清华大学刘万里课题组发表PNAS文章

　　

2016年1月13日，清华大学生命科学学院刘万里课题组在(Proceedings of the National Academy of Science)在线发表题为《利用光控抗原系统探究B细胞活化过程中抗原探索行为终止和抗原受体聚类拣选分子机制》(Utilization of a photoactivatable antigen system to examine B cell probing termination and the B cell receptor sorting mechanisms during B cell activation)的研究论文，报道了B细胞识别外来抗原后，在跨膜信号转导过程中的一系列早期分子事件。

配体-受体的识别触发跨膜信号转导是淋巴细胞普遍采用的活化起始路径。本论文结合光控抗原系统和高速高分辨率的活细胞单分子荧光成像技术，系统解析了B细胞受体（抗原受体或者BCR）与抗原识别后触发的BCR微簇体成熟和免疫突触形成等一系列精细分子事件。

研究人员发现静息态B细胞本能性的进行持续性的探索行为试图寻找能够被它识别的抗原。BCR与抗原的识别能够在极短的时间内（4秒）终止抗原探索行为，并高效启动BCR聚集，形成BCR微簇体，随后BCR微簇逐渐成熟并定向汇聚到抗原呈递面，形成免疫突触，最终驱动B淋巴细胞进入完全活化状态。这一生物学过程的发生完全依赖于BCR对抗原的特异性识别。细胞质膜上的其他受体分子，如MHC-I等并没有随着细胞的活化而聚集成簇，说明BCR微簇体以及免疫突触是开放、但有选择性的细胞质膜结构，藉此特性B细胞在其活化过程中能够对BCR进行精密的聚类拣选。

较于前人的研究，利用光控抗原系统，该论文更为细致的捕捉观察B细胞接受抗原刺激前后，BCR微簇体的布朗运动速率由快变慢，运动矢量性由无规则的二维布朗运动转变为定向主动运输这一微观的活细胞上的生物学过程。上述实验结果为系统研究B细胞在抗原识别活化的早期分子事件，提供了强有力的技术支持和理论基础。

清华大学生命科学学院2013级博士生王静以及清华大学化学系2011级博士生唐姗为本文的共同第一作者，清华大学生命科学学院刘万里研究员和清华大学化学系刘磊教授为本文的共同通讯作者。该研究由国家自然科学基金委、科技部、青年****、清华大学免疫学研究所和清华-北大生命科学联合中心提供经费支持。

B淋巴细胞的抗原探索行为和抗原受体在免疫突触中的聚类拣选行为

（A）光控抗原系统和高速高分辨率的活细胞单分子荧光成像技术结合研究淋巴细胞活化

（B）B细胞抗原探索行为以及抗原识别后触发的免疫突触形成

（C）B细胞抗原识别后极短的时间内BCR微簇体形成及成熟

原文摘要：

Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.