The NK receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells

Natural killer cells (NK) are a first line of immune defense to eliminate infected, transformed and stressed cells by releasing cytotoxic granules. NK activation is controlled by the balance of signals transmitted by activating and inhibitory receptors but activating receptor engagement is required to trigger cytotoxicity. The activating receptor NKp46, encoded by the NCR1 gene, is expressed by virtually all NK cells and is the most evolutionarily ancient NK receptor. NKp46 plays a major role in NK recognition of cancer cells, since NKp46 blocking antibodies potently inhibit NK killing of many cancer targets. Although a few viral, fungal and soluble host ligands have been identified, the endogenous cell-surface ligand of this important activating NK receptor is unknown. Here we show that NKp46 recognizes and is activated by the P-domain of externalized calreticulin (ecto-CRT). CRT, normally localized to the ER, translocates to the cell surface during ER stress and is a hallmark of chemotherapy-treated dying cancer cells that induce an immune response (immunogenic cell death, ICD). NKp46 caps with ecto-CRT in NK immune synapses formed with ecto-CRT-bearing target cells. ER stress, induced by ZIKV infection, ICD-causing chemotherapy drugs and some senescence activators, externalizes CRT and triggers NKp46 signaling. NKp46-mediated killing is inhibited by CRT knockout or knockdown or anti-CRT antibodies and is enhanced by ectopic expression of GPI-anchored CRT. NCR1/Ncr1-deficient human and mouse NK are impaired in killing ZIKV-infected, ER-stressed, and senescent cells and cancer cells that endogenously or ectopically express ecto-CRT. Importantly, NKp46 recognition of ecto-CRT controls the growth of B16 melanoma and RAS-driven lung cancer in mouse models and enhances tumor-infiltrating NK degranulation and cytokine secretion. Thus, ecto-CRT is a danger-associated molecular pattern (DAMP) that is an endogenous NKp46 ligand that promotes innate immune elimination of ER-stressed cells.

459 NK cells activation and recruitment to irradiated tumors is increased in the presence of IL-15

BackgroundFocal radiotherapy (RT) promotes tumor infiltration by conventional dendritic cells type 1 (cDC1), an effect dependent on radiation’s ability to induce type I interferon (IFN-I) secretion. We recently demonstrated that peritumoral s.c. IL-15, while ineffective by itself, synergized with RT, inducing complete regression of the irradiated tumor and long-term protective memory in two murine carcinomas models (TSA, MCA-38) (1). These responses were abrogated in the absence of CD8 T cells or cDC1. Detailed investigations in the TSA model showed that, whereas IL-15 alone had no effects on cDC1, it did significantly increase intratumoral cDC1 numbers and expression of costimulatory molecules CD80 and CD86 induced by RT (1). In addition to CD8 T cells, IL-15 activates NK cells, which have also been implicated in cDC1 tumor recruitment (2). Thus, we hypothesized that NK cells may play a role in the synergy between radiation therapy and IL-15.MethodsTo test this hypothesis, BALB/c mice were injected with TSA mammary carcinoma cells and treated with RT (8Gy X3) and daily subcutaneous injections of IL-15 (5µg). Tumors were excised at day 18 and analyzed by immunostaining for NKp46+ cells on tumor sections and flow cytometry after tumor dissociation.ResultsThe number of intra-tumoral NKp46+ NK cells was significantly higher (p<0.005) in mice treated with IL-15 as compared to control. Whereas RT itself had no effect, it further increased NK cell numbers above what was achieved by IL-15 alone (p<0.05). In addition, tumor infiltrating NK cells expressed higher levels of CD137/4-1 BB, an effect largely driven by IL-15. Finally, NK cells depletion by anti-asialo GM1 before initiation of the treatment abrogated the enhanced cDC1 infiltration in tumors of mice treated with RT + IL-15, and the therapeutic effect of the combination.ConclusionsOur results strongly suggest a role for NK cells in the anti-tumor immune response induced by the combination of RT and IL-15. We are currently working to confirm the role of NK cells by using a complementary approach of engineering TSA cells to overexpress CLEC2D/Clr-b, the ligand for the inhibitory NKR-P1 NK receptor (3,4). Data obtained will improve current knowledge about the interaction of RT with IL-15 and support a rationale strategy for translation to the clinic.ReferencesPilones KA, Charpentier M, Garcia-Martinez E, et al. Radiotherapy cooperates with IL15 to induce antitumor immune responses. Cancer Immunol Res 2020;8(8):1054–1063. doi:10.1158/2326-6066.CIR-19-0338Böttcher JP, Bonavita E, Chakravarty P, et al. NK Cells stimulate recruitment of cDC1 into the tumor microenvironment promoting cancer immune control. Cell 2018;172(5):1022–1037.e14. doi:10.1016/j.cell.2018.01.004Carlyle JR, Jamieson AM, Gasser S, Clingan CS, Arase H, Raulet DH. Missing self-recognition of Ocil/Clr-b by inhibitory NKR-P1 natural killer cell receptors. Proc Natl Acad Sci U S A 2004;101(10):3527–3532. doi:10.1073/pnas.0308304101Williams KJ, Wilson E, Davidson CL, et al. Poxvirus infection-associated downregulation of C-type lectin-related-b prevents NK cell inhibition by NK receptor protein-1B. J Immunol 2012;188(10):4980–4991. doi:10.4049/jimmunol.1103425