Seminars in Immunology 15 (2003) 307–315 Linking molecular and cellular events in T-cell activation and synapse formation Michelle Krogsgaard, Johannes B. Huppa, Marco A. Purbhoo, Mark M. Davis ∗ Howard Hughes Medical Institute and the Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305-5323, USA Abstract The complex sequence of events in which T cells recognize foreign entities on other cells is not well understood. However, the development of new techniques and approaches in both the molecular and cellular aspects of this problem have provided significant insights into the mechanisms of T-cell recognition and synapse formation. In particular, we have a clearer picture of T-cell sensitivity, the role of co-stimulation in formation of the immunological synapse, and how TCR signaling acts to maintain synapse structure and potentiate the T cells over many hours of engagement. We also are aware of new complexities in the way T-cell receptor molecules bind peptide-MHC (pMHC) ligands and what that may mean for TCR scanning, cross-reactivity, and activation. Ultimately, we want to integrate these cellular aspects of T-cell recognition with key features of the molecular interactions that drive specific events. © 2003 Elsevier Ltd. All rights reserved. Keywords: T-cell receptor; Peptide-MHC; T-cell activation; T-cell recognition; Immunological synapse 1. Introduction Cytotoxic and helper T lymphocytes are continually sur- veying other cells and tissues for foreign or aberrant pep- tides bound to major histocompatibility complex (MHC) molecules (reviewed in Ref. [1]). While much is known about the various molecular and cellular interactions that are important for a successful outcome, the specific sequence of events and mechanisms by which T cells become activated and by which immunological synapses form (and for what purpose) remains obscure. Here, we discuss recent cellular and biochemical data that we have generated in an effort to derive a better understanding of how T cells are able to scan APCs efficiently and what contributes to T-cell sensitivity and robust synapse formation. 2. Initiation of T-cell activation by peptide-MHC (pMHC) Previous work has suggested that T cells are capable of recognizing small numbers of pMHC on cell surfaces, in the range of 1–400 per average APC [2–7]. However, these studies are fraught with uncertainty as they depend on aver- age values with no firm idea of what percentage of the APC ∗ Corresponding author. Tel.: +1-650-723-7962; fax: +1-650-498-7771. E-mail address: mdavis@cmgm.stanford.edu (M.M. Davis). surface is seen by T cell. To overcome these uncertainties, we have directly visualized individual pMHC complexes on live cells to precisely quantify how many pMHC complexes are necessary for both the initiation of T-cell activation and synapse formation [8]. This method involves pulsing APCs with N-terminally biotinylated peptides extending out of the MHC. These are then detected with saturating concentra- tions of streptavidin coupled to phycoerythrin (PE) (Fig. 1). As PE is such a strong fluorophore, we are able to count the number of pMHC complexes that a T cell recognizes on the surface of an APC using three-dimensional (3D) fluo- rescence microscopy and simultaneously follow the level of intracellular Ca 2+ . These experiments show that T cells can detect even a single peptide ligand, although about 10 pep- tides are necessary to reach a maximal Ca 2+ response and form a stable synapse [8]. One interesting observation in this study is that if CD4 is blocked with an antibody (or Fab), the threshold T-cell activation is lowered to 25–30 pMHC complexes, indicat- ing that CD4 plays a critical role in the sensitivity of T-cell recognition. Based on this effect and the large number of endogenous pMHCs capable of interacting with some TCRs (see below), we have proposed a “pseudodimer” model of early T-cell activation where at low antigen levels TCR, CD4, and both agonist and endogenous ligands interact to- gether to activate T cells [8,9]. As illustrated in Fig. 2, this model suggests that a TCR binding to an agonist peptide can use its associated CD4 molecule to bind to an adjacent en- 1044-5323/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2003.09.002