Tyro3 facilitates cytoplasmic delivery of extracellular vesicle contents and antigen presentation via major histocompatibility complex class I in dendritic cells.

Extracellular vesicles (EVs) are membrane-bound particles that mediate intercellular communication and are being explored as carriers for antigen delivery in cancer immunotherapy. However, the molecular mechanisms underlying the uptake of antigen-carrying EVs by dendritic cells (DCs) remain unclear. EV uptake is influenced by the recognition of EV surface components by receptors on recipient cells. Phospholipids, including phosphatidylserine (PS), are common structural components of EV membrane. We therefore focused on the TAM receptor tyrosine kinases Tyro3, Axl, and Mertk, which are expressed in antigen-presenting cells and engage PS-exposing membranes through the bridging ligands Growth arrest-specific 6 (Gas6) and Protein S (Pros1). Using the DC2.4 dendritic cell line as a model system, we investigated the involvement of TAM receptors in the uptake of small EVs (sEVs) and subsequent antigen presentation. We found that Tyro3 binding to sEVs was enhanced by Gas6 and Pros1, and that Tyro3 also associated with phosphatidylserine (PS) and phosphatidylglycerol (PG) through these bridging ligands. Among TAM receptors, only Tyro3 markedly enhanced sEV internalization in DC2.4 cells. Deletion analysis indicated that the immunoglobulin C2-type domain (IG2c domain) of Tyro3 is required for efficient sEV association and uptake. Moreover, Tyro3-mediated internalization enabled cross-presentation of sEV-delivered antigens and activation of CD8+ T cells. This function required a 46-amino acid intracellular region of Tyro3, which we designated the Tyro3 antigen presentation-related domain (TAPD); deletion of TAPD impaired cross-presentation of sEV-delivered antigens. Finally, we observed an increase in the frequency of Tyro3 ⁺ DCs in the spleens of tumor-bearing mice. These findings suggest that Tyro3 mediates sEV uptake and antigen cross-presentation in DCs and may represent a candidate molecular target for EV-based cancer immunotherapy.