Regulation of Natural Killer Cell Ligands by Poxvirus Infection
Natural Killer (NK) cells are a major part of the innate immune defence against viral infection and tumours. However, our understanding of how NK cells can be exploited for antiviral and antitumor therapy is still in its infancy. We are interested in the interplay between NK cells and Vaccinia Virus (VACV) since VACV is a basis of vaccine design and has the potential as an oncolytic virus for tumor therapy. Currently, we are focused on how VACV affects the ligands of NK receptors belonging to
... tors belonging to the NKR-P1 family. This family of C-type lectin-related receptors contains members that can stimulate or inhibit NK cell function. We first studied the impact of VACV and Ectromelia virus (ECTV) infection on expression of the mouse CLEC2D protein, Clr-b, and Clr-b mediated protection from mouse NK cells. We observed a loss of Clr-b cell surface protein upon infection of murine cell lines and bone marrow derived macrophages with VACV and ECTV. The reduction of Clr-b is more rapid than MHC class I, the prototypic ligand of NK cell inhibitory receptors. Reduction of Clr-b requires an active viral infection but not expression of late viral genes. The loss of Clr-b mRNA appears to be delayed behind loss of Clr-b surface protein. Finally, Clr-b mediated protection from NK cells is lost following VACV infection. Subsequently, we studied the influence of VACV infection of the expression of human CLEC2D and CLEC2D mediated protection from human NK cells. Human CLEC2D is constitutively expressed on transformed cells and it is believed that cells may need to be stimulated to induce expression. We observed a rapid increase in CLEC2D cell surface protein as detected by 4C7 antibody staining upon VACV infection of human cell lines that gradually decreased over time. The increase of 4C7 staining requires an active infection with the presence of an early viral protein, however, a late viral protein enhances the increase in the 4C7 reactive iii protein. We discovered that all cells may express an isoform of the 4C7 reactive protein, and that they all contain an intracellular pool of the 4C7 reactive protein. Although VACV infection causes the upregulation the 4C7 reactive protein, it does not increase surface expression of isoform 1 of CLEC2D which is reported to bind to NKR-P1A (85). Given the lack of increase in isoform 1, NK cells did not recognize infected cells through NKR-P1A. My results suggest that Clr-b is another mechanism of the missing self recognition system that may act earlier than MHC I recognition in rodents. In contrast, in humans, the function of CLEC2D is still undergoing investigation and my results suggest that intracellular pools of a 4C7 reactive protein may be manipulated by VACV infection. However the function of the CLEC2D isoforms involved in the infection remains an open question. Collectively, these data augment our knowledge in viral evasion and host response to infection. Further studies of this process could facilitate improved vaccine design and cancer therapy that currently uses VACV as a platform. iv The thesis is dedicated to my father (Vernon P. Williams) and the memory of my mother (Rose Jasmine Warner-Williams) who taught me patience, dedication and that hard work is the only way to get ahead in this world. Along with my brothers and sisters, my entire family has shown me love and support that helped me through some very long and arduous times. This thesis is also dedicated to my Godparents and numerous friends both near and far, who believed in me and were always there with a good pep talk and a glass of wine. v ACKNOWLEDGMENTS My thanks and appreciation goes to my supervisor Dr. Deborah N. Burshtyn. She has been a wealth of wisdom and support during my time at the University of Alberta. Without her understanding and sometime patience, I would not have made it as far as I did.