Down-regulation of the NKG2D receptor is differentially controlled by MICA and ULBP2 ligands

The activating NKG2D receptor on human NK cells mediates “induced self recognition” in that its ligands are up-regulated by stressed or diseased cells. Evidence collected in the past years demonstrated that chronic exposure to NKG2DLs induces receptor down-modulation. The aim of this study was to evaluate whether different NKG2D ligands (NKG2DLs), namely MICA and ULBP2, are equivalent in their capacities to down-modulate the surface receptor expression on human NK cells. We analyzed the rate and kinetics of NKG2D down-modulation in primary cultured NK cells and in the NKL NK cell line upon stimulation with the Ba/F3 cell line stably overexpressing comparable levels of MICA or ULBP2 by FACS and fluorescence microscopic analysis. Although both ligands were able to reduce NKG2D expression, exposure to MICA over-expressing target cells resulted in a more rapid and efficient receptor down-modulation and in a more pronounced impairment of NKG2D-dependent cytotoxicity. MICA-experienced NK cells also showed a higher tyrosine phosphorylation of the ubiquitin ligase c-Cbl. Furthermore, ligand-induced receptor down-modulation affected the total NKG2D cellular levels, suggesting that the internalized receptor complexes were mainly subjected to degradation. All together these results demonstrate that NKG2D down-regulation is influenced by the nature of its ligand and suggest a different contribution of the ubiquitin pathway in the control of NKG2D internalization and degradation in MICA- versus ULBP2-experienced cells. Understanding the mechanisms of ligand-induced NKG2D down-modulation will be helpful to prevent evasion from NK cell-mediated immune response.