Presenting exogenous antigen to T cells

Antigen-processing experiments can be performed with a wide variety of antigen-presenting cells (APCs). An important technical distinction for some types of experiments is whether the APCs are adherent or nonadherent, since this dictates the procedures that must be used to wash the cells as the medium is changed. This unit describes approaches that can be used for adherent cells (e.g., macrophages or fibroblasts) or nonadherent cells (e.g., B lymphoma cell lines), or both, using similar cell numbers. The protocols utilize T hybridomas to detect expression of peptide-MHC complexes, since these cells provide the most convenient, consistent, and flexible T cell readout systems for these purposes. If desired, antigen-specific T cell clones can be used in lieu of T hybridoma cells, but T cell clones often give poorer responses than T hybridomas to fixed APCs due to fixation-induced loss of costimulator function.