Method: To investigate the involvement of SLP-76 in Ly49D signal transduction, SLP-76 phosphorylation and clustering at the plasma membrane was examined in Ly49D-activated NK cells. Furthermore, the role of SLP-76 in NK cell signal transduction, activation, and development was assessed in SLP-76-knockout (KO) compared to wildtype (WT) NK cells.
Result: When NK cells were activated through Ly49D, SLP-76 was phosphorylated and recruited to the plasma membrane. Furthermore, SLP-76 was required for optimal signal transduction through Ly49D as SLP-76 KO NK cells exhibited diminished ERK and Akt phosphorylation compared to WT NK cells. This correlated with decreased IFNg production and granule exocytosis by SLP-76 KO NK cells. Although NK cells from SLP-76 KO mice appeared developmentally mature based on expression of late maturation markers, 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 perturbed development, SLP-76 was inducibly deleted in NK cells after maturation. Despite normal Ly49 receptor expression, NK cells inducibly deleted of SLP-76 still displayed 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. As this process depends on membrane-resident adaptor molecules LAT and NTAL in T cells and mast cells, we tested whether LAT and NTAL were similarly crucial for SLP-76 function in NK cells. Like SLP-76 KO NK cells, LAT/NTAL double KO (DKO) NK cells displayed significant functional defects, suggesting that LAT/NTAL may be required for SLP-76 activation. Surprisingly, membrane recruitment and phosphorylation of SLP-76 were intact in LAT/NTAL DKO NK cells following Ly49D stimulation. Cellular proliferation was also independent of LAT/NTAL, but dependent on SLP-76 expression.
Conclusion: These data suggest that NK cells use a novel LAT/NTAL-independent pathway leading to SLP-76 phosphorylation and membrane recruitment. This novel pathway leads to distinct NK cell effector functions. Together, these results demonstrate a critical role of SLP-76 in NK cell activation downstream of multiple signaling pathways emanating from the Ly49D activating receptor.
Disclosure: R. May, None; C. J. Hsu, None; M. Okumura, None; G. A. Koretzky, Rigel Pharma, 5, Pfizer Inc, 5 ; T. Kambayashi, None.