Phosphoproteomic analysis of successive Jurkat CD19-CAR generations reveals TCRζ-driven signalling

Although chimeric antigen receptor (CAR) T cell therapy has revolutionised individualised cancer therapies for relapsed/refractory lymphomas, low long-term retention due to basal signalling (antigen-independent activation in the absence of cognate antigen) and off-target toxicity limit the broad applicability of CAR-T products. During CAR development, researchers use model systems, like Jurkat T cells (Jurkats), to screen intracellular signalling arrangements based on their ability to activate (e.g., CD69 expression) and withstand repeated antigen encounters. Although Jurkats are standard for CAR screening, the mapping of CAR generations to Jurkat-specific pTyr networks relative to key TCR nodes and CD69 readouts is not well defined, blurring how hierarchical signalling drives activation. Here, we investigated how costimulation influenced tyrosine phosphorylation cascades using LC-MS/MS based phosphotyrosine (pY) proteomics and CD69 expression in the presence of small molecule inhibitors of key TCR signalling regulators. We found that including TCRζ (CD3ζ; gene CD247) in first (ζ-CAR), second (28ζ-CAR and BBζ-CAR), and third (28BBζ-CAR) generation CARs largely determined pY signalling, irrespective of costimulation. Further, we showed that the phosphatase activity of PTPN22 and SHP-1 were largely negligible for activation of CARs, but indiscriminate inhibition of phosphatases using pervanadate (PV) selectively activated BBζ-CARs without antigen encounter. Finally, we found that selective, partial inhibition of Itk using Soquelitinib reduced basal CD69 expression in CAR-Jurkat cells while maintaining their ability to activate in response to antigen. These data suggest that TCRζ determines the pY signalling profile and that Itk drives basal activation of CD19-CAR Jurkats, which may impact evaluation of new CAR designs in CAR-Jurkat screens.

嵌合抗原受体（chimeric antigen receptor, CAR）T细胞疗法已革新了复发/难治性淋巴瘤的个体化癌症治疗，但因基础信号传导（即同源抗原缺失时发生的抗原非依赖性激活）导致的长期留存率低下，以及脱靶毒性，限制了CAR-T产品的广泛应用。在CAR研发过程中，研究者常以Jurkat T细胞（Jurkats）这类模型系统为工具，根据细胞的激活能力（如CD69分子表达）与耐受反复抗原刺激的特性，筛选细胞内信号传导通路的最优排布。尽管Jurkats是CAR筛选的标准模型，但目前尚未明确CAR代次与Jurkat特异性磷酸酪氨酸（phosphotyrosine, pTyr）网络（结合关键T细胞受体（T cell receptor, TCR）节点及CD69检测指标）之间的对应关系，这模糊了分级信号传导如何调控细胞激活的机制。本研究基于液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）的磷酸酪氨酸（pTyr）蛋白质组学技术，结合CD69表达检测，在关键TCR信号传导调控因子的小分子抑制剂存在的条件下，探究了共刺激信号如何影响酪氨酸磷酸化级联反应。研究发现，在第一代（ζ-CAR）、第二代（28ζ-CAR与BBζ-CAR）及第三代（28BBζ-CAR）CAR中引入TCRζ（CD3ζ；基因CD247），基本决定了pY信号传导的特征，且不受共刺激信号的影响。进一步研究表明，PTPN22与SHP-1的磷酸酶活性对CAR的激活基本无影响，但使用过钒酸盐（pervanadate, PV）非特异性抑制磷酸酶时，可在无抗原接触的情况下选择性激活BBζ-CAR。最后，本研究发现，使用索奎替尼（Soquelitinib）选择性部分抑制Itk，可降低CAR-Jurkat细胞的基础CD69表达，同时保留其对抗原刺激产生激活反应的能力。上述数据表明，TCRζ决定了pY信号传导的特征谱，而Itk介导了CD19-CAR Jurkat细胞的基础激活，这一发现可能影响CAR-Jurkat筛选模型中新型CAR设计的评估工作。