Endogenous ligand of the NK activating receptor NKp46

Natural killer cells (NK) act as a first line of defense against infection and cancer. NK activation is controlled by a balance of signals transmitted by activating and inhibitory NK receptors. The activating receptor NKp46 is considered the major activating receptor in natural cytotoxicity against autologous, allogeneic, and xenogeneic target cells. NKp46 is conserved in mammals and virtually ubiquitously expressed on NK, suggesting it is functionally important. Mice deficient in Ncr1, the gene encoding NKp46, are impaired in tumor immune surveillance, have more severe influenza A, metapneumovirus, reovirus, and fusobacterium infections, and graft versus host disease. The endogenous ligand of this important activating NK receptor is not known, despite decades of searching. Our preliminary work suggests that NKp46 recognizes calreticulin (CRT), which is normally found inside the endoplasmic reticulum (ER) but gets transferred to the cell surface in ER-stressed cells or tumor cells treated with some cancer drugs. An NKp46-Ig fusion protein specifically pulls down cell-surface CRT (ecto-CRT) and preliminary data using surface plasmon resonance indicate specific binding. Knocking down CALR, the gene encoding for CRT, or adding blocking antibodies to CRT inhibits NKp46-mediated NK killing. Some chemotherapeutic agents and radiotherapy induce an immunostimulatory type of programmed cell death in cancer cells, known as immunogenic cell death (ICD), which removes surviving tumor cells after treatment. The mechanism behind ICD has been linked to ecto-CRT, which serves as an "eat me"/phagocytic signal for dendritic cells, which then activate anti-tumor CD8+ T cells by cross-priming. Here we hypothesize that NKp46 recognizes ecto-CRT, and that NK recognition of ER-stressed cells via NKp46 interaction with ecto-CRT plays an important role in ICD and NK-mediated immune defense more generally. In preliminary data, infection with ZIKV, which replicates in the ER and causes ER stress, and treatment of tumor cell lines with ICD-inducing chemotherapy drugs induces ecto-CRT and NKp46-dependent NK killing. Moreover, CRT-coated tumor cells become NK cell targets, and NCR1 knockout in an NK cell line strongly inhibits its killing of targets with exposed CRT. To test our hypotheses, we will first confirm that ecto-CRT is a ligand for NKp46 by using biophysical methods to characterize ecto-CRT and NKp46 binding and identifying the regions of CRT responsible for the interaction. Genetic manipulation or blocking of NKp46 on NK and of ecto-CRT on tumor targets will examine the role of this receptor-ligand interaction in NK recognition, immune synapse formation and functional responses to infected and tumor cells. NK killing of untreated tumor cells and tumor cells treated with ICD-inducing and noninducing drugs will be compared. The in vivo role of NKp46 in controlling ZIKV infection and mouse tumors (without and with chemotherapy) will be assessed by comparing viral levels and tumor growth, NK cell infiltration of tumors and survival in wild-type and Ncr1-/- mice.