Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/ KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.killer cell immunoglobulin-like receptors | MHC class I

Word count: 7316 One sentence summary:Movement of sub-synaptic vesicles rich in LAT is constrained by protein microclusters at T cell immune synapses, dependent on LAT residues important for its binding to SLP-76 and consistent with a role for vesicular LAT in T cell signal transduction.

Despite the increasingly widespread use of single-molecule localization microscopy (SMLM) in biology, the extent to which the spatial organization of proteins influences signaling is not easy to quantify. Clus-DoC is a novel analysis method that combines cluster detection and colocalization analysis for SMLM data.