Name: the use of fluorescent target arrays for assessment of t cell responses in vivo
Uploaded: 2014-06-19T15:00:00
Description: Watch this Scientific Journal Video about The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo at JoVE.com

这项工作是由项目资助＃1010395（BQ与CP）和＃525431（CR）和项目资助＃455395（CP）来自澳大利亚，澳大利亚的中心为肝炎和HIV病毒学意向书的国家健康和医学研究委员会的支持2012赠款（CR和RJJ）和戈登和格莱特牧夫座基金会（BQ和CR）的资助。我们要感谢HARPREET Vohra保持和迈克尔Devoy其出色的保养JCSMR流式细胞实验室，澳大利亚癌症研究基金会生物分子资源设施，JCSMR，澳大利亚国立大学，用于多肽合成，和大卫博伊尔博士，CSIRO动物健康实验室，澳大利亚Geelong提供家长HIV疫苗的股票。

注：此协议下使用小鼠据澳大利亚国立大学动物实验伦理委员会的小鼠的指导方针和处理颈椎脱位处死。 1，染料和肽制剂<ol><li>染料的制备 注：染料被预稀释的不同浓度，以允许细胞在离散荧光强度标记。 CFSE是用在通过3.5倍连续稀释制成浓度7，和CTV和CPD用于在6个不同的浓度，通过3.7倍连续稀释制成（ 见表2-4）。股票的浓度在表2-4中列出的?...

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的FTA为基础的检测的优点在于，它们允许从流动的单一宿主动物术&gt; 250可行的和全功能的靶细胞群的判别。这提供了复杂性的一个级别一直未能前体内流式细胞仪为基础的分析。这是在上面所示的2动物实验，其中反应7不同的病毒表位，6的浓度可在6次重复同时在一个单一的动物允许将用于体内的T细胞应答所确定的参数如AUC和EC 50监视高亮。 除了 ​​?...

T细胞发挥的适应性免疫应答的核心作用，并且通常针对操作在免疫治疗。通过分泌细胞因子，调节免疫力的许多方面，也可以直接帮助B细胞制造抗体外来抗原的CD4 +效应T细胞反应。 CD8 +细胞毒性T细胞（CTL）还可以以外来抗原通过分泌细胞因子以及打在直接杀伤细胞中表达外来抗原核心作用作出响应。发起这些T细胞的效应子功能的基本相互作用涉及T细胞受体（TCR）与细胞表面上显示的MHC分子外来肽的相互作用。 CD4 + T细胞识别的抗原呈递细胞和CD8 + T细胞的MHC-II类分子的肽显示的认识上是通常显示在微生物感染细胞的MHC-I类分子的肽显示。 为了以评估的作用的T细胞在免疫反应中发挥，其效应子功能是通过可靠和灵敏的技术来测量它是必不可少的。 T细胞反应评估常用的方法包括; MHC class-I/II/peptide四聚体反应;细胞因子的产生通过ELISPOT和细胞内细胞因子染色;并通过51 Cr释放测定杀伤能力。这些测定法，但是，通常是进行先体外后体内与体外刺激，或提供有限的洞察T细胞的功能。理想的情况下，测量T细胞应答时，将所发生的，与无处理的T细胞，从而避免通过体外刺激，可能会发生变化的功能参数是有利的，以评估它们的原位 ， 在体内 。一些最常用的体内 T细胞功能测定法中使用是基于测量的CTL介导的杀伤脉冲与I类MHC结合肽的靶细胞，所列举的体内通过他们的检测通过与重要的染料如CFSE荧光标记。虽然这些类型的测定可以监视的CTL介导的杀伤的目标时，它们发生在体内 ，他们以前有一个相对有限的能力来评估杀灭的多个目标呈现不同浓度和不同类型的肽表位，这是必需的，以允许定性参数，例如作为功​​能性抗体亲抗原性和特异性表位变体的交叉反应性进行评估。这些检测也没有提供对CD4 + T细胞介导的反应的任何信息。 为了克服许多与用于评估的T细胞反应当前方法的局限，我们最近开发了一种基于荧光的目标阵列（自由贸易协定），它同时允许T细胞反应的监测对&gt; 250靶细胞在一个动物由多重检测流式细胞仪1，2。自由贸易协定是由淋巴细胞标记的SEV像CFSE，CTV和CPD活体染料允许产生独特的荧光&gt; 250细胞团ERAL浓度和组合。由于这些细胞保持活力和功能齐全的，它们可以被注射到动物，让监控它们的相互作用与效应性T细胞在体内3。例如，在FTA细胞簇可与MHC I类结合肽进行脉冲，让抗原特异性的CTL介导的杀死靶细胞1的评估。另外，在FTA细胞团，也可与MHC II类结合肽脉冲，使抗原特异性T辅助细胞（T H）通过评估活化（通过激活标记物如CD69，CD44和/或评估活动的评估CD62L的自由贸易协定中的B细胞轴承同源肽2）。因为超过250个目标可以同时检测，也可以对测量脉冲具有n个多靶细胞簇的CTL和T H的反应umerous肽在不同浓度及加入许多重复的。因此，自由贸易区法提供的体内 T细胞效应反应评估了前所未有的水平。 在这里，我们详细描述了自贸区的建设和展示如何将它们应用到评估在体内的 T细胞反应。该过程描述通过使用三个重要的染料，包括6重复脉冲与MHC I类和II-结合肽42细胞团组成的252分辨细胞簇一个自贸区的建设。 42细胞簇标记出现在10ml锥形底管，它是有帮助的，如表1中所示来放置这些在一个试管架上。该方法可用于更小的数字可辨别的集群根据需要通过降低标记的量来调节各染料进行1。 我们强调检测的实用程序，通过展示它如何衡量RESPONS通过重组痘病毒接种在一个小队列小鼠的多个表位产生的ES。这显示了自由贸易协定法可用于通过，测量累计反应和功能分别亲和力，曲线下面积（AUC）评估和产生半数最大反应（EC 50）所需的有效肽浓度测量的使用面积。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">RPMI</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">R8758</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fetal calf serum</td>
			<td style="width:71px;">Serana</td>
			<td style="width:119px;">FCS-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CTV</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C34557</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CFDA, SE</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C1157</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">CPD</td>
			<td style="width:71px;">eBioscience</td>
			<td style="width:119px;">65-0840-90</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">anti-B220 PerCp-Cy5.5</td>
			<td style="width:71px;">eBioscience</td>
			<td style="width:119px;">110730</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">anti-CD69 brilliant violet 605</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">104529</td>
			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:216px;">PKH-26</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
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			<td height="22" style="height:22px;">EQUIPMENT:</td>
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			<td height="43" style="height:43px;width:216px;">Material Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments (optional)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Vortex</td>
			<td style="width:71px;">Scientific Industries Inc&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="64" style="height:64px;width:216px;">Flow cytometer (Fortessa or equivalent with blue, red and violet laser source)</td>
			<td style="width:71px;">BD Bioscience</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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the use of fluorescent target arrays for assessment of t cell responses in vivo

