Multimodal profiling of HER2/neu-specific TCR-T cells reveals clonotype-dependent activation programs underlying differential cytotoxic efficacy

Adoptive T cell therapy targeting tumor-associated antigens offers a promising avenue for cancer immunotherapy, yet the mechanisms underlying functional heterogeneity among T cell receptors recognizing the same antigen remain poorly understood. Here, we comprehensively profile three HER2/neu-specific T cell receptor constructs using integrated multimodal analysis that combines in vitro 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 displayed 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. In silico interactomics also revealed a high probability of communication between APC-like NKT cells and CD4⁺ T cells, potentially forming a cooperative activation network that sustains effector function. Bulk transcriptomic and secretomic profiling showed that Construct #3 couples cytolytic gene expression with the secretion of anti-tumor cytokines and effector molecules. Together, these findings propose a mechanistic link between anti-HER2/neu T cell receptor-driven transcriptional programming and therapeutic efficacy, illustrating how multi-omic approaches can inform the rational design of potent T cell receptor-based therapies.