这项工作是在 Labex 总监察办项目 (n°ANR-11-LABX-0016-01) 的范围内实现的, 这是 "Investissements 艾文莉" 法国政府方案的一部分, 由国家情报局 (ANR-11-LABX-0016-01) 管理, IHU-Cesti 项目也由 "Investissements 艾文莉 "法国政府项目, 由法国国家研究机构 (ANR-10-IBHU-005) 管理。IHU-Cesti 项目也得到了南特 Métropole 和 Région 支付 de la 卢瓦尔的支持。

作者声明他们没有竞争的金融利益。

将总脾移植到新移植的受体中是一种有效的方法, 可以通过治疗来检测诱导或强化的调节细胞的存在。宿主辐照诱导的瞬态淋巴细胞促进细胞在移植后的存活和耐受性的建立。此外, 致死照射留下时间的细胞与耐受属性转换为新的调节细胞在免疫重建, 一种现象称为传染性耐受34。通常, 在观察公差时, 首先研究描述 CD4+CD25高Foxp3+CD127低Tregs。但是, ?...

大鼠心脏移植是一种可靠的器官移植模型, 用于评估耐受性诱导治疗, 破译耐受诱导和维持的机制, 并具有诱导功能性和显性调控细胞的潜能。下面的协议描述了一个完全不匹配的异位心脏移植从刘易斯1W 捐赠鼠 (卢 1 w, RT1u) 到刘易斯1A 受体鼠 (卢. 1 a, RT1a)。在这种嫁接组合中, 急性排斥反应迅速 (在大约7天), 可以很容易地通过通过触诊的腹部进行移植物跳动测量。在这里, 我们提出了三项协议, 以诱导耐受性心脏移植大鼠。在这些模型中, 公差是由不同的调节细胞类型诱导和/或维持的。首先, 阻断 CD40-CD40L 的相互作用与腺病毒编码 CD40Ig (AdCD40Ig) 诱导的 CD8+ Tregs 能够诱导耐受性时输转移到辅助嫁接收件人1。此外, AdCD40Ig-treated 接收者中的 CD8+细胞 (带有 anti-CD8α抗体) 的损耗产生了 Bregs 和 RegMCs2。CD8 的深层分析+ Tregs 属性突出显示了定义为 interleukin-34 (IL-34) 和 Fibroleukin-2 (FGL-2)3、4、5、6 的几个免疫调节分子的关键作用..而过度表达的 IL-34 (与 AAV 载体) 诱导 Tregs 通过生成 RegMCs, 过度表达 FGL-2 诱导 Bregs, 在复杂的调控细胞网络的基础。
由于慢性排斥发展缓慢, 是长期的, 需要 in-depth 分析来区分耐受性与慢性排斥。移植通常被评估为细胞浸润, 纤维化, 血管壁增厚和补充 C4d 沉积由 immunohistology7。虽然组织学方法需要动物的牺牲或移植活检, 这里我们描述了一个简单的方法来评估不同的特点, 耐受同种异体: 调节细胞的出现和功能和 anti-donor 特异抗体反应的血液样品流式细胞仪 (在这里, 我们使用荧光活化细胞分类 (外地))。
对同种异体骨移植后的耐受性的维持通常与诱导的调节细胞8有关。在过去的几十年中, 研究的重点是 CD4+Tregs 一致的特点, 他们的关键标记 Foxp3+, CD25高, 和CD12710911。同样, 多个标记被归因于 CD8+ Tregs, 如 CD122+、CD28、CD45RC低、PD1+和赫利俄斯+ 1、12、13、,15,16,17. 多年来, GITR、CTLA4 和细胞因子 (IL-10、TGFβ、IL-34、IL-35、FGL-2) 的表达还与 t 配置文件3、4、6、13等相关联. 18、19、20、21。然而, 新兴的调控细胞群体, 如 Bregs, RegMCs, 或 NKTregs, 缺乏相关的特定标记。实际上, Bregs 主要报告为未成熟的 CD24+单元格, CD27 表达式不明确, 有时生成 IL-10、TGFβ或颗粒 B22、23、24。髓细胞谱系的复杂性需要多个标记的组合来定义它们的调节或促炎症轮廓, 如 CD14、CD16、CD80、CD86、CD40、CD209a 或 CD16325,26。最后, 报告了一些标记来识别 NKTregs, 如 CD11b+, CD27+, TGFβ+, 但是需要更多的研究来进一步型描述它们27,28,29 ,30,31,32。因此, 需要抑制活动的证据, 使进一步的表型描述合法化, 新的生物标志物, 新的免疫调节介质, 并扩大范围, 以新的细胞疗法。
我们提出两种互补的方法来评价细胞的抑制活性。首先,体外方法包括培养具有标记效应 t 细胞的抑制细胞, 由异基因供体细胞抗原呈递细胞 (apc) 在6天内不同比例的刺激, 并分析效应 t 细胞增殖反映了捐助者定向的免疫抑制。治疗后的大鼠细胞可以直接与单纯的大鼠和处理移植大鼠的细胞进行比较, 以抑制活性 (或其他调控细胞的数量), 在一系列抑器: 效应比。此外, 这种方法不需要任何移植, 并取得的结果在6天内。其次,体内方法包括将预期的调节细胞从被处理的老鼠转移到新近辐照的接枝受体。处理天真大鼠的 B 细胞、髓细胞或 T 细胞通常无法抑制急性排斥反应, 并在过继转移时延长移植成活, 而接受治疗的受体强化抑制作用的细胞具有这些特性1,2,3,4,33。淋巴细胞诱导受照射的接受者建议允许输转移细胞保持不受血液稳态的影响, 更容易掌握 anti-donor 免疫应答。对于这两种方法,体外利用同种异体第三方 apc 或体内将抑制细胞转移到接受 third-party 心脏移植的受体中, 可以分析 anti-donor 特异性。而在体内方法需要大量的细胞, 但表现不佳的细胞亚群可以更容易地评估为抑制活动体外33。
体液反应也可以测量, 以评估耐受状态和控制的定向抗体反应的施主抗原。的确, 耐受性的特征是缺乏体液对捐献者的反应, 而是对受体对新抗原的体液反应和记忆反应的保存能力的保护。首先, 同种检测的原理是基于受体抗体在捐赠细胞类型与移植接受者血清的孵育后对供体细胞的识别。其次, 对外源性抗原的体液反应可以通过对帽血 (KLH) 与完全弗氏佐剂乳化的长期耐受受体的刺激进行评估。在免疫接种后, 可分别检测4和13天的特异 IgM 和 IgG 抗体, 与酶联免疫吸附试验 (ELISA)34。第三, 免疫记忆反应的保存可以通过注射异种红细胞 (红细胞) 在-7 天和 +3 的移植和红细胞染色与受体血清收集在 +8 和 +17 天后移植。所有这些方法都可以通过使用特定的二级抗体来鉴定免疫球蛋白亚型, 并通过酶联免疫吸附法或几个小时快速获得1.5 小时以内的结果。
最后, 这些协议是为移植模型的特点而设计的, 在一定程度上可以应用于自身免疫疾病模型。该方法的原理可以转换为所有物种。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LEW.1W and LEW.1A rats</td>
			<td>Janvier Labs, France</td>
			<td></td>
			<td>8 weeks old,&#160;</td>
		</tr>
		<tr>
			<td>BN third party donor rats</td>
			<td>Janvier Labs, France</td>
			<td></td>
			<td>8 weeks old,&#160;</td>
		</tr>
		<tr>
			<td>name</td>
			<td>company</td>
			<td>catalogue number</td>
			<td>comments</td>
		</tr>
		<tr>
			<td>reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AdCD40Ig</td>
			<td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td>
			<td></td>
			<td>home made plasmids</td>
		</tr>
		<tr>
			<td>IL34-AAV</td>
			<td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td>
			<td></td>
			<td>home made plasmids</td>
		</tr>
		<tr>
			<td>FGL2-AAV</td>
			<td>Viral Vector Core, INSERM UMR 1089, Nantes, France</td>
			<td></td>
			<td>home made plasmids</td>
		</tr>
		<tr>
			<td>anti-TCR</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>R7/3 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD25</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX39 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD8</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX8 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD45RA</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX33 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD161</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>3.2.3 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD11b/c</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX42 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-TCRgd</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>V65 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD45RC</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX22 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD4</td>
			<td>Hybridoma from European Collection of Cell Culture, Salisbury, U.K</td>
			<td>OX35 clone</td>
			<td>Home made culture, purification and fluororophore coupling</td>
		</tr>
		<tr>
			<td>anti-CD45R</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#554881, His24 clone</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgG-FITC</td>
			<td>Jackson ImmunoResearch Laboratories, INC, Baltimore, USA</td>
			<td>#112-096-071</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgG1</td>
			<td>Serotec</td>
			<td>#MCA 194</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgG2a</td>
			<td>Serotec</td>
			<td>#MCA 278</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgG2b</td>
			<td>Serotec</td>
			<td>#MCA 195</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat IgM-FITC</td>
			<td>Jackson ImmunoResearch Laboratories, INC, Baltimore, USA</td>
			<td>#115-095-164</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin HRP</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#554066</td>
			<td></td>
		</tr>
		<tr>
			<td>KLH</td>
