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>

<ol><li>Xavier, R. J., Podolsky, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unravelling+the+pathogenesis+of+inflammatory+bowel+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Unravelling the pathogenesis of inflammatory bowel disease.</a> Nature. 448 (7152), 427-434 (2007).</li>
<li>Cho, J. H., Brant, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+insights+into+the+genetics+of+inflammatory+bowel+disease%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Recent insights into the genetics of inflammatory bowel disease.</a> Gastroenterology. 140 (6), 1704-1712 (2011).</li>
<li>Jostins, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host-microbe+interactions+have+shaped+the+genetic+architecture+of+inflammatory+bowel+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.</a> Nature. 491 (7422), 119-124 (2012).</li>
<li>Powrie, F., Leach, M. W., Mauze, S., Caddle, L. B., Coffman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotypically+distinct+subsets+of+CD4%2B+T+cells+induce+or+protect+from+chronic+intestinal+inflammation+in+C.+B-17+scid+mice%5BTitle%5D)+AND+%22Int+Immunol%22%5BJournal%5D)">Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice.</a> Int Immunol. 5 (11), 1461-1471 (1993).</li>
<li>Ostanin, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+cell+transfer+model+of+chronic+colitis%3A+concepts%2C+considerations%2C+and+tricks+of+the+trade%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade.</a> Am J Physiol Gastrointest Liver Physiol. 296 (2), 135-146 (2009).</li>
<li>Ma, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Routine+habitat+change%3A+a+source+of+unrecognized+transient+alteration+of+intestinal+microbiota+in+laboratory+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Routine habitat change: a source of unrecognized transient alteration of intestinal microbiota in laboratory mice.</a> PLoS One. 7 (10), e47416(2012).</li>
<li>Read, S., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+inflammatory+bowel+disease+in+immunodeficient+mice+by+depletion+of+regulatory+T+cells%5BTitle%5D)+AND+%22Curr+Protoc+Immunol%22%5BJournal%5D)">Induction of inflammatory bowel disease in immunodeficient mice by depletion of regulatory T cells.</a> Curr Protoc Immunol. Chapter 15 (Unit 15 13), (1999).</li>
<li>Maillard, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Wiskott-Aldrich+syndrome+protein+is+required+for+the+function+of+CD4%28%2B%29CD25%28%2B%29Foxp3%28%2B%29+regulatory+T+cells%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">The Wiskott-Aldrich syndrome protein is required for the function of CD4(+)CD25(+)Foxp3(+) regulatory T cells.</a> J Exp Med. 204 (2), 381-391 (2007).</li>
<li>Hegazi, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Carbon+monoxide+ameliorates+chronic+murine+colitis+through+a+heme+oxygenase+1-dependent+pathway%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway.</a> J Exp Med. 202 (12), 1703-1713 (2005).</li>
<li>Kole, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+I+IFNs+regulate+effector+and+regulatory+T+cell+accumulation+and+anti-inflammatory+cytokine+production+during+T+cell-mediated+colitis%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Type I IFNs regulate effector and regulatory T cell accumulation and anti-inflammatory cytokine production during T cell-mediated colitis.</a> J Immunol. 191 (5), 2771-2779 (2013).</li>
<li>Kobayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NFIL3-deficient+mice+develop+microbiota-dependent%2C+IL-12%2F23-driven+spontaneous+colitis%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">NFIL3-deficient mice develop microbiota-dependent, IL-12/23-driven spontaneous colitis.</a> J Immunol. 192 (4), 1918-1927 (2014).</li>
<li>Steinbach, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innate+PI3K+p110delta+Regulates+Th1%2FTh17+Development+and+Microbiota-Dependent+Colitis%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Innate PI3K p110delta Regulates Th1/Th17 Development and Microbiota-Dependent Colitis.</a> J Immunol. 192 (8), 3958-3968 (2014).</li>
<li>Kobayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NFIL3+is+a+regulator+of+IL-12+p40+in+macrophages+and+mucosal+immunity%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">NFIL3 is a regulator of IL-12 p40 in macrophages and mucosal immunity.</a> J Immunol. 186 (8), 4649-4655 (2011).</li>
<li>Leach, M. W., Bean, A. G., Mauze, S., Coffman, R. L., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+bowel+disease+in+C.B-17+scid+mice+reconstituted+with+the+CD45RBhigh+subset+of+CD4%2B+T+cells%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Inflammatory bowel disease in C.B-17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells.</a> Am J Pathol. 148 (5), 1503-1515 (1996).</li>
<li>Powrie, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+Th1+responses+prevents+inflammatory+bowel+disease+in+scid+mice+reconstituted+with+CD45RBhi+CD4%5BTitle%5D)+AND+%22T+cells.+Immunity%22%5BJournal%5D)">Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4.</a> T cells. Immunity. 1 (7), 553-562 (1994).</li>
<li>Read, S., Malmstrom, V., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+T+lymphocyte-associated+antigen+4+plays+an+essential+role+in+the+function+of+CD25%28%2B%29CD4%28%2B%29+regulatory+cells+that+control+intestinal+inflammation%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation.</a> J Exp Med. 192 (2), 295-302 (2000).</li>
<li>Rogers, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+divergence+in+gastrointestinal+microbiota+in+physically-separated+genetically+identical+mice%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice.</a> Sci Rep. 4, 5437(2014).</li>
<li>Fukata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+myeloid+differentiation+factor+88+%28MyD88%29+is+required+for+CD4%2B+T+cell+effector+function+in+a+murine+model+of+inflammatory+bowel+disease%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease.</a> J Immunol. 180 (3), 1886-1894 (2008).</li>
<li>Kurtz, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+adenosine+regulates+colitis+through+effects+on+lymphoid+and+nonlymphoid+cells%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">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/pubmed?term=((Cutting+edge%3A+Critical+role+for+A2A+adenosine+receptors+in+the+T+cell-mediated+regulation+of+colitis%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((A+protective+role+for+human+IL-10-expressing+CD4%2B+T+cells+in+colitis%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((Carboxylated+glycans+mediate+colitis+through+activation+of+NF-kappa%5BTitle%5D)+AND+%22B.+J+Immunol%22%5BJournal%5D)">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/pubmed?term=((IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">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><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>12 mm x 75 mm 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>26 G 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 &amp;#956;m 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>&amp;beta;-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% &amp;beta;-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 colspan="4">[header]</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% &amp;beta;-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

16.5K 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

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

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

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

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

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

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

科研

免疫与感染

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

活小鼠肠道移植物对宿主病的诱导及微内窥镜评价

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

在 Vivo 增强的古特-霍姆调节 T 细胞诱导

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

医学

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

白细胞介素10缺陷小鼠模型中粪便微生物群移植的治疗评估

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

诱导小鼠肠道炎症由效应CD4的过继转移

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移植物抗宿主病的诱导和