A multipronged Tα1 reset of CD8+ T cell cytotoxicity against breast cancer.

Thymosin α1 (Tα1) is an endogenous thymic peptide that enhances immune competence through activation of T cells, dendritic cells, and innate immune pathways. However, its direct impact on CD8+ T cell-mediated antitumor immunity in breast cancer remains unclear. In this study, CD8+ T cells isolated from peripheral blood of ten healthy donors were cultured under unstimulated, CD3/CD28-stimulated, Tα1-treated, or exhaustion-rescue conditions to evaluate cytotoxic activity against MDA-MB-231 breast cancer cells and CD44+ cancer stem-like cells (CD44+ CSC-like cells). Tα1 significantly enhanced CD8+ T cell-mediated apoptosis, suppressed tumor cell proliferation, and increased granzyme B secretion beyond CD3/CD28 stimulation alone. In exhausted T cells, Tα1 partially restored effector function and reduced PD-1, TIM-3, and LAG-3 expression. Complementary transcriptomic analysis using a compact four-gene Tα1 Response Index (Tα1-RI: TLR9, TLR2, IRF1, NLRC5) in TCGA-BRCA (n = 1,112) confirmed positive correlations with antigen presentation and cytotoxic programs and enrichment in CD8-like T cells in single-cell datasets. Collectively, these findings demonstrate that Tα1 enhances CD8+ T cell cytotoxicity while alleviating exhaustion, supporting its potential as an adjunct immunomodulator for improving immune surveillance in breast cancer.