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Background Anti-tumor immunity is deficient in aggressive triple-negative breast cancers (TNBC). Impaired antigen presentation, driven by reduced number and function of cross-presenting dendritic cells (cDC1s), is a major barrier to effective T cell-mediated immunity. Flt3 ligand (Flt3L) expands cDC1s, CD40 agonists (CD40a) activate antigen-presenting cells, and anthracycline chemotherapy promotes immunogenic tumor cell death. We hypothesized that combining these agents would enhance anti-tumor immunity.Methods EO771, AT3, or 4T1 TNBC cells were implanted orthotopically in female mice. Triplet therapy consisted of Doxil D1, Flt3L D1-5, and CD40a D11, 14, 17 after tumors reached 50 mm 3. Tumor growth and survival were recorded thrice weekly. Immune profiling was performed with flow cytometry, single-cell RNA sequencing (scRNA-seq), and bulk TCR sequencing (TCR-seq). Mechanistic dependence was tested by depleting CD8+, CD4+, and Foxp3+ regulatory T cells (Tregs) as well as by blocking lymphocyte egress with FTY720.Results Triplet therapy significantly delayed tumor progression and extended median survival compared to chemotherapy alone or doublet combinations. Compared to chemotherapy alone or doublet combinations, triplet therapy markedly increased intra-tumoral CD8+ T cell infiltration. Enhanced CD8 infiltration was accompanied by reduced expression of exhaustion markers (LAG-3 and TIM-3) and elevated activation, as evidenced by increased PD-1 single-positive T cells. scRNA-seq and TCR-seq of intra-tumoral T cells further demonstrated a reduction in exhausted CD8+ T cells and clonal expansion of T cells. Notably, blockade of lymphocyte egress using FTY720 did not compromise therapeutic efficacy, suggesting that pre-existing intra-tumoral T cells are sufficient to mediate the anti-tumor response. CD8+ T cell depletion partially attenuated the therapeutic effect, implying a contributory role of other immune effector cells. Co-depletion with CD4+ T cells completed eradicated effect. Interestingly, depletion of CD4+ T cells alone or of Tregs significantly enhanced treatment efficacy resulting in tumor free mice, indicating that Tregs limit the full therapeutic potential of the triplet regimen.Conclusions CD40a-based triplet therapy induces a potent and localized T cell-driven anti-tumor response, characterized by reduced exhaustion, enhanced stem-like properties, and selective clonal expansion of T cells. These findings have led to a first-in-human clinical trial in metastatic breast cancer ( NCT05029999), establishing a foundation for future combinatorial immunotherapies.