NKG2D ligation relieves 2B4-mediated NK-cell self-tolerance in mice

Along with MHC class I (MHCI), 2B4 provides nonredundant NK-cell inhibition in mice. The immunoregulatory role of 2B4 has been increasingly appreciated in models of tumor and viral infection, however, the interactions among 2B4, MHCI, and other activating NK-cell receptors remain uncertain. Here, we dissect the influence of two distinct inhibitory pathways in modulating NK-cell-mediated control of tumors expressing strong activating ligands, including RAE-1γ. In vitro cytotoxicity and in vivo peritoneal clearance assays using MHCI⁺CD48⁺ (RMA-neo), MHCI⁺CD48⁺RAE-1γ (RMA-RAE-1γ), MHCI^-CD48⁺ (RMA-S-neo), and MHCI^-CD48⁺RAE-1γ (RMA-S-RAE-1γ) tumor lines demonstrated that NKG2D activation supersedes the inhibitory effect of both 2B4- and MHCI-mediated immune-tolerance systems. Furthermore, 2B4KO mice subcutaneously challenged with RMA-neo and RMA-S-neo exhibited reduced tumor growth and significantly prolonged survival compared with WT mice, implying that 2B4 is constitutively engaged in the NK-cell tolerance mechanism in vivo. Nevertheless, the inhibitory effect of 2B4 is significantly attenuated when NK cells encountered highly stressed tumor cells expressing RAE-1γ, resulting in an immune response shift toward NK-cell activation and tumor regression. Therefore, our data highlight the importance of the 2B4-mediated inhibitory system as an alternate self-tolerance mechanism, whose role can be modulated by the strength of activating receptor signaling within the tumor microenvironment.