NKp44 Triggers NK Cell Activation through DAP12 Association That Is Not Influenced by a Putative Cytoplasmic Inhibitory Sequence

Abstract

NKp44 (NCR2) is a member of the natural cytotoxicity receptor (NCR) family that is expressed on activated human NK cells. We dissected structural attributes of NKp44 to determine their contributions to receptor function. Our results demonstrate that surface expression and NK cell activation by NKp44 is mediated through noncovalent association with the immunoreceptor tyrosine-based activation motif-containing protein, DAP12. Physical linkage to DAP12 requires lysine-183 in the NKp44 transmembrane domain. Intriguingly, the cytoplasmic domain of NKp44 also contains a sequence that matches the immunoreceptor tyrosine-based inhibitory motif (ITIM) consensus. By expressing a chimeric receptor in an NK-like cell line, we found that this ITIM-like motif from NKp44 lacks inhibitory capacity in a redirected cytotoxicity assay. The NKp44 cytoplasmic tyrosine was efficiently phosphorylated in the chimeric receptor upon treating the cells with pervanadate, but it was unable to recruit ITIM-binding negative effector phosphatases. We also generated NK-like cell lines expressing epitope-tagged wild-type or tyrosine to phenylalanine mutant (Y238F) versions of NKp44 and compared their capacities to induce activation marker expression, promote IFN-γ production, or stimulate target cell cytotoxicity. We did not detect any tyrosine-dependent reduction or enhancement of NK cell activation through wild-type vs. Y238F mutant NKp44. Finally, the cytoplasmic tyrosine-based sequence did not provide a docking site for the AP-2 clathrin adaptor, nor did it potentiate receptor internalization. In summary, all activating properties and surface expression of NKp44 are mediated through its association with DAP12, and the putative ITIM in the NKp44 cytoplasmic domain does not appear to attenuate activating function.

Novel Insights on Human NK Cells’ Immunological Modalities Revealed by Gene Expression Profiling

“…In agreement with previous observations, inhibitory receptor KIR3DL2 mRNA was preferentially expressed in unactivated CD56 dim CD16 ϩ NK cells (9), while activating receptor KIR2DL4 mRNA was specifically transcribed in unactivated CD56 bright CD16 Ϫ and in vitro-activated CD16 ϩ NK, but not unactivated CD56 dim CD16 ϩ NK (14). Our results also confirm L-selectin mRNA-specific expression on CD56 bright CD16 Ϫ NK cells (6), preferential expression of granzyme B in CD56 dim CD16 ϩ NK cells regardless of their activation state (9), and specific expression of the NK lysis receptor NKp44 transcript following activation (15). Finally, it was important to assess whether protein expression of the corresponding gene products of interest correlated with the transcriptional differences in these genes.…”

DAP12-Based Activating Chimeric Antigen Receptor for NK Cell Tumor Immunotherapy

“…24) (25). In the lymphoid lineage, DAP12 is expressed in NK cells and associates with activating receptors such as the C-type lectin receptor NKG2C (26), the natural cytotoxicity receptor NKp44 (27), and the short-tailed KIR3DS1 (28) and KIR2DS1/2/5, respectively (29)(30)(31). In particular, NGK2C is the dominant activating NK cell receptor for controlling CMV infection in both humans and mice (32)(33)(34).…”

NCR2 (natural cytotoxicity triggering receptor 2)

“…The groove appears wide enough to host a sialic acid or an elongated branched ligand. The cytoplasmic tail of NKp44 also contains a tyrosine sequence resembling a tyrosine-based inhibitory motif (Cantoni et al, 1999;Campbell et al, 2004). This motif is functional and inhibits the release of cytotoxic agents and IFN-γ (Rosental et al, 2011).…”

“…The identities of activating ligands are currently unknown, but bind NKp44 to induce activating signals through Dap12 (Vitale et al, 1998;Cantoni et al, 1999;Campbell et al, 2004). NKp44 inhibits NK cell function through recognition of cell surface exosomal PCNA which colocalizes with HLA I (Rosental et al, 2011;Horton et al, 2013 …”

“…Surface expression of the receptor and signalling physically requires the transmembrane accessory protein DAP12 which bears Immunoreceptor Tyrosine Activation Motifs (Cantoni et al, 1999). NKp44 activates NK cells through DAP12 linked directly to Lysine 183 in the transmembrane domain (Campbell et al, 2004). NKp44 inhibits NK cells through a tyrosine-based inhibitory motif located in the cytoplasmic tail of the receptor (Rosental et al, 2011).…”