991 - Dual Signaling Pathways Dependent Upon the Adaptor Protein SLP-76 Lead to Distinct Natural Killer Cell Effector Functions

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Rebecca May1, Chih-Jung Hsu1, Mariko Okumura1, Gary A. Koretzky2 and Taku Kambayashi1, 1University of Pennsylvania, Philadelphia, PA, 2University Pennsylvania, Philadelphia, PA
Presentation Number: 991

Background/Purpose: Natural killer (NK) cells are innate immune cells that provide a defense against intracellular pathogens and tumors by displaying cytotoxicity and producing immune-activating cytokines. NK activation is regulated by the expression of activating receptors that are finely counterbalanced by inhibitory receptors. Although mechanisms by which activating signals are blocked by inhibitory receptors are well defined, the proximal signaling pathways that lead to NK effector function are incompletely understood. Thus, we aimed to dissect the proximal signaling pathways downstream of the Ly49D activating receptor and focused our studies on SLP-76, an adaptor molecule which is important in mediating signals downstream of activating receptors in many hematopoietic cell types.

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.

Keywords: natural killer (NK) cells and signal transduction

Disclosure: R. May, None; C. J. Hsu, None; M. Okumura, None; G. A. Koretzky, Rigel Pharma, 5, Pfizer Inc, 5 ; T. Kambayashi, None.