研究免疫突触形成过程中B细胞中的细胞动力学
Summary
本文描述了在IS形成过程中描述B淋巴细胞细胞极化事件的两种方法。第一,涉及细胞器招募的定量和突触膜的细胞骨架重组。第二种是生化方法,用于描述中心体组成的变化,这种物质对免疫突触发生极化。
Abstract
B细胞受体(BCR)对表面束缚抗原的识别触发免疫突触(IS)的形成,其中信号和抗原吸收均得到协调。IS 的形成涉及动态行为因重塑,并伴有偏振招募到中源体和相关细胞内细胞器(如溶酶体和Golgi设备)的突触膜。作用素重塑的初始阶段允许B细胞增加其细胞表面,并最大化在突触中收集的抗原-BCR复合物的数量。在某些情况下,当B细胞识别与刚性表面相关的抗原时,这个过程与地合体体招募和分泌相结合,从而促进抗原提取。被利用的抗原被内化成专门的内乳糖体隔间,用于加工成肽,这些肽被加载到主要组织相容性复合物II(MHC-II)分子上,以便进一步呈现给T帮助细胞。因此,研究与IS的形成相关的细胞器动力学对于理解B细胞是如何被激活的关键。在本文中,我们将讨论成像和用于研究细胞内细胞器定位和细胞骨架重组的变化的成像和生化技术,这些变化与B细胞中IS的形成有关。
Introduction
B淋巴细胞是适应性免疫系统的重要组成部分,负责产生针对不同威胁和入侵病原体的抗体。抗体的产生效率取决于B细胞在可溶性或表面束缚形式1、2中获取、处理和呈现抗原的能力。BCR对附着细胞表面的抗原的识别,导致形成称为IS3,4的紧密细胞间接触。在此动态平台中,均发生依赖 BCR 的下游信令和抗原内化到内生体隔间中。被吸收的抗原被处理并组装到MHC-II分子上,然后呈现给T淋巴细胞。生产性B-T相互作用,称为B-T细胞合作,允许B淋巴细胞接收适当的信号,促进其分化为产生抗体的血浆细胞或记忆细胞8。
B细胞的抗原提取有两种机制。第一种依赖于来自溶酶体的蛋白酶的分泌,这些蛋白酶在突触裂5、6处进行招募和融合。第二,取决于Myosin IIA介导的拉力,触发含有的抗原的内化到克拉辛涂层坑7的抗原的阴道。抗原提取模式依赖于发现抗原的膜的物理特性。然而,在这两种情况下,B细胞经历两个主要的重塑事件:行为细胞骨架重组和细胞器的极化到IS。Actin 细胞骨架重塑涉及初始扩散阶段,其中突触膜上的活性因子相关突起增加与抗原接触的表面。随后是收缩阶段,其中BCR与抗原结合集中在IS的中心,由分子马达和反应因子细胞骨架重塑8,9,10的协同作用, 11.细胞器的极化也依赖于细胞骨架的重塑.例如,通过相关行为因的局部去聚合,中心体与原子核脱钩,从而允许将这种细胞器重新定位到IS5,12。在B细胞中,将中心体重新定位到一个细胞杆(IS)将溶酶体招募到突触膜中,分泌后可促进表面束状抗原6的提取和/或处理。在IS招募的裂氧体富含MHC-II,这有利于在内皮腔中形成肽-MHC-II复合物,提交给T细胞13。此外,也观察到Golgi仪器被密切招募到IS14,这表明从分泌途径的Golgi衍生的囊泡可以参与抗原提取和/或处理。
总之,在突触形成期间,细胞内细胞器和细胞骨架在B细胞中重新排列是允许其进一步激活所需的高效抗原采集和处理的关键步骤。在这项工作中,我们介绍了如何在B细胞中执行成像和生化分析的详细方案,以研究与IS形成相关的细胞内重塑。这些技术包括:(i) 用抗原涂层珠和抗原涂层盖玻片激活的B细胞的免疫荧光和图像分析,允许对动员到IS和(ii)的细胞内成分进行可视化和定量。)通过在蔗糖梯度上超离心分离B细胞中中中质体富集的成分,从而能够识别与中质体相关的蛋白质,从而可能参与调节细胞极性。
Protocol
Representative Results
Discussion
我们描述了一个综合的方法,研究B淋巴细胞如何重新组织其细胞内结构,以促进IS的形成。这项研究包括使用成像技术来量化细胞器的细胞内分布,如在B细胞激活期间,如中位体、Golgi装置和乳索体,以及它们如何与IS极化。此外,我们描述了一种生物化学方法,用于研究B细胞激活后中体组合物的变化。
为了促进在B细胞中形成IS,我们使用固定抗原(抗原涂层珠子)代替可溶性免疫复合物,…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
M.-I.Y.由FONDECYT#1180900的研究资助。J.I.、D.F.和J.L.得到国家委员会研究金的支持。我们感谢智利天主教大学的大卫·奥索里奥进行录像和编辑。
Materials
| IIA1.6 (A20 variant) mouse B-lymphoma cells | ATCC | TIB-208 | Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface without FcγIIR |
| 100% methanol | Fisher Scientific | A412-4 | |
| 10-mm diameter cover glasses thickness No. 1 circular | Marienfield-Superior | 111500 | |
| 2-mercaptoethanol | Thermo Fisher Scientific | 21985023 | |
| Alexa 488 fluor- donkey ant-rabbit IgG | LifeTech | A21206 | 1:500 dilution recomended but should be optimized |
| Alexa Fluor 546 goat anti-Rabbit IgG | Thermo Fisher Scientific | A-11071 | 1:500 dilution recomended but should be optimized |
