Longitudinal Plasma Proteomics Distinguishes Primary Immune Tolerance from Chronic Graft-versus-Host Disease After Pediatric allo-HSCT

Chronic graft-versus-host disease (cGvHD) remains a major cause of morbidity after pediatric allogeneic hematopoietic stem cell transplantation (HSCT), but the early plasma molecular features associated with primary immune tolerance (PIT) versus cGvHD are incompletely understood. We performed longitudinal LC-MS plasma proteomics in 32 pediatric allo-HSCT recipients sampled at Months 1, 3, 6, 9, and 12 after transplantation, quantifying 4,702 proteins. Across timepoint-specific differential-abundance analyses, Month 3 showed the strongest observed PIT-cGvHD proteomic separation and was selected for deeper exploratory characterization. At Month 3, eight proteins were higher in PIT at BH-FDR <0.05, including the 19S proteasome ATPases PSMC1, PSMC3 and PSMC5, the chaperonin subunit CCT3, the ubiquitin-conjugating enzyme UBE2O, and APEH, RAN and NME1, pointing to differences in protein degradation and folding pathways. Time-resolved pathway analysis showed PIT-associated immune, immunoglobulin, and proteostasis programs at distinct post-transplant intervals, whereas Month-3 cGvHD-associated profiles were enriched for platelet activation, cell adhesion, transmembrane transport, and metabolic processes. Outcome-blinded clustering of the 500 most temporally variable proteins resolved three descriptive trajectory patterns: a transient Month-3 peak, a Month-3 nadir with late recovery, and an early-high/late-decline pattern. The distribution of Month-3 PIT–cGvHD protein effects differed across outcome-blinded trajectory clusters, providing an internally consistent temporal framework for the Month-3 signal. These exploratory findings nominate Month 3 as a potentially informative interval of molecular divergence after pediatric allo-HSCT and identify pathway-level features of PIT and cGvHD for prospective validation.