Effects of complementarity determining region mutations on the affinity ofan alpha/beta T cell receptor: Measuring the energy associated with CD4/CD8 repertoire skewing

It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (K (d)similar to 10(-4) to 10(-7) M) are the result of biological selection ra ther than an intrinsic affinity limitation imposed by the TCR framework. Us ing a soluble version of the 2C TCR, we have used complementarity determini ng region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We repo rt that several mutants at positions lying within CDR3 alpha and CDR2 beta showed increased affinities for pMHC compared with the wildtype receptor. A dditionally, we have investigated whether V alpha mutations that have been implicated in the phenomenon of CD8(+) repertoire skewing achieve this skew ing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenoty pe, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of GD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand inter actions above a minimal energy threshold. Together, the results (a) demonst rate that engineering higher affinity TCRs is feasible, and (b) provide TCr -pMHC energy values associated with CD4/CD8 repertoire skewing.