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Biochemical and Functional Characterization of Inhibitory Leukocyte Immune-Type Receptors in the Channel Catfish (Ictalurus punctatus)

  • Author / Creator
    Montgomery, Benjamin Christian Sivert
  • A balance of intracellular signaling events control immune cell functions. In innate immunity, many of these signals are transmitted by subsets of cell surface proteins known as immunoregulatory receptors. Through their recognition of a diverse array of molecules these receptors effectively translate extracellular cues into immune cell responses. These effector responses are vital for the elimination of pathogens and the destruction of damaged and/or infected host cells. In general, the ability of immunoregulatory receptors to promote or abrogate cellular responses is directly linked with their ability to transmit stimulatory and/or inhibitory signals. These signals are responsible for the direct immune cell-mediated elimination of pathogens, such as viruses and bacteria, and simultaneously protect healthy host tissue from autoimmune reactions. Co-expression of stimulatory and inhibitory immunoregulatory receptor-types by immune cells is common in all vertebrates but the majority of the research into understanding these complex networks has focused on mammals. Recently, it has been shown that immune cells in non-mammalian vertebrates, such as teleost (i.e. bony fish), express larger repertoires of immunoregulatory proteins but evidence as to how they are involved in mediating cellular signaling and controlling effector responses remain unknown. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) represent a large polymorphic gene family with members that possess the characteristics of putative stimulatory and inhibitory immunoregulatory proteins. The goal of my thesis work was to determine whether putative inhibitory IpLITR-types were bona fide functional receptors and to examine the signaling molecules/pathways involved in their activity. Using standard cellular transfection procedures, co-immunoprecipitations, site- directed mutagenesis, and adaptation of a vaccinia virus-based expression system for displaying IpLITRs on the surface of freshly isolated mouse natural killer (NK) cells, I discovered that two putative IpLITR-types, termed IpLITR1.2a and IpLITR1.1b, are both functional inhibitory receptors. I also determined that these IpLITRs effectively diminish cellular responses using two different mechanisms. Specifically, both IpLITR1.1b and IpLITR1.2a abrogated NK cell-mediated killing using ‘classical’ inhibitory tyrosine- based signaling motifs within their cytoplasmic regions, which recruited inhibitory phosphatases. Unlike IpLITR1.2a, IpLITR1.1b has additional tyrosine residues encoded within the membrane proximal region of its cytoplasmic tail that are not contained within known inhibitory motifs. Interestingly, one of these unique tyrosines abrogated the killing response of NK cells. However, unlike what I showed for IpLITR1.2a this was via a phosphatase-independent mechanism. These results highlight the conserved aspects of immunoregulatory receptor signaling in vertebrates while simultaneously revealing subtle nuances that may contribute towards a greater understanding of cellular effector responses.

  • Subjects / Keywords
  • Graduation date
    2013-06
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3N92M
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biological Sciences
  • Specialization
    • Physiology, Cell and Developmental Biology
  • Supervisor / co-supervisor and their department(s)
    • Stafford, James (Biological Sciences)
  • Examining committee members and their departments
    • Magor, Kathy (Biological Sciences)
    • Barry, Michele (Medical Microbiology and Immunology)
    • Wilson, Melanie (Microbiology)
    • Barreda, Daniel (Biological Sciences)