这项工作是支持由美国国立卫生研究院（5U01CA137070）的赠款和美国癌症协会（RSG - 09 - 070 - 01 - LIB），一个癌症研究所研究员奖（MK），美国心脏协会前博士后研究（KM），和的教育和科学奖学金（APG）的西班牙部。 我们想感谢博士尼古拉Restifo和指压于博士（NIH / NCI）的协议的有益的建议。

1。准备逆转录病毒载体构建<ol><li>小组克隆利益的T细胞受体（TCR）基因逆转录病毒载体（图1，例如向量，PMSG 7，pMIGII 5,8，从Cellbiolabs pMXs） 。的TCRα和β链基因应该是相同的启动子控制下的同一载体上，以确保平等的表达。如果感兴趣的TCR是人类，人类不断域需要将鼠标不断域9更换。我们的观察是，全长人类电阻温度系数并不稳定表达对小鼠T细胞。 </li><li>准备高质量的质粒DNA用Qiagen公司马克西准备试剂盒或类似的产品。质粒的浓度应在约1微克/μL。 </li></ol> 2。转染和T细胞隔离日-1（板包装细胞） <ol><li>打跑铂- E（PLAT - E Cellbiolabs）用胰蛋白酶- EDTA的逆转录病毒包装细胞，用DMEM媒体和压制。 </li><li>离心5分钟在1000 XG的细胞。吸上清，悬浮细胞沉淀的DMEM媒体。 </li><li>确定细胞数量和0.6x10 6 /毫升稀释的细胞。板10毫升细胞悬浮在一个10mm的多聚赖氨酸涂层的组织培养板和一个细胞培养箱中过夜增长在37℃，5％的CO 2。 </li></ol> 0天（转） <ol start="4"><li>第二天早晨，检查光显微镜下的细胞。应约80％融合的细胞。 </li><li>轻轻地从板中删除媒体。一次1X PBS清洗细胞，并没有青霉素，链霉素（PEN /链球菌）添加10毫升的预热，新鲜的DMEM媒体。 （注：笔/链球菌可以干扰与转） </li><li>准备转染复杂。 准备组合A和B如下： <table border="0"><tbody><tr><td>混合：质粒DNA </td><td> 9微克</td></tr><tr><td> PCL - ECO辅助质粒</td><td> 6.3微克</td></tr><tr><td> OptiMEM </td><td> 1.5毫升</td></tr><tr><td colspan="2"></td></tr><tr><td>混合物B：Lipopfectime 2000 </td><td> 60微升</td></tr><tr><td> OptiMEM </td><td> 1.5毫升</td></tr></tbody></table> 孵育5分钟A和B分别在室温 混合A和B一起轻轻为20分钟，在室温下孵育形式转复杂。 </li><li>慢慢地滴入开发平台- E细胞的混合物（共3毫升）。轻轻摇动板来回的转染混合物均匀地分发。 </li><li>细胞在37 ° C / 5％的CO 2在细胞培养箱。 </li><li> 6-8小时后，取代病毒生产的新鲜DMEM培养基用10 mL（与PEN /链球菌）的媒介。 </li></ol>涂层板与抗体<ol start="10"><li>小鼠T细胞病毒转导前需要激活。有各种方法来激活T细胞。在这里，我们用板的约束反CD3e和抗CD28抗体。准备1微克/毫升抗CD3e和2微克/毫升无菌PBS的抗CD28抗体的混合物。 </li><li>分送250μL抗体的混合物，每孔24孔组织培养板。该板块可以涂在一夜之间在4 ° C或2小时37 ° C。 </li></ol>从小鼠脾脏单细胞悬浮液的制备<ol start="12"><li>收获小鼠脾和转移的RPMI培养基。 </li><li>将单元格中的过滤器的脾。捣碎成一个5厘米的组织培养板的注射器柱塞脾。用无菌PBS冲洗掉细胞过滤器的细胞。 </li><li>离心1000 XG，5分钟。 </li><li>弃上清。在确认裂解缓冲液悬浮细胞沉淀（2％毫升脾）。孵育2〜3分钟在室温。添加20毫升的RPMI培养基，离心5分钟XG 1000。 </li><li>弃上清。悬浮细胞沉淀在无菌PBS。