SLP-76 Is Required for Optimal Natural Killer Cell Activation and Ly49 Receptor Acquisition

Abstract 103

Natural killer (NK) cells are innate immune cells that defend the host from intracellular pathogens and tumors. Regulation of NK cell activation involves the expression of activating receptors that are finely counterbalanced by inhibitory MHC class I-binding receptors (e.g., Ly49 family members), which allow NK cells to achieve self-tolerance. How the precise signaling pathways leading from activating receptors contribute to effector function and inhibitory receptor acquisition by NK cells are not well understood. Thus, we aimed to dissect the proximal signaling pathways downstream of the Ly49D activating receptor and focused our studies on SLP-76 (SH2 containing leukocyte protein of 76kD), an adaptor molecule which is important in mediating signals downstream of ITAM-containing activating receptors in a variety of hematopoietic cell types. When NK cells were activated through Ly49D, SLP-76 was phosphorylated and recruited to the plasma membrane. SLP-76 was required for optimal signal transduction through Ly49D as SLP-76 knockout (KO) NK cells exhibited diminished ERK (extracellular signal-regulated kinase) and Akt phosphorylation compared to wildtype (WT) NK cells. These biochemical defects correlated with decreased IFNγ and TNFα production, and granule exocytosis by SLP-76 KO NK cells compared to WT NK cells. Although NK cells from SLP-76 KO mice appeared developmentally mature based on expression of late maturation markers DX5 and CD11b, we noted a selective defect in the acquisition of Ly49 family member inhibitory and activating receptors in SLP-76 KO NK cells. Since the defective function of SLP-76 KO NK cells might be related to their perturbed development, SLP-76 was inducibly deleted in NK cells after full maturation. Such NK cells displayed normal Ly49 receptor expression but still exhibited defective IFNg production and granule exocytosis, suggesting that SLP-76 plays an important role in Ly49D-mediated NK cell function. We next explored the mechanisms by which SLP-76 relocalizes from the cytosol to the plasma membrane and is subsequently phosphorylated. As this process depends on membrane-resident LAT (linker of activation of T cells) family adaptor molecules (LAT1 and LAT2) in T cells and mast cells, we tested whether LAT1 and LAT2 were similarly crucial for SLP-76 function in NK cells. Like SLP-76 KO NK cells, LAT1/LAT2 double KO (DKO) NK cells displayed significant functional defects. Surprisingly, however, membrane recruitment and phosphorylation of SLP-76 were intact in LAT1/LAT2 DKO NK cells following Ly49D stimulation. Moreover, a SLP-76 mutant that is unable to bind to LAT1/LAT2 was also recruited to the plasma membrane following Ly49D stimulation. Together, these results point towards the existence of a novel alternative signaling pathway leading to SLP-76 activation in NK cells. This alternative pathway may be important during NK cell development, since LAT1/LAT2 DKO NK cells displayed only a mild defect in Ly49 receptor acquisition compared to SLP-76 KO NK cells, underscoring the significance of this LAT1/LAT2-independent pathway. Together, these results demonstrate a critical role for a LAT1/LAT2-dependent and independent pathway leading to SLP-76 in NK cell activation and development.