Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: MECHANISMS IMPAIRING DNAM1-MEDIATED NK CELL ANTI-TUMOR RESPONSE.

Natural Killer (NK) cells express a wide array of activating receptors that cooperate in driving anti-tumor immune response. Among them, NKG2D and DNAM1, are able to recognize ligands poorly expressed on normal cells but upregulated on transformed cells. NKG2D binds MICA, MICB and ULBP proteins, whereas DNAM1 ligands, PVR and Nectin2, are shared with the inhibitory receptors TIGIT and TACTILE. During tumor progression several mechanisms impair NK cell anti-tumor response: on one side, NK cell activating receptors decrease their surface expression, after a chronic exposure to their ligands; on the other side, tumor cells shed or retain intracellularly activating ligands. Thus, understanding how NK cell receptors and their ligands are regulated is crucial for the development of new therapeutic anti-cancer approaches aimed at improving NK cell-mediated tumor clearance. While several data are available on the regulation of NKG2D/NKG2DL axis, little is currently known about DNAM1 and its ligands. The aims of this PhD thesis are to investigate whether post-translational modifications affect the expression of DNAM1Ls on tumor cells and to identify novel mechanism(s) regulating DNAM1 surface expression and functionality on primary human NK cells. We focused on Multiple Myeloma (MM), a hematological tumor in which malignant plasma cells proliferate in the bone marrow and we found that DNAM1Ls are preferentially expressed as intracellular pool, suggesting the involvement of post-translational modifications in the regulation of their expression. Indeed, we demonstrated that PVR and Nectin2 undergoes Sumoylation and Ubiquitination, respectively. We found that SUMOylation of PVR promotes its subcellular localization and Ub-pathway is responsible for intracellular retention of Nectin2 and its degradation on MM cells, rendering them less susceptible to DNAM1-mediated cytotoxicity by NK cells. Moreover, using as cell system transfectants overexpressing either the DNAM1 ligand PVR or the NKG2D ligand MICA, we observed that a persistent stimulation with membrane-bound ligands induces a selective down-modulation of their respective receptors, thus impairing NK cell cytotoxic activity. Surprisingly, while DNAM1 down-modulation only affects DNAM1-mediated NK cell killing, NKG2D down-modulation results in the impairment of both NKG2D and DNAM1 functionality. Thus, we investigated the mechanisms underlying DNAM1 hypo-responsiveness demonstrating that a persistent NKG2D stimulation up-regulates TIGIT expression and promotes a concomitant reduction of DNAM1-mediated signaling pathways leading to cytotoxicity. Collectively, our data elucidate new molecular mechanisms that regulate DNAM1L expression, identifying novel pathways that can be targeted to increase NK cell capability to kill tumors and reveal a potential interplay between NKG2D- and DNAM-mediated functions that may impact on NK cell anti-tumor response.