Small size of membrane-tethered ligands regulates phosphatase segregation in γδ TCR triggering

Abstract

Abstract Ligand recognition by the T cell antigen receptor (TCR) is central to effective immunity. Unlike αβ T cells, γδ T cells do not recognize pMHC antigens or use coreceptors CD4/8. Instead they recognize invariant membrane-tethered ligands, which are upregulated at sites of inflammation and infection. The substantial structural similarities between αβ and γδ TCRs prompted us to investigate whether they share triggering mechanisms. However, recent studies show that binding-induced γδ TCR conformational changes are not induced by physiological ligands, leaving open the possibility that other mechanisms are involved. We investigated the stress-inducible murine surface protein T22, that is recognized by the G8 γδ TCR - since detailed biophysical and structural information is available for this system. Here we tested whether ‘size-based’ kinase-phosphatase segregation (also known as the kinetic-segregation model) mediates G8 TCR signal initiation. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation and IL-2 release. Electron microscopy showed that elongated T22/G8 leads to increased intermembrane spacing at the immunological synapse. Conversely, truncation of the large CD45 ectodomain attenuated triggering, pointing to a critical dependence on ‘close contact’ for phosphatase exclusion. To establish the physiological relevance of our findings, we confirmed that T22 elongation abrogates triggering in primary G8 γδ T cells, and using a second γδ TCR (KN6). We are currently completing investigations on the role of kinetic-segregation in hapten-specific and human γδ TCR triggering. Taken together, our findings establish that γδ TCR triggering occurs primarily through the kinetic-segregation mechanism.