玻片破碎，梳理出脾成单细胞悬液。通过一个70微米的过滤器过滤细胞悬浮到50ml锥形管中，并冲洗过滤用5ml完全培养基中。放置最多5脾脏在一个50ml锥形管中。 </li><li>离心细胞，在450×g离心7分钟。浇关闭或通过真空抽吸到废物容器弃上清。 </li><li>轻轻重悬细胞于每裂解液脾5毫升以裂解红血细胞，在室温下10分钟。加完整的媒体等体积（每脾5ml）中的管中。 </li><lI&gt;离心细胞，在450×g离心7分钟。浇关闭或通过真空抽吸到废物容器弃上清。 </li><li>轻轻重悬细胞于10ml标记缓冲液。 </li><li>细胞计数台盼蓝排斥。 <ol><li>除去20微升细胞悬浮液，并加入到180微升台盼蓝到微量离心管中并充分混合。 5分钟后，添加10微升标记的细胞，以血球和在显微镜下计数非蓝色的细胞。确定存活细胞的总数。丢弃电池/台盼蓝组合。 </li></ol></li><li>离心细胞在10ml标记缓冲液中，在450×g离心7分钟。浇关闭或通过真空抽吸到废物容器弃上清。 </li></ol> 3.富集CD4 + T细胞的注：按照制造商的说明在本节中使用的特定产品。 <ol><li>轻轻悬浮细胞，以20×10 6个细胞/ ml，在寒冷的标签单细胞悬浮液ING缓冲。 </li><li>添加5μl的每1×10 6个细胞的生物素化的CD4 T细胞富集的抗体。孵育细胞在冰上15分钟。 </li><li>加入10倍体积标记缓冲液中。离心细胞，在450×g下7分钟。小心吸全部采用真空吸进废物容器上清。 </li><li>彻底涡旋链霉亲和共轭磁性粒子。添加5μl的每1×10 6个细胞的颗粒。 </li><li>调匀而是轻轻。保持混合物在6-12℃下进行30分钟。 </li><li>添加标记缓冲液至20-80×10 6细胞/ ml的浓度。发送上行至1.0ml每12×75mm的圆底试管标记的细胞（称为“阳性馏分管”）。 </li><li>放置在磁铁每个正分数管6-8分钟。 </li><li>与正馏分管仍然在磁铁，小心地从阳性馏分管与玻璃巴斯德吸管转移上清至新的无菌50ml锥形管（referred，来作为“富集级分”）。这种富集的馏分含有CD4 + T细胞。要小心，不要破坏吸引到磁铁的标记细胞。 </li><li>留在标记缓冲液的相同体积的正馏分管悬浮细胞如在步骤3.6通过上下吹打大力。将积极分数管回磁铁6-8分钟。 </li><li>阳性馏分管仍然在磁铁，小心地从阳性馏分管与玻璃巴斯德吸管转移上清液（富集馏分，CD4 +），以无菌50ml锥形管中，从步骤3.8而不破坏附着到磁体的细胞。 </li><li>重复步骤3.9-3.10增加获得CD4 + T细胞的产率。使用富集级分（CD4 +细胞）继续协议。 </li></ol> 4.标签和分类 ​​细胞7 <ol><li>离心富集的细胞在450×g离心7分钟。浇过或真空suct弃上清离子进入废物容器。 </li><li>重悬的细胞在1ml标记缓冲液。去除细胞等分计算和台盼蓝染色，以评估细胞活力按照步骤2.9。 </li><li>标记缓冲液的体积加至10×10 6个细胞/ ml;如果细胞是已经&lt;10×10 6，离心机在450×g离心7分钟，弃上清通过倾倒或关闭真空抽吸到废物容器中，并标记缓冲液的添加量至10×10 6个细胞/ ml。 <ol><li>设置的各约5-10×10 5个细胞的等分试样单独对未染色，同种型染色，并且单染色的对照细胞在微量离心管中。 </li></ol></li><li>添加5微克/毫升的CD4-FITC和1微克/毫升CD45RB-PE到细胞中。加同种型对照染色和单污渍以相同的浓度到合适的离心管中的等分试样。拌匀，但轻轻地，并培育冰上避光30分钟。 </li><li>加入10倍体积标记缓冲液的细胞和控制秒。离心机450 XG 7分钟。弃上清通过真空抽吸到废物容器。 </li><li>在标记缓冲液悬浮体积步骤4.5。离心机450 XG 7分钟。弃上清通过真空抽吸到废物容器。 </li><li>在标记缓冲液重悬，以10×10 6个细胞/ ml。通过70微米的过滤器进入FACS管传递细胞。置于冰上避光，直到准备流式细胞仪。 </li><li>建立和确定的细胞分选与未染色细胞和单染控制适当的补偿。 </li><li>排除不能存活的细胞与正向和侧向散射设门（ 图1A）。成立了CD4 +和CD45RB +细胞亚型染色控制门控。门上的细胞CD4 +人口。 </li><li>排序CD4 +细胞进入CD45RB 高 CD45RB 低的人群使用一个简单的柱状图PE染色细胞转化为管用2毫升全媒体。该CD45RB 高 </suP&gt;人口代表的CD4 + CD45RB +细胞（CD45RB 高 ）的最高40％，并且CD45RB 低人口是最低的20％的CD4 + CD45RB +细胞（CD45RB 低 ; 图1B）。 </li></ol><img alt="图1" src="/files/ftp_upload/52533/52533fig1.jpg" /> 图1：在FACS分析仪曲线的CD4 + CD45RB T细胞群的代表性流（A - C）的 FITC CD4-和从通过FACS分选入供体C57BL / 6小鼠的PE CD45RB染色的脾的CD4 + CD45RB 高和CD4 +。 CD45RB 低 T细胞群。 （A）双峰事件被排除在前进散点图。 （B）的淋巴细胞进行门控，在正向和侧向散射图。 （C）CD4 + </SUP&gt; T细胞被门控，和（D）的CD4 + CD45RB + T细胞上绘制的PE与事件直方图。 CD4 + CD45RB low细胞被认为是CD45RB +细胞的最低的20％。 CD4 + CD45RB 高细胞被定义为CD45RB +细胞的最高的40％。 <ol start="11"><li>运行的FACS机器上的每个细胞群体的等分试样来评估群体的纯度。 </li><li>离心分选的细胞在450×g离心7分钟。重悬在1ml PBS中。去除细胞等分计算和台盼蓝染色，以评估细胞活力按照步骤2.9。 </li></ol> 5.细胞注射到收件人<ol><li>重悬分选的细胞以4×10 6个细胞/ ml（CD45RB 高 ）或2×10 6个细胞/ PBS中毫升（CD45RB 低 ）。 </li><li>每个收件人转移100微升CD45RB 高细胞对新的无菌管。添加100微升PBS每收件人该管。因此，每只动物的总注射体积是200微升，和每受体细胞的总量为4×10 5个CD4 + CD45RB 高幼稚T细胞。 </li><li>如果实验组接受调节性T细胞的需要，每个收件人转移100微升CD45RB 高细胞对新的无菌管。加入100微升每收件人CD45RB 低细胞对相同的管中。每个动物的总注射量为200微升; CD45RB 高的比率：CD45RB low细胞是2：1。 </li><li>注入100微升CD45RB 高或CD45RB的高 / 低 CD45RB的CD4 +细胞腹膜内成每个收件人的腹部（总200微升）的右侧和左侧。 </li></ol>在收件人动物6.监测病情进展<ol><li>为了评估该受体动物的临床状况，对于以下paramete分配骨料临床得分在注射当天RS 8，此后每周，和在处死时： <ol><li>确定通过测量重量损失浪费：0 - 没有体重减轻; 1 - 0.1-10％的损失的初始体重; 2 - 10％以上的损失初始体重（ 图2A）的。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> 图2：炎症的临床和病理总额转移的迹象野生型CD4 + CD45RB 高 T细胞进入RAG1后出现- / -和NRDKO受体小鼠11（A）NRDKO收件人5告负的初始体重平均水平10％。周后转移，而RAG1- / -收件人未在此时显示出疾病的临床症状。每个点代表的初始体​​重为队列±SEM的平均百分比。 **，P &lt;0.005。 （B）中的一些小鼠发展严重的肠道炎症，这表现在直肠脱垂的存在。这是直肠脱垂的NRDKO受体小鼠的代表图片。 （C）的眼观从冒号既RAG1 - / -和NRDKO受体小鼠增厚，缩短与从RAG1冒号- / -和NRDKO小鼠没有T细胞过继转移。从NRDKO受体小鼠冒号显示严重的炎症，增加结肠重量（数据未显示）。 <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li><ol start="2"><li>通过将动物干净的容器确定大便质量，直到它已通过便血：0 - 无; 1 - 大便软; 2 - 水样和/或便血。 </li><li>确定动物的一般健康受疾病的下列迹象的存在：0 - 不弯腰驼背的姿势，毛的皮毛，或皮肤损伤; 1 - 以下目前的任何一个：弯腰驼背的姿势，毛的皮毛，或皮肤损伤。 </li><li>确定直肠脱垂存在：0 - 缺席; 1 -本（ 图2B）。 </li></ol></li><li>通过CO牺牲动物2吸入颈椎脱位时，他们已经失去了他们的出发体重20％，或期望的时间点，以先到者为准。临床疾病通常是显而易见的，在开始5周后饱食。 </li><li>评估疾病严重程度如前所述5,7。 <ol><li>分配临床得分如在步骤6.1 8。 </li><li>测量结肠长度和重量（ 图2C）。 </li><li>执行炎症的组织学分析5。 </li><li>在肠组织外植体培养9确定自发细胞因子的表达，肠系膜淋巴结10，和/或血清11。 <ol><li>简单地说，对于植文化9，牺牲后删除冒号，开放纵向和干净PBS。孵育在轨道摇床设定以250rpm在完整的媒体为30分钟，在室温下冒号。 </li><li>剁的组织切成小块并在完全培养基在37℃下放置24小时。收集上清液，并使用为每100mg组织中的细胞因子，通过ELISA定量。 </li></ol></li><li>执行的T细胞表型和/或功能10 体外表征。 </li></ol></li></ol>

