NK cells mediate important anti-tumor and anti-viral innate immune responses and can contribute to potent graft-versus-leukemia responses in allogeneic stem cell transplants. Unlike CTLs, where the crucial recognition event driving activation is mediated by a single receptor, the unique aB TCR expressed on that particular cell, NK cells must function by integrating a complex set of signals from the diverse array of activating and inhibitory NK receptors engaged upon interrogation of target cells. The ultimate goal of these studies is to understand the mechanism of NK cell signal integration. To achieve this long-term goal, we have begun with the C-type lectin-like NK receptor NKG2D, first determining receptor, ligand (MICs, ULBPs, RAE-ls) and complex crystal structures, characterizing interaction affinities, kinetics and thermodynamics by SPR and analyzing the interfaces computationally - all leading to an understanding of recognition and the determinants underlying structure and function.
In Aim 1, we propose to complete these studies with specific mutations to confirm our recognition model, crystallographic and SPR analyses of the uniquely divergent ligands MIC-A*004 and ULBP4, and by determining the structural constraints on NKG2D signal transduction through the ectodomain, using mutagenesis and cell-based activation assays. We next propose to extend these studies to the rest of the NKG2x-CD94 receptor family, having already analyzed additional structures of their ligand (HLA-E) and the correlation between its thermal stability, expression and affinity.
In Aim 2, we propose crystallographic and continuing SPR analyses of the structures, recognition machinery, affinities and kinetics of NKG2x-CD94-HLA-E interactions, concurrent with cell-based studies of the relative strengths of the signals delivered by the various activating and inhibitory CTLD NK receptors in driving activation. Completing Aims 1 and 2 will result in fully characterizing the extracellular components of the CTLD arm of the NK cell signal integration mechanism, and allow us to rationalize those parameters with measurements of relative signal strengths. Finally, in Aim 3, we propose to study other receptors that interact with MICs: V81 y8 TCRs. Having expressed soluble forms of three different MIC-responsive y8 TCRs, we propose SPR interaction studies (including whether NKG2D-TCR-MIC interactions are cooperative, anti-cooperative or independent) and crystallographic analyses to characterize the complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI048675-09
Application #
7576177
Study Section
Special Emphasis Panel (ZRG1-SSS-F (03))
Program Officer
Leitner, Wolfgang W
Project Start
2000-12-01
Project End
2011-02-28
Budget Start
2009-03-01
Budget End
2011-02-28
Support Year
9
Fiscal Year
2009
Total Cost
$359,090
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
Finton, Kathryn A; Strong, Roland K (2012) Structural insights into activation of antiviral NK cell responses. Immunol Rev 250:239-57
Xu, Bin; Pizarro, Juan C; Holmes, Margaret A et al. (2011) Crystal structure of a gammadelta T-cell receptor specific for the human MHC class I homolog MICA. Proc Natl Acad Sci U S A 108:2414-9
Correnti, Colin; Clifton, Matthew C; Abergel, Rebecca J et al. (2011) Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities. Structure 19:1796-806
Bandaranayake, Ashok D; Correnti, Colin; Ryu, Byoung Y et al. (2011) Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Res 39:e143
Kaiser, Brett K; Pizarro, Juan Carlos; Kerns, Julie et al. (2008) Structural basis for NKG2A/CD94 recognition of HLA-E. Proc Natl Acad Sci U S A 105:6696-701
McBeth, Christine; Seamons, Audrey; Pizarro, Juan C et al. (2008) A new twist in TCR diversity revealed by a forbidden alphabeta TCR. J Mol Biol 375:1306-19
Vigdorovich, Vladimir; Miller, A Dusty; Strong, Roland K (2007) Ability of hyaluronidase 2 to degrade extracellular hyaluronan is not required for its function as a receptor for jaagsiekte sheep retrovirus. J Virol 81:3124-9
Andersen-Nissen, Erica; Smith, Kelly D; Bonneau, Richard et al. (2007) A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin. J Exp Med 204:393-403
Holmes, Margaret A; Paulsene, Wendy; Jide, Xu et al. (2005) Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration. Structure 13:29-41
Kaiser, Brett K; Barahmand-Pour, Fariba; Paulsene, Wendy et al. (2005) Interactions between NKG2x immunoreceptors and HLA-E ligands display overlapping affinities and thermodynamics. J Immunol 174:2878-84

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