There has been considerable progress in the past decade on understanding the structure and function of T cell receptors (TCRs). The structures often a(3 TCR:peptide/MHC complexes have been solved and binding studies have shown that these interactions exhibit low affinities and slow on-rates. Structures and biophysical measurements indicate that the TCR has considerable flexibility, in the CDR3 loops in particular. Despite these studies, many important questions concerning T cell recognition and signaling still remain. For example, the energetic contribution of different peptide residues to TCR binding remains to be examined in a comprehensive way. Conversely, limited information is available on how regions of the TCR outside of the CDR3 loops contribute to peptide binding and specificity. It is also not at all clear what mechanism accounts for the conserved diagonal docking mode of TCRs. Related to this, the molecular basis of MHC restriction and alloreactivity for distinctly different MHC alleles are not understood (e.g. to what extent are specific TCR residues and/or TCR flexibility involved in these processes?). Finally, it remains enigmatic whether the a?TCR undergoes conformational changes in pepMHC distal regions upon pepMHC binding, and if so, if this movement contributes to signaling. We have developed a yeast display technology to produce stabilized, higher affinity TCRs that will allow us to explore these questions. ? The project will use the class I restricted 2C TCR that recognizes, as strong agonists, a syngeneic complex SIYR/Kb and an allogeneic complex QL9/Ld. We will also use the class II restricted TCR 3.L2 that recognizes Hb/l-Ek. The 3.L2 TCR was recently engineered, in collaboration with Paul Allen's lab, to produce high-affinity mutants, enabling crystallographic analysis of a 3.L2 TCR mutant:Hb/l-Ek complex (with Daved Fremont).
The specific aims of the project are: 1) To measure the contribution of individual peptide residues to binding by selected high-affinity TCR mutants. Using a high-affinity TCR against SIYR/Kb (KD = 16 nM), QL9/Ld (KD = 6 nM), and Hb/l-Ek (KD = 25 nM), we will determine the energetic contribution of each peptide residue. 2) To characterize the structure, flexibility, and binding mechanisms of selected TCR mutants. The wild type 2C:QL9/Ld alloantigen complex and several high-affinity TCRs (liganded and unliganded) will be examined structurally (with Chris Garcia); various binding studies will explore the mechanisms of the interactions. 3) To explore the role of TCR extracellular domain interactions in T cell signaling. Mutations at V:C and Va:V? interfaces will be introduced into a T cell transfection system to examine their possible influence on T cell activation. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM055767-09A1
Application #
7034440
Study Section
Cellular and Molecular Immunology - B (CMI)
Program Officer
Marino, Pamela
Project Start
1997-05-01
Project End
2009-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
9
Fiscal Year
2006
Total Cost
$293,920
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Adams, Jarrett J; Narayanan, Samanthi; Birnbaum, Michael E et al. (2016) Structural interplay between germline interactions and adaptive recognition determines the bandwidth of TCR-peptide-MHC cross-reactivity. Nat Immunol 17:87-94
Narayanan, Samanthi; Kranz, David M (2013) The same major histocompatibility complex polymorphism involved in control of HIV influences peptide binding in the mouse H-2Ld system. J Biol Chem 288:31784-94
Stone, Jennifer D; Chervin, Adam S; Aggen, David H et al. (2012) T cell receptor engineering. Methods Enzymol 503:189-222
Engels, Boris; Chervin, Adam S; Sant, Andrea J et al. (2012) Long-term persistence of CD4(+) but rapid disappearance of CD8(+) T cells expressing an MHC class I-restricted TCR of nanomolar affinity. Mol Ther 20:652-60
Aggen, D H; Chervin, A S; Schmitt, T M et al. (2012) Single-chain V?V? T-cell receptors function without mispairing with endogenous TCR chains. Gene Ther 19:365-74
Stone, Jennifer D; Chervin, Adam S; Schreiber, Hans et al. (2012) Design and characterization of a protein superagonist of IL-15 fused with IL-15R? and a high-affinity T cell receptor. Biotechnol Prog 28:1588-97
Stone, Jennifer D; Artyomov, Maxim N; Chervin, Adam S et al. (2011) Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs. J Immunol 187:6281-90
Adams, Jarrett J; Narayanan, Samanthi; Liu, Baoyu et al. (2011) T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex. Immunity 35:681-93
Aggen, David H; Chervin, Adam S; Insaidoo, Francis K et al. (2011) Identification and engineering of human variable regions that allow expression of stable single-chain T cell receptors. Protein Eng Des Sel 24:361-72
Stone, Jennifer D; Aggen, David H; Chervin, Adam S et al. (2011) Opposite effects of endogenous peptide-MHC class I on T cell activity in the presence and absence of CD8. J Immunol 186:5193-200

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