在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化
1Department of Urology, South China Hospital, Medical School,Shenzhen University, 2Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering,Shenzhen University Medical School, 3Cancer Center, Daping Hospital & Army Medical Center of PLA,Third Military Medical University, 4Guangdong Provincial Key Laboratory of Immune Regulation and Immunotherapy, School of Laboratory Medicine and Biotechnology,Southern Medical University, 5Institute of Immunology,Third Military Medical University, 6Dermatology Hospital,Southern Medical University, 7Institute of Immunological Innovation and Translation,Chongqing Medical University
Summary
目前的研究展示了评估病毒特异性 TFH 细胞的早期命运承诺和操纵这些细胞中基因表达的方案。
Abstract
滤泡辅助性 T （TFH） 细胞被认为是一种独立的 CD4+ T 细胞谱系，可帮助同源 B 细胞产生高亲和力抗体，从而建立长期体液免疫。在急性病毒感染期间，病毒特异性 TFH 细胞的命运承诺在感染早期就确定了，早期分化的 TFH 细胞的研究对于了解 T 细胞依赖性体液免疫和优化疫苗设计至关重要。在这项研究中，使用急性淋巴细胞性脉络丛脑膜炎病毒 （LCMV） 感染的小鼠模型和具有特异性识别 LCMV 糖蛋白表位 I-AbGP66-77 的 CD4+ T 细胞的 TCR 转基因 SMARTA （SM） 小鼠，我们描述了基于流式细胞术染色的病毒特异性 TFH 细胞的早期命运承诺的程序。此外，通过利用 SM CD4 + T 细胞的逆转录病毒转导，还提供了操纵早期分化病毒特异性 TFH 细胞中基因表达的方法。因此，这些方法将有助于探索病毒特异性 TFH 细胞早期定型机制的研究。
Introduction
遇到不同的病原体或威胁，初始 CD4+ T 细胞通过分化为具有特殊功能的各种辅助性 T （TH） 细胞亚群来定制其免疫反应1。在急性病毒感染的情况下，很大一部分幼稚 CD4+ T 细胞分化为滤泡辅助性 T （TFH） 细胞，为 B 细胞提供帮助 2,3。与其他 CD4+ TH 细胞亚群（例如，TH1、TH2、TH9 和 TH17 细胞）不同，TFH 细胞表达大量 CXCR5，这是 B 细胞归巢趋化因子 CXCL13 的趋化因子受体，使 TFH 细胞能够迁移到 B 细胞滤泡中。在 B 细胞滤泡中，TFH 细胞协助同源 B 细胞启动和维持生发中心反应，从而能够快速产生高亲和力抗体和长期体液记忆 2,3。
急性病毒感染后，病毒特异性 TFH 细胞的早期命运承诺发生在 72 小时内 4,5，并受转录抑制因子 B 细胞淋巴瘤-6 （Bcl-6）5,6,7,8 的控制，该细胞是控制 TFH 细胞命运决定的“主调节因子”。Bcl-6 的缺乏会严重减弱 TFH 细胞的分化，而异位 Bcl-6 的表达会显著促进 TFH 细胞的命运决定。除 Bcl-6 外，多个分子还参与指导早期 TFH 细胞命运定型。转录因子 TCF-1 和 LEF-1 通过诱导 Bcl-6 启动 TFH 细胞分化 9,10,11。Bcl-6 和 TCF-1 对 Blimp1 的抑制是早期 TFH 细胞命运承诺所必需的11,12。STAT1 和 STAT3 也是早期 TFH 细胞分化所必需的13。此外，组蛋白甲基转移酶 EZH214,15 和 m6A 甲基转移酶 METTL316 的表观遗传修饰有助于稳定 TFH 细胞转录程序（尤其是 Bcl6 和 Tcf7），从而引发早期 TFH 细胞命运承诺。虽然在理解早期 TFH 细胞命运承诺的转录和表观遗传调控方面取得了进展，包括上述分子和其他分子，在别处3 中总结，但以前未知的分子仍有待了解。
在急性淋巴细胞性脉络丛脑膜炎病毒 （LCMV） 感染的小鼠模型中，过继转移的同基因 TCR 转基因 SMARTA （SM） CD4+ T 细胞，特异性识别 LCMV 糖蛋白表位 I-AbGP66-77，在病毒感染期间经历 TFH 或 TH1 细胞分化。这种 TFH/TH1 分叉分化模式支持 SM/急性 LCMV 感染模型在研究病毒特异性 TFH 细胞生物学方面的进步。事实上，SM/急性 LCMV 感染模型已广泛应用于 TFH 细胞研究领域，并在 TFH 细胞生物学的里程碑性发现中发挥了至关重要的作用。这包括将上述 Bcl-6 鉴定为 TFH 细胞的谱系定义转录因子 5,6，以及其他重要的转录因子（例如，Blimp-16、TCF-1/LEF9、10、11、STAT1/STAT313、STAT517、KLF218 和 Itch19）指导 TFH 细胞分化、转录后调节 （例如，TFH 细胞分化的 METTL316 和 miR-17~9220），TFH 细胞记忆和可塑性 21,22，以及针对 T FH 细胞的合理疫苗接种策略（例如，硒23）。
