New evidence that complement activation and NLRP3 inflammasome signaling work together to cause cytokine release syndrome after CAR-T cell therapy

Abstract Background: Cytokine release syndrome (CRS) is a common and potentially serious complication of chimeric antigen receptor T-cell (CAR-T) therapy in hematologic cancers. Although the role of cytokines in CRS is well known, the specific upstream immune triggers—including the involvement of the complement system and inflammasome activation—are less clear. Increasing evidence indicates that innate immune processes such as complement activation and pyroptosis contribute to the systemic inflammation seen in CRS, but detailed mechanistic data from patient groups are limited. Hypothesis:We propose that patients who develop CRS show synchronized activation of the complement system and the NLRP3 inflammasome, leading to systemic inflammation, pyroptosis, and cytokine storm during the early post-infusion period of CAR-T therapy.Materials and Methods: Twenty-one patients with B-cell cancers receiving CAR-T therapy were enrolled and divided into CRS-positive (n=15) and CRS-negative (n=14) groups. Blood plasma and PBMC samples were collected on Day 0 (infusion), Day +7, +14, and +21. We conducted: 1) quantitative proteomics (label-free LC-MS/MS) with FDR-adjusted significance testing (p < 0.05), 2) multiplex cytokine analysis (IL-1β, IL-6, IL-8, IFN-γ, TNF-α, Gasdermin 3), complement factor measurement (C3, C4, C5, C9, C1s, CFI, CFH, C1-INH), 4) caspase-Glo® 1 inflammasome activity assay, and 5) qRT-PCR on PBMCs for NLRP3, CASP1, GSDMD, and IL1β expression. Results: CRS-positive patients showed significant activation of both classical and alternative complement pathways. On Days +7 and +14, circulating levels of complement proteins C3, C4, C5, C9, and regulators (CFH, CFI, C1-INH) were significantly higher in CRS-positive patients (p < 0.01 vs. CRS-negative). Proteomics confirmed these increases, showing strong enrichment of the “terminal complement cascade” and “regulation of complement activation” pathways. Caspase-Glo® 1 tests revealed that CRS-positive patients had a 4.2-fold increase in caspase-1 activity compared to CRS-negative controls (p < 0.001), peaking on Day 7. Gene expression levels of NLRP3, CASP1, and IL1b rose by 2.5 to 3.2 times in PBMCs from CRS-positive patients (p < 0.01), consistent with inflammasome activation. Gasdermin D, a marker of pyroptotic cell death, was notably increased in plasma during CRS episodes, supporting a mechanistic link between inflammasome activation and membrane pore formation. Cytokines IL-1β, IL-6, and TNF-α were higher in CRS-positive patients and closely linked with caspase-1 activity (r > 0.7, p < 0.001) and complement levels, suggesting a feedback loop between complement-induced inflammation and inflammasome-driven cytokine production. Finally, combined pathway analysis of proteomics and cytokine data revealed co-enrichment of innate immunity pathways including “interleukin-1 processing,” “inflammasome activation,” “platelet degranulation,” and “complement activation.” Specific biomarker signatures (C3, C5, Caspase-1, Gasdermin D, IL-1β) accurately distinguished CRS-positive patients and could aid in risk-based monitoring. Conclusion: Our findings strongly indicate that CRS in CAR-T–treated patients result from early, sustained activation of the complement system and NLRP3 inflammasome. These processes lead to pyroptosis, cytokine release, and systemic inflammation. This mechanistic understanding emphasizes the role of innate immune regulation in CAR-T-related toxicities and suggests that therapies targeting the complement or inflammasome pathways may reduce CRS severity and improve patient outcomes. The identification of complement and pyroptosis biomarkers sets a foundation for developing precise monitoring tools and targeted interventions in future CAR-T treatments.