CD4−CD16+ NK cells showed upregulation of the activation marker NKG2D (Fig. 5A and Galunisertib concentration D) after co-incubation with iTreg cells. While the inhibitory KIRs CD158a and CD158b were not modulated on NK cells (Fig. 5B), the expression of perforin was clearly enhanced after co-culture with iTreg cells (Fig. 5C and D). These data indicate that iTreg cells are able to activate NK cells resulting in the upregulation of NKG2D and perforin in the absence of IL-2 pre-stimulation. In summary, our findings thus far demonstrate that iTreg cells impair IL-2-mediated NK activation, provided that NK cells have no target cell contact. In contrast, target cell-induced NK

activation is enhanced by iTreg cells. In the final series

of experiments, we investigated NK activation induced by a combination of IL-2 and target cell contact. Under these conditions, NK degranulation was induced from 8 to 26% (p=0.02) compared with resting NK cells (Fig. 6). Induced Treg cells further promoted this NK cell function compared with IL-2-activated NK cells with tumors alone (from 26 to 54%; p=0.01, Fig. 6). In contrast, nTreg cells did not further modulate degranulation of IL-2-activated NK cells towards target cells. In agreement with previous reports, we found that IL-2 induces activation of primary human peripheral blood NK cells resulting in upregulation of activating receptors, NKG2D and NKp44, as well as increased degranulation and IFN-γ secretion

21, 22. These effects were significantly impaired in the presence of tumor iTreg cells, nTreg cells or TGF-β. Our results are in agreement with published Alectinib reports, where other types of Treg cells were described to suppress NK cell functions under various experimental conditions, in most instances in a TGF-β-dependent N-acetylglucosamine-1-phosphate transferase manner 11, 12, 19, 23–27. Unexpectedly, we found that degranulation and the subsequent tumoricidal activity of naive NK cells were enhanced by iTreg cells. iTreg cell-enhanced cytotoxicity of NK cells was perforin- and FasLigand-dependent, while death receptor TRAIL was not involved. Consistent with the upregulation of activating receptors NKG2D and NKp44, the expression of inhibitory KIRs CD158a and CD158b on NK cells remained at basal levels in the co-culture with tumor iTreg cells. In conjunction, these data suggest that tumor iTreg cells negatively interfere with IL-2-mediated NK-cell activation, while the IL-2-independent activation of NK cells by target cell contact is augmented in the presence of iTreg cells. Importantly, the activation of NK cells by a combination of IL-2 and target cell contact is further promoted in the presence of iTreg cells. It is well established that NK cells activated by IL-2 are highly cytolytic to many tumor targets and thus NK cell-activating cytokines like IL-2 are frequently incorporated into current immunotherapeutic strategies and clinical trials 28.