Unphosphorylated tyrosines mediate PD-1 inhibition of T cell signaling condensate formation

Abstract

T cell receptor (TCR) engagement stimulates formation of a biomolecular condensate, including the scaffold protein linker of activated T cells (LAT). We investigated whether the immune-inhibitory receptor programmed cell death protein 1 (PD-1) inhibits by impeding TCR-induced LAT condensation. In vitro reconstitutions revealed that the cytoplasmic domain of PD-1 directly inhibited LAT condensation. This inhibitory interaction required tyrosine residues in the two inhibitory motifs of PD-1. Contrary to current working models, tyrosine phosphorylation eliminated PD-1 inhibition. Replacing tyrosine with tryptophan reconstituted PD-1 inhibition in T cells, confirming that phosphorylation does not mediate this effect. These findings support the concept that hydrogen bond–donating aromatic residues within inhibitory motifs provide the primary mechanism that explains how inhibitory receptors disrupt signaling networks and block immune activation.