Cell:重磅,中外团队联合发文,有望设计全新CAR-T疗法

2020-07-30 CPMMR 生物探索

研究通过开发基于质谱的绝对定量蛋白质组新方法,揭示了T细胞受体-共受体复合物酪氨酸在不同抗原刺激下的动态磷酸化修饰全貌,解析了不同CD3链ITAM结构域磷酸化特征的奥秘,有助于设计全新的CAR-T疗法

2020年7月29日,北京大学医学部精准医疗多组学研究中心黄超兰团队,中科院上海生化与细胞所许琛琦团队、美国加州大学圣地亚哥分校惠恩夫团队,联手在Cell上发表了题为“Multiple signalingroles of CD3ε and its application in CAR-T cell therapy”的论文,该研究通过开发基于质谱的绝对定量蛋白质组新方法,揭示了T细胞受体-共受体(TCR-CD3)复合物酪氨酸在不同抗原刺激下的动态磷酸化修饰全貌,解析了不同CD3链ITAM结构域磷酸化特征的奥秘,从中发现了其中一条亚基CD3ε的单磷酸化新功能,有望助力于设计全新的CAR-T疗法。

TCR-CD3复合物在T细胞的发育、激活及对病原的免疫反应中起着决定性作用。这一重要作用来自于CD3链胞内端的免疫受体酪氨酸激活基序(Immunoreceptortyrosine-based activation motif-ITAM)。而ITAM的多样性功能主要取决于其结构域的酪氨酸(Tyrosine)磷酸化,比如招募SYK激酶家族蛋白ZAP70进而激活下游的信号传导。另外,ITAM的功能也被广泛应用在对嵌合抗原受体(CAR)的研究中。其中CD3ζ亚链便常用于构建CAR-T细胞疗法抗肿瘤活性,但其他CD3链的功能以及是否可应用于CAR的设计还有待进一步开发。

深入探索 CD3 ITAM的酪氨酸动态磷酸化模式可为全面理解不同CD3链的功能提供核心信息。TCR-CD3受体复合物有10个ITAM结构域分布着20个磷酸化位点,在时间分辨率下实现对全部磷酸化位点的同时定量分析在技术上极具挑战性。

为了直观比较不同TCR刺激下的磷酸化模式,精确绘制出TCR所有酪氨酸磷酸化的动态过程,黄超兰团队开发了一种新颖的绝对定量方法Targeted-IP-Multiplex-Light-Absolute-Quantitative Mass Spectrometry(TIMLAQ-MS)。该方法区别于目前报道的蛋白组绝对定量手段,不需要加入同位素重标的合成肽段,而是巧妙地利用串联质量标签(TMT),设计将6个标准样品和4个分析样品混合起来作为内标。标准样品为不同浓度梯度的合成非重标磷酸化/非磷酸化CD3肽(A)和从未经抗原刺激的T细胞中通过IgG抗体免疫沉淀下来的背景蛋白(B)的混合物;用数据依赖采集(Data-dependent acquisition,DDA)结合平行反应监测(Parallel reaction monitoring, PRM)的方式获得抗原刺激下,TCR-CD3免疫沉淀(IP)复合物中不同酪氨酸位点的磷酸化/非磷酸化在不同时间点的定量结果。TIMLAQ 成功绕过了以前的定量方法中通常使用的同位素重标记肽,既节约了成本,又有效降低了方法的复杂性和数据采集误差,进一步提高了定量准确性,最终可完全实现在一次测量中对不同时间点全部ITAM磷酸化修饰的绝对定量,描绘TCR-CD3复合物的酪氨酸动态磷酸化修饰全貌。

利用这一方法鉴定到在不同的TCR刺激条件下,CD3各亚基主要表现为双磷酸化修饰模式,而唯独CD3ε则呈现出单磷酸化修饰模式。此前研究表明,双磷酸化的ITAM与激酶家族蛋白ZAP70有很强的结合而激活下游信号传导,而单磷酸化的ITAM则表现出很低的结合性。新研究中的这一特殊的新发现驱使研究人员进一步深入探索CD3ε在TCR通路中的新潜在功能。结果显示,单磷酸化的CD3ε可通过专门募集抑制性Csk激酶减弱TCR信号传导,说明TCR中既有激活基元又有抑制基元,总体呈现为一种自制的信号传导机制。作者团队进一步深入研究,发现一旦将CD3ε细胞质结构域整合到第二代CAR中,CD3ε的ITAM结构域可以通过募集Csk减少CAR-T细胞因子的产生,而CD3ε的BRS结构域则可以通过募集p85促进CAR-T细胞的持久性。总体而言,将CD3ε应用于CAR的设计可显着提高CAR-T细胞的抗肿瘤活性。

黄超兰教授是北京大学医学部精准医疗多组学研究中心主任,北京大学医学部基础医学院长聘副教授,北京大学生命科学联合中心研究员,曼彻斯特大学荣誉教授。近年来,黄超兰教授带领团队积极开发基于质谱的蛋白质组学新方法,实验室拥有国际领先的仪器、技术和方法,致力于为生物学和临床研究中遇到的难题提供最有质量保证的全面蛋白质组和质谱技术手段。

原始出处:
Wei Wu,Qiuping Zhou,Takeya Masubuchi,et al.Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy.Cell.Published:July 29, 2020DOI:https://doi.org/10.1016/j.cell.2020.07.018

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    2020-10-26 维他命
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    2020-08-01 xxxx1054

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