Transient Transfection and T Cell Activation in the Assessment of Endoplasmic Reticulum Aminopeptidase 1 and 2...

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play a critical role in antigen processing by trimming N-terminally extended peptides within the ER, thereby shaping the repertoire of peptides presented by Major Histocompatibility Complex Class I (MHC I) molecules. Polymorphic variants in these enzymes give rise to functionally distinct allotypes, influencing peptide trimming efficiency and specificity, modulating CD8+ T cell responses in both health and disease. This chapter outlines a cellular model system for assessing ERAP1 and ERAP2 peptide trimming activity, using peptide-specific T cell activation as a surrogate readout. By employing transient transfection and co-culture with either T cell hybridoma (B3Z) or cytotoxic T lymphocytes (CTL), this approach enables the evaluation of peptide processing efficiency of ERAP1/2 based on the presentation and recognition of optimally generated MHC I-restricted epitopes.