Complementarity-Determining Region 1 Sequence Requirements Drive Limited Vα Usage in Response to Influenza Hemagglutinin 307–319 Peptide

Abstract

We have developed a T cell activation-based system that allows for the selection of TCRs with defined peptide/MHC specificities from libraries in which complementarity-determining region (CDR) sequences have been randomized by in vitro mutagenesis. Using this system, we have explored the sequence requirements for CDR1 and CDR2 of the TCR α-chain in a human T cell response characterized by restricted Vα and Vβ usage. Libraries of T cells expressing receptors built on the framework of a TCR specific for the influenza virus peptide hemagglutinin 307–319 presented by HLA-DR4, but with random sequences inserted at CDR1α or CDR2α, were selected for response to the same peptide/MHC ligand. A wide variety of CDR2α sequences were found to be permissive for recognition. Indeed, >25% of T cell clones chosen at random displayed a significant response. In contrast, a similar challenge of a randomized CDR1α library yielded only the parental sequence, and then only after multiple rounds of selection. T cell clones cross-reactive on closely related HLA alleles (subtypes of DR4) could be isolated from randomized libraries, but not clones restricted by more distantly related alleles such as HLA-DR1. These results indicate that, in the context of this T cell response, the structural requirements for recognition at CDR1α are significantly more restricted than at CDR2α. This system for mutation and selection of TCRs in vitro may be of use in engineering T cells with defined specificities for therapeutic applications.