Abstract

A key challenge in improving T cell-mediated immunotherapies is defining the factors that regulate functional versus exhausted T cell fates. Through multi-round in vivo CRISPR screens in chronic lymphocytic choriomeningitis virus Clone 13 (LCMV Cl13) infection and transcription factor (TF) benchmarking, we identified Krüppel-like factor 2 (KLF2) as a top TF driving CX3CR1 + effector-like exhausted cell (Tex eff-like ) differentiation. Overexpression of KLF2 converted CX3CR1⁻ cells into Tex eff-like cells by direct engagement of key loci. Conversely, loss of KLF2 increased inhibitory receptor expression and redirected cells toward terminal exhaustion. However, early after infection, KLF2 deficiency yielded increased CD8 + T cell accumulation and improved viral control. This effect was, in part, mediated by TOX and improved T cell localization with dendritic cells. Additional deletion of PD-1 further enhanced viral control but induced severe immunopathology. Collectively, these findings identify KLF2 as a central regulator of the Tex eff-like program and underscore exhaustion features as checkpoints balancing antiviral immunity and immunopathology.