Unraveling\nof Phosphotyrosine Signaling Complexes\nAssociated with T Cell Exhaustion Using Multiplex Co-Fractionation/Mass\nSpectrometry

T cell exhaustion, characterized by the upregulation\nof inhibitory\nreceptors and loss of effector functions, plays a crucial role in\ntumor immune evasion. This study utilizes a high-throughput, reproducible,\nand robust integrated ion-exchange chromatography–tandem mass\ntag (IEC-TMT) platform, coupled with a complex-centric quantification\nalgorithm, to thoroughly profile phosphotyrosine (pTyr) protein complex\nchanges during T cell exhaustion. The platform’s high reproducibility\nis evidenced by >0.94 correlation and a median coefficient of variation\nof 0.25 among quantified complexes in HeLa cell biological replicates.\nThis high-throughput approach allowed analysis of 312 fractions within\n2 days, identifying 268 pTyr protein complexes from the T cell exhaustion\nmodel. Robust quantification of 28 complexes revealed 12 exhibiting\nsignificant abundance alterations in exhausted T cells, notably impacting\nlysosomal and endoplasmic reticulum-associated complexes. RTN4, a\nsubunit of the newly identified PPI204 protein complex, is upregulated\nin exhausted T cells. Its knockdown reversed T cell exhaustion, enhancing\nantitumor immunity. These findings provide novel insights into the\nmolecular mechanisms of T cell exhaustion and propose RTN4 as a potential\ntherapeutic target.