The initiation of adaptive immune responses requires the interactions of T cells with antigen presenting cells (APC) in the context of an immunological synapse (IS). Naive T cell responses are dependent on the engagement of CD28 and CTLA-4 by CD86 and CD80, respectively amplifying and dampening the antigen specific signal. CD80 and CD86 cosignaling molecules display three major domains: a membrane distal IgV-like domain, a membrane proximal IgC-like domain and an intracellular domain. Crystallographic data has shown that only the IgV domain of CD80 and CD86 physically interacts with CTLA-4. However, extensive mutational analyses have also implicated the IgC domain in receptor binding and in the overall function of these molecules. The role of CD80 and CD86 within the IS and their exact molecular structure remains to be elucidated. The work presented in this thesis employs wild type, mutant, deleted and chimeric forms of CD80 and CD86 to characterize the role of their domains in molecular structure, receptor binding and overall cosignaling function in an antigen specific cellular interaction system. CD80 and CD86 are shown to be associated to the AFC cytoskeleton. A highly conserved K4 motif within CD86 is shown to be a cytoskeletal association motif. Moreover, CD86 is shown to physically interact with ERM proteins. Only cytoskeleton-linked CD86 localizes at the IS and induce IL-2 production. CD80 and CD86 molecular organization is clearly established using cytometry-based fluorescence resonance energy transfer (FCET) and biochemical approaches. CD80 exists as a mixed monomeric and dimeric population and CD86 as a monomer in live cells. The crucial role of CD80 and CD86 IgC domain in multimerization is revealed. Importantly, the molecular structure of these molecules correlates with their binding properties and cosignaling function. A functional picture of CD80 and CD86 domains emerges where the IgV is responsible for receptor binding, the IgC domain impacts dimerization, and the intracellular domain functionally links these proteins to the cytoskeleton. The findings presented in this thesis certainly contribute to the general understanding of cosignaling protein interactions and functions and may facilitate the design of structure-based immunotherapeutics.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.103159 |
| Date | January 2006 |
| Creators | Girard, Tanya. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | © Tanya Girard, 2006 |
| Relation | alephsysno: 002584324, proquestno: AAINR32353, Theses scanned by UMI/ProQuest. |
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