In the previous year, we had reported the three-dimensional structure of the MHC/CD8alpha beta complex, a structure that had eluded scientists for many years. The initial conclusion from this study revealed the topology of the location of the CD8beta chain with respect to alpha, and posed several important functional questions related to CD8 dependent signaling. Our plan in the past year was to: 1) develop systems for the expression of the human CD8 alpha beta;2) to exploit such protein for binding and structural studies;3) to extend our observations on the mouse CD8 alpha beta molecule to include the structurally plastic stalk region of the CD8 alpha beta heterodimer. With considerable effort, we now have engineered human CD8 alpha/beta in several mammalian cell expression systems. Additional efforts to understand important aspects of the TCR/MHC interaction have been more productive. We have recently completed the 2.0 X-ray crystallographic structure of an MHC-I (H-2Dd/P18 peptide)/B4.2.3 TCR complex, which allows us to examine in detail questions relating to whether or not the TCR has inherent reactivity toward the MHC, independent of the bound peptide. To this end, we have used the high resolution three-dimensional structure of the complex to serve as the basis for targeted mutagenesis of the TCR to further evaluate quantitatively the rules that govern the TCR/MHC interaction in quantitative terms. Specifically, the structure suggests that CDR3 regions of the TCR alpha and beta chains primarily are involved in molecular contacts with the antigenic peptide, and that CDR1 and CDR2 regions of both chains function in a complementary fashion to interact with the MHC-I molecule, in this case, H-2Dd. A set of carefully considered deletion and substitution mutants of the TCR CDR3 regions have been made to explore the possibility that some of these molecules will retain reactivity with MHC-I despite eliminating or changing reactivity with the bound peptide. Efforts to understand the structure and function of the Treg expressed molecule, GARP, also known as LRRC 32, are designed to;1) understand the fundamental structure of GARP, predicted to be a leucine rich repeat protein;2) evaluate the interact of GARP with the latent form of TGF-beta;3) develop monoclonal antibodies to both human and mouse forms of GARP for functional and further structural studies. To these ends, we have successfully engineered the expression of both human and mouse GARP proteins in Drosophila S2 cells. Human latent TGF-beta (LAT) has also been expressed in CH0-Lec cells, a cell line deficient in the addition of terminal carbohydrates. Using surface plasmon resonance, we have measured the binding constant for the GARP/Latent TGF beta interaction. In collaboration with the Shevach laboratory, utilizing our recombinant human and mouse GARP, we are developing monoclonal antibodies to both human and mouse GARP. Antibodies to both the mouse and human molecules have been obtained, and are now being characterized. These antibodies should permit the characterization of T cells that express GARP on their cell surface and will be essential in understanding the interactions of GARP with latent TGF-beta. Our expertise in evaluating molecular interactions by SPR has allowed a direct comparison of the binding affinity of a panel of mouse anti-SEB monoclonal antibodies with chimeric antibodies engineered to have human constant regions. Our results show clearly that the chimeric antibodies have essentially the same affinity for SEB that the original mouse antibodies have. In a recent series of experiments we have contributed to the characterization of the MHC-I molecule, HLA-B*5701, which, when bound to an anti=retroviral drug, abacavir, can cause severe acute hypersensitivity. We have successfully engineered HLA-B*5701, and have obtained protein crystals for diffraction studies.

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Voss, Oliver H; Murakami, Yousuke; Pena, Mirna Y et al. (2016) Lipopolysaccharide-Induced CD300b Receptor Binding to Toll-like Receptor 4 Alters Signaling to Drive Cytokine Responses that Enhance Septic Shock. Immunity 44:1365-78
Murakami, Y; Tian, L; Voss, O H et al. (2014) CD300b regulates the phagocytosis of apoptotic cells via phosphatidylserine recognition. Cell Death Differ 21:1746-57
Subramanian, Naeha; Natarajan, Kannan; Clatworthy, Menna R et al. (2013) The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation. Cell 153:348-61
Norcross, Michael A; Luo, Shen; Lu, Li et al. (2012) Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity. AIDS 26:F21-9
Revilleza, Maria Jamela; Wang, Rui; Mans, Janet et al. (2011) How the virus outsmarts the host: function and structure of cytomegalovirus MHC-I-like molecules in the evasion of natural killer cell surveillance. J Biomed Biotechnol 2011:724607
Tilahun, Mulualem E; Kwan, Alan; Natarajan, Kannan et al. (2011) Chimeric anti-staphylococcal enterotoxin B antibodies and lovastatin act synergistically to provide in vivo protection against lethal doses of SEB. PLoS One 6:e27203
Tilahun, Mulualem E; Rajagopalan, Govindarajan; Shah-Mahoney, Nalini et al. (2010) Potent neutralization of staphylococcal enterotoxin B by synergistic action of chimeric antibodies. Infect Immun 78:2801-11
Wang, Rui; Natarajan, Kannan; Margulies, David H (2009) Structural basis of the CD8 alpha beta/MHC class I interaction: focused recognition orients CD8 beta to a T cell proximal position. J Immunol 183:2554-64
Tang, Xiaobin; Narayanan, Sriram; Peruzzi, Giovanna et al. (2009) A single residue, arginine 65, is critical for the functional interaction of leukocyte-associated inhibitory receptor-1 with collagens. J Immunol 182:5446-52
Margulies, David H (2009) Antigen-processing and presentation pathways select antigenic HIV peptides in the fight against viral evolution. Nat Immunol 10:566-8

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