Phospho-mimetic CD3ε variants prevent TCR and CAR signaling

• DOI: 10.3389/fimmu.2024.1392933
• Publication Date: 2024-05-08T04:34:23Z
• Authors: Nadine M. Woessner; Simon M. Brandl; Sara Hartmann; Wolfgang W. Schamel; Frederike A. Hartl; Susana Minguet
• Keywords: CD3 Complex; T-Lymphocytes; Immunology; Receptors, Antigen, T-Cell; Immunoreceptor Tyrosine-Based Activation Motif; Lymphocyte Activation; phospho-mimetic; Jurkat Cells; 03 medical and health sciences; TCR - T cell receptor; Humans; CAR (chimeric antigen receptor); Phosphorylation; Adaptor Proteins, Signal Transducing; 0303 health sciences; Receptors, Chimeric Antigen; ZAP-70 Protein-Tyrosine Kinase; RC581-607; LCK; HEK293 Cells; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Receptor-CD3 Complex, Antigen, T-Cell; CD3epsilon; Immunologic diseases. Allergy; Protein Binding; Signal Transduction

Introduction Antigen binding to the T cell antigen receptor (TCR) leads phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) CD3 complex, and thereby activation. The CD3ε subunit plays a unique role in TCR by recruiting kinase LCK adaptor protein NCK prior ITAM phosphorylation. Here, we aimed investigate how individual tyrosines impacts signalosome. Methods We mimicked irreversible tyrosine substituting glutamic acid for residues ITAM. Results Integrating phospho-mimetic variants into complete TCR-CD3 complex resulted reduced signal transduction, which was partially compensated involvement other ITAMs. By using novel Chimeric Receptor (CAR) variants, avoided any compensatory effects ITAMs complex. demonstrated that prevented transduction upon CAR engagement. Mechanistically, substitution at N-terminal residue (Y39E) significantly reduces TCR. In contrast, mutation C-terminal (Y50E) abolished recruitment TCR, while increasing binding. Double C- (Y39/50E) allowed ZAP70 bind, but interaction with NCK. Conclusions data demonstrate dynamic is essential orchestrate optimal signaling highlights key signalosome tune transduction.