T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand Kaushik Choudhuri 1 , David Wiseman 1 , Marion H. Brown 1 , Keith Gould 2 * & P. Anton van der Merwe 1 * The binding of a T-cell antigen receptor (TCR) to peptide antigen presented by major histocompatibility antigens (pMHC) on anti- gen-presenting cells (APCs) is a central event in adaptive immune responses 1,2 . The mechanism by which TCR–pMHC ligation initiates signalling, a process termed TCR triggering, remains controversial 3–5 . It has been proposed 6–8 that TCR triggering is promoted by segregation at the T cell–APC interface of cell-surface molecules with small ectodomains (such as TCR–pMHC and accessory receptors) from molecules with large ectodomains (such as the receptor protein tyrosine phosphatases CD45 and CD148). Here we show that increasing the dimensions of the TCR–pMHC interaction by elongating the pMHC ectodomain greatly reduces TCR triggering without affecting TCR–pMHC ligation. A similar dependence on receptor–ligand complex dimensions was observed with artificial TCR–ligand systems that span the same dimensions as the TCR–pMHC complex. Interfaces between T cells and APCs expressing elongated pMHC showed an increased intermembrane separation distance and less depletion of CD45. These results show the importance of the small size of the TCR–pMHC complex and support a role for size- based segregation of cell-surface molecules in TCR triggering. Antigen recognition by T cells involves simultaneous interactions between many T-cell surface receptors and their ligands on APCs or target cells 9 . TCR–pMHC and various accessory receptor–ligand interactions that contribute to TCR triggering span a relatively short intermembrane distance (,14 nm), whereas receptor protein tyrosine phosphatases known to inhibit TCR triggering, including CD45 and CD148, have much larger ectodomains 1,10,11 . This led to the hypothesis that size-dependent segregation of the TCR and its accessory molecules from inhibitory molecules with large ecto- domains is required for TCR triggering 6–8 . A key prediction of this hypothesis is that TCR triggering is dependent on the dimensions of the TCR–pMHC interaction. In order to test this prediction, we exploited a recently described method 12 to produce a murine pMHC class I complex as a single- chain trimer (SCT) fusion protein (Fig. 1a and Supplementary Methods). The SCT consists of the ovalbumin-derived peptide SIINFEKL (pOVA), b 2 -microglobulin and the H-2K b MHC class I heavy chain, joined by two flexible glycine/serine linker sequences (Fig. 1a). The insertion of spacers containing two, three or four immunoglobulin superfamily (IgSF) domains into the membrane- proximal stalk region of the SCT allowed us to produce elongated forms of H-2K b molecules (SCT-CD2, SCT-CD22 and SCT-CD4; Fig. 1a). A key advantage of using SCTs is that intracellular assembly is less dependent on the cis-interactions in the endoplasmic reticu- lum (for example, with tapasin and TAP) required for assembly of native peptide–MHC I complexes (ref. 13). Consequently, assembly and peptide loading are unlikely to be affected by domain insertions. A further advantage of this system is the availability of a monoclonal antibody (25-D1.16) specific for the pOVA/H-2K b complex 14 , which allows the detection and quantification of cell-surface pOVA/H-2K b . As a control, we expressed a single-chain dimer (SCD) fusion protein, which contained just the b 2 -microglobulin and the H-2K b heavy chain joined by a glycine/serine linker (Fig. 1a). The SCT construct is stably expressed on CHO cells, as determined by staining with the 25-D1.16 antibody (Supplementary Fig. 1). SCT-transfected CHO cells were used to stimulate the pOVA/H-2K b - specific murine T-cell hybridoma B3Z, and activation was monitored by measurement of interleukin-2 (IL-2) secretion. CHO cells expres- sing SCTs were at least as efficient at stimulating B3Z cells as CHO cells expressing equivalent levels of SCD loaded with exogenous pOVA (pOVA/SCD), indicating that covalent linkage of pOVA to H-2K b in the SCT does not abrogate TCR recognition (Supplemen- tary Fig. 1). When constructs encoding the elongated SCTs were transfected into CHO cells, they produced proteins of the expected size (Supplementary Fig. 2a), all of which were recognized at the cell surface by the pOVA/H-2K b -specific monoclonal antibody (Supplementary Fig. 2b). To directlycompare stimulation efficiencies of the SCTs, CHO cells expressing each SCT were sorted for similar surface ligand densities by flow cytometry (Supplementary Fig. 2b), and varying numbers of these CHO APCs were used to stimulate B3Z cells. CHO APCs expressing elongated SCTs were much less effective at activating B3Z cells, as measured by IL-2 secretion (Fig. 1b). Moreover, their ability to activate B3Z cells decreased with increasing ectodomain size, with SCT-CD4 being the least effective (Fig. 1b). When APCs expressing ,10-fold lower surface density of SCTs or elongated SCTs were compared, the differences were even more pronounced, with only SCT-expressing CHO APCs stimulating detectable IL-2 secretion (Supplementary Fig. 3). A similar dependence on the size of pMHC was observed when the SCT-transfected APCs were used to stimulate CD8 þ T cells from mice transgenic for the OT1 TCR, which is identical to the B3Z TCR 15 and therefore also recognizes pOVA/H-2K b . Only SCT- and SCT-CD2-expressing APCs stimulated IL-2 production (Fig. 1c) and cell proliferation (Supplementary Fig. 2c) in a secondary stimulation; SCT-CD22 and SCT-CD4 had no effect. As IL-2 secretion and proliferation are very late consequences of TCR triggering, we next investigated whether proximal TCR signals were also dependent on pMHC dimensions. One of the earliest events in TCR signal transduction is increased phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR-associated homodimeric z-chains (TCR-z) (ref. 9). When B3Z T cells were incubated with APCs expressing SCT, there was a robust increase in TCR-z phosphorylation, which peaked within 5 min (Fig. 1d), consistent with previous studies of the kinetics LETTERS 1 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. 2 Department of Immunology, Wright-Fleming Institute, Imperial College London, Norfolk Place, London W2 1PG, UK. *These authors contributed equally to this work. Vol 436|28 July 2005|doi:10.1038/nature03843 578 © 2005 Nature Publishing Group