Genetic Investigation of MHC-independent Missing-self Recognition by NK Cells

NK cells are innate lymphocytes involved in the elimination of transformed, infected, stressed, antibody-coated and transplanted cells. The goal of this thesis, as well as the interest of our laboratory is to explore the MHC-independent “missing-self” branch of NK recognition, and more specifically, the NKR-P1B:Clr-b receptor-ligand system and its role in bone marrow transplantation. In the first results chapter, we generated hybridoma clones producing monoclonal antibodies against another inhibitory receptor in the Nkrp1 receptor family, NKR-P1G via the CELLISA assay. Our results demonstrate that CELLISA is a cost-effective, efficient, and high-throughput screening protocol, and the generation of the α-NKR-P1G 1H8 hybridoma clone enables characterization of the mouse Nkrp1-Clr receptor-ligand system via in vitro reporter cell and transfection approaches. Next, we show that the Nkrp1-Clr receptor-ligand system exhibits substantial allelic polymorphisms in NKR-P1B, NKR-P1C, and Clr-c loci between three common laboratory inbred mouse strains (C57BL/6, BALB/c and 129); however, no differential gene content has been observed in these loci in contrast to the Ly49 and MHC-I loci in mice. Furthermore, through the usage of BWZ reporter cell assays, we reveal several new cognate receptor-ligand pairs in addition to the previously reported NKR-P1B:Clr-b and NKR-P1F: Clr-g interactions; they are: NKR-P1F: Clr-c; NKR-P1F: Clr-d; NKF-P1G: Clr-d; NKR-P1G: Clr-f; NKR-P1G: Clr-g. In the final results chapter, we show that Clr-b–/– bone marrow cells are rejected by the recipients’ NKR-P1B+ NK cells. Importantly, through the usage of a 7-colour membrane labeling technique on the donor cells, we can simultaneously compare the rejection of Clr-b–/– grafts to WT cells and different variants of MHC-I-deficient bone marrow cells in a single animal. Up to 30-40% of Clr-b–/– bone marrow cells were rejected, which is similar to the rejection phenotype for H-2Db–/– bone marrow cells. Importantly, this rejection is reverted fully or partially (in B6 vs. Swiss recipients) in NK-depleted (total or NKR-P1B+ NK subset depleted) recipients. Collectively, these results extend our understanding of the Nkrp1-Clr receptor-ligand family by revealing additional Nkrp1-Clr receptor-ligand pairs; and that the missing-self recognition of Clr-b in vivo is instrumental in determining the prognosis of bone marrow transplantation.Ph