The Role of TRAIL and TRAIL receptors in NKp46-Mediated Cytotoxicity Against ER-Stressed Cancer Cells

Abstract

Natural killer (NK) cells play a critical role in anti-tumor immunity through the recognition and elimination of malignant cells. The activating receptor NKp46 has been implicated in cytotoxicity of many cancer targets, but the molecular mechanisms underlying this process remain incompletely understood. Previous studies have proposed ER-stress induced ecto-calreticulin (ecto-CRT) as a ligand for NKp46, whereas more recent evidence suggests that NKp46 recognizes TRAIL receptors through in cis interaction with membrane-bound TRAIL. The aim of this thesis was to elucidate the molecular mechanisms by which TRAIL and TRAIL receptors contribute to NKp46-mediated recognition and killing of ER-stressed cancer cells and to identify structural determinants of the NKp46–TRAIL interaction. ER stress and immunogenic cell death were induced in cancer cell lines using anthracyclines and oxaliplatin. NK-cell responses were assessed using degranulation assays in combination with NKp46 blockade, TNFSF10 (TRAIL) knockdown, and CRT blockade. In parallel, AlphaFold-generated structural models of the NKp46–TRAIL complex were analyzed using computational alanine scanning and binding-energy calculations, in order to identify key interaction determinants. Immunogenic cell death-inducing treatments resulted in the co-upregulation of calreticulin and TRAIL receptors, particularly TRAIL-R2. Functional studies demonstrated that TRAIL was required for efficient NKp46-mediated degranulation against both untreated and ER-stressed cancer cells. NKp46 blockade and TNFSF10 knockdown markedly reduced NK-cell activation, and the effect of NKp46 blockade was largely lost in the absence of TRAIL expression. In contrast, CRT blockade did not significantly impair NK-cell degranulation, suggesting that TRAIL receptor recognition constitutes the dominant mechanism of NKp46-dependent cytotoxicity under conditions where TRAIL is present. Complementary structural modeling of the NKp46–TRAIL complex identified recurrent energetic hotspot residues on NKp46, including Tyr 100 and Arg 70, although the low confidence of AlphaFold-predicted interfaces highlights the need for experimental validation. Together, these findings support a model in which TRAIL and TRAIL receptors mediate NKp46-dependent NK-cell recognition of ER stressed cancer cells. The study provides mechanistic insight into NKp46-dependent tumor surveillance and identifies potential targets for engineering improved NK-cell receptor–ligand interactions for cancer immunotherapy.