<p>The central challenge in triple-negative breast cancer (TNBC) immunotherapy is to identify novel mechanism-derived strategies for anti-programmed death-1 (PD-1) resistance and efficiently assess their efficacy and safety in humans. Understanding the intricate heterogeneity of the tumor microenvironment and its impact on treatment could guide the initiation of proof-of-concept clinical trials. Here, integrating single-cell transcriptome of 44 treatment-naive patients with TNBC, we unveiled an association between intrapatient mast cell heterogeneity and clinical benefit of PD-1 blockade. Upon independent parallel validation in 484 patients with TNBC, high levels of breast tissue antigen-presenting mast cells (apMCs) were associated with enhanced anti-PD-1 efficacy. Mechanistically, apMCs largely located within tertiary lymphoid structures and were efficient in performing presentation and cross-presentation of antigens and expressed co-stimulatory molecules. Conditional deletion of antigen-presenting machinery in mast cells dampened tumor-reactive T cells. A widely prescribed allergy medication, cromolyn, was identified to mobilize apMC-mediated T cell immunity and sensitize tumors to PD-1 blockade. We subsequently initiated a phase 2 clinical trial in female patients with anti-PD-1-refractory metastatic TNBC. Here we report the results of the cromolyn arm (cromolyn plus anti-PD-1 backbone). The prespecified primary endpoint of this arm was met, with a confirmed objective response rate of 50.0%. Our study defines a crucial role of mast cells in cancer immune control, identifies an apMC-directed approach to overcome anti-PD-1 resistance and highlights a reverse-translational framework that offers conceptual advances in precision immuno-oncology with direct implications for clinical therapy. ClinicalTrials.gov identifier: <a href="https://clinicaltrials.gov/study/NCT05076682?tab=table">NCT05076682</a>.</p>