NKG2D down-modulation on human NK cells is followed by DNAM-1 hypo-responsivenss

Natural killer (NK) cells are cytotoxic innate lymphocytes that represent the first line of defense against viral infections and tumor growth. NK cell activation is regulated by activating receptors able to recognize self-molecules up-regulated in stress conditions and inhibitory receptors that mainly bind to major histocompatibility complex class-I (MHC-I) molecules to prevent lysis of normal cells. Among activating receptors, NKG2D and DNAM-1 play a pivotal role in anticancer immune responses since they bind ligands upregulated on transformed cells. The interaction of both NKG2D and DNAM-1 with their ligands promotes a rapid receptor downmodulation that mainly occurs through internalization and lysosomal degradation. Particularly, NKG2D downmodulation has been associated to an exhausted phenotype characterized by down-modulation of the cytolytic machinery and upregulation of inhibitory receptors on both human and murine NK cells. However, our knowledge of the consequences of NKG2D engagement is still incomplete. The aim of the study is to investigate if NKG2D downmodulation could impair DNAM-1 functionality. Primary human NK cells were co-cultured with MICA-transfectants, purified, and used as effector cells in a cytotoxicity assay toward Ba/F3 cell line stably overexpressing MICA and PVR. MICA-experienced NK cells resulted impaired in NKG2D-mediated killing and showed a marked reduction of DNAM-1-triggered cytotoxicity. Analysing the different stages of cellular cytotoxicity by confocal microscopy, we found that MICA-experienced NK cells retained the ability to induce LFA-1 conformational change upon PVR recognition while they fail to polarize perforin-containing granules toward the BaF/3-PVR contact site. We verified the expression of DNAM-1-related inhibitory receptors, CD96 and TIGIT, and found that while CD96 expression remains untouched, TIGIT resulted upregulated in MICA-experienced NK cells both at protein and mRNA levels, as demonstrated by employing FACS and Real-Time PCR analyses. We performed cytotoxicity assays and experiments of confocal microscopy in the presence of anti-TIGIT blocking antibody demonstrating TIGIT contribution in inhibiting the killing of Ba/F3-PVR and a in perforin polarization impairment. However, impairment of MICA-experienced NK cell ability to lyse PVR-expressing target cells was only partially reverted by TIGIT blocking Ab, suggesting that besides TIGIT other mechanisms are responsible for defective lysis. Thus, MICA-experienced NK cells were cross-linked with anti-DNAM-1 antibody and signaling events leading to NK cell killing were assessed. Immunoblotting of cell lysates showed that, Vav1 and Akt phosphorylation resulted almost unaltered. However, DNAM-1 mediated activation of Erk1/2 and Pyk2 was reduced demonstrating that NKG2D stimulation directly affects some of the DNAM-1 activating signals leading to cell cytotoxicity. Collectively, these results demonstrate that NKG2D engagement on human NK cells impairs DNAM-1-mediated killing through two different converging mechanisms: by the upregulation of the checkpoint inhibitory receptor TIGIT, that in turn suppresses DNAM-1-mediated cytotoxic function, and by direct inhibition of DNAM-1-promoted signaling. Our results highlight a novel interplay between NKG2D and DNAM-1/TIGIT receptors that may facilitate neoplastic cell evasion from NK cell-mediated clearance.