Abstract
Cytokine release syndrome (CRS) is a common and potentially life-threatening complication following chimeric antigen receptor T-cell (CAR-T) therapy. Despite its clinical importance, the systemic metabolic and structural effects of CRS remain poorly understood. Although pro-inflammatory cytokine networks have been linked to CRS, the downstream impacts on protein-level immune regulation, lipid remodeling, and membrane stability are largely unexplored. A multi-omics approach could help identify predictive biomarkers, clarify mechanisms of toxicity, and guide future treatments. Hypothesis:CRS involves widespread changes in immune, metabolic, and membrane-related pathways that can be understood through integrated proteomic and lipidomic analysis of patient samples collected over time during CAR-T therapy and serve as a tool to predict this complication.Methods: Plasma samples from CAR-T–treated patients were gathered at baseline (Day 0, infusion time) and at Days 7, 14, and 21 post-infusion. Quantitative proteomics was conducted using label-free LC-MS/MS with FDR correction (q<0.05), and untargeted lipidomics was performed using ultra high-resolution 7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometry. Differential expression and functional annotation (Reactome, Gene Ontology) were applied across different timepoints and stratified by CRS status. Results: Proteomic analysis revealed dynamic changes in inflammatory, metabolic, and homeostatic proteins over time. On Day 7 post-infusion, 79 proteins showed significant alterations (46 increased, 33 decreased), with 91 changes on Day 14 (70 increased, 21 decreased), and 98 on Day 21 (54 increased, 44 decreased). Patients with CRS exhibited a notable increase in acute-phase and complement-related proteins—including CRP, Haptoglobin, Complement C9, Vitronectin, and Complement Factor I—alongside a decrease in mitochondrial electron transport components (e.g., NDUFA8, NDUFS6) and lipid-binding regulators. Functional analysis highlighted activation of key pathways related to i) innate immune signaling and complement activation, ii) platelet degranulation and hemostasis, iii) heme scavenging and IGF transport regulation, and iv) post-translational protein phosphorylation and mitochondrial translation. Simultaneously, lipidomic studies identified consistent disturbances in lipid metabolism and membrane integrity in CRS-positive patients. Notably, phosphatidylserine (PS 37:0) and phosphatidylglycerol (PG 41:0) were significantly reduced, indicating increased apoptotic cell clearance and mitochondrial stress. These lipids are vital for immune recognition and energy production, respectively. Sulfatides (ST 35:0 and ST 37:1), which influence immune signaling and membrane stability, were depleted—pointing to sphingolipid imbalance and heightened cellular stress. Also decreased were ceramide phosphate (CerP 20:1) and plasmenyl-phosphatidylethanolamine (plasmenyl-PE 38:4), which are important for membrane fluidity, redox defense, and inflammatory signaling—likely reflecting lipid peroxidation and rapid consumption in activated immune cells. Conversely, CRS-positive patients showed increased levels of saturated fatty acids (e.g., palmitic acid), phosphatidic acid (PA 24:1), and phosphatidylinositol (PI 28:1), possibly indicating compensatory mechanisms for immune activation, membrane turnover, and bioenergetic stress. The combined proteomic and lipidomic results revealed a consistent pattern of membrane remodeling, immune overactivation, and oxidative stress, with clear changes over time during therapy. Conclusion: This study offers the first comprehensive proteo-lipidomic analysis of CRS in CAR-T recipients, identifying linked disruptions in inflammatory signaling, mitochondrial function, and lipid remodeling. The coordinated reduction of immunoregulatory lipids (PS, PG, sulfatides) and mitochondrial proteins, along with increased levels of acute-phase and complement factors, reflects systemic immune-metabolic stress specific to CRS. These multi-omic signatures provide a mechanistic basis for biomarker-driven monitoring, prevention, and proposed treatment strategies in patients receiving CAR-T cell therapy.