目前的研究描述了获取病毒特异性 TFH 细胞早期命运承诺的可重复方法，包括 （1） 建立适用于访问早期分化 TFH 细胞的急性 LCMV 感染的 SM 嵌合体小鼠模型，（2） 对与早期分化 TFH 细胞相关的分子进行流式细胞术染色，以及 （3） 在 SM CD4+ 中进行基于逆转录病毒载体的基因操作T 细胞。这些方法将有助于调查病毒特异性 TFH 细胞的早期命运承诺的研究。
Protocol
所有动物实验均按照第三军医大学机构动物护理和使用委员会批准的程序进行。本研究使用了以下小鼠品系：C57BL/6J （B6） 小鼠（男女），年龄 6 至 8 周龄，体重 25-30 克;CD45.1+SM TCR转基因小鼠（B6 CD45.1 × SM TCR转基因），两性，年龄6-8周龄，体重25-30 g;和 CXCR5-GFP CD45.1+SM TCR 转基因小鼠 （B6 CD45.1 × SM TCR 转基因 × CXCR5-GFP 敲入），两性，年龄 6 至 8 周，体重 25-30 g。转基因物种是在先?…
Representative Results
急性 LCMV 感染期间早期分化病毒特异性 TFH 细胞的特征为了探测病毒特异性 TFH 细胞的早期命运承诺，将特异性识别 LCMV GP 表位 I-AbGP66-77 的幼稚同源 SM CD4 + T 细胞过继转移到 CD45.2 + C57BL/6 受体中。第二天，这些接受者被静脉注射高剂量的急性缓解的 LCMV Armstrong 感染（图 1A）。感染后第 3 天，通过流式细胞术分?…
Discussion
自从发现 TFH 细胞在帮助 B 细胞方面的特殊功能以来，TFH 细胞领域的研究一直备受关注。累积的研究表明，TFH 细胞分化是一个多阶段和多因素的过程30，其中 TFH 细胞命运承诺在早期确定5。因此，更好地了解早期分化 TFH 细胞的潜在机制对于 TFH 细胞生物学和合理的疫苗设计至关重要。在此，我们提供了通过利用…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了中国国家自然科学基金（X.C. 的第 32300785 号）、中国国家创新人才博士后计划（第 1 号）的资助。BX20230449 至 X.C.），以及国家科技重大专项（编号 2021YFC2300602 至 L.Y.）。
Materials
| 0.25% Trypsin-EDTA | Corning | 25-052-CI | |
| 4% Paraformaldehyde Fix Solution, 4% PFA | Beyotime | P0099-500mL | |
| 70 μm cell strainer | Merck | CLS431751 | |
| Alexa Fluor 647 anti-mouse TCR Vα2 (clone B20.1) | Biolegend | 127812 | 1:200 dilution |
| Alexa Fluor 700 anti-mouse CD45.1 (clone A20) | Biolegend | 110724 | 1:200 dilution |
| APC anti-mouse CD25 (clone PC61) | Biolegend | 101910 | 1:200 dilution |
| B6 CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mouse | The Jackson Laboratory | 002014 | |
| BeaverBeads Streptavidin | Beaver | 22321-10 | |
| Biotin anti-mouse F4/80 Antibody (clone BM8) | Biolegend | 123106 | 1:200 dilution |
| Biotin Rat anti-mouse CD11c (clone N418) | Biolegend | 117304 | 1:200 dilution |
| Biotin Rat anti-Mouse CD19 (clone 6D5) | Biolegend | 115504 | 1:200 dilution |
| Biotin Rat anti-Mouse CD8a (clone 53-6.7) | Biolegend | 100704 | 1:200 dilution |
| Biotin Rat anti-mouse NK-1.1 (clone PK136) | Biolegend | 108704 | 1:200 dilution |
| Biotin Rat anti-mouse TER-119/Erythroid Cells (clone TER-119) | Biolegend | 116204 | 1:200 dilution |
| bovine serum albumin, BSA | Sigma | A7906 | |
| Brilliant Violet 421 anti-T-bet (clone 4B10) | Biolegend | 644816 | 1:100 dilution |
| Brilliant Violet 605 anti-mouse CD279 (PD-1) (clone 29F.1A12) | Biolegend | 135220 | 1:200 dilution |
| C57BL/6J (B6) mouse | The Jackson Laboratory | 000664 | |
| CXCR5-GFP knock-in reporter mouse | In house; the CXCR5-GFP knock-in mouse line was generated by the insertion of an IRES-GFP construct after the open reading frame of Cxcr5. | ||
