2026/03/10 by Ting Yang, Min Gao, Yufang Sun +4 · 1 voice
Medicine · Biochemistry, Genetics and Molecular Biology · #CAR-T cell therapy research #Signaling Pathways in Disease #Protein Degradation and Inhibitors
paper · doi:10.1093/abt/tbag010
openalex publication_date 2026/03/10 · openalex created_date 2026/03/13 · openalex updated_date 2026/06/18
Abstract Background Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic malignancies but remains limited by on-target, off-tumor toxicity and the lack of precise mechanisms to restrain aberrant activation. Inhibitory CARs (iCARs) offer a logic-gated strategy to suppress unwanted immune responses, yet optimization of inhibitory signaling modules has been hindered by an incomplete understanding of immunoreceptor tyrosine-based inhibitory motif (ITIM) diversity. Methods We established a systematic screening platform to evaluate ITIM-containing intracellular domains (ICDs) from 35 candidate receptors. Using nuclear factor of activated T cells (NFAT) reporter assays across multiple activation contexts, we quantified the ability of each domain to counteract iimmunoreceptor tyrosine-based activating motif (ITAM)-mediated signaling. Intracellular truncated analyses tested the requirement of intact cytoplasmic motifs, and the most potent ITIMs were incorporated into iCAR architectures to assess their capacity to inhibit CAR-driven effector responses. Results We identified 12 ITIM domains that strongly suppressed NFAT signaling across diverse activation states. Their inhibitory activity required intact ITIM motifs. When integrated into iCAR constructs, these domains conferred markedly greater suppression of CAR-induced activation compared with conventional programmed cell death protein 1 (PD-1)-based designs. Among them, the SIGLEC9-derived ITIM exhibited the most potent inhibition, significantly reducing CAR-T effector function and protecting target cells from cytotoxicity in vitro. Conclusions We identified distinct intracellular modules that effectively suppress CAR-mediated activation, with the SIGLEC9-derived ITIM exhibiting the strongest inhibition. Leveraging domain-specific inhibitory strength enables precise and modular control of T-cell activation, thereby improving the safety, specificity, and tunability of next-generation cellular immunotherapies.