Unraveling of Phosphotyrosine Signaling Complexes Associated with T Cell Exhaustion Using Multiplex Co-Fractionation/Mass Spectrometry

T cell exhaustion, characterized by the upregulation
of inhibitory
receptors and loss of effector functions, plays a crucial role in
tumor immune evasion. This study utilizes a high-throughput, reproducible,
and robust integrated ion-exchange chromatography–tandem mass
tag (IEC-TMT) platform, coupled with a complex-centric quantification
algorithm, to thoroughly profile phosphotyrosine (pTyr) protein complex
changes during T cell exhaustion. The platform’s high reproducibility
is evidenced by >0.94 correlation and a median coefficient of variation
of 0.25 among quantified complexes in HeLa cell biological replicates.
This high-throughput approach allowed analysis of 312 fractions within
2 days, identifying 268 pTyr protein complexes from the T cell exhaustion
model. Robust quantification of 28 complexes revealed 12 exhibiting
significant abundance alterations in exhausted T cells, notably impacting
lysosomal and endoplasmic reticulum-associated complexes. RTN4, a
subunit of the newly identified PPI204 protein complex, is upregulated
in exhausted T cells. Its knockdown reversed T cell exhaustion, enhancing
antitumor immunity. These findings provide novel insights into the
molecular mechanisms of T cell exhaustion and propose RTN4 as a potential
therapeutic target.

以抑制性受体上调及效应功能丧失为特征的T细胞耗竭（T cell exhaustion）在肿瘤免疫逃逸中发挥关键作用。本研究采用高通量、可重复且稳定可靠的整合型离子交换色谱-串联质量标签（ion-exchange chromatography–tandem mass tag, IEC-TMT）平台，结合以复合物为中心的定量算法，对T细胞耗竭过程中的磷酸酪氨酸（phosphotyrosine, pTyr）蛋白复合物变化进行全面表征。该平台的高重复性可通过海拉（HeLa）细胞生物学重复样本中定量复合物的相关系数>0.94、变异系数中位数为0.25得到验证。此高通量分析方法可在2天内完成312个组分的检测，从T细胞耗竭模型中鉴定出268个pTyr蛋白复合物。对28个复合物的准确定量显示，其中12个在耗竭T细胞中存在丰度显著变化，这些变化显著影响溶酶体及内质网相关蛋白复合物。新鉴定的PPI204蛋白复合物的亚基RTN4在耗竭T细胞中表达上调。敲低RTN4可逆转T细胞耗竭，增强抗肿瘤免疫。本研究结果为T细胞耗竭的分子机制提供了全新见解，并提出RTN4可作为潜在治疗靶点。