As the antigen receptor, the T cell receptor (TCR)-CD3 complex contains a panel of immunoreceptor tyrosinebased activation motifs (ITAMs) in the CD3 subunits to transduce diverse antigen signals, but the mechanism underlying TCR signaling versatility remains poorly understood.
In a study published in Molecular Cell, a research team led by Prof. XU Chenqi from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences, in collaboration with Prof. SHI Xiaoshan from Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and Prof. WANG Haopeng from ShanghaiTech University, unraveled the structural and functional heterogeneity of the major CD3ζ signaling component, and revealed a key regulatory mechanism driven by electrostatic interactions between membrane lipids and Basic Residue-rich Sequences (BRS) in CD3ζ. The research team further identified insufficient phosphorylation of CD3ζ as a contributing factor to T cell exhaustion.
The CD3ζζ homodimer is a key signaling subunit of the TCR-CD3 complex. Each of its intracellular domains contains three ITAMs, contributing to 60% of the phosphorylatable tyrosine sites in the TCR. Recent cryo-EM studies have reported the structures of free and ligand-bound TCR-CD3 complexes, including the parts of the extracellular and transmembrane domains. The CD3 cytoplasmic domains, however, are missing in these complex structures, which is likely due to their disordered and highly dynamic nature.
It follows that filling in this knowledge gap is essential for a complete understanding of how extracellular ligand binding induces intracellular TCR-CD3 signaling. To overcome the structural challenges posed by the "disordered and dynamic" nature of the CD3ζ cytoplasmic domain, the research team employed a lipid bilayer (bicelle) system that mimics the acidic phospholipid environment of physiological membranes. Combined with solution NMR spectroscopy, they successfully resolved the dynamic conformation of the CD3ζ cytoplasmic domain. Structural analysis revealed that the three ITAM motifs of CD3ζ exhibit a gradual increase of membrane insertion from the N- to the C-terminus: the N-terminal ITAM1 has the shallowest membrane insertion, ITAM2 is intermediate, and ITAM3 is the deepest. Notably, the level of ITAM membrane insertion is primarily regulated by the interaction between its adjacent BRS and lipids, rather than the ITAM sequence itself. This finding clarifies the molecular basis of the structural heterogeneity of CD3ζ ITAMs.
To investigate the functional impact of differential ITAM membrane insertion, the research team analyzed its phosphorylation patterns using techniques such as novel targeted quantitative mass spectrometry. It was found that in an acidic lipid environment, ITAM phosphorylation efficiency is negatively correlated with its membrane insertion depth. Specifically, ITAM1, with the shallowest membrane insertion, is most accessible to LCK kinase and thus phosphorylated most rapidly, resulting in sequential phosphorylation from the N- to the C-terminus. This sequence was abolished in both neutral lipid environments and BRS mutants, demonstrating that the electrostatic interaction of "BRS-lipid" is the core driver of the sequential phosphorylation of CD3ζ.
The research team further investigated the impact of chronic TCR stimulation, a scenario relevant to cancer and chronic infection, on CD3ζ phosphorylation. The results indicate that the phosphorylation pattern of CD3ζ undergoes significant changes during T cell exhaustion—the C-terminal ITAM3 displays faster decay kinetics of phosphorylation than the N-terminal ITAM1, leading to the accumulation of large amounts of "partially phosphorylated" CD3ζ in cells, which in turn causes insufficient TCR signaling. This finding provides a new explanation for the mechanism of T cell exhaustion: in addition to "extrinsic" immune checkpoint molecules (such as PD-1, LAG3, etc.), "intrinsic" insufficient phosphorylation of the TCR itself is also an important cause of T cell dysfunction. In various human tumors, this signaling attenuation is closely associated with T cell exhaustion.
In summary, this study employed an interdisciplinary approach integrating biophysics, biochemistry, and immunology to resolve the dynamic structure of the CD3ζ cytoplasmic domain in a physiological acidic membrane environment. It elucidated the electrostatically regulated sequential phosphorylation pattern of ITAMs and revealed a novel mechanism of T cell exhaustion, providing new insights for immunotherapy.
Reference: https://www.cell.com/molecular-cell/fulltext/S1097-2765(25)00746-4
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