确定细胞数为5分钟，离心1000 XG在4 ° C。进入T细胞的分离。 </li></ol>小鼠T细胞的分离和激活<ol start="17"><li>根据产品说明书（CD8 + T细胞分离试剂盒从美天旎生物技术），磁标签的细胞。 </li><li>放置在米LS列（美天旎生物技术）agnetic领域。应用到的列标记的细胞悬液。收集流量通过（丰富小鼠CD8 + T细胞）。列洗净，用3 mL缓冲三次，根据产品说明书并结合与以前流过。 </li><li>反CD3e和反CD8a抗体染色的细胞，并通过流式细胞仪分析（图2a）。对于一个典型的分离，细胞总数的8-10％可以被孤立。 〜90％，分离出的细胞CD8 + T细胞。 </li><li>离心机在1000 XG，5分钟的细胞。上清液。重组人IL - 2（rhIL2，20毫微克/毫升）在1 × 10 6 /毫升，重悬细胞沉淀于RPMI培养基。 </li><li>之前加入的细胞，取出板（准备在2.11以前）抗体的解决方案。每孔用无菌PBS和删除PBS冲洗。 </li><li>加入1毫升细胞悬液，每孔。置于37 ° C / 5％CO 2细胞培养箱中板。 </li></ol> 3。 （2和第3天天）感染T细胞激活<ol><li>经过2天的病毒生产，在开发平台- E细胞板的介质应变成黄色。病毒上清转移到15 mL锥形管。添加10毫升的新鲜DMEM培养基为进一步的病毒生产板（可选）。 </li><li>离心5分钟，以去除细胞碎片在1000 XG病毒上清。小心病毒上清转移到一个新的管。保留一些液体的试管底部，不打扰细胞碎片。 </li><li>从板材到50 mL锥形管收集激活T细胞。确定细胞数量。保存到一个单独的管为阴性（未转）控制使用的一些细胞。离心机在1000 XG 5分钟，弃上清。 </li><li>重悬在10 6细胞每1毫升20毫微克/毫升rhIL2和10微克/毫升硫酸鱼精蛋白在病毒上清的细胞沉淀。加入1 ml的细胞悬液，每孔24孔板。 rhIL2和硫酸鱼精蛋白的相同数额相同的细胞密度控制在RPMI培养基管重悬细胞。添加细胞相同的板。 </li><li>包装用塑料薄膜板。在2000年的XG离心90分钟，在32 ° C，没有刹车。 </li><li>离心后，取出塑料薄膜。 1毫升新鲜的RPMI培养基添加20 ng / mL的rhIL2每口井。把板的孵化器。 </li><li> （可选）的表达水平较高的TCR可以通过收集病毒上清，重复3天的感染过程。 </li></ol> 4。扩展的细胞和转染效率的评价<ol><li>检查每日转导的T细胞。拆分在必要时在RPMI培养基rhIL2 1:3比例的细胞。不要让细胞长满（中等变为黄色）。 </li><li>在6或第7天天，染色与抗体和MHC四聚体，以评估对T细胞表面的TCR的表达特定的TCR细胞。使用未转导的T细胞作为对照（图2c）。 </li><li>导的T细胞，准备进一步的下游应用。 </li></ol> 5。代表性的成果它往往是有利的，净化前转导的T细胞亚群，虽然脾可以不净化转。使用商业磁珠（图2a），可以得到高纯度的T细胞亚群。 要评估的T细胞上的电阻温度系数的表达水平，可用于特定抗体TCRα或β链。通常30％至80％的细胞可以表达转导TCR（图2b）根据TCR的基因和病毒滴度。 MHC四聚体为特定的TCR也可用于进一步验证功能表达的电阻温度系数（图2c）。 <img src="/files/ftp_upload/2307/2307fig1.jpg" alt="图1" /> 图1。 T细胞受体基因的例子构造子克隆到逆转录病毒载体 。全长α和β链基因的自我裂解2A肽连接器8挂钩。 <img src="/files/ftp_upload/2307/2307fig2.jpg" alt="图2" /> 图2。成功分离和小鼠T细胞的转导。范 ​​例（一）使用CD8a + T细胞分离试剂盒（美天旎生物技术公司）和反CD3e和反CD8a抗体染色脾CD8 + T细胞隔离。隔离CD8 + T细胞的转R6C12的TCR具体为人类GP100：209 -217 4肽与抗人Vbeta 8抗体（b）和HLA - A2kb GP100四聚体（C）染色。

