High-throughput bead-based cytokine profiling reveals dynamic immune signatures and identifies candidate exploratory biomarkers in CAR-T therapy

Abstract

Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of refractory B-cell malignancies, yet severe cytokine release syndrome (CRS) and unpredictable long-term outcomes remain major clinical challenges. Here, we developed a rapid, scalable, 96-well plate-based strategy for the high-throughput preparation of lanthanide-encoded classifier beads. This approach substantially increases production throughput, from several types per day to 96 types within 8 hours, enabling sensitive multiplexed cytokine profiling by mass cytometry. By applying this platform to 136 longitudinal plasma samples from 32 patients with diffuse large B-cell lymphoma receiving CD19-directed CAR-T therapy, we achieved fg mL−1-level sensitivity and broad dynamic ranges across 14 key cytokines. Longitudinal profiling revealed distinct cytokine dynamics between patients with low- and high-grade CRS. Baseline TGF-β1 showed a significant univariate association with subsequent high-grade CRS (p = 0.001) and inferior event-free survival (p = 0.02). These findings demonstrate the utility of a scalable bead-based platform for high-resolution immune monitoring in CAR-T therapy and identify baseline TGF-β1 as a candidate exploratory biomarker associated with CRS severity and clinical outcome. This approach provides a practical tool for sensitive and high-throughput cytokine profiling and deeper understanding of immune responses in immunotherapy.