Methodology for in vitro Assessment of Human T Cell Activation and Blockade

Methods to test both the functionality and mechanism of action for human recombinant proteins and antibodies in vitro have been limited by multiple factors. To test the functionality of a recombinant protein or antibody, the receptor, the receptor-associated ligand, or both must be expressed by the cells present within the in vitro culture. While the use of transfected cell lines can circumvent this gap, the use of transfected cell lines does not allow for studying the native signaling pathway(s) modulated by the specific recombinant protein or antibody in primary cells. The present protocol utilizes sort purified CD14⁺ monocytes and T cells, both CD4⁺ T cells and CD8⁺ T cells, from healthy donors in a co-culture system. This methodology is particularly relevant for testing recombinant proteins or antibodies that are putative therapeutics for the treatment of autoimmune disease and cancer. While the current protocol focuses on co-cultures containing B7-H4 expressing monocytes plus either autologous CD4⁺ T cells or CD8⁺ T cells, the protocol can be modified for the user’s specific needs.