作者衷心感谢鲍里斯Reizis在哥伦比亚大学的微生物学和免疫学系博士的有益讨论。我们也希望在康奈尔大学兽医学院，感谢冯富珍博士Bynoe，罗德曼Getchell博士和Deequa Mahamed从微生物学和免疫学系与流式细胞仪和视频编辑的协助下，黄路为技术援助。作者还希望感谢魏斯实验室杜哈明和魏斯实验室的成员，他们的手稿和视频制作，严格审查和斯蒂芬妮Yazinski育种和基因分型p53基因- / -小鼠。在康奈尔大学机构动物护理和使用委员会规定的准则和法规的规定进行动物实验。这项研究是由美国农业部部，合作国家研究，教育和推广服务，动物健康和疾病研究计划（GED和RSW）和美国国立卫生研究院拨款R03 HD058220和R01CA108773（RSW）提供的资金支持部分。视频制作杜哈明魏斯实验室的人员使用的内部设施。

1。肿瘤剖析<ol><li>肿瘤清扫干净的纸巾或一次性手术悬垂放在解剖台上，喷以70％的乙醇。 </li><li>本议定书小鼠常规安乐死的CO 2。安乐死鼠标放置在解剖台上，和动物两侧喷用乙醇。鼠标是在地方上的夹层板，使用四个或更多的推引脚通过插入腿和喷用70％乙醇彻底。 </li><li>从耻骨头的基地使用无菌剪刀，同时也避免了潜在的肌肉和渗透到体腔，在中线的皮肤被切断。 </li><li>打开腹腔，用无菌剪刀和镊子，小心不要损坏内部器官的一双新。 </li><li>通过解剖和偏转左肋揭示不损害血管的肿瘤，打开胸腔。 </li><li>从其他器官中分离出来的肿瘤，转移到一块冷PBS总的肿瘤约40-50％（约1.5 × 0.8 × 0.8厘米）保持在冰上。 </li><li>肿瘤的剩余部分可以保存为其他实验可以在这个时候进行一个完整的尸体剖检。 </li></ol> 2。培养淋巴瘤细胞<ol><li>喷雾管中，用70％乙醇和转移到了一个生物安全柜的解剖肿瘤。 </li><li> 10毫升培养液中添加150毫米无菌培养皿。 </li><li>转移到培养皿中，用吸管轻轻吸一块肿瘤。 </li><li>弯曲两个无菌18号针头面临斜角方。 </li><li>游离的肿瘤，使用弯曲的针。 </li><li>涡流的菜，请点击轻轻地放在一个角度，一个侧面收集细胞悬液，同时避免了大块肿瘤。 </li><li>吸50 mL锥形管的细胞悬液和转让，同时避免了大块肿瘤。 </li><li>洗净同一管板与10毫升的媒介和转移，同时避免大块肿瘤。 </li><li>混合轻轻的淋巴细胞和基质细胞组成的细胞悬液，并转移到了T - 25的组织培养瓶和10毫升到5毫升的T - 75的组织培养瓶中。轻轻漩涡的烧瓶。 </li><li>使用剩余的细胞悬浮于2.5毫升的最终体积（在使用几种类型的文化船只不同密度的培养细胞最大限度地建立一个T -细胞株的可能性）2，4，10100和1000倍稀释。 </li><li>板2.0毫升每稀释到6孔板个别井。轻轻旋流板。 </li><li>在加湿空气中的CO 2气氛中孵育48 h后的细胞。 </li></ol> 3。饲养淋巴瘤细胞<ol><li>孵化48小时后，添加5毫升的中型的T - 75烧瓶烧瓶的T - 25和10毫升介质沿墙，干扰最小的细胞。 </li><li>加入2毫升培养基，每孔的墙沿，干扰最小的细胞的6孔板。 </li><li>孵育细胞额外的72小时</li></ol> 4。传代淋巴瘤细胞<ol><li>初步通过<ol><li>在孵化第五天结束时，在1:1的比例传代细胞，同时保留原瓶和板（约3x106 /毫升的浓度保持在第一段落的细胞密度，促进建立一个成功的连续细胞系）。 <ol class="lalpha"><li>的T - 25烧瓶，转移到一个新的中等5毫升容量瓶中含有5 ml的细胞悬液。添加到原瓶5毫升的中型。 </li><li>的T - 75烧瓶中，转移到一个新的含有10毫升中等的容量瓶的10 ml的细胞悬液。添加到原瓶10毫升介质。 </li><li>对于6孔板，每口井从传输到相应的一个新的含有2毫升培养基板，每孔井2 ml的细胞悬液。添加到每一个原来的水井以及2毫升培养基。 </li></ol></li><li>所有额外的72小时孵育瓶和盘子根据我们的经验，大多数细胞系建立了从这些通道之一的文化（松散贴壁细胞悬浮聚合的附着的基质细胞的存在是一个已建立的细胞株的早期迹​​象）。 </li><li>对于较少或没有松散贴壁细胞悬浮聚合的文化，饲料仔细中期每72小时更换约50％的烧瓶或板为了避免去除细胞的原始文化，文化的船只罚站和不受干扰至少5分钟，让在底部的上清液中期之前温柔的愿望重力细胞泥沙。 </li></ol></li><li>维护建立的细胞株<ol><li>建立的细胞株传代成新瓶和盘子。对于T - 25和T - 75烧瓶中，加入5毫升的原件或通过暂停到5毫升中等。对于6孔板中，加入7 mL的容量瓶中的T - 25中型3毫升的原件或通过一个细胞悬液。饲料和更换新鲜培养液中删除的卷保留原或通过一种文化。 </li><li>通道设立了​​新的T - 25培养瓶细胞株。细胞系有一个倍增时间为18-24小时。文化是经常保持在1 - 3x106 /毫升的密度，每3天传代。以前通过文化需要的量添加到新的介质，以弥补10毫升。饲料和保留了原有的文化。 </li><li>几个段落后，非贴壁细胞会自我维持不贴壁的基质细胞的贴壁细胞数量较少，每个通道进行。除了这一点，可以保持文化的非组织培养治疗的T - 25培养瓶。 </li></ol></li></ol> 5。冻结和恢复淋巴瘤细胞株<ol><li>新鲜配制RPMI 1640培养液含20％胎牛血清和10％DMSO的培养基中的细胞系被冻结。 </li><li>从一个48小时的T - 25瓶（约1.5x10 6细胞/ mL）的旧文化的细胞在室温下5分钟1500转离心。丢弃上清后，细胞重新悬浮在2毫升冷冻结介质。两个1.0毫升细胞悬液卷从每个烧瓶放置在冷冻小瓶，立即冻结在-80 ° C，并转移到液态氮的翌日。 </li><li>从液氮保存的细胞恢复快速解冻，resuspending 10 mL新鲜培养液中的内容。另外，刚刚解冻的细胞在温暖的培养基上进行了一次洗涤之前删除二甲基亚砜10毫升的新鲜培养液resuspending。细胞培养随后通过前至少72 h。 </li></ol> 6。代表性的成果 自动取款机- / - T细胞线使用此协议，并指定DWJ - ATM - 1与抗CD3 - FITC，抗CD4 - APC和抗- CD8 - PE和流式细胞仪分析（图1）染色的特点。超过90％的T细胞，CD3，CD4和CD8 +三重阳性。 <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="图1" /> 图1:使用该协议所开发的T -细胞系的表征。 / - -建立ATM的细胞表面标记的细胞株指定DWJ - ATM - 1的特点是染色与抗CD3 - FITC，抗CD4 - APC，抗- CD8 - PE和流式细胞仪分析。简单地说，1X10 6新鲜细胞清洗一次冷PBS（1500转/ 5分），孵育4 ° C为10分钟与200μL抗体鸡尾酒（每个抗体1:200稀释1％BSA的PBS。细胞可以立即进行检查，而新鲜或固定为以后的分析。固定细胞准备加入缓冲的福尔马林在中立在终浓度为4％（体积/体积），并在4 ° C孵育30分钟，离心收获，（1500转/ 5分钟），重悬于200μL1％BSA的PBS。共1 × 10 4细胞进行了分析使用BD流式细胞仪刀魂分析仪和CellQuest软件（BD公司，加利福尼亚州圣何塞） 。

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	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

