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

Abstract

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.Keywords: 2B4 r Natural killer cells r NKG2D r self-tolerance Additional supporting information may be found in the online version of this article at the publisher's web-site Recent work has revealed the existence of an alternate NK-cell inhibitory system, independent of MHCI molecules, which provides an added level of self-tolerance [6]. Of the non-MHC binding receptors, 2B4 (CD244) is ubiquitously expressed on all NK cells [7,8] and provides inhibition against CD48-expressing hematopoietic and tumor cells [9]. 2B4 is a member of signaling lymphocyte activation molecule (SLAM) receptor family that has been shown to exert both activating and inhibitory roles in NK cells [10]. In mature human NK cells, 2B4 functions primarily as an activating and costimulatory receptor, but it can also act as an inhibitory receptor in developing NK cells to ensure self-tolerance against neighboring hematopoietic compartments [11]. By contrast, the major role of 2B4 in murine NK cells has been shown to be inhibitory both in vitro and in vivo [6,9,12]. 2B4-deficient mice demonstrate enhanced killing of CD48 + tumor cells by 2B4KO NK cells over that by wild-type NK cells [9] and more efficient regression of tumors in vivo [13], suggesting that murine 2B4 functions as an inhibitory receptor. In addition, 2B4 inhibits NK-cell production of IFN-γ in recognition of CD48 + tumor targets. These data demonstrate that the mouse 2B4/CD48 pair provides an important MHCI-independent inhibitory system for NK-cell self-tolerance. We have shown previously that...

NK cell education via nonclassical MHC and non-MHC ligands

“…Murine 2B4 is primarily considered to be a non-MHC-I-dependent inhibitor of NK cells, especially in the absence of its adapter SAP (SLAM-associated protein), as it inhibits NK cells from killing CD48-sufficient tumor cells or allogeneic cells. 24, 25, 26 Given that NK cells from either 2B4 knockout mice or CD48 knockout mice fail to reject CD48-deficient tumor cells, 24, 27 this 2B4-CD48 engagement is necessary for NK cell-mediated recognition of missing self-CD48 targets and thus represents a MHC-I-independent education pathway.…”

“…Recent studies demonstrate that either MHC-I- or Ly49-deficient mice contain a normal number of terminally mature NK cells. 1, 11 In addition, hypo-responsive NK cells from mice deficient in the expression of H2M3, CD1d1, 2B4, CD48, SLAMF6, NKRP1-B, Clr-b or even TIGIT, 26, 32, 35, 69, 70, 71 which are either nonclassical MHC-dependent or non-MHC-dependent receptors, likewise undergo normal development and exhibit regular expression of activating receptors, for example, NKG2D and activating Ly49 receptors. Thus, although functionally impaired, uneducated NK cells develop normally and exhibit the characteristics of a mature NK cell.…”

CD244 regulates both innate and adaptive immune axes in melanoma by inhibiting autophagy-mediated M1 macrophage maturation

“…CD244 interacts with CD48 (SLAMF2), the expression of which is restricted to hematopoietic cells. Although extensive studies by Veillette et group [51][52][53][54] and our group 13,[55][56][57][58][59] have revealed the importance of CD244 as a self-tolerance receptor on NK cells, the role of SLAM family receptors on monocytes/macrophages has only recently been recognized 27,29,40,60,61 . Chen et al demonstrated that SLAMF7 promotes phagocytosis of hematopoietic tumor cells in macrophages in the absence of SIRPa-CD47 interactions 35 .…”

WhatSAP — 2B4 sends mixed messages in the absence of SAP

“…However, mouse 2B4 can also mediate inhibitory signals when there is a high level of 2B4 expression, heavy receptor cross-linking, and low SAP expression levels [25]. Reminiscent of the finding by Meazza et al, this inhibitory effect of mouse 2B4 does not interfere with NKG2Dmediated NK-cell activation [26].…”