<ol>
	<li>Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+T+cells+'see'+antigen.">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li><li>Berg, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li><li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li><li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li><li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li><li>Szymczak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li><li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin.">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li><li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li></ol>

几个步骤，以达到最佳效果的关键。开发平台- E包装细胞系应保持在健康状态。如果有必要，可以传代细胞与1微克/毫升嘌呤，10微克/毫升杀稻瘟逆转录病毒结构蛋白的表达，以防止损失的DMEM培养基几次。细胞在转染当天，应〜80％融合。太少或太多的细胞可能会减少病毒滴度。该病毒的质量可以检查测试可以很容易导的细胞株。由于电阻温度系数要求CD3复合物在细胞表面的表达，我们经常使用58α-/β-杂交瘤细胞10（不表达内源性的T细胞受体α或β链的，但不表达CD3复合物的细胞系）。 1毫升病毒上清转导细胞时，我们获得近100％，杂交瘤细胞的转染效率。最后，T细胞被激活和增殖，因为逆转录病毒只能感染积极分裂的细胞。 这里介绍的方法产生抗原特异性T细胞有几个限制。首先，这是很难达到100％，小学小鼠T细胞的转导效率。部分细胞不表达利益的TCR。二，转导的TCRα和β链，可以mispair与内源性电阻温度系数，这可能会减少利益的TCR的表达水平。此外，mispaired电阻温度系数可能导致在体内 11的自身免疫性疾病。最后，有只导的T细胞可用于有限的时间框架。经过一周左右，除非被重新刺激细胞凋亡的细胞进行。然而，细胞可能会耗尽而失去功能与重复重新刺激。

媒体： DMEM培养基（DMEM培养液10％热灭活胎牛血清+ PEN /链球菌+ L -谷氨酸+丙酮酸钠+非必需氨基酸）。 DMEM培养基（DMEM培养基相同，但没有加入笔/链球菌），无笔/链球菌。 的RPMI培养基（RPMI 10％热灭活胎牛血清+ L - GLU +非必需氨基酸+β- Mercaphoethanol +钢笔/链球菌+丙酮酸钠）。

retroviral transduction of t-cell receptors in mouse t-cells

在免疫系统的T细胞受体（电阻温度系数）发挥核心作用。在T细胞表面的电阻温度系数，可以特异性识别肽抗原，抗原提呈细胞（APCs）1提出。这种认识导致激活T细胞和一系列功能的结果（如细胞因子的产生，靶细胞的杀伤）。了解电阻温度系数的职能作用是至关重要的，利用免疫系统的力量来治疗各种免疫相关疾病（如癌症或自体免疫）。 研究在小鼠模型的电阻温度系数，它可以通过多种方式完成，这是方便。制作TCR转基因小鼠模型是昂贵和费时，目前还只是一个数量有限的可用 2-4 。另外，与小鼠的抗原特异性T细胞可产生骨髓嵌合体。此方法还需要几个星期，和需要的专业知识 5 。逆转录病毒转导电阻温度系数为T细胞在体外激活鼠标是一个快速和相对容易的方法来获取所需的肽- MHC的特异性T细胞。抗原特异性T细胞可在一周内产生任何下游应用中使用。学习导的T细胞也有直接应用到人类免疫，人类T细胞过继转移与特定抗原的电阻温度系数转导是一个新兴的癌症治疗6战略。 在这里，我们提出了一个协议retrovirally在体外激活的小鼠T细胞转导电阻温度系数到的。可用于人类和小鼠的TCR基因。携带特定的T细胞受体基因的逆转录病毒感染小鼠抗CD3和抗CD28抗体激活T细胞的生成和使用。 在体外扩增后，转导的T细胞流式细胞仪分析。

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

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

我们提出了一个协议，以产生抗原特异性的小鼠的T细胞，用逆转录病毒转导

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 ...

retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

Research

JoVE Journal

Immunology and Infection

34.6K 次观看.  New York University School of Medicine. 我们提出了一个协议，以产生抗原特异性的小鼠的T细胞，用逆转录病毒转导

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

科研

免疫与感染

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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&amp;#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 ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

小鼠肾-居民CD8+T 细胞的分离，用于流细胞测量分析

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

全谱流式细胞术分析原代小鼠T细胞受体诱导的钙内流

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...