系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠
Summary
The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.
Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.
Introduction
系统性红斑狼疮（SLE）是一种复杂的自身免疫性疾病用抗核抗体（ANA）的生产和肾小球肾炎prototypically特征。许多其他的后遗症，包括皮肤，心肺，肝和病变与疾病在一些个体相关联。在美国的流行率估计相差很大，从150,000-1,500,000 1,2，特别高的发病率在女性和少数族裔3。虽然SLE的病因已经很难辨别，它被认为是来自各种遗传和环境因素，在系统性自身免疫其中高潮的相互作用出现。
许多动物模型已被用于研究导致病情发作和进展的因素。 SLE的经典小鼠模型包括遗传上易感小鼠品系包括NZB x NZW的F1模型及其NZM衍生物中，MLR / lpr狼疮应变，并且BXSB /雅阿应变，和可诱导系统，例如作为pristane和慢性移植物抗宿主病（慢性GVHD）模型4。自身抗体产生的GVHD模型早期的报告中使用的各种小鼠品系或仓鼠株父到F1转移5 – 8;用于研究狼疮样疾病更常见的方法目前包括DBA / 2父→（C57BL / 6×DBA / 2）F 1，和这里描述的BM12传递模型。每个模型都有自己的警告，但他们一般都有一个共同的一套与人类疾病的临床特点相关的功能。在小鼠模型中最经常报告的参数包括脾肿大，淋巴结病，肾炎，ANA生产，并在细胞水平，T滤泡辅助细胞（TFH），生发中心（GC）B细胞和浆细胞的扩张。
H2 – – AB1 BM12 / KhEgJ（BM12）小鼠，应变我的诱导BM12模型由淋巴细胞从IA BM12 B6（C）过继转移实现dentical到C57BL / 6除了上MHC II类3个氨基酸的取代，插入IA b的C57BL / 6（B6）小鼠。捐助者的CD4 T细胞受援国的装甲运兵车Alloactivation导致慢性GVHD的症状非常类似系统性红斑狼疮。具体地，这些包括扩展供体来源的TFH，扩大受者来源的GC B细胞和浆细胞，和生产抗核抗体，包括抗dsDNA，抗单链DNA，抗染色质，以及抗红细胞抗体9。随着时间的推移，受体小鼠发展与在肾小球，间质性的IgG沉积物，和肾脏10的血管区域相关性肾炎。我们最近证明，类似于人类疾病，也有对I型IFN在该模型11中的关键作用。值得注意的是，对于人的SLE的定义标准包括肾炎的SLE兼容的抗dsDNA的存在下发展的抗体12，这两者都是该小鼠模型的显着的特征。
有SE在自发模型BM12模型veral优势。经典模型发展SLE状标志自发依赖任一杂交小鼠品系，近交小鼠品系不上B6背景，或在B6背景大的基因位点，这使交叉敲除或以其它方式转基因小鼠困难且耗时。与BM12诱导模型中，遗传修饰的小鼠可用作任一供体或受体，从而允许更快速鉴定的细胞区室，其中特定基因可能是疾病重要的。此外，在BM12模型疾病发展的速度要快得多，只需要2个星期，直到抗核抗体的外观相比，数月为最自发模型。此外，在对比的是开发疾病在不同时间点的自发模型中，疾病发作和进展中的BM12→B6模型高度同步。这允许适当尺寸的组群，可以B中的代Ë用于介入或治疗策略在疾病发展的任何阶段。
下面是用于通过过继转移BM12淋巴细胞到C57BL / 6小鼠，或遗传变异体上的B6背景发起SLE状自身免疫的详细协议。此外，我们描述了流式细胞染色方案，用于枚举TFH，气相色谱B细胞和浆细胞，细胞类型与人类疾病相关的。重要的是，这些协议也可以被用于表征疾病在SLE最小鼠模型，并确定TFH，气相色谱B细胞和浆细胞中的其他疾病模型。
Protocol
Representative Results
Discussion
的BM12诱导模型是研究SLE的细胞和分子过程相对容易且有效的方式。过继转移的CD4 T细胞直接针对自身抗原的慢性活化导致TFH，气相色谱B细胞和浆细胞的可通过流来测量仪，如这里所描述的积累。采用这种模式可以方便快捷地询问候选基因和新疗法的自身免疫生发中心的过程这类似于那些发生在SLE患者，最终支配自身抗体的病理积累的作用今后的研究。此外，这里所描述的流式细胞仪分析可用于研…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by the Lupus Research Institute, NCI grant CA138617, NIDDK grant DK090978, Charlotte Schmidlapp Award (to E.M.J.), and the Albert J. Ryan Fellowship (to J.K.). We are grateful for the support and instrumentation provided by the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center, supported in part by NIH AR-47363, NIH DK78392 and NIH DK90971.
Materials
| B6.SJL-Ptprca Pepcb/BoyJ | The Jackson Laboratory | 001162 | CD45.1+ BoyJ mouse strain |
| B6(C)-H2-Ab1bm12/KhEgJ | The Jackson Laboratory | 001162 | Bm12 mouse strain |
| FastDigest PsuI | Life Technologies | FD1554 | Restriction digest enzyme for genotyping |
| 1X RBC Lysis Buffer | eBioscience | 00-4333-57 | |
| IMDM | GE Healthcare | SH30228.01 | |
| Plasma Separation Tube (PST) | BD | 365974 | Blood collection tube with Dipotassium EDTA |
| Serum Separation Tube (SST) | BD | 365967 | Blood collection tube with Clot activator / SST Gel |
| Ficoll | GE Healthcare | 17-1440-02 | High density cell separation solution |
| Lympholyte-M | Cedarlane | CL5030 | High density cell separation solution |
| GL-7-biotin | eBioscience | 13-5902-82 | |
| Streptavidin-BUV395 | BD | 564176 | |
| CD138-BV421 | BioLegend | 142508 | |
| CD4-BV510 | BioLegend | 100559 | |
| TCRβ-BV605 | BD | 562840 | |
| CD45.1-BV711 | BioLegend | 110739 | |
| CD45.2-FITC | BioLegend | 109806 | |
| PD-1-PE | BioLegend | 135206 | |
| CD19-PerCP | BioLegend | 115532 | |
| Fas-PE-Cy7 | BD | 557653 | |
| CXCR5-APC | BioLegend | 145506 | |
| Fixable Viability Dye ef780 | eBioscience | 65-0865-18 | |
| CD4-BV421 | BioLegend | 100443 | |
| 1.2 ml FACS tube inserts, racked | USA Scientific | 1412-1400 | |
| BD Falcon™ Round-Bottom Tubes | BD | 352017 |
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