Name: quantification of proliferating human antigen-specific cd4+ t cells using carboxyfluorescein succinimidyl ester
Uploaded: 2019-06-04T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester at JoVE.com

这项工作得到了:青少年糖尿病研究基金会的支持[JDRF 5-CDA-2014-210-A-N](S.M.)。国家健康和医学研究委员会(NHMRC GNT123586)(S.M.),糖尿病澳大利亚研究信托千年奖(Y17M1-MANS)(S.M.),维多利亚州政府运营基础设施支助计划(S.M.,A.D.,E.T.,M.S.)和NHMRC研究生奖学金APP1094337和JDRF博士补发奖学金(硕士)。

所有受试者在采集外周血之前都给予知情同意。圣文森特霍斯普蒂亚尔(HREC-A 135/08和HREC-A 161/15)对使用PBMC的实验给予伦理批准。
1. 试剂制备
<ol><li>
人类T细胞培养基
	<ol>
<li>准备RP-5培养PBMC,包括RPMI 1640、1x非必需氨基酸、L-丙烯-谷肽(2 mM)、青霉素(100 U/mL)/链霉素(0.1毫克/mL)和5%的人类血清(PHS)。</li>
	</ol>
</li>
	<li>
CFSE 库存解决方案
	<ol>
<li>通过在+9 mL DMSO?...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> Journal of Immunological Methods. 171, 131-137 (1994).</li><li>Mannering, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> Journal of Immunological Methods. 283, 173-183 (2003).</li><li>Quah, B. J. C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Carboxyfluorescein+Diacetate+Succinimidyl+Ester+(CFSE)+to+Monitor+Lymphocyte+Proliferation.">The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation.</a> Journal of Visualized Experiments. , 4-7 (2010).</li><li>Ten Brinke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+T-cell+responses+in+translational+studies:+Optimization+of+dye-based+proliferation+assay+for+evaluation+of+antigen-specific+responses.">Monitoring T-cell responses in translational studies: Optimization of dye-based proliferation assay for evaluation of antigen-specific responses.</a> Frontiers in Immunology. 8, 1-15 (2017).</li><li>Gillespie, G. M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+TCR+Conservation+and+Altered+T+Cell+Cross-Reactivity+Characterize+a+B*57-Restricted+Immune+Response+in+HIV-1+Infection.">Strong TCR Conservation and Altered T Cell Cross-Reactivity Characterize a B*57-Restricted Immune Response in HIV-1 Infection.</a> Journal of Immunolgy. 177, 3893-3902 (2006).</li><li>Tynan, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Resolution+Structures+of+Highly+Bulged+Viral+Epitopes+Bound+to+Major+Histocompatibility+Complex+Class+I:+Implications+for+t-cell+receptor+engagement+and+t-cell+immunodominance.">High Resolution Structures of Highly Bulged Viral Epitopes Bound to Major Histocompatibility Complex Class I: Implications for t-cell receptor engagement and t-cell immunodominance.</a> Journal of Biological Chemistry. 280, 23900-23909 (2005).</li><li>Blanchard, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+aminopeptidase+associated+with+antigen+processing+defines+the+composition+and+structure+of+MHC+class+I+peptide+repertoire+in+normal+and+virus-infected+cells.">Endoplasmic reticulum aminopeptidase associated with antigen processing defines the composition and structure of MHC class I peptide repertoire in normal and virus-infected cells.</a> Journal of Immunology. 184, 3033-3042 (2010).</li><li>Glanville, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specificity+groups+in+the+T+cell+receptor+repertoire+repertoire.">Identifying specificity groups in the T cell receptor repertoire repertoire.</a> Nature. 547, 94-98 (2017).</li><li>Wooldridge, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricks+with+tetramers:+how+to+get+the+most+from+multimeric+peptide+MHC.">Tricks with tetramers: how to get the most from multimeric peptide MHC.</a> Immunology. 126, 147-164 (2009).</li><li>Mannering, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+cloning+human+autoantigen-specific+T+cells.">An efficient method for cloning human autoantigen-specific T cells.</a> Journal of Immunological Methods. 298, 83-92 (2005).</li><li>So, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proinsulin+C-peptide+is+an+autoantigen+in+people+with+type+1+diabetes.">Proinsulin C-peptide is an autoantigen in people with type 1 diabetes.</a> Proceedings of the National Academy of Sciences of the United States of America. 115, (2018).</li><li>Hui-Yuen, J., Mcallister, S., Koganti, S., Hill, E., Bhaduri-Mcintosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+Epstein-Barr+Virus+Growth-transformed+Lymphoblastoid+Cell+Lines.">Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines.</a> Journal of Visualized Experiments. , 2-7 (2011).</li><li>Alexander-Miller, M. 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基于CFSE的增殖是检测和枚举抗原特异性人类CD4+T细胞的简单而可靠的方法。此前已经证明,使用CFSE的最佳浓度对于最佳结果4至关重要。过多的CFSE会废除增殖,而太少不允许分离和未分割的细胞被区分。相比之下,相对较高的CFSE浓度(5.0μM)用于标记纯化的鼠T细胞3。由于批次之间的变异性,建议对每批进行定位,以确定最佳浓度。我们使用当前批次的浓度(10 nM)...

