Protocol for analyzing tumor-infiltrating bystander CD8+ T cells in mice using adoptive transfer of P14 TCR-transgenic CD8+ T cells.

While tumor-specific CD8+ T cells become functionally exhausted within the tumor microenvironment (TME), virus-specific bystander memory CD8+ T (TBYS) cells are abundant and represent valuable immunotherapy targets. Here, we present a protocol to analyze tumor-infiltrating virus-specific TBYS cells in murine tumor models using P14 CD8+ T cells. We describe steps for monitoring long-term in vivo behavior of P14 TBYS cells and characterizing their differentiation trajectories and functional heterogeneity. For complete details on the use and execution of this protocol, please refer to Lin et al.1.