Single-cell transcriptomics identifies multiple pathways underlying antitumor function of TCR- and CD8αβ-engineered...

Transgenic coexpression of a class I-restricted tumor antigen-specific T cell receptor (TCR) and CD8αβ (TCR8) redirects antigen specificity of CD4⁺ T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4⁺ T cells recognize target cells, but the transcriptional basis for their acquired antitumor function remains elusive. We, therefore, interrogated redirected human CD4⁺ and CD8⁺ T cells by single-cell RNA sequencing and characterized them experimentally in bulk and single-cell assays and a mouse xenograft model. TCR8 expression enhanced CD8⁺ T cell function and preserved less differentiated CD4⁺ and CD8⁺ T cells after tumor challenge. TCR8⁺CD4⁺ T cells were most potent by activating multiple transcriptional programs associated with enhanced antitumor function. We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation- and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.