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. Despite considerable effort to engineer human CD8 alpha beta for high level expression in bacteria, we were unable to obtain CD8 alpha beta heterodimers, though we did succeed in making human CD8 alpha alpha homodimers. Our lack of success in the effective high level expression of human CD8 alpha beta has thus limited our ability to pursue this project further. Further efforts in the past year have been devoted to efforts to extend these observations to the human system, where expression of CD8alpha/beta in a form amenable to structural, binding, and functional studies has been difficult. Other approaches to address the precise structure of the CD8 stalk region, using monoclonal antibodies directed against the stalk region alone to stabilize the structure have not yet proved fruitful. 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 are now being 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 3.8.2.1 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. In addition, we have had the opportunity to explore the relationship of antibody affinity to therapeutic effect in a model system developed by the Goldsby laboratory. Here, a broad array of mouse anti-SEB antibodies have been developed, and their effects in preventing the toxic effects of SEB have been characterized in a mouse system. 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.

Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2011
Total Cost
$554,120
Indirect Cost
City
State
Country
Zip Code
Frey, Blake F; Jiang, Jiansheng; Sui, Yongjun et al. (2018) Effects of Cross-Presentation, Antigen Processing, and Peptide Binding in HIV Evasion of T Cell Immunity. J Immunol 200:1853-1864
Margulies, David H (2018) How MHC molecules grab citrullinated peptides to foster rheumatoid arthritis. J Biol Chem 293:3252-3253
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
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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

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