| DMEM 10% medium | DMEM medium containing 10% FBS | ||
| DMEM medium | Gibco | 11885092 | |
| EDTA | Sigma | E9884 | |
| FACSFortesa | BD Biosciences | ||
| Fetal bovine serum, FBS | Sigma | F8318 | |
| FlowJo (version 10.4.0) | BD Biosciences | ||
| Foxp3/Transcription Factor Staining Buffer Set | Invitrogen | 00-5523-00 | The kit contains three reagents: a. Fixation/Permeabilization Concentrate (4X); b. Fixation / Permeabilization Diluent; c. Permeabilization Buffer. |
| Goat Anti-Rat IgG Antibody (H+L), Biotinylated | Vector laboratories | BA-9400-1.5 | 1:200 dilution |
| Invitrogen EVOS FL Auto Cell Imaging System | ThermoFisher Scientific | ||
| Isolation buffer | FACS buffer containing 0.5% BSA and 2mM EDTA | ||
| LCMV GP61-77 peptide (GLKGPDIYKGVYQFKSV) | Chinese Peptide Company | ||
| LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation | Life Technologies | L10199 | 1:200 dilution |
| MigR1 | addgene | #27490 | |
| NaN3 | Sigma | S2002 | |
| Opti-MEM medium | Gibco | 31985070 | |
| pCL-Eco | addgene | #12371 | |
| PE anti-mouse CD69 (clone H1.2F3) | Biolegend | 104508 | 1:200 dilution |
| PE Mouse anti-Bcl-6 (clone K112-91) | BD Biosciences | 561522 | 1:50 dilution |
| Phosphate buffered saline, PBS | Gibco | 10010072 | |
| Polybrene | Solarbio | H8761 | |
| Purified Rat Anti-Mouse CXCR5 (clone 2G8) | BD Biosciences | 551961 | 1:50 dilution |
| Rat monoclonal PerCP anti-mouse CD4 (clone RM4-5) | Biolegend | 100538 | 1:200 dilution |
| recombinant murine IL-2 | Gibco | 212-12-1MG | |
| Red Blood Cell Lysis Buffer | Beyotime | C3702-500mL | |
| RPMI 1640 medium | Sigma | R8758 | |
| RPMI 2% | RPMI 1640 medium containing 2% FBS | ||
| SMARTA (SM) TCR transgenic mouse | SM TCR transgenic line in our lab is a gift from Dr. Rafi Ahmed (Emory University). Additionally, this mouse line can also be obtained from The Jackson Laboratory (stain#: 030450). | ||
| Staining buffer | PBS containing 2% FBS and 0.01% NaN3 | ||
| Streptavidin PE-Cyanine7 | eBioscience | 25-4317-82 | 1:200 dilution |
| TCF1/TCF7 (C63D9) Rabbit mAb (Alexa Fluor 488 Conjugate) | Cell signaling technology | 6444S | 1:400 dilution |
| TFH cell staining buffer | FACS buffer containing 1% BSA and 2% mouse serum | ||
| TransIT-293 reagent | Mirus Bio | MIRUMIR2700 |
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Cite This Article
Lin, Y., Yue, S., Yang, Y., He, J., Yang, X., Ye, L., Chen, X. Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice. J. Vis. Exp. (206), e66752, doi:10.3791/66752 (2024).