			<td>Sigma Aldrich, St. Louis, USA</td>
			<td>#9013-72-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td></td>
			<td>Phosphate Buffer Solution without calcium and magnesium,&#160;</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma, Saint-Louis, USA</td>
			<td>#9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB substrate reagent kit</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#555214</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTraceTM CFSE cell proliferation kit</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium 1X</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#31870-025</td>
			<td></td>
		</tr>
		<tr>
			<td>penicilline streptomycine</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes Buffer</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>non essential amino acids</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#11360-039</td>
			<td></td>
		</tr>
		<tr>
			<td>2 beta mercaptoethanol</td>
			<td>Sigma, Saint-Louis, USA</td>
			<td>#M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Proliferation Dye eFluor&#174; 450 Cell</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#65-0842-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma, Saint-Louis, USA</td>
			<td>#G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher&#160;Scientific Inc, USA</td>
			<td>#D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche Diagnostics, Germany</td>
			<td>#11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma, Saint-Louis, USA</td>
			<td>#E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Fresenius Kabi</td>
			<td>#B230561</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic dynabeads</td>
			<td>Dynal, Invitrogen</td>
			<td>#11033</td>
			<td>Goat anti-mouse IgG</td>
		</tr>
		<tr>
			<td>One Comp eBeads</td>
			<td>Ebiosciences, San Diego, USA</td>
			<td>#01-1111-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Naplbuphine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Terramycine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Freund&#39;s adjuvant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer lactate</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Refer to the institutional guidelines</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis solution</td>
			<td></td>
			<td></td>
			<td>Dilute 8,29g NH4Cl (Sigma, Saint-Louis, USA A-9434), 1g KHCO3 (Prolabo 26 733.292) and 37.2mg EDTA (Sigma, Saint-Louis, USA E5134) in 800ml H2O. Adjust pH to 7.2-7.4 and complete to 1L with H2O.</td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td></td>
			<td></td>
			<td>Dilute 1g collagenase in 500 ml RPMI-1640 + 5 ml Hepes + 2% FCS</td>
		</tr>
		<tr>
			<td>PBS-FCS (2%)-EDTA (0.5%)</td>
			<td></td>
			<td></td>
			<td>Add 5 mL EDTA 0,1M (Sigma, Saint-Louis, USA E5134) and 20ml FCS to 1ml PBS 1X</td>
		</tr>
		<tr>
			<td>CFSE (Vybrant CFDA SE Cell Tracer Kit Invitrogen)</td>
			<td></td>
			<td></td>
			<td>Dilute 50&#181;g (=1 vial) of CFDA SE (component A) in 90&#956;l DMSO (component B) solution to obtain a 10mM stock solution. Then, dilute stock solution at 1/20 000 in PBS 1X to obtain a 0.5&#956;M solution</td>
		</tr>
		<tr>
			<td>complete medium for coculture</td>
			<td></td>
			<td></td>
			<td>500ml complete RPMI-1640 medium with 5 ml Penicillin (80 unit/ml)-Steptomycin (80 mg/ml), 5 ml L-Glutamine, 5 ml Non Essential Amino Acids (100X), 5ml Pyruvate Sodium (100mM), 5 ml HEPES buffer (1M), 2.5 ml b mercaptomethanol (7 ml of 2-bmercaptoethanol stock diluted in 10 ml RPMI), 10% FCS</td>
		</tr>
		<tr>
			<td>name</td>
			<td>company</td>
			<td>catalog number</td>
			<td>comments</td>
		</tr>
		<tr>
			<td>equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>falcon 50ml</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#227261</td>
			<td></td>
		</tr>
		<tr>
			<td>falcon 15ml</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#188271</td>
			<td></td>
		</tr>
		<tr>
			<td>sieve</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning plastic culture dishes</td>
			<td>VWR, Pessac</td>
			<td>#391-0439</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#181;m and 60&#181;m tissue filters</td>
			<td>Sefar NITEX, Heiden, Switzerland</td>
			<td>#03-100/44 and #03-60/35</td>
			<td></td>
		</tr>
		<tr>
			<td>96 wells U bottom plates for coculture</td>
			<td>&#160;Falcon U-bottom Tissue Culture plate, sterile, Corning</td>
			<td>#353077</td>
			<td></td>
		</tr>
		<tr>
			<td>96 wells V bottom plates for FACS staining</td>
			<td>ThermoScientifique, Danemark</td>
			<td>#249570</td>
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			<td>Nunc Maxisorb</td>
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			<td>seringue for spleen crush</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td>#309649</td>
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			<td>ELISA reader</td>
			<td>SPARK 10M, Tecan, Switzerland</td>
			<td>SPARK 10M, Tecan, Switzerland</td>
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			<td>bain marie&#160;</td>
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			<td>X rays irradiator</td>
			<td>Lincolshire, England</td>
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			<td>solar agitator</td>
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			<td>FACS Canto II</td>
			<td>BD Biosciences, Mountain View, CA</td>
			<td></td>
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			<td>FACS Aria II</td>
			<td>BD Biosciences, Mountain View, CA</td>
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in vitro and in vivo assessment of t, b and myeloid cells suppressive activity and humoral responses from transplant recipients

