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Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Systematic functional screening of immunoreceptor tyrosine-based inhibitory motif domains identifies potent
Ting Yang1,2, Min Gao1,2, Yufang Sun3
1Department of Immunology, School of Basic Medical Sciences, Health Science Center, Peking University, Beijing 100191, China.
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.
Insights
Researchers identified novel inhibitory signaling domains for chimeric antigen receptor (CAR) T-cell therapy. The SIGLEC9-derived inhibitory motif offers potent control, enhancing CAR T-cell safety and specificity.
Area of Science:
- Immunology
- Cellular Therapy
- Cancer Research
Background:
- Chimeric antigen receptor (CAR) T-cell therapy shows promise for hematologic malignancies.
- Current CAR T-cell therapies face limitations including on-target, off-tumor toxicity and uncontrolled activation.
- Inhibitory CARs (iCARs) aim to mitigate these issues, but optimizing inhibitory signaling requires understanding immunoreceptor tyrosine-based inhibitory motif (ITIM) diversity.
Purpose of the Study:
- To systematically screen and identify potent ITIM domains for enhancing iCAR function.
- To evaluate the inhibitory capacity of diverse ITIMs in counteracting CAR T-cell activation.
- To develop improved iCAR architectures for safer and more specific cellular immunotherapies.
Main Methods:
- Established a screening platform to test 35 ITIM-containing intracellular domains (ICDs).
- Utilized nuclear factor of activated T cells (NFAT) reporter assays to quantify inhibitory signaling.
- Incorporated top-performing ITIMs into iCAR constructs to assess inhibition of CAR-driven effector functions.
Main Results:
- Identified 12 ITIM domains that effectively suppressed NFAT signaling.
- Demonstrated that intact ITIM motifs are crucial for inhibitory activity.
- Showcased iCARs with novel ITIMs providing superior suppression compared to PD-1-based designs.
- The SIGLEC9-derived ITIM exhibited potent inhibition, reducing CAR-T effector function and protecting target cells in vitro.
Conclusions:
- Identified distinct intracellular modules for suppressing CAR-mediated activation, with SIGLEC9-derived ITIM being the most potent.
- Domain-specific inhibitory strength allows precise, modular control of T-cell activation.
- These findings pave the way for safer, more specific, and tunable next-generation cellular immunotherapies.
