Generation of human and murine exhausted CD8⁺ T cells in vitro

T cell exhaustion is a state of dysfunction that can occur due to persistent exposure to antigens, such as in the tumor microenvironment. The progressive loss of effector functions in exhausted T cells can lead to resistance to immune checkpoint inhibitors and adoptive cell immunotherapies. Improving our understanding of the exhaustion process is thus crucial for optimizing the clinical outcomes of immunotherapy. A significant hurdle in this area is obtaining an adequate quantity of exhausted T cells. One solution could be the in vitro production of exhausted T cells by mimicking exhaustion-induced conditions. We present a simple, repeatable, flow cytometry-assisted method for generating exhausted CD8⁺ T cells from both human and mouse sources. This flexible protocol works with various cell sources and activation methods. Our results confirm the production of dysfunctional CD8⁺ T cells, akin to those in mouse tumor models and patient tumor samples. This methodology could help identify genes involved in the exhaustion process and serve as a platform for finding agents capable of altering, reversing, or accelerating this dysfunctional state. By using both mouse and human models, we increase the adaptability of the method, making it a powerful instrument for assessing potential substances with immunotherapeutic utility.