Expression of Ligands for the NKG2D Activating Receptor are Linked to Proliferative Signals

NKG2D is a stimulatory receptor expressed by natural killer cells and subsets of T cells. The receptor recognizes a set of self-encoded cell surface proteins that are usually not displayed on the surface of healthy cells but are often induced in transformed and infected cells. NKG2D engagement activates or enhances the cell killing function and cytokine production programs of NK cells and certain T cells. Emerging evidence suggests that different ligands are to some extent regulated by distinct signals associated with disease states, thus enabling the immune system to respond to a broad range of disease-associated stimuli via a single activating receptor. The research presented in this thesis demonstrated that at least one of the murine NKG2D ligands, RAE-1 ε (gene: Raet1e), is transcriptionally activated by signals associated with cell proliferation. Primary cultured fibroblasts from normal tissue, which did not express RAE1 in vivo, were induced to express large amounts of cell surface RAE-1 ε upon culture in vitro. RAE-1 ε induction was associated with increased Raet1e transcription. Inhibitor and other experiments showed that RAE-1 ε induction was dependent on sustained cell proliferation induced by growth factors, but was not dependent on a variety of other pathways, including the DNA damage response, oxidative stress or serum components other than growth factors. In vivo, correlative, evidence showed that RAE-1 ε was also displayed on rapidly proliferating brain cells in early embryos, but was extinguished at later stages of brain development as cell proliferation slowed.In line with these findings, analysis showed that the Raet1e promoter was more active in proliferating cells than quiescent cells. In silico analysis of the Raet1e promoter for potential binding sites for transcription factors associated with cell cycle regulation revealed multiple putative binding sites for E2F family members. Chromatin immunoprecipitation studies demonstrated that the Raet1e promoter was bound in vivo by E2F family members in proliferating cells. Overexpression of activating E2F family members induced endogenous Raet1e transcripts in nonproliferating cells and transactivated a Raet1e promoter reporter plasmid. Transactivation was strongly inhibited if two putative E2F sites in the promoter were mutated. Collectively, the data show that transcription factors that regulate cell proliferation regulate the transcriptional activation of cell surface ligands that target transformed and infected cells for destruction by NK cells and T cells. E2F family members are often mutated in cancer and proliferative signals often accompany viral infections. On the other hand, many healthy cells undergo proliferation without induction of RAE-1 on the cell surface, so it appears that proliferation by itself is not always sufficient to induce RAE-1 expression at the cell surface. Other stress pathways activated during tumorigenesis or in infected cells are likely to work together with the proliferative signal documented here to ensure that cell surface expression of NKG2D ligands is restricted to unhealthy cells