Name: adoptive immunotherapy of inkt cells in glucose-6-phosphate isomerase (g6pi)-induced ra mice
Uploaded: 2020-01-31T12:30:00
Description: Watch this Scientific Journal Video about Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice at JoVE.com

本研究得到国家自然科学基金（国家自然科学基金）（81771755）、河北省高校科技重点研究项目（ZD2017009）和河北大学医学实验中心动物实验室的支持。我们感谢他们的支持。

所有实验小鼠（共150只）为健康的雄性DBA/1小鼠，6~8周大（20.0~1.5克），在特定的无病原体（SPF）环境中饲养。建模前没有特殊处理。实验分为健康对照组（15只小鼠）、模型对照组（15只小鼠）和细胞治疗组（55只小鼠）。这项研究得到了河北大学动物福利伦理委员会的批准。
1. 构建疾病模型
<ol><li>复制 RA 动物模型<ol>
<li>重量为1.75毫克的hG6PI 325-339和hG6PI 469-483碎片，并将其?...

<ol>
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iNKT细胞是特殊的T细胞，桥接先天和适应性免疫，主要从CD4+/CD8+ 胸腺细胞开发。iNKT细胞具有不同的免疫调节功能，通过直接接触和分泌不同细胞因子23与其他免疫细胞相互作用，影响树突细胞（DC）、巨噬细胞、嗜中性粒细胞、B细胞、T细胞和NK细胞分化和发育2 4。β-GalCer是从海绵中提取的经典iNKT细胞特异性?...

风湿性关节炎（RA）是一种自身免疫性疾病，其特征是慢性、渐进性侵入性，发病率为0.5-1%，发病率为1，2。潜在的发病机制归因于自动反应CD4+和CD8+T细胞的异常增殖，表现为CD4+IFN++和CD4+IL-17A+T细胞比例增加，CD4+IL-4+CD4+CD4+CD25+FoxP3+T细胞比例增加。 因此，炎症细胞因子的分泌增加，过度的炎症反应破坏人体免疫系统的本生平衡和耐受功能。此外，穿透关节的助剂T淋巴细胞（Th）1细胞会加重炎症反应和关节损伤。因此，抑制过度的炎症反应和恢复免疫耐受性和免疫平衡是治疗RA3，4的关键。
iNKT细胞同时具有NK细胞和T细胞的功能和特性。iNKT细胞具有独特的、不变的T细胞受体（TCR）+链，具有有限的TCR+链表5，并识别抗原呈现细胞表面的主要组织相容性复合物（MHC）I类分子CD1d呈现的糖脂抗原。三三等人6日检测出大量iNKT细胞和功能缺陷，包括RA在内的许多自身免疫性疾病。Aurore等人7表明，iNKT细胞对维持自身免疫耐受性有积极作用，当iNKT细胞的数量和功能恢复时，疾病得到缓解。此外，Miellot-Gafsou等人8日发现，iNKT细胞不仅可使疾病发生，而且增加了疾病的进展。这些相互矛盾的结果表明，iNKT细胞是异质T细胞，不同子集的功能可能逆转。在RA的临床研究中，iNKT细胞的频率与疾病活性9的分数相关。结果还证实，在RA患者中，iNKT的频率降低，CD4+IFN-α+T细胞子集数量增加，炎症细胞因子IFN-α和TNF-α的分泌水平增加10，11。此外，Sharif等人12日调查了1型糖尿病（T1D），发现INKT细胞的选择性输注能调节炎症细胞因子IL-4的表达，保持免疫耐受性，并防止1型糖尿病的发展。因此，采用特定iNKT细胞的输注或针对iNKT细胞的活化可增加RA患者的iNKT细胞水平，这在RA治疗中是一个突破。
细胞免疫疗法目前备受关注，已广泛应用于癌症治疗。然而，iNKT细胞是罕见的，异质免疫调节细胞（只占PBMC总数的0.3%）13，这限制了潜在的临床应用。这些细胞主要分为三个亚群：1）iNKT1细胞，具有高表达的原发性白血病锌指蛋白（PLZF）和T-盒转录因子（T-bet）;2） 具有PLZF和GATA结合蛋白3（GATA3）中间表达的iNKT2细胞;3）iNKT17细胞，具有PLZF和视网膜相关孤立核受体（ROR）的低表达，即分泌IFN-*、IL-4和IL-17 14。激活的iNKT细胞分泌Th1、Th2和Th17样细胞因子，这些细胞因子决定了iNKT细胞15的不同免疫调节作用。iNKT细胞各亚群特异性活化的免疫调节和免疫治疗效果不同。因此，选择具有抗炎功能的iNKT细胞（主要是iNKT2）的特定表型，以调节人体的免疫反应，可以纠正RA的免疫不平衡和免疫障碍。
建立理想的动物模型对RA发病机制的治疗和研究具有重要意义。目前，最常用的和成熟的动物模型包括胶原蛋白引起的关节炎，辅助性关节炎，酶性关节炎，和多糖引起的关节炎16-17。然而，没有一个模型可以完全模拟人类RA的所有特征。II型胶原蛋白诱发关节炎（CIA）是一种经典的关节炎模型。CIA是通过对II型胶原蛋白特异性单克隆抗体的小鼠进行免疫接种，反映该疾病模型的抗体依赖性。Benurs等人描述了一个对葡萄糖-6-磷酸异构酶（G6PI）有全身免疫反应的模型，该模型在易感小鼠菌株18、19中诱导外周对称多关节炎。在这个模型中，关节炎的发展依赖于T细胞、B细胞和先天免疫18、19、20。Horikoshi21发现，在 CD4+ T 细胞和细胞因子（即 IL-6 和 TNF-α）方面，通过免疫 DBA/1 小鼠的 G6PI 多肽片段产生的 RA 模型比 CIA 模型更类似于人类 RA。为了增加对TCR识别部位的刺激作用，利用G6PI（hGPI325-339和hGPI469-483）的混合多肽片段对DBA/1小鼠进行免疫，以构建RA小鼠模型。这种方法的成功率可以很高，因为hGPI325-339和hGPI469-483是I-A q受限T细胞反应的免疫主导。因此，该模型可以模拟RA患者22的CD4+T细胞和iNKT细胞缺陷的增殖。RA免疫病理学的基础研究为我国进一步深入研究奠定了基础。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 647 Mouse Anti-PLZF</td>
			<td>BD</td>
			<td>563490</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Anti-PE MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-048-801</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Columns</td>
			<td>Miltenyi</td>
			<td>MS</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Cryogenic Centrifuge</td>
			<td>Beckman</td>
			<td>Allegra&#174; X-15R</td>
			<td>America</td>
		</tr>
		<tr>
			<td>DiR</td>
			<td>Thermo Fisher Scientific</td>
			<td>D12731</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Embedding Center</td>
			<td>Tianjin Aviation Electromechanical Co., Ltd.</td>
			<td>BMJ-1</td>
			<td>China</td>
		</tr>
		<tr>
			<td>FITC Hamster Anti-Mouse TCR &#946; Chain</td>
			<td>BD</td>
			<td>553170</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD</td>
			<td>Accuri C6</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Freund&#39;s complete adjuvant</td>
			<td>Sigma</td>
			<td>F5881</td>
			<td>America</td>
		</tr>
		<tr>
			<td>hGPI325-339 (IWYINCFGCETHAML)</td>
			<td>Karebay Biochem</td>
			<td>18062202</td>
			<td>China</td>
		</tr>
		<tr>
			<td>hGPI469-483 (EGNRPTNSIVFTKLT)</td>
			<td>Karebay Biochem</td>
			<td>18062203</td>
			<td>China</td>
		</tr>
		<tr>
			<td>In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>caliper IVIS lumina II</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Ionomycin Calcium</td>
			<td>Cayman</td>
			<td>10004974</td>
			<td>America</td>
		</tr>
		<tr>
			<td>KRN7000</td>
			<td>AdipoGen</td>
			<td>AG-CN2-0013</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Mouse CD1d Tetramer-PE</td>
			<td>MBL</td>
			<td>TS-MCD-1</td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>Mouse percoll</td>
			<td>Solarbio</td>
			<td>P8620</td>
			<td>China</td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Olympus</td>
			<td>Olympus-II</td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>PerCP-CyTM5.5 Mouse anti-ROR-&#978;t</td>
			<td>BD</td>
			<td>562683</td>
			<td>America</td>
		</tr>
		<tr>
			<td>PerCP-CyTM5.5 Mouse anti-T-bet</td>
			<td>BD</td>
			<td>561316</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Pertussis toxin</td>
			<td>Sigma</td>
			<td>P7208</td>
			<td>America</td>
		</tr>
		<tr>
			<td>phorbol esters</td>
			<td>Cayman</td>
			<td>10008014</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>BD</td>
			<td>555899</td>
			<td>America</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Biological Industries</td>
			<td>01-100-1ACS</td>
			<td>Israel</td>
		</tr>
		<tr>
			<td>Th1/Th2/Th17 cytokines kit</td>
			<td>BD</td>
			<td>560485</td>
			<td>America</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>Leica EM UC6</td>
			<td>Germany</td>
		</tr>
	</tbody>
</table>

adoptive immunotherapy of inkt cells in glucose-6-phosphate isomerase (g6pi)-induced ra mice

风湿性关节炎（RA）是一种复杂的慢性炎症性自身免疫性疾病。这种疾病的发病机制与不变的自然杀手T（iNKT）细胞有关。活性RA的患者存在较少的iNKT细胞，有缺陷的细胞功能，和Th1的极化过度。在这项研究中，使用hGPI325-339和hGPI469-483肽的混合物建立了RA动物模型。iNKT细胞通过体内诱导和体外纯化获得，然后输入RA小鼠进行采用免疫治疗。体内成像系统（IVIS）跟踪显示，iNKT细胞主要分布在脾脏和肝脏中。细胞治疗后的第12天，病情进展明显减缓，临床症状得到缓解，胸腺中iNKT细胞的丰度增加，胸腺中iNKT1的比例下降，TNF-α、IFN-α和IL-6水平下降。血清减少。iNKT细胞的接受免疫疗法恢复了免疫细胞的平衡，纠正了身体的过度炎症。

关节指数得分和爪子厚度增加建模后。与对照组相比，RA模型组在建模后6天开始出现红色肿胀，逐渐加重。在14天，脚踝关节的红色肿胀达到顶峰，随后逐渐缓解。爪子的厚度变化类似（P < 0.05） （图 1）。
炎症细胞渗透在建模后显著增加。病理结果表明，RA模型小鼠脚踝间阴膜组织炎症细胞的渗透程度不同。高峰炎症发生在第14天后建模（<strong c...

Watch this Scientific Journal Video about Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice at JoVE.com

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

该协议使用G6PI混合肽构建类风湿关节炎模型，更接近CD4+T细胞和细胞因子中的人类类风湿性关节炎模型。高纯度不变天然杀伤性T细胞（主要是iNKT2），具有特定的表型和功能，通过体内诱导和体外纯化获得，用于采用免疫治疗。

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

我们的协议描述了一种构建 RA 模型的方法。其成功率超过93%，其发病机制取决于T细胞、B细胞和先天免疫，这使得研究人员能够从全球免疫角度更好地研究RA的发病机制。然后，我们使用具有免疫调节作用的 INKT 细胞进行治疗。INKT细胞通过体内引入对治疗效果良好，为 INKT 细胞的临床应用提供基础数据支持，真正具有 INKT 细胞的临床应用价值。该技术的最大优点是操作简单，易于复制。演示程序将是陈胜德，高翔和张景南，研究生从我实验室。首先，重达 1.75 毫克的 HGPI325 至 339 和 HGPI469 至 483 片段，并将它们溶解在 5.25 毫升 4 摄氏度的三蒸馏水中。在 50 摄氏度的水浴中溶解完全弗伦德的辅助剂。将 5.25 毫升的离心管中再抽出 5.25 毫升，冷却以使用。将HG6PI溶液和全弗伦德佐剂溶液的混合物放入人工乳化装置中，并连接两个玻璃注射器。在冰浴中，以每分钟十到二十次的恒定速度和频率推动注射器，以完全乳化混合物肽溶液，并完成Freund的辅助剂溶液。然后，将乳液液滴保持在水中十分钟。接下来，将乳化的HG6PI的150微升注入小鼠的尾根皮下。立即和48小时后，注射200毫克百日咳毒素到小鼠内腹。现在，注射正常的DBA1小鼠内没有高斯瑟在剂量0.1毫克每公斤体重。建模三天后，麻醉后，分离小鼠的脾脏。通过切割和研磨脾脏在 200 网筛中，准备单个细胞悬架。用 PBS 清洗电池悬浮液。在200倍g下离心5分钟，然后丢弃上一代。用一毫升全血组织溶液重新悬浮细胞。加入三毫升小鼠淋巴细胞分离介质。然后在室温下以300倍g离心20分钟。为了净化 INKT 细胞，将 10 个 10 微升的 4 摄氏度 PBS 重新向第七细胞中，加入 10 微升装装 CD1 四面体 PE，并在 4 摄氏度下在黑暗中孵育 15 分钟。用 PBS 清洗细胞两次，并在 80 微升 PBS 中重新暂停。加入20微升的抗PE微珠，在4摄氏度下孵育20分钟。用 PBS 清洗两次，用 500 微升 PBS 重新暂停细胞。将分拣柱放在 MACS 分拣机的磁场中，然后用 500 微升 PBS 冲洗。将准备好的单元格悬浮液添加到分拣列中，收集流通过，然后用 PBS 缓冲液冲洗三次。移除磁场，将 PBS 缓冲液添加到分拣列中。以恒定压力快速推动柱塞，将标记的电池推入收集管。并获得纯化的 INKT 细胞。使用自动单元格计数器计数。要识别 INKT 细胞表型，首先，从纯化的 INKT 细胞中向第六细胞服用 1 倍十倍，并在 50 微升 PBS 中重新发送。在单一正向控制管中加入0.5微升的α高瑟PE CD1四分之一，或10微升的菲特克TCRβ。添加 0.5 微升的 α 高斯珀 PE CD1 四分之一，在样品管中加入 10 微升 FITC TCR 测试版。在黑暗中在摄氏四度下孵育三十分钟。之后，在PBS中清洗细胞，然后以200倍g离心5分钟。丢弃上经剂，加入一毫升FoxP3固定渗透工作溶液。在黑暗中4摄氏度下孵育细胞45分钟。然后，加入1倍渗透工作溶液1毫升，在室温下以500倍g离心细胞5分钟。丢弃上一杯。添加一个亚历克萨弗尔647小鼠抗 PLZF 的微升和 PerCP Cy5.5 小鼠抗 Tbet 的微升，在黑暗中的室温下孵育 30 分钟。接下来，添加两个微升渗透缓冲液工作溶液，并在200倍g下离心，进行5分钟的清洁。丢弃上一杯。将细胞重新在500微升PBS中，通过流式细胞测量。本研究与对照组相比，RA模型组脚趾在建模后开始出现红肿，并逐渐加重。14天后，脚踝关节的红色肿胀达到峰值，随后逐渐缓解。病理结果表明，RA模型小鼠脚踝突触组织中炎症细胞的渗透程度不同。峰值炎症发生在建模后的第 14 天。在RA模型组中，亲炎细胞因子的血清水平在建模后11天和14天显著增加。而抗炎细胞因子显著减少。这一数字表明，正常小鼠的 INKT2 速率约为 5%。在体内诱导后，INK2的速率约为82%。MACS 纯化后，INK2 的速率超过 92%。在这里，我们展示一种乳化我们在RA中看到的主要多肽的方法。乳化液滴在水中保存10分钟。结果正在分散。此协议是治疗RA引发的特殊NKT细胞的新附加程序。可应用于细胞免疫治疗或其他自身免疫性疾病和肿瘤。该模型可刺激RA患者CD4 T细胞和NKT细胞缺陷的整体增殖，为RA免疫通路深入研究奠定了基础。在此协议中，百日咳毒素在建模过程中通过间向注射。请穿实验服和一次性手套。并携带所有操作标准，以防止刺伤感染。

Research

JoVE Journal

Immunology and Infection

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

免疫与感染

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

制备肿瘤抗原负载成熟树突状细胞免疫治疗

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Intralymphatic Immunotherapy and Vaccination in Mice

淋巴免疫治疗和疫苗接种小鼠

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

CAR性T细胞过继治疗的护理标准胶质母细胞瘤的上下文生成

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...