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.