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.

We use single-molecule localization microscopy and novel analysis to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and non-triggered TCR-CD3 complexes. For example, 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. Both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels.

In summary, we propose a model in which 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.