Sterile inflammation can be initiated by innate immune recognition of markers of tissue injury termed damage-associated molecular patterns (DAMPs). DAMP recognition by dendritic cells (DCs) has also been postulated to lead to T cell responses to foreign antigens in tumors or allografts. Many DAMPs represent intracellular contents that are released upon cell damage, notably after necrosis. In this regard, we have previously described DNGR-1 (CLEC9A) as a DC-restricted receptor specific for an unidentified DAMP that is exposed by necrotic cells and is necessary for efficient priming of cytotoxic T cells against dead cell-associated antigens. Here, we have shown that the DNGR-1 ligand is preserved from yeast to man and corresponds to the F-actin component of the cellular cytoskeleton. The identification of F-actin as a DNGR-1 ligand suggests that cytoskeletal exposure is a universal sign of cell damage that can be targeted by the innate immune system to initiate immunity.

DNGR-1 is a C-type lectin receptor that binds F-actin exposed by dying cells and facilitates cross-presentation of dead cell-associated antigens by dendritic cells. Here we present the structure of DNGR-1 bound to F-actin at 7.7 Å resolution. Unusually for F-actin binding proteins, the DNGR-1 ligand binding domain contacts three actin subunits helically arranged in the actin filament, bridging over two protofilaments, as well as two neighboring actin subunits along one protofilament. Mutation of residues predicted to mediate ligand binding led to loss of DNGR-1-dependent cross-presentation of dead cell-associated antigens, formally demonstrating that the latter depends on F-actin recognition. Notably, DNGR-1 has relatively modest affinity for F-actin but multivalent interactions allow a marked increase in binding strength. Our findings shed light on modes of actin binding by cellular proteins and reveal how extracellular detection of cytoskeletal components by dedicated receptors allows immune monitoring of loss of cellular integrity.

SummaryConventional type 1 DCs (cDC1s) excel at cross-presentation of dead cell-associated antigens partly because they express DNGR-1, a receptor that recognizes exposed actin filaments on dead cells. In vitro polymerized F-actin can be used as a synthetic ligand for DNGR-1. However, cellular F-actin is decorated with actin-binding proteins, which could affect DNGR-1 recognition. Here, we demonstrate that myosin II, an F-actin-associated motor protein, greatly potentiates the binding of DNGR-1 to F-actin. Latex beads coated with F-actin and myosin II are taken up by DNGR-1+ cDC1s, and antigen associated with those beads is efficiently cross-presented to CD8+ T cells. Myosin II-deficient necrotic cells are impaired in their ability to stimulate DNGR-1 or to serve as substrates for cDC1 cross-presentation to CD8+ T cells. These results provide insights into the nature of the DNGR-1 ligand and have implications for understanding immune responses to cell-associated antigens and for vaccine design.