ADAM17, a Novel Metalloproteinase, Mediates CD16 and CD62L Shedding in Human NK Cells and Modulates IFNγ Responses

Abstract 2184

NK cells play a key role in graft versus leukemia (GvL) effect in myeloid leukemia and in controlling viral infections, yet the optimal way to exploit their therapeutic potential is unknown. While the CD56^BrightCD16⁻ and CD56^DimCD16⁺ NK cell subsets exhibit differential killing and cytokine production, less is known about the CD56^DimCD16⁻ population. After overnight incubation in serum containing media in the absence of cytokines, approximately 6 % of the CD56^Dim population are negative for the expression of FcRgIII (CD16) (n=8). Loss of CD16 has been reported to be mediated by metalloproteinase (MMP) activity, but the mechanism of this activity is poorly understood and the precise MMP involved in this activity has not been characterized. Moreover, it is not known whether the CD56^DimCD16⁻ NK cell subset, which might be generated by MMP cleavage, is functionally active. We first compared CD56^bright, CD56^dimCD16⁺ and CD56^dimCD16⁻ cells in functional assays. The CD56^DimCD16⁻ cells exhibit the highest level of CD107a and IFNy production after redirected triggering of activating receptors (2B4, NKp46, alone or in combination) or by exposure to K562 target cells. Stimulation by IL-12/IL-18/IL-15 overnight and CD16/2B4 redirected lysis in a 4-hour assay potently induced CD16 down-regulation. We next explored the role of a novel MMP, ADAM17 (a disintegrin and MMP-17), also called TACE (TNFα cleaving enzyme), a transmembrane protein capable of ectodomain protein shedding. NK cells from normal healthy donors expresser higher levels of ADAM17 (MFI 3330 ±217.9) compared to resting T-cells (MFI 626.8 ±62.06) p=<0.001. We then explored the function of NK cells with and without the presence of an ADAM17 specific inhibitor (10nM). Inhibition of ADAM17 resulted in significant abrogation of CD16 loss specifically implicating ADAM17 as the metalloproteinase sheddase on NK cells. Inhibition of ADAM17 had no effect on stimulation of activating receptor or K562 induced CD107a degranulation. In marked contrast, ADAM17 inhibition lead to enhanced IFNy production upon CD16 (±2B4) activation. ADAM17 inhibition also prevented the shedding of CD62L, an important lymph node homing receptor. To get a better understanding of ADAM17 in early reconstituting NK cells in vivo, we studied NK cells early after transplantation, a setting where we have recently reported potent IFNy production defects. We found that ADAM17 expression on NK cells 28 days after umbilical cord blood transplantation was higher than the normal donor NK cells, but these levels approached normal by day 60. Increased ADAM17 expression on post allo-transplant NK cells was associated with a higher level of CD16 and CD62L down-regulation, which was not found on NK cells early after auto transplantation. In functional assays, ADAM17 inhibition led to the increased CD16 expression and better IFNγ response with redirected stimulation through CD16 and 2B4 on NK cells early after the transplantation. In conclusion, this is the first report showing that human NK cells express ADAM17, which is responsible for the activation induced shedding of CD16 and CD62L. We also demonstrate ADAM17 modulates IFNy production, but has little effects on killing. Adam 17 expression is higher early after cord blood transplantation, is associated with a higher CD16 and CD62L down-regulation and suggests the developmental regulation of ADAM17 on NK cells that reconstitute in vivo. ADAM17 induced CD16 and CD62L down-regulation could impair antibody dependent cellular cytotoxicity (ADCC) and homing of the NK cells to lymph nodes early after allogeneic HCT. ADAM17 inhibitors, being developed clinically as anti-cancer agents, may alter the homing of NK cells and enhance ADCC to therapeutics antibodies.