Neoadjuvant chemotherapy remains the standard of care for high-risