通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症
Summary
在该协议中，描述了肠道微生物群抗原特异性T细胞过继转移结肠炎模型。从CBir1 TCR转基因小鼠中分离出CD4 + T细胞。这些对于免疫优势性肠道微生物群抗原CBir1鞭毛蛋白具有特异性，其被转移到受体 Rag1-/- 小鼠中，导致肠道炎症。
Abstract
随着发病率的增加，炎症性肠病（IBD）是影响胃肠道的慢性疾病，给个人和社会带来了相当大的健康和经济负担。因此，研究IBD发病机制和发展的机制至关重要。这里描述了肠道微生物群抗原特异性T细胞转移结肠炎模型。CBir1 鞭毛蛋白已被公认为实验性结肠炎和克罗恩病患者的免疫优势性肠道细菌抗原。CBir1 TCR转基因naϊve CD4 + T细胞，对CBir1鞭毛蛋白具有特异性，可在过继转移到免疫缺陷 的Rag1-/- 小鼠后诱导慢性结肠炎。通过组织病理学评估疾病的严重程度。还测定了固有结肠层中的CD4 + T细胞表型。该模型与IBD的发展非常相似，IBD为研究驱动IBD发病机制和测试治疗IBD的潜在药物提供了理想的小鼠模型。
Introduction
炎症性肠病（IBD），主要包括克罗恩病（CD）和溃疡性结肠炎（UC），其特征是胃肠道的慢性复发缓解炎症，影响全球数百万人1。IBD的发展和发病机制涉及几个因素，包括遗传易感性、肠道微生物群、免疫反应、饮食和生活方式2。然而，IBD的确切机制仍未完全了解。
其中一个特别感兴趣的是肠道微生物群与宿主免疫反应在调节肠道炎症方面的相互作用3。肠道微生物群提供一系列免疫刺激分子和抗原，可以激活免疫反应4。虽然效应T细胞和调节性T细胞（Tregs）之间的平衡对于维持肠道稳态至关重要，但肠粘膜CD4 + T细胞对肠道微生物群抗原的过度反应会导致肠道炎症5，6，7。作为一种免疫优势性肠道微生物群抗原，CBir1 鞭毛蛋白与人 CD8，9 的发病机制有关。此外，CBir1 TCR转基因（Tg）T细胞的转移在免疫缺陷小鼠6中诱导肠道炎症，与人类IBD非常相似，表明这种T细胞转移模型有助于研究人类IBD的机制。
这项工作描述了通过通过收养转移CBir1 TCR Tg naϊve CD4 + T细胞并评估疾病严重程度来诱导Rag1-/-小鼠结肠炎的详细方案。此外，还显示了预期的结果，并讨论了程序和故障排除的关键步骤，这将有助于研究人员研究肠道炎症发病机制并测试治疗IBD的潜在药物。
Protocol
所有动物程序均根据德克萨斯大学医学分会的动物使用和护理委员会进行。CBir1 TCR Tg小鼠由阿拉巴马大学伯明翰分校的Charles Elson博士提供。CBir1 TCR Tg小鼠可以是雌性或雄性，但应该在8-12周。C57BL / 6背景上的 Rag1-/- 小鼠是从杰克逊实验室获得的10。 Rag1-/- 小鼠必须是性别和年龄匹配的，可以使用雄性或雌性，但应该在8-12周。整个协议总结如图 <strong c…
Representative Results
从成年CBir1 TCR Tg小鼠中分离出每个脾脏约5 x 106个CBir1 TCR Tg naϊve CD4 + T细胞。CBir1 TCR Tg naϊve CD4 + T细胞的转移在受体Rag1-/-小鼠中诱导慢性结肠炎。细胞转移后，监测临床体征以评估肠道炎症的进展，包括体重减轻，粪便稠度和驼背姿势。正如预期的那样，小鼠在细胞转移后三周左右开始减肥，并且在细胞转移后六周体重达到原始重量的80%-85%左右（<strong class=…
Discussion
虽然每个步骤对于该结肠炎模型的可重复性都至关重要，但有几个关键步骤。受体Rag-/-小鼠应接受足够的活的naϊve CD4 + T细胞以诱导肠道炎症。我们使用脾脏来分离幼稚的CD4 + T细胞而不是MLN。因为MLNs中幼稚CD4 + T细胞的产量远低于脾脏。CD62L在幼稚T细胞中高度表达，CD44和CD25是T细胞的活化标志物13，14。在这项研究…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院拨款DK125011，AI150210和DK124132，德克萨斯大学系统STAR奖（Y.C.）和德克萨斯大学加尔维斯顿分校（W.Y.）的James W. McLaughlin奖学金基金的部分支持。图 1 是使用 BioRender.com 创建的。
Materials
| 0.22 µm vacuum-driven disposable bottle top filter | MilliporeSigma | SCGPS05RE | |
| 100x Penicillin-Streptomycin | Corning | 30-002-CI | |
| 100-µm strainer | BD Biosciences | 352360 | |
| 3-mL Transfer Pipette | Fisherbrand | 13-711-9CM | |
| Anti-Mouse CD16/32 | Biolegend | 101302 | |
| Anti-Mouse CD25-Percp/Cy5.5 | Biolegend | 102030 | |
| Anti-mouse CD3-Percp/Cy5.5 | Biolegend | 100327 | |
| Anti-Mouse CD4 APC | Biolegend | 100516 | |
| Anti-Mouse CD4 Magnetic Particles | BD Biosciences | 551539 | |
| Anti-Mouse CD4-BV421 | Biolegend | 100544 | |
| Anti-Mouse CD62L-PE | Biolegend | 104408 | |
| Anti-Mouse Foxp3-PE | ThermoFisher | 12-5773-82 | |
| Anti-Mouse IFNγ-FITC | Biolegend | 505806 | |
| Anti-Mouse IL-17A-PE/Cy7 | Biolegend | 506922 | |
| Automated Cell Counter | Bio-rad | TC20 | |
| Brefeldin A | BD Biosciences | 555029 | |
| BSA | Fisher Bioreagents | BP1600-1 | |
| C tube | Miltenyi | 130-093-237 | |
| Cell Separation Magnet | BD Biosciences | 552311 | |
| Collagenase IV | Sigma-Aldrich | C5138 | |
| DAPI | Sigma-Aldrich | D9542 | |
| Dissociator Machine | Miltenyi | 130-096-427 | |
| DNase I | Sigma-Aldrich | ||
| EDTA | Corning | 46-034-CI | |
| EDTA (0.5 M, PH 8.0) | Corning | 46-034-CI | |
| FBS | R&D Systems | S11550 | |
| Flow cytometer | BD Biosciences | LSD Fortessa | |
| Heat Lamp | CoverShield | BR40 | |
| Hematoxylin and Eosin (H&E) Stain Kit | Abcam | ab245880 | |
| Insulin Syringes | BD Biosciences | 329412 | |
| Ionomycin | ThermoFisher | I24222 | |
| Live/dead Fixable Near-IR Dead Cell Stain kit | ThermoFisher | L10119 | |
| MaxQ 6000 Incubated/Refrigerated Stackable Shakers | ThermoFisher | SHKE6000 | |
| NH4Cl | Thermo Scientific | A687-500 | |
| Percoll | GE Healthcare | 17-0891-01 | |
| Phorbol-12-myristate 13-acetate | Sigma-Aldrich | P8139 | |
| RPMI 1640 Medium | Cytiva HyClone | SH3002702 | |
| Sorter | BD Biosciences | Arial Fusion | |
| Tissue Automatic Processor | ThermoFisher | STP120 | |
| Tissue Embedding/Processing Cassette | Fisher Healthcare | 22048142 | |
| Tris Base | Thermo Scientific | BP154-1 | |
| True-Nuclear Transcription Factor Buffer Set (including Perm Buffer) | Biolegend | 424401 |
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Citazione di questo articolo
Yang, W., Yu, T., Cong, Y. Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice. J. Vis. Exp. (178), e63293, doi:10.3791/63293 (2021).