移植的主要关注是通过诱导调节细胞来达到特定的耐受性。对公差机制的理解需要可靠的模型。在这里, 我们描述模型的耐受性心脏移植的大鼠, 诱导阻断 costimulation 信号或通过上调的免疫调节分子通过基因转移。这些模型中的每一个都允许在体内生成调节细胞, 如调节性 T 细胞 (Tregs)、调节 B 细胞 (Bregs) 或调控髓细胞 (RegMCs)。在这篇手稿中, 我们描述了两种互补的协议, 它们被用来识别和定义体外和体内调节细胞活动, 以确定它们在耐受性诱导和维护中的责任.首先, 一个体外抑制性试验允许以剂量依赖性的方式快速识别具有抑制能力的免疫细胞, 并可用于进一步分析, 如细胞因子的测定或细胞毒性。第二, 从耐受治疗的接受者的细胞移植到新近辐照的接枝受体, 突出了这些细胞在控制移植定向免疫应答和/或转换新的调节细胞方面的耐受特性 (称为传染性耐受)。这些方法不限于具有已知表型标记的细胞, 并且可以扩展到任何细胞群。此外, 捐助者定向 allospecificity 调节细胞 (这一领域的一个重要目标) 可以通过使用第三方供体细胞或移植或体外或体内来评估。最后, 为了确定这些调节细胞的特定耐受能力, 我们提供了评估体液 anti-donor 抗体应答的协议和接受者对新的或前已知抗原进行体液反应的能力。所描述的公差模型可用于进一步刻画调控细胞, 识别新的生物标志物和免疫调节分子, 并适应其他移植模型或啮齿动物或人类自身免疫性疾病。

对 apc (图 1)、响应单元和 Tregs 同时 (图 2) 或单独 (图 4) 和任何其他假定的管理单元 (图 3) 进行排序后的抑制活动的评估可以通过 CFSE 亮度的测量 (图 5) 直接注入调节细胞和体外完成体内。受体对捐献者的体液反应 (图 6) 或外...

Watch this Scientific Journal Video about In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients at JoVE.com

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

在这里, 我们提出了一个诱导耐受性的移植, 并评估体外和体内的抑制能力的不同细胞亚群从接受者和免疫状态的受体向施主或外源性抗原。

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-vitro-vivo-assessment-t-b-myeloid-cells-suppressive-activity

Research

JoVE Journal

Immunology and Infection

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

14.0K 次观看.  INSERM, Université de Nantes. 在这里, 我们提出了一个诱导耐受性的移植, 并评估体外和体内的抑制能力的不同细胞亚群从接受者和免疫状态的受体向施主或外源性抗原。

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

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

纯净小鼠的B细胞的诱导和评估类开关重组

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

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 ...

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 ...

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

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 immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

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

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

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 ...

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 ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

的分离，分化和人类的定量分泌抗体的B细胞从血液:酶联免疫斑点作为体液免疫的功能读出

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

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

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

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

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

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 ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...