在这个协议中，我们提供了详细的程序，建立从两个不同的鼠标基因型的小鼠的T 细胞系;自动取款机- / - [FVB/N- ATM tm1Led / A]和p53 - / - [129/S6- Trp53 tm1Tyj / J]。 。通过使用这个协议中，共有6（满分7次尝试）自动取款机- / -三（5次尝试）P53 - / -小鼠的T细胞系已经建立在我们的实验室。 / - -证明该细胞系DWJ - ATM - 1，ATM细胞株有代表性的数据由一个细胞的人口，其中大于90％的细胞表达CD3 +，CD4和CD8表面标记证实，克隆的T细胞可使用我们的协议成立。 小鼠T细胞系的发展有报道 3,6,7;然而，为建立这些线路的详细协议并没有被描述以前。虽然只有两个鼠标基因型在我们的实验室小鼠的T细胞线的推导，我们相信，这些程序适用于自发T细胞淋巴瘤，其他如KRAS基因缺陷的小鼠品系中的一个广泛，以及其他可能动物物种。小鼠T细胞株有潜在的应用理解在免疫学和肿瘤学的基本问题。目前，我们正在利用这些细胞株在宿主 - 病原体相互作用和genotoxin细胞毒作用，包括细胞周期停滞和细胞凋亡的机制特征的研究。鉴于小鼠T细胞株的份额，如PTEN和FBXW7缺失的基因的改变与人类T细胞急性淋巴细胞白血病和淋巴瘤共同，它们特别适合于研究在癌症生物学8，并可能有进一步的应用，快速前在体外免疫学和癌症化疗药物的临床疗效筛选。无论这些细胞株，可在同源或免疫功能低下小鼠体内传播尚未确定。 对比以前的协议，其中的T -细胞系成功建立了3个月6个，开发利用当前协议的细胞株，建立在一个月内（通过2-8之间） 。我们发现，存在较松散贴壁细胞附着的基质细胞聚集建立的细胞株是一个重要的的早期迹象。为了让小鼠T细胞能无限期地传播，我们也发现，重要的是，离开最初的松散贴壁细胞聚集在喂食不受干扰。此外，在第一段落的细胞密度维持在约3X10 6 /毫升的浓度，还促进成功地建立了一个连续的细胞株。根据我们的经验，在使用几种类型的文化船只的不同密度的培养细胞，建立一个T -细胞株的可能性最大化。大多数T细胞系建立了从T - 75培养瓶或6孔板和10或100倍稀释，这对应约1 × 10 7或1 × 10 6细胞/ ml 。一个先前的协议，建议在5 × 10 6，7，1 X10和2 × 10 7 6孔板每孔细胞培养细胞，喂养后7天（7）。我们观察，初步培养后48小时，并通过72小时喂5天，是最佳的。不再等待前一次投料和通过，将导致细胞失去其可行性，并停止分裂。 最后一个参数，我们发现，在体外对小鼠T细胞成功繁殖的关键是，可以支持快速的细胞生长在培养基中的FBS。 FB的不同地段和供应商应开始前确定一个合适的试剂T细胞系的推导的筛选。如果小鼠T细胞株筛选，水井24孔板放置在浓度为1 × 10 6每毫升含有刀豆蛋白A（1μg/毫升），以刺激细胞快速增长试验介质的胸腺淋巴细胞的主要文化是足够的。在FB的最佳来源存在，细胞的数量应该增加一倍每隔18至24小时

