Genetic Regulation of Natural Killer cell Homeostasis and Function

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Natural Killer (NK) cells are a population of cytotoxic innate lymphocytes with a crucial role in protection against tumors and virus infections. Studies of rare patients and mouse mutants with germ line mutations leading to NK cell deficiencies have illuminated fundamental mechanisms of NK cell development and function. It is notable that certain NK cell depleting mutations impact on cell mechanisms present in every cell such as DNA replication or ribosome assembly. The latter are known as ribosomopathies, which are genetic diseases caused by mutation in ribosome structural proteins or maturation factors. Ribosomopathies show characteristically tissue specific effects, in part due to the p53-mediated cell-death pathway that can be activated by free ribosomal proteins. This discovery grant will confront the issue of why NK cells appear to be more susceptible than other lymphocytes to defects in ribosomes hoping to illuminate new molecular determinants of NK cell development and homeostatic requirements. In a recent genome-wide chemical (ENU) screen for altered response to mouse cytomegalovirus (MCMV)infection, we have identified a pedigree showing severe susceptibility to MCMV due to NK cell-specific deficiency. A recessive stop-gain mutation (C567X) in the translational GTPase guanine nucleotide binding protein-like 1 (Gnl1) gene dictated an NK cell-specific phenotype characterized by 1) lack of homeostatic proliferation and decreased cell survival in vivo; 2) blockade of cell cycle in response to the homeostatic cytokine IL-15 ex vivo; 3) inflammatory proliferation during MCMV infection. GNL1 is predicted to encode a ribosome assembly factor the dysfunction of which results in p53-dependent cell death. Characterization of the immune compartment in p53 and Gnl1 doubly-deficient mice showed a full rescue of Gnl1(C367X) deficits in NK cell numbers and survival. Based on these results we hypothesized that p53 activation is a driving mechanism for NK cell abnormalities in homeostasis and survival in Gnl1-deficient mice. To test this hypothesis, we propose a multipronged approach involving 1) the use of bone marrow chimera mice, and adoptive transfer of cell sorted cell populations, to determine the immunological compartments affected by Gnl1 deficiency; 2) the identification of transcriptional networks disrupted in Gnl1(C367X) NK cells by examining cytosolic and polysome-associated transcripts, using RNA sequencing; 3) the screening and validating GNL1 binding partners to ultimately situate GNL1 in a biochemical pathway. This research program will further our understanding of NK cell development and homeostasis while producing cutting-edge results about tissue-specific functions of ribosome-associated proteins. The proposal will enable a graduate student and a post-doctoral fellow to learn advanced immunological, genomic and biochemical techniques, thus providing an ideal setting for training of highly qualified personnel.