CRISPR/Cas9‐Mediated Gene Knockout Reveals a Nonredundant Role for p16 ^INK4A in Controlling TCR‐Dependent and Independent CD8 T Cell Expansion

ABSTRACT

The possibility of enhancing T cell function by deleting specific genes represents a long‐sought goal in preclinical studies and ultimately for clinical applications. Using CRISPR/Cas9 genome editing, we report that, in human cytotoxic CD8 T cell clones, the cell cycle checkpoint gene CDKN2A , encoding p16 ^INK4A , plays a nonredundant role in controlling T cell receptor (TCR)‐dependent and independent cell expansion. Deletion of CDKN2A dramatically enhanced antigen‐driven and homeostatic proliferation, while preserving effector functions. In contrast, the deletion of other cell cycle inhibitors ( CDKN1B , CDKN2C , and CDKN2D ), alone or in combination, had no impact on T cell proliferation. We also report that mediator complex subunit 12 (MED12) and the E3 ubiquitin ligase CBL‐B deletions did not affect proliferative capacity of CD8 T cell clones. Interestingly, deletion of the negative regulator of Ras signaling, RASA2, increased antigen sensitivity and cytotoxic activity, while not improving in vitro expansion. Collectively, these findings reveal a unique and critical nonredundant role for p16 ^INK4A in regulating CD8 T cells. Deletion of CDKN2A offers a promising strategy to enhance CD8 T cell expansion ex vivo, thereby improving TCR discovery pipelines and, potentially, therapeutic applications.