Abstract
This protocol uses the Tg4 Nr4a3-Tocky mouse model to recalibrate T cell activation thresholds and reveals the role that immune checkpoints play in controlling T cell activation. The example approach here uses flow cytometry to characterize quantitative and qualitative changes in splenic CD4+ T cells reactivated in the presence of anti-PD1 immunotherapy. The protocol is optimized for studying anti-PD1 pathway blockade only. The protocol is not compatible with cellular fixation, and T cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Elliot et al. (2021).