Multimodal delineation of a layer of effector function among exhausted CD8 T cells in tumors

Undescribed functional axes may intersect with the trajectory of T cell exhaustion (T_EX) to contribute to the antitumoral functions of CD8 T cells. By leveraging fluorescent transcriptional reporting of the T cell activation marker Cd69, we defined a classifier for potent versus suboptimal CD69⁺ activation states arising from T cell stimulation. In tumors, this delineation provided an additional functional readout among T_EX subsets, marked by enhanced effector molecule production. The more potent Cd69-TFP^hi state was the most prominent in a T cell-mediated tumor clearance model, displaying increased engagement and superior tumor cell killing. Simultaneous analysis of gene and protein expression in human head and neck tumors enabled a similar strategy to identify Cd69RNA^hiCD69⁺ cells with enhanced functional features compared with Cd69RNA^loCD69⁺ cells among intratumoral CD8 T cell subsets. Thus, refining the T cell functional landscape in tumors potentiates the identification of rare, potent effectors that could be leveraged for improving cancer treatment.