Transcriptomic Characterization of TCR-NK Cells Reveals Challenges Associated with CD3ζ Overexpression

Objective: TCR-engineered NK cells represent a promising strategy for targeting intracellular tumor antigens while overcoming the risk of TCR alpha/beta chain mispairing inherent to TCR-engineered T cells. Previous work showed that CD3ζ overexpression in TCR-NK cells did not improve antigen-specific cytotoxicity and instead increased non-specific background reactivity. This study aims to investigate the molecular alterations associated with this phenotype by examining the transcriptomic profile of engineered NK-92 cells. Materials and Methods: Wild-type and genetically modified NK-92 cell lines expressing a tyrosinase-specific TCR in the presence or absence of CD3ζ overexpression were analyzed. Bulk RNA sequencing was followed by differential gene-expression analysis, hierarchical clustering, multidimensional scaling, and pathway enrichment analysis. CD28 surface expression was assessed by flow cytometry, and activation-associated signaling responses were evaluated by western blotting after CD3/CD28 stimulation. Results: CD3ζ-overexpressing NK-92 cells formed distinct transcriptomic clusters and displayed broader gene expression changes. Expression of CD3δγε alone or in combination with tyrosinase-specific TCR resulted in comparatively limited transcriptional divergence from the empty-vector control, whereas CD3ζ inclusion shifted enriched pathways toward lymphocyte activation, cytotoxicity, and PI3K-AKT-related signaling. CD3ζ- overexpressing cells also exhibited increased CD28 surface expression and altered phosphorylation patterns, most notably involving AKT in response to CD3/CD28 stimulation. Conclusion: CD3ζ overexpression is not a neutral modification in TCR-NK cell engineering. It is associated with broad transcriptional remodeling and activation-associated signaling changes that may provide a molecular context for the previously observed increase in non-specific activity. These findings support TCR-NK engineering strategies that preserve endogenous CD3ζ regulation while introducing only the missing CD3δγε components required for functional TCR expression.