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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=Extracellular+adenosine+regulates+colitis+through+effects+on+lymphoid+and+nonlymphoid+cells.">Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells.</a> Am J Physiol Gastrointest Liver Physiol. 307 (3), 338-346 (2014).</li><li>Naganuma, 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=Cutting+edge:+Critical+role+for+A2A+adenosine+receptors+in+the+T+cell-mediated+regulation+of+colitis.">Cutting edge: Critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis.</a> J Immunol. 177 (5), 2765-2769 (2006).</li><li>Ranatunga, D. 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=A+protective+role+for+human+IL-10-expressing+CD4++T+cells+in+colitis.">A protective role for human IL-10-expressing CD4+ T cells in colitis.</a> J Immunol. 189 (3), 1243-1252 (2012).</li><li>Srikrishna, G., 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=Carboxylated+glycans+mediate+colitis+through+activation+of+NF-kappa.">Carboxylated glycans mediate colitis through activation of NF-kappa.</a> B. J Immunol. 175 (8), 5412-5422 (2005).</li><li>Wang, F., 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=IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction.">IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction.</a> Gastroenterology. 131 (4), 1153-1163 (2006).</li></ol>

The authors declare that they have no competing financial interests.

在这里，我们描述了一个一步一步的协议，通过过继转移CD4 + CD45RB + T细胞的免疫缺陷小鼠诱导小鼠结肠的炎症。我们使用的C57BL / 6供体脾和同系RAG1 - / -受体小鼠，尽管其它菌株（ 例如 ，BALB / c小鼠，129S6 / SvEv，非肥胖糖尿病（NOD））免疫缺陷的和遗传模型（ 例如 ，SCID， 的Rag2 - / - ），也可以使用4,14-16。它是公认的背景品系?...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

有可用于研究人炎性肠疾病（IBD），每个都有其自己的优点和缺点的发病许多不同的动物模型。我们在这里描述通过过继转移的同源脾的CD4 + CD45RB 高 T细胞的成T和B细胞缺陷型受体小鼠启动的一个实验性结肠炎模型。在CD4 + CD45RB 高 T细胞群在很大程度上是由幼稚效应细胞能够诱导的慢性肠道炎症，非常类似于人类IBD的关键环节。这种方法可以被操纵来研究疾病发作和进展的方面。此外，它可以被用来研究先天，自适应的功能，以及调节免疫细胞群，和环境暴露的作用，也就是说 ，该微生物群，在肠道炎症。在这篇文章中，我们阐述了诱导结肠炎一步一步的协议的方法。这INC吕代的成功开发实验性结肠炎的研究目的这个小鼠模型所需要的关键技术环节的视频演示。

约10×10 6 CD4 + CD45RB 高Tg从成年C57BL / 6小鼠捐助10脾脏细胞是可靠的隔离。这个数目将根据患者的年龄的供体小鼠的应变和研究者的熟练程度而有所不同。当4×10 5只C57BL / 6的CD4 + CD45RB 高 T细胞转移到C57BL / 6 RAG1 - / -受体小鼠，疾病的临床症状出现围绕周5后饱食或更快，如果小鼠在遗传上易感更严重的疾病（ 图2，3）11,12。</sup...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

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 ...

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

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

16.2K 次观看.  University of North Carolina at Chapel Hill. 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.

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

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

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 ...

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.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

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 ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

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

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

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

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

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

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

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

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