检测和研究抗原特异性T细胞的能力在细胞介导免疫研究中非常重要。然而,这样做对于自身抗原特异性CD4和T细胞反应尤其具有挑战性,后者非常弱,难以检测。用于检测抗原特异性淋巴细胞增殖的常用方法是[3H]-胸腺素,这是一种放射性标记的核苷酸,并结合在增殖细胞的DNA中。虽然[3H]-胸腺酰胺测定可以检测DNA合成,但这种方法是细胞分裂的间接测量,因为DNA合成可以独立于线粒体分裂(即,在基因复制和凋亡1期间)启动。由于细胞抗原特异性增殖可导致大量细胞凋亡,导致抗原特异性增殖的潜在高估,使这一问题更加复杂。此外,[3H]-胸腺素法不为扩散淋巴细胞提供花样信息,如CD4+或CD8+在受抗原肽刺激的PBMC中系生增殖。
2003年,我们发布了第一个使用CFSE的染料稀释测定,称为CFSE增殖测定3,4。CFSE 是一种荧光染料,通过与细胞内溶酶残留物形成共价键,与细胞内蛋白质形成稳稳结合。由于CFSE标记的蛋白质在子细胞3之间平均分配,分裂细胞可以通过流动细胞测定来区分未分割的细胞,这也允许淋巴细胞群的定量型型。事实上,细胞从CFSE染色时开始经历的分裂数量可以测量到一定程度5。最近,许多类似的染料,如细胞跟踪紫罗兰增殖染料(VPD)和细胞跟踪染料已经开发出来,它们的工作方式相似6。该协议侧重于 CFSE,但这些原则同样适用于其他相关染料。
肽-MHC四聚体染色是检测和克隆抗原特异性CD8+T细胞的广泛使用的方法。这是一个公认的方法7,8,9,10;然而,基于四元体的克隆需要现有的表位/MHC限制知识,每个表位都需要自己的四元体11,这限制了发现和克隆新型表位特异性T细胞的范围。基于CFSE的增殖可用于肽、蛋白质或细胞裂物。本文所述的协议既简单又坚固,响应CD4+T细胞可分类,用于下游功能和生化表征测定12,13。

<table>
	<tbody>
		<tr>
			<td>Anti-human CD4-AlexaFluor647</td>
			<td>Biolegend</td>
			<td>317422</td>
			<td>RRID:AB_2716180</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein succinimidyl ester (CFSE)</td>
			<td>ThermoFisher</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (1x)</td>
			<td>Gibco</td>
			<td>35050</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A H1N1 (PR8) Matrix protein 1</td>
			<td>Sino Biological</td>
			<td>40010-V07E</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential amino acids (1x)</td>
			<td>Gibco</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin (1x)</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Pooled human serum</td>
			<td>Australian Red Cross</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Proinsulin C-peptide PI33-63</td>
			<td>Purar Chemicals</td>
			<td>N/A</td>
			<td>Custom made synthetic peptide</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetanus Toxoid protein</td>
			<td>Statens Serum Intitut</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of proliferating human antigen-specific cd4+ t cells using carboxyfluorescein succinimidyl ester

所述是一种简单的体外染料稀释方法,用于测量人类外周血单核细胞(PBMC)中抗原特异性CD4+T细胞增殖。稳定、无毒的荧光染料(如卡博基氟辛苏奇尼酰基酯(CFSE)的开发,通过减少荧光染色(通过流式细胞测定)来区分稀有的抗原特异性T细胞。与替代方法不同,此方法具有以下优点:(i) 对低频 T 细胞非常敏感,(ii) 不需要抗原或表位的知识,(iii) 可以分析响应细胞的表型,以及 (iv) 可行、有反应的细胞可以排序,并用于进一步分析,如T细胞克隆。

用破伤风类毒素蛋白对人PBMC进行体外刺激:在破伤风类毒素存在的情况下,PBMC被染色,并在破伤风类毒素下被刺激7天。几乎所有的捐赠者都表现出强烈的T细胞对破伤风类毒素的反应,因为他们已经接种了疫苗,这使得破伤风类毒素是一种有用的阳性控制抗原。图2在三联中显示,CD4+ T细胞从未刺激的PBMC中增殖最小(±12 CFSE暗细胞;<strong class="x...

