T cell receptor (TCR) development in the presence of the tumour antigen, as in the case of human-derived TCRs, or in the absence of the tumour antigen, as in the case of mouse-derived TCRs, results in differences in peptide sensitivity. Such human leukocyte antigen (HLA) class I-restricted TCRs and HLA class II-restricted TCRs each selected in the presence or absence of the human tumour antigen were subject of the project. During the outgoing phase, biochemical experiments and structural analyses were conducted at Monash University in Clayton, Australia. The alpha- and beta-chains of fourteen TCRs were expressed in bacteria, and the TCRs were folded and purified by hydrophobic-interaction chromatography, anion-exchange chromatography, and S200 size-exclusion chromatography. HLA class I molecules and HLA class II molecules were expressed in bacteria and mammalian cells, respectively, and also purified. X-ray diffraction patterns of the crystals for three pHLA classs I molecules and one pHLA class II molecule were recorded at the MX2 beamline of the Australian Synchrotron. Surface plasmon resonance (SPR) binding analysis was performed and analysed for ten TCRs in total, where the peptide-HLA (pHLA) was the ligand and the TCRs were the analyte. During the return phase the data were analysed and were described in two research articles. The above-mentioned differences in peptide sensitivity can now be evidenced and explained by the binding parameters such as association kinetics, dissociation kinetics, and affinity. Evaluation of the data showed that mouse-derived TCRs and human-derived TCRs show different affinities for the pHLA and the TCRs differ in their kinetics of binding to the pHLA.
