Cytotoxic T lymphocytes (CTL), or CD8⁺ T cells, are responsible for clearing infected or diseased cells from the body. Diseased cells label themselves through presentation of non-self, or antigenic, peptides on the exterior of the cell. Both healthy and infected cells present peptides on the surface. These self and non-self peptides are presented by the class I major histompatibility complex molecule, or MHC. The class I peptide-MHC (pMHC) complex is the ligand for the T cell receptor (TCR), which is expressed on the surface of CTLs. TCR discrimination between self and non-self pMHC is the critical event in determining whether or not the CTL will be activated and lyse the antigen presenting cell. It remains unclear through what mechanism(s) the recognition of different pMHC by the TCR translates into qualitatively different activation signals in the T cell.

The AHIII12.2 (AHIII) TCR recognizes and is activated by its cognate pMHC, p1049 in HLA-A2(A2) Activation of AHIII T cells is inhibited by the peptide p1058 when presented by A2. Through mutation of residues in both the peptides and MHC, we present here further support for the affinity model of T cell triggering, that AHIII T cell activation correlates with TCR-pMHC binding affinity. Interestingly, mutations in the peptide seem to be more deleterious to binding than mutations in the MHC. Crystal structures of A2 mutants bound to the AHIII TCR were determined, and showed that the K66A mutation in the MHC can influence the conformation of a TCR CDR3 loop, which is generally thought of as being restricted to peptide recognition. Thermodynamic analysis of AHIII-p1049/A2(K66A) binding confirmed that the loop movement results in a loss of H-bonds. Thermodynamic analysis of the AHIII TCR binding to wild-type p1049/A2 determined that binding occurs through favorable enthalpy and favor entropy, which contradicts the favored thermodynamic TCR-pMHC binding model of enthalpically driven and entropically apposed binding. Finally, we present here a streamlined protocol to express and purify soluble TCR from singly isolated T cells directly ex vivo.