NMR Footprinting Of Activating And Non-activating Monoclonal Antibodies On CD3 Indicate Dynamic Quaternary Structure Changes In The TCR Complex

The T cell receptor (TCR) mediates antigen recognition and T cell activation via its dimeric αβ, CD3eγ, CD3eδ and CD3ζζ subunits, however, a structural mechanism relating both functions has remained elusive. Here, we determine the NMR footprints on CD3eγ of one non-agonist and two agonist anti-CD3 monoclonal antibodies (mAbs). The data indicate changes of the site-specific binding topology and the TCR quaternary structure upon activation. NMR cross-saturation and chemical shift mapping showed that agonist and non-agonistic mAbs have distinct binding sites on the CD3eγ heterodimer. Agonistic mAbs bind to the membrane distal CD3e lobe, whereas a non-agonist mAb targets the cleft between CD3e and CD3γ causing a non-native quaternary structure in TCRβ-CD3eγ module. Subsequent biological experiments confirmed that the difference in cell triggering is not linked to mAb affinity or CD3e binding stoichiometry per TCR but to the difference in the binding epitope on CD3eγ. More importantly, an Fab that stabilizes an intact TCRβ-CD3eγ module inhibits antigen-dependent activation. These findings indicate that a dynamic but coordinated receptor quaternary structure change in T cell receptor is important for T cell activation, which offer new insights into functional integration within multi-subunit receptors and may guide design of immunosuppressive mAbs devoid of agonist activity.