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.