| Alexa Fluor 647-conjugated phalloidin | Thermo Fisher Scientific | A21238 | 1:500 dilution recomended but should be optimized |
| Amaxa Nucleofection kit V | Lonza | VCA-1003 | Follow the manufacturer's directions for mixing the transfection reagents with the DNA |
| Amaxa Nucleofector model 2b | Lonza | AAB-1001 | Program L-013 used |
| Amino- Dynabeads | ThermoFisher | 14307D | |
| Anti-pericentrin | Abcam | ab4448 | 1:200 dilution recomended but should be optimized |
| Anti-rab6 | Abcam | ab95954 | 1:200 dilution recomended but should be optimized |
| Anti-sec61 | Abcam | ab15575 | 1:200 dilution recomended but should be optimized |
| BSA | Winkler | BM-0150 | |
| CaCl2 | Winkler | CA-0520 | |
| Culture plate T25 | BD | 353014 | |
| Fiji Software | Fiji col. | ||
| Fluoromount G | Electron Microscopy Science | 17984-25 | |
| Glutamine | Thermo Fisher Scientific | 35050061 | |
| Glutaraldehyde | Sigma | G7651 | |
| Glycine | Winkler | BM-0820 | |
| Goat-anti-mouse IgG antibody | Jackson ImmunoResearch | 315-005-003 | IIA1.6 positive ligand |
| Goat-anti-mouse IgM antibody | Thermo Fisher Scientific | 31186 | IIA1.6 negative ligand |
| HyClone Fetal bovine serum | Thermo Fisher Scientific | SH30071.03 | Heat inactivate at 56 oC for 30 min |
| KCl | Winkler | PO-1260 | |
| Leica SP8 TCS microscope | Leica | ||
| NaCl | Winkler | SO-1455 | |
| Nikon Eclipse Ti-E epifluorescence microscope | Nikon | ||
| Parafilm | M | P1150-2 | |
| Paraformaldehyde | Merck | 30525-89-4 | Dilute to 4% with PBS in a safety cabinet, use at the moment |
| Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | Liquid |
| Polybead Amino Microspheres 3.00μm | Polyscience | 17145-5 | |
| Poly-L-Lysine | Sigma | P8920 | Dilute with sterile water |
| Rabbit anti- alpha tubulin antibody | Abcam | ab6160 | 1:1000 dilution recommended but should be optimized |
| Rabbit anti mouse lamp1 antibody | Cell signaling | 3243 | 1:200 dilution recomended but should be optimized |
| Rabbit anti-cep55 | Abcam | ab170414 | 1:500 dilution recomended but should be optimized |
| Rabbit Anti-gamma Tubulin antibody | Abcam | ab16504 | 1:1000 for Western Blot |
| RPMI-1640 | Biological Industries | 01-104-1A | |
| Saponin | Merck | 558255 | |
| Sodium pyruvate | Thermo Fisher Scientific | 11360070 | |
| Sucrose | Winkler | SA-1390 | |
| Triton X-100 | Merck | 9036-19-5 | |
| Tube 50 ml | Corning | 353043 |
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