监测T细胞应答在体内的功能是对我们的免疫应答的理解的发展和免疫疗法的设计很重要。在这里，我们描述了使用的荧光目标阵列（FTA）技术，它利用活体染料，如羧基荧光素琥珀酰亚胺酯（CFSE），紫激光激发的染料（CellTrace紫罗兰：CTV）和红色激光激发的染料（细胞增殖染料eFluor 670：CPD ）以组合性标签小鼠淋巴细胞为&gt; 250分辨荧光细胞簇。这些自由贸易协定内细胞团可以是脉冲与主要组织相容性复合体（MHC）I类和MHC II类结合肽，从而作为靶细胞对CD8分别+和CD4 + T细胞。这些FTA细胞保持存活，功能齐全的，并且因此可以被施用到小鼠体内，以允许的CD8 + T细胞介导的杀伤FTA靶细胞和CD4 + T细胞预谋HEL的评估FTA B细胞的靶细胞的对实时体内通过流式细胞术。因为&gt; 250靶细胞可以一次进行评估，该技术允许对多个抗原表位的T细胞应答的若干浓度和在多次重复的监控。因此，该技术可以在两个定量（ 如累积响应的幅度）和定性（ 例如 ，功能性抗体亲抗原性和响应的表位的交叉反应性）水平测量T细胞应答。本文中，我们描述了如何将这些自由贸易协定的构造，并给予他们如何可以应用于评估诱导的重组痘病毒疫苗的T细胞反应的例子。

由于使用了FTA测定的一个例子，一个BALB / c小鼠进行免疫接种的重组牛痘病毒（VV）的表达HIV-I表位（VV-HIV）和响应的HIV-CTL表位，加格，加格MUT，的env和聚合物，该VV CTL表位F2L和F2L MUT和HIV-T H细胞表位，加格钍（如2所述）通过使用252参数FTA测定法（ 图1B）进行评估。加格（GAG MUT）和F2L（F2L MUT）的表位变异并不表示在VV-HIV载体，因此对这些表位的反应是反射性的表位变?...

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The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

监测体内详细的T细胞应答的能力，对于我们的免疫应答的理解的发展非常重要。在这里，我们描述了评估同时采用流式细胞仪&gt; 250参数体内 T细胞检测使用荧光目标阵列（自由贸易协定）的。

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Research

JoVE Journal

Immunology and Infection

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

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

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 ...

科研

免疫与感染

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

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

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 ...

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

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 ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...