CD96-mediated internalisation of ligand CD155 as a novel mechanism for immune regulation

CD96 is a member of the immunoglobulin superfamily including TIGIT and CD226 that bind to a shared ligand, CD155. This family of co-receptors and its ligands are dysregulated in various autoimmune conditions and cancers, highlighting therapeutic potential. While TIGIT and CD226 are recognised co-inhibitory and co-stimulatory receptors respectively, the function of CD96 remains incompletely defined. Here, we assessed CD96-CD155 interaction and downstream trafficking to define a novel CD96 mechanism of action. Using primary human T cells and engineered cell models, we demonstrate that CD96 mediates uptake and internalisation of both soluble and cell-associated CD155. CRISPR-Cas9-mediated receptor knockout in primary human T cells revealed that CD155 uptake was uniquely dependent on CD96, but not TIGIT or CD226, identifying CD96 as the dominant mediator of CD155 internalisation in human T cells. This uptake process was dependent on active receptor cycling, which was partially facilitated by the CD96 cytoplasmic domain. Furthermore, CD96 variant 2 exhibited enhanced ligand binding and uptake efficiency compared with variant 1. Mechanistically, internalised CD155 trafficked to lysosomal compartments and associated with autophagy-related proteins, consistent with degradative processing. These findings reveal a previously unrecognised mechanism by which CD96 may regulate ligand availability through ligand internalisation and trafficking, with potential implications for T cell function and immune regulation. This process may promote changes in the immunoregulatory balance and could inform new targeted therapeutic development.