Watch this Scientific Journal Video about Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester at JoVE.com

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

这里介绍的是一个协议,用于测量增殖CD4+ T细胞,以响应抗原蛋白或肽使用染料稀释。这种测定对稀有抗原特异性T细胞特别敏感,可以进行改性,以利于抗原特异性细胞的克隆。

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

该协议为研究CD4T细胞响应提供了一个平台，这些反应通常很弱，难以检测。此技术允许在不事先了解抗原表示的情况下检测、枚举和隔离 CD4T 细胞。主要优点是分析CD4T细胞，这些细胞通常是与自身免疫性疾病（如1型糖尿病）相关的罕见人群。从健康献血者那里获得外周血样本后，至少以一至两个比例稀释PBS中的血液，然后将35毫升的血液分层到15毫升密度梯度介质中。按密度梯度离心分离细胞，并去除产生的顶部血浆层约20毫升。然后收集白细胞层小心，以避免红细胞颗粒和洗细胞三次新鲜PBS。计数后，将细胞稀释至每毫升PBS浓度的1倍至第六位外周血单核细胞或PBMC。将每个对照的300微升细胞加入到单独的10毫升管中。用PBS给每个管浇注后，通过离心沉淀细胞，以每毫升RP5中浓度的10倍至第六细胞重新悬浮细胞。然后板100微升细胞每个控制到三个井的96孔板包含100微升RP5介质每孔。将盘子放在细胞培养箱中七天。接下来，将剩余的 PBMC 转移到新的 50 毫升管中，并在管侧添加一微升 CFSE 工作库存溶液。快速反转管几次混合，将细胞放在细胞培养箱中五分钟。在孵育结束时，用5毫升RP5介质进行反应，通过离心收集细胞。每毫升新鲜RP5介质中，每毫升将颗粒以1倍10至第六PBMC重新暂停，并将一毫升细胞加入每根10毫升的管中进行测试。然后，在含有100微升RP5介质的96孔板井的三口井中，每口每口添加100微升的抗原，并辅以每口井的稀释抗原。将盘子放在细胞培养箱中七天。为了染色CD4+T细胞群进行流动细胞测量分析，在培养结束时，将整个细胞体积从每个井转移到单独的荧光活性细胞分选或FACS管中。用一毫升PBS洗涤每个样品，并辅以0.1%的胎儿小牛血清或FCS。将颗粒在适当的抗人 CD4 抗体中重新在 100 微升 PBS 加 FCS 中。在4摄氏度下孵育细胞20分钟，防止光线。在孵育结束时，将样品用一毫升新鲜 PBS 加 FCS 每管清洗，然后将颗粒重新在 100 微升新鲜 PBS 加 FCS 中。在分析之前，在每个管中加入一微升的碘化丙胺，以便排除死细胞。根据淋巴细胞的向前和侧面散射区域对淋巴细胞进行门控。要排除凋亡细胞，请将正向散射区域与碘化负细胞进行栅极化，并使用未污染的细胞为非荧光细胞设置电压基线。设置 CD4 抗体荧光和 CFSE 信号的电压，使每个荧光信号低于 1000。使用单色控制 CFSE 和 CD4 样本，使用与未污染的电池设置的电压确认每种颜色的正荧光信号。由于CFSE和碘化丙二具有一些光谱重叠，因此调整补偿，从CFSE荧光中减去碘化荧光，直到CFSE仅样品在碘化丙胺通道中不产生信号。分析所有样本后，计算细胞分裂指数，方法是将分裂细胞的数量除以抗原刺激组的 5000 个未分裂的细胞，除以不培养的不抗原细胞中每 5000 个未分裂的细胞的均数。几乎所有捐赠者在体外刺激后都表现出对破伤风类毒素的强烈T细胞反应，因为捐赠者已经接种了疫苗，使破伤风类毒素成为有用的阳性对抗抗原。然而，未刺激的PBMC中CD4+T细胞的增殖是最小的。在用人类亲胰岛素C-肽刺激7天后，在1型糖尿病患者外周血中可以检测到亲胰岛素C-肽特异性CD4+T细胞。PBMC受甲型流感病毒基质蛋白刺激也表现出增殖。综合起来，这些数据表明，可以使用全长蛋白质和短合成肽进行测定。可以使用细胞分选流细胞计执行此方法的 FACS 组件。使用细胞分拣机，可以在单个细胞级别分离抗原特异性T细胞，用于下游分析。这种方法允许在早期发病的1型糖尿病患者中发现CD4T细胞反应。利用其他方法分析这些罕见的T细胞群会带来各种技术挑战。

Research

JoVE Journal

Immunology and Infection

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

科研

免疫与感染

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

羧基荧光素二醋酸琥珀酰亚胺酯（CFSE）用于监视淋巴细胞增殖

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion of Human Peripheral Blood  ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells using Zoledronate

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

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 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

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

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

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, ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4+ T Cells Using a TGF-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-containing Protocol

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...