Optimization of the T2 HLA-A2 shift assay for testing of the biological activity of immunotherapies

Abstract T2 is a lymphoma-derived cell line that express low amounts of HLA-A2 on the cell surface due to TAP deficiency and can only present exogenous peptides. Binding of exogenous peptides to HLA-A2 stabilizes the HLA-A2- peptide complexes and can be detected using immunofluorescence staining. The T2 cell line is commonly used for identification and analysis of novel HLA-A2-restricted CTL epitopes in the assay known as T2 shift. In this study we evaluated parameters that may influence the T2 shift assay performance and considered new applications of this method for testing of the biological activity of peptide-based immunotherapies. Method optimization was performed using HLA-A2-restricted peptide SurA2.M alone and in the context of a peptide-based immunotherapy drug, DPX-Survivac. DPX-Survivac is a lipid nanoparticle-based formulation that contains five peptides aiming to elicit a CTL response against tumor cells presenting the survivin peptides on HLA class I molecules. Results indicated that performance of the T2 shift assay was highly sensitive to cell passaging intervals, pH variations, cell concentrations and β2-microglobulin concentrations. Discrimination of the T2 cells by either size or granularity did not affect the results for the T2 shift assay. We determined the linear response range for the SurA2.M peptide to be 30-110 μg/mL in the T2 shift assay. The T2 shift assay can detect SurA2.M within the DPX-Survivac matrix. In conclusion, we found that optimal culturing conditions of T2 cells are critical for the T2 shift assay reproducibility and linearity and should be carefully considered if the assay is to be used for quantitative or semi-quantitative evaluation of the peptide activity alone or as a part of an immunotherapeutic drug.