<ul><li>冰桶</li><li> 70％的乙醇</li><li>覆盖铝箔和引脚的泡沫块组成的夹层板</li><li>两套小无菌剪刀和镊子</li><li>无菌150毫米2培养皿（赛默飞世尔科技，沃尔瑟姆，马，猫＃08-757-13） </li><li>无菌18表针</li><li> 50 ml锥形管（康宁公司，康宁纽约猫＃430829） </li><li> 10％的福尔马林</li><li>组织培养瓶<ul><li> T - 25（TPP，Trasadingen，瑞士，CAT＃90025） </li><li> T - 75（TPP，Trasadingen，瑞士，CAT＃90075） </li></ul></li><li> 6孔板（康宁公司康宁公司，纽约;猫＃3506） </li><li>无菌磷酸盐缓冲液pH值7.2（PBS） </li><li>培养基<ul><li> RPMI 1640培养液，与25毫米的HEPES，200毫米以下补充L -谷氨酰胺（龙沙，Walkersville，MD;猫＃12 - 115F）: <ul><li> 10％热灭活（56℃，30分钟）小牛血清（FBS，Hyclone公司，UT斯达康，洛根＃SH30088.03猫） </li><li> 1％青霉素和链霉素（Mediatech，弗吉尼亚州马纳萨斯的猫＃30 - 002 - CI） </li><li> 1％的不重要的氨基酸（猫＃25 - 025 - CI Mediatech） </li><li> 55毫米2β-巯基乙醇（Sigma公司，圣路易斯，密苏里州，猫＃M752） </li></ul></li></ul></li><li>二甲基亚砜（DMSO; Calbiochem，拉霍亚，CA;猫＃317275） </li><li>抗体（eBioscience公司，加利福尼亚州圣迭戈） <ul><li>抗CD3 - FITC（货号＃11-0031-85） </li><li>抗CD4 - APC（货号＃17-0041-83） </li><li>抗- CD8 - PE（货号＃12-081-85） </li></ul></li><li>牛血清白蛋白（BSA;生命科技，大岛，NY;猫＃11018-017） </li><li>伴刀豆球蛋白A（Sigma公司猫＃C5275） </li><li> 1.8毫升冷冻瓶（NUNC，丹麦的罗斯基勒，猫＃368632） </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

建立的细胞系是一个重要的的研究工具，它可以减少使用实验动物的研究。某些菌株的转基因小鼠， 如 ATM - / - 和 p53 - / -胸腺淋巴瘤不断发展早在生活中1,2，因此，可以作为一个可靠的来源小鼠T细胞株的推导。在这里，我们建立了小鼠胸腺淋巴瘤的T细胞系的发展，而不需要添加白细胞介素1,3以前的协议来描述一个详细的协议。从年龄在3至6个月的小鼠肿瘤收获，最早表现的基础上，弯腰驼背的姿势观察可见肿瘤，呼吸困难，穷人疏导，并在敏感品系1,4浪费。我们已经成功地建立了几个使用此协议的T细胞系和近交系的自动取款机- / - [FVB/N- 自动柜员机tm1Led / J] 2 和p53 - / - [129/S6- Trp53 tm1Tyj / J] 5只。我们进一步表明，既定的T细胞的人口超过90％表示CD3，CD4和CD8。与稳定建立的细胞株，使用本议定书所产生的T细胞已传了一年多。

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

在这个视频中，我们展示了一个协议来建立小鼠胸腺淋巴瘤细胞株。通过这个协议，我们已经成功地建立了几个从ATM / T细胞线 - 和p53 - / - 小鼠胸腺淋巴瘤。

胸腺淋巴瘤的T细胞系的推导 ATM - / -</sup P53 - / -</sup鼠标

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

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Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

12.2K 次观看.  Cornell University. 在这个视频中，我们展示了一个协议来建立小鼠胸腺淋巴瘤细胞株。通过这个协议，我们已经成功地建立了几个从ATM / T细胞线 - 和p53 - / - 小鼠胸腺淋巴瘤。

视频: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

科研

免疫与感染

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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

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

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

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

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

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

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

制备及胸腺器官切片文化中的应用

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

胸腺淋巴瘤的T细胞系的推导 ATM^{- / - P53^{- / -鼠标}}

摘要

探索更多视频

此视频中的章节

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

T细胞受体在小鼠T细胞的逆转录病毒转导

考试流式细胞仪检测胸腺阳性和阴性选择

T细胞实时成像实时信号复合物的形成

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

对T细胞推导

鼠标朴素CD4

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

制备及胸腺器官切片文化中的应用

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

胸腺淋巴瘤的T细胞系的推导 ATM - / - P53 - / -鼠标

摘要

探索更多视频

此视频中的章节

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

T细胞受体在小鼠T细胞的逆转录病毒转导

考试流式细胞仪检测胸腺阳性和阴性选择

T细胞实时成像实时信号复合物的形成

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

对T细胞推导

鼠标朴素CD4

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

制备及胸腺器官切片文化中的应用

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

胸腺淋巴瘤的T细胞系的推导 ATM^{- / - P53^{- / -鼠标}}