Multimodal profiling of Her2/neu-specific TCR-T cells reveals clonotype-dependent activation programs underlying differential cytotoxic efficacy

Here, we comprehensively profile three Her2/neu-specific TCR constructs using integrated multimodal analysis that combines cytotoxicity assays, single-cell multiomics, bulk transcriptomics, cytokine profiling, and in vivo tumor modeling.

Despite identical antigen specificity, the constructs exhibit a functional hierarchy: Construct #3 displays potent cytotoxicity in vitro and potent tumor regression in vivo, whereas Constructs #1 and #2 were less effective. Single-cell proteotranscriptomic analysis shows that tumor cell encounter drives activation of cytotoxic CD8⁺ T effector cells and transdifferentiation of NKT cells into APC-like cells. Interactomics also revealed high communication probability between APC-like NKT cells and CD4⁺ T cells, potentially forming a cooperative activation network that sustains effector function.