Name: in vitro and in vivo assessment of t, b and myeloid cells suppressive activity and humoral responses from transplant recipients
Uploaded: 2017-08-12T14:00:00.000+00:00
Duration: 18 min 48 s
Description: 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

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><tbody><tr><td>&lt;strong&gt;animals&lt;/strong&gt;</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,&amp;nbsp;</td></tr><tr><td>BN third party donor rats</td><td>Janvier Labs, France</td><td></td><td>8 weeks old,&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;name&lt;/strong&gt;</td><td>&lt;strong&gt;company&lt;/strong&gt;</td><td>&lt;strong&gt;catalogue number&lt;/strong&gt;</td><td>&lt;strong&gt;comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;reagents&lt;/strong&gt;</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-TCRab</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&amp;nbsp;Scientific Inc, USA</td><td></td><td>Phosphate Buffer Solution without calcium and magnesium,&amp;nbsp;</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>CellTrace&lt;sup&gt;TM&lt;/sup&gt; CFSE cell proliferation kit</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#C34554</td><td></td></tr><tr><td>RPMI 1640 medium 1X</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#31870-025</td><td></td></tr><tr><td>penicilline streptomycine</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#15140-122</td><td></td></tr><tr><td>Hepes Buffer</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#15630-056</td><td></td></tr><tr><td>non essential amino acids</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>#11140-035</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fisher&amp;nbsp;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&amp;reg; 450 Cell</td><td>Thermo Fisher&amp;nbsp;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&amp;nbsp;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&#039;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&amp;micro;g (=1 vial) of CFDA SE (component A) in 90&amp;mu;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&amp;mu;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>&lt;strong&gt;name&lt;/strong&gt;</td><td>&lt;strong&gt;company&lt;/strong&gt;</td><td>&lt;strong&gt;catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;equipments&lt;/strong&gt;</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&amp;micro;m and 60&amp;micro;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>&amp;nbsp;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><td></td></tr><tr><td>96 wells flat bottom ELISA plates</td><td>Nunc Maxisorb</td><td></td><td></td></tr><tr><td>seringue for spleen crush</td><td>BD Biosciences, Mountain View, CA</td><td>#309649</td><td></td></tr><tr><td>ELISA reader</td><td>SPARK 10M, Tecan, Switzerland</td><td>SPARK 10M, Tecan, Switzerland</td><td></td></tr><tr><td>centrifuge</td><td></td><td></td><td></td></tr><tr><td>bain marie&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>X rays irradiator</td><td>Lincolshire, England</td><td>Faxitron CP160</td><td></td></tr><tr><td>solar agitator</td><td></td><td></td><td></td></tr><tr><td>FACS Canto II</td><td>BD Biosciences, Mountain View, CA</td><td></td><td></td></tr><tr><td>FACS Aria II</td><td>BD Biosciences, Mountain View, CA</td><td></td><td></td></tr><tr><td>magnet</td><td>Thermo Fisher&amp;nbsp;Scientific Inc, USA</td><td>12302D</td><td></td></tr></tbody></table>

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 ...

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Immunology and Infection

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

14.1K 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

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

科研

免疫与感染

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

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 deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

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

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

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 ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

癌症研究

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

跨体内迟发型变态反应检测方法的抗原特异性调节

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

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 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

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

摘要

探索更多视频

此视频中的章节

人类承认免疫失衡的早期国家禁止作为筛检工具

移植物抗宿主病的诱导和

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

cd8⁺ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

跨体内迟发型变态反应检测方法的抗原特异性调节

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

荧光目标阵列的T细胞反应评价中的应用

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

使用成像流式细胞仪T细胞同种异体反应性测定

活体细胞毒性试验研究肿瘤特异性 CD8⁺ t 细胞反应中的免疫调节

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

摘要

探索更多视频

此视频中的章节

人类承认免疫失衡的早期国家禁止作为筛检工具

移植物抗宿主病的诱导和

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

跨体内迟发型变态反应检测方法的抗原特异性调节

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

荧光目标阵列的T细胞反应评价中的应用

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

使用成像流式细胞仪T细胞同种异体反应性测定

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

cd8⁺ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

活体细胞毒性试验研究肿瘤特异性 CD8⁺ t 细胞反应中的免疫调节