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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
注意: 这里的所有协议都是由一个道德委员会批准的, 应该以不育的方式进行。 大鼠同种异体心脏移植模型的耐受性1.Generation 卢 1 w 到卢1异体移植手术 麻醉 1 w 大鼠使用异氟醚-O2吸入, 在5分钟后补充 1% N2O 将动物放在背卧处, 并用控告消毒腹部以执行开胸手术 (即, 切开胸腔的胸膜空间)。 钳的劣势和上级络静脉, 结扎他?…
Representative Results
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…
Discussion
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, ?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| animals | |||
| LEW.1W and LEW.1A rats | Janvier Labs, France | 8 weeks old, | |
| BN third party donor rats | Janvier Labs, France | 8 weeks old, | |
| nom | company | catalogue number | comments |
| reagents | |||
| AdCD40Ig | Viral Vector Core, INSERM UMR 1089, Nantes, France | home made plasmids | |
| IL34-AAV | Viral Vector Core, INSERM UMR 1089, Nantes, France | home made plasmids | |
| FGL2-AAV | Viral Vector Core, INSERM UMR 1089, Nantes, France | home made plasmids | |
| anti-TCR | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | R7/3 clone | Home made culture, purification and fluororophore coupling |
| anti-CD25 | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | OX39 clone | Home made culture, purification and fluororophore coupling |
| anti-CD8 | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | OX8 clone | Home made culture, purification and fluororophore coupling |
| anti-CD45RA | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | OX33 clone | Home made culture, purification and fluororophore coupling |
| anti-CD161 | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | 3.2.3 clone | Home made culture, purification and fluororophore coupling |
| anti-CD11b/c | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | OX42 clone | Home made culture, purification and fluororophore coupling |
| anti-TCRgd | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | V65 clone | Home made culture, purification and fluororophore coupling |
| anti-CD45RC | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | OX22 clone | Home made culture, purification and fluororophore coupling |
| anti-CD4 | Hybridoma from European Collection of Cell Culture, Salisbury, U.K | OX35 clone | Home made culture, purification and fluororophore coupling |
| anti-CD45R | BD Biosciences, Mountain View, CA | #554881, His24 clone | |
| anti-rat IgG-FITC | Jackson ImmunoResearch Laboratories, INC, Baltimore, USA | #112-096-071 | |
| anti-rat IgG1 | Serotec | #MCA 194 | |
| anti-rat IgG2a | Serotec | #MCA 278 | |
| anti-rat IgG2b | Serotec | #MCA 195 | |
| anti-rat IgM-FITC | Jackson ImmunoResearch Laboratories, INC, Baltimore, USA | #115-095-164 | |
| streptavidin HRP | BD Biosciences, Mountain View, CA | #554066 | |
| KLH | Sigma Aldrich, St. Louis, USA | #9013-72-3 | |
| PBS 1X | Thermo Fisher Scientific Inc, USA | Phosphate Buffer Solution without calcium and magnesium, | |
| Tween 20 | Sigma, Saint-Louis, USA | #9005-64-5 | |
| TMB substrate reagent kit | BD Biosciences, Mountain View, CA | #555214 | |
| CellTraceTM CFSE cell proliferation kit | Thermo Fisher Scientific Inc, USA | #C34554 | |
| RPMI 1640 medium 1X | Thermo Fisher Scientific Inc, USA | #31870-025 | |
| penicilline streptomycine | Thermo Fisher Scientific Inc, USA | #15140-122 | |
| Hepes Buffer | Thermo Fisher Scientific Inc, USA | #15630-056 | |
| non essential amino acids | Thermo Fisher Scientific Inc, USA | #11140-035 | |
| Sodium pyruvate | Thermo Fisher Scientific Inc, USA | #11360-039 | |
| 2 beta mercaptoethanol | Sigma, Saint-Louis, USA | #M3148 | |
| Cell Proliferation Dye eFluor® 450 Cell | Thermo Fisher Scientific Inc, USA | #65-0842-85 | |
| Glutamine | Sigma, Saint-Louis, USA | #G3126 | |
| DAPI | Thermo Fisher Scientific Inc, USA | #D1306 | |
| Collagenase D | Roche Diagnostics, Germany | #11088882001 | |
| EDTA | Sigma, Saint-Louis, USA | #E5134 | |
| NaCl 0.9% | Fresenius Kabi | #B230561 | |
| Magnetic dynabeads | Dynal, Invitrogen | #11033 | Goat anti-mouse IgG |
| One Comp eBeads | Ebiosciences, San Diego, USA | #01-1111-42 | |
| Betadine | Refer to the institutional guidelines | ||
| Isoflurane | Refer to the institutional guidelines | ||
| Naplbuphine | Refer to the institutional guidelines | ||
| Terramycine | Refer to the institutional guidelines | ||
| Buprenorphine | Refer to the institutional guidelines | ||
| Meloxicam | Refer to the institutional guidelines | ||
| Complete Freund's adjuvant | |||
| Rompun | Refer to the institutional guidelines | ||
| Ringer lactate | Refer to the institutional guidelines | ||
| Ketamine | Refer to the institutional guidelines | ||
| Red blood cell lysis solution | 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. | ||
| Collagenase D | Dilute 1g collagenase in 500 ml RPMI-1640 + 5 ml Hepes + 2% FCS | ||
| PBS-FCS (2%)-EDTA (0.5%) | Add 5 mL EDTA 0,1M (Sigma, Saint-Louis, USA E5134) and 20ml FCS to 1ml PBS 1X | ||
| CFSE (Vybrant CFDA SE Cell Tracer Kit Invitrogen) | Dilute 50µg (=1 vial) of CFDA SE (component A) in 90μl DMSO (component B) solution to obtain a 10mM stock solution. Then, dilute stock solution at 1/20 000 in PBS 1X to obtain a 0.5μM solution | ||
| complete medium for coculture | 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 | ||
| nom | company | catalog number | comments |
| equipments | |||
| falcon 50ml | BD Biosciences, Mountain View, CA | #227261 | |
| falcon 15ml | BD Biosciences, Mountain View, CA | #188271 | |
| sieve | |||
| Corning plastic culture dishes | VWR, Pessac | #391-0439 | |
| 100µm and 60µm tissue filters | Sefar NITEX, Heiden, Switzerland | #03-100/44 and #03-60/35 | |
| 96 wells U bottom plates for coculture | Falcon U-bottom Tissue Culture plate, sterile, Corning | #353077 | |
| 96 wells V bottom plates for FACS staining | ThermoScientifique, Danemark | #249570 | |
| 96 wells flat bottom ELISA plates | Nunc Maxisorb | ||
| seringue for spleen crush | BD Biosciences, Mountain View, CA | #309649 | |
| ELISA reader | SPARK 10M, Tecan, Switzerland | SPARK 10M, Tecan, Switzerland | |
| centrifuge | |||
| bain marie | |||
| X rays irradiator | Lincolshire, England | Faxitron CP160 | |
| solar agitator | |||
| FACS Canto II | BD Biosciences, Mountain View, CA | ||
| FACS Aria II | BD Biosciences, Mountain View, CA | ||
| magnet | Thermo Fisher Scientific Inc, USA | 12302D |
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Citer Cet Article
Bézie, S., Usal, C., Guillonneau, C. In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients. J. Vis. Exp. (126), e55510, doi:10.3791/55510 (2017).