High affinity T cell receptors from yeast display libraries block T cell activation by superantigens.
The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.
Kieke
M C
MC
Department of Biochemistry, University of Illinois, Urbana, IL, 61801, USA.
Sundberg
E
E
Shusta
E V
EV
Mariuzza
R A
RA
Wittrup
K D
KD
Kranz
D M
DM
D001665:Binding Sites; D019020:Directed Molecular Evolution; D004768:Enterotoxins / Q000737:chemistry / Q000276:immunology; D005434:Flow Cytometry; D008213:Lymphocyte Activation; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D012995:Solubility; D013211:Staphylococcus aureus / Q000737:chemistry / Q000276:immunology; D018089:Superantigens / Q000737:chemistry / Q000276:immunology*; D020349:Surface Plasmon Resonance; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
D004768:Enterotoxins; D019151:Peptide Library; D011948:Receptors, Antigen, T-Cell; D018089:Superantigens; C034959:enterotoxin C, staphylococcal