Role and Regulation of NKG2D Ligands on Tumors and Associated Cells

Natural Killer (NK) cells are crucial effectors of the immune response to tumors and infections. NK cells recognize and respond to diseased cells using receptors that bind to cell surface molecules that distinguish unhealthy cells from normal tissue. NK activation leads to the release of cytotoxic granules and pro-inflammatory cytokines such as interferon-gamma. The capacity of NK cells to respond to a given target cells is modulated by signals from the cellular environment, often via steady-state interactions of the same receptors used to respond to target cells. The interplay among environmental cues, ligands on target cells, and other factors that regulate NK responses are of ongoing interest within the fields of immunology and cancer biology./nNKG2D is one important activating receptor expressed by all NK cells and subsets of T cells. NKG2D recognizes a set of ligands that are absent from most healthy cells but are upregulated on many tumors and infected cells. Acute ligation of NKG2D by ligand(s) on target cells transmits a strong activating signal that can trigger the NK cell response. Interactions between NKG2D and its ligand(s) on tumor cells have been shown to confer protection for the host, but NKG2D has also been found in some circumstances to negatively regulate NK responses to infections and tumors. The molecular and cellular mechanisms that distinguish the beneficial anti-tumor effects of NKG2D vs. its detrimental effects remain unclear. In addition, the signals that mediate expression of NKG2D ligands are incompletely understood./nA major finding described in this dissertation is that NKG2D constitutively interacts with one of its ligands, RAE-1ε, at steady state in healthy mice. This interaction causes constitutive internalization of the NKG2D receptor and transmits a signal that causes global desensitization of NK cells to acute stimulation. Genetic or antibody-mediated interruption of host RAE-1ε / NKG2D interactions elevates NK responsiveness and enhances anti-tumor NK responses in vitro and in vivo. Bone marrow chimera assays and flow cytometry were used to identify endothelial cells in secondary lymphoid tissue as the most likely source of RAE-1ε responsible for steady-state NKG2D engagement and NK desensitization. We also find that endothelial cells infiltrating multiple transplant and spontaneous mouse tumor models show a super-induction of RAE-1ε relative to naïve mice. These findings suggest that inhibiting NKG2D ligands on endothelial cells may be a promising new avenue of immunotherapy./nThe second major focus of this dissertation is the expression of the NKG2D ligand RAE-1δ on tumor-associated macrophages. RAE-1δ but not other NKG2D ligands was induced on macrophages infiltrating several transplant and spontaneous tumor models. We find that tumor cell production of the cytokine colony-stimulating factor-1 (CSF-1) ligates the CSF-1 receptor on tumor-associated macrophages to initiate a PI3Kα-dependent signaling cascade leading to induction of mRNA and cell surface expression of RAE-1δ./nWe also present data here on the role of NK cells and NKG2D in the autochthonous, genetically engineered “KP” cancer model driven by inducible expression of a Kras oncogene and deletion of the Trp53 tumor suppressor gene. NKG2D was found to provide modest protection against KP sarcomas and lung cancer, and KP sarcoma cells were found to express the NKG2D ligands RAE-1δ and RAE-1ε. Interestingly, antibody-mediated depletion of NK cells did not have an effect on KP sarcoma incidence, whereas genetic deletion of conventional αβ T cells showed a trend towards accelerated and increased incidence of KP sarcoma./nFinally, we describe the successful generation of a conditional KO mouse targeting loxP sites to the Raet1e gene, which encodes the RAE-1ε protein. CRISPR/Cas9 and two high-quality guide RNAs were used to target loxP insertion to intronic sequences flanking the 2nd and 3rd protein-coding exons in Raet1e, and germline transmission of the targeted allele was confirmed. These mice were crossed to the Cdh5-Cre-ERT2 mouse strain, which allows for inducible deletion of RAE-1ε in endothelial cells. This represents and important tool to study interactions between endothelial RAE-1ε and NK cells, and will facilitate the tissue-specific deletion of RAE-1ε in other research contexts./nThe studies presented in this dissertation serve to clarify pleiotropic effects of the NKG2D system, examine a novel mechanism of NKG2D ligand induction relevant for tumor immunology, provide preliminary evidence of a protective role for NKG2D in the KP spontaneous tumor model, and describe the generation of new conditional RAE-1ε-KO mouse for future experiments. These findings add substantial insight and advancement for the field of NK cell biology and cancer immunology, and may suggest novel strategies for the treatment of cancer.

Advisor: Raulet, David H