Silensilencing Human NKG2D, DAP10 and DAP12 Reduces Cytotoxicity of in Vivo Activated CD8+ T and NK Cells.

Abstract

NKG2D is a cell surface receptor expressed on NK cells, CD8+ T cells, γδ T cells and macrophages. NKG2D is activated by stress-induced ligands MICA and MICB, and UL16 binding proteins ULBP1, ULBP2 and ULBP3 which are expressed on target cells modified by infection, transformation or stress. Binding of NKG2D on NK cells results in an activation signal that bypasses or overrides inhibitory signals from major histocompatibility complex (MHC) class I specific receptors, resulting in cytotoxic granule content release and target cell killing. NKG2D on T cells co-stimulates effector T cell function. Signaling is achieved by selective expression of the adaptor proteins DAP10 and DAP12 which form distinct associations with NKG2D isoforms in different cell types. Previously we have shown that CD8+ T cells activated with INF-γ, anti CD3 MAB and IL2 express NKG2D and share functional and phenotypic properties with NK cells. Activated CD8+ T cells up-regulate NKG2D as well as DAP10 and DAP12 during ex-vivo activation and expansion which correlated with cytotoxic function. We demonstrated that CD8+ T cell-mediated cytotoxicity is MHC1 independent and that NKG2D monoclonal antibodies directed against NKG2D reduced cytotoxicity. To further elucidate the role of NKG2D signaling in CD8+ T cell-mediated cytotoxicity, six 21-nucleotide small interfering RNAs (siRNA) were designed against multiple sites within the NKG2D gene. The greatest suppression of NKG2D expression was observed with the siRNA targeting BP1546-1567 (siRNA #6) located between exon 8 and 9. FACS analysis, northern and western blots demonstrated that effecter cells transfected with siRNA #6 incorporated into the pLV ThM lentiviral vector resulted in >90% (P <.0001) suppression of NKG2D expression after 72 hrs. In both CD8+ T cells and NK cells, suppression of NKG2D resulted in a >90% decrease in cytotoxicity against multiple myeloma tumor cell line targets (RPMI and U266). We also performed experiments using cells derived from the peripheral blood of ovarian cancer patients and autologous tumor targets derived from ascites. Similar to the results obtained using CD8+ T cells and NK cells against multiple myeloma cell lines, suppressing NKG2D expression in activated CD8+ T Cells resulted in >90% decrease in cytotoxicity against the autologous tumor targets.To characterize the role of DAP10 and DAP12 adaptor molecules in NKG2D cytotoxic signaling, we generated three siRNA molecules spanning DAP10 and four siRNA molecules for DAP12. Using northern blot analysis, >90% suppression of DAP 10 was observed when CD8+ T cells and NK cells were treated with the siRNA targeting BP356-377. A similar level of silencing of DAP12 was observed using a siRNA targeting BP 323-344 of DAP 12. Suppression of DAP10 expression in CD8+ T cells and NK cells resulted in a decrease in cytotoxicity of more than ~75% (P .0003) compared to untreated cells or cells treated with a control siRNA, siRNA against murine CD8. Suppressing DAP12 expression also resulted in significant reduction in cytoxicity, however was consistently less than that of DAP10. When DAP10 and DAP12 ware simultaneously silenced in CD8+ T and NK cells, an ~82% (P <.0001) reduction in cytotoxicity was observed. While DAP 10 is known to play a key role in NKG2D signaling, other adaptor proteins such as DAP12, also are involved in activated and expanded CD8+ T cell cytotoxicity.