Reconstructing the TCR

Science Signaling  10 Jul 2012:
Vol. 5, Issue 232, pp. ec185
DOI: 10.1126/scisignal.2003380

T cell activation requires tyrosine phosphorylation of the T cell receptor (TCR) after it binds a peptide-bound major histocompatability complex (pMHC), which is presented to the T cell by an antigen-presenting cell (APC). James and Vale explored phosphorylation of the TCR subsequent to binding of the pMHC (referred to as “triggering”) by introducing genes encoding the TCR and its regulatory proteins into nonimmune human embryonic kidney cells (HEK-1G4 cells) and reconstituting TCR triggering when these cells interacted with Raji B cells (the APCs). The minimal components required to recapitulate a quiescent TCR system were the α and β subunits of the TCR, Lck, which phosphorylates the TCR immunoreceptor tyrosine-based activation motifs (ITAMs) on the CD3ζ subunit; zeta-chain–associated protein kinase 70 (ZAP70), which associates with the phosphorylated ITAMs and mediates downstream signaling events; CSK, which phosphorylates and deactivates Lck; CBP, which localizes CSK to the plasma membrane; and CD45, a transmembrane protein with an extended extracellular domain and an intracellular phosphatase domain that dephosphorylates Lck and CD3ζ. The authors used the membrane localization of ZAP70 fused to green fluorescent protein (ZAP70-GFP) as a proxy for TCR triggering. When HEK-1G4 cells interacted with pMHC-bearing APCs, ZAP70-GFP fluorescence was enriched in and fluorescent protein-tagged CD45 was excluded from the zone of contact. The authors used an induced proximity assay in which the intracellular ITAM domain of CD3ζ of the TCR was replaced with the protein FKBP, which binds to its partner FRP, which was fused to the ITAM-domain–containing cytoplasmic fragment of CD3ζ (CD3ζ-FRP), only in the presence of rapamycin. CD45 was excluded from the interaction site between the cells reconstituted with this chimeric TCR and the APCs in the absence of rapamycin and CD3ζ-FRP, but ZAP70 recruitment did not occur unless CD3ζ-FRP was present and rapamycin was added. Chimeric CD45 with its extracellular domain replaced with that of CD2, an adhesion protein that binds a protein present on the cell surface of APCs, was not excluded from the interface between the interacting cells, and ZAP70 was not recruited to the zone of contact. Replacement of the CD45 extracellular domain with that of CD86 (CD86ExCD45Int), a transmembrane protein the same size as CD2, resulted in exclusion of these chimeric proteins from the zone of contact. But when the APCs had the binding partner for CD86, CD86ExCD45Int was not excluded from the zone of contact. Thus, it appears that CD45 is excluded from the zone of contact because it does not have a binding partner on the APC, not due to its size. In a modified transcellular version of the induced proximity assay, the authors then emulated pMHC interaction with the TCR by presenting FRP fused to a transmembrane protein on the surface of the APCs instead of pMHC to HEK-1G4 cells with FKBP fused to the extracellular portion CD3ζ at the cell surface instead of the TCR. Addition of rapamycin recapitulated the exclusion of CD45 and recruitment of ZAP70. These results were consistent with a model in which the binding energy from pMHC interaction with the TCR drives exclusion of CD45 from the interface between interacting cells, allowing the TCR phosphorylated by Lck to accumulate and recruit ZAP70.

J. R. James, R. D. Vale, Biophysical mechanism of T-cell receptor triggering in a reconstituted system. Nature 487, 64–69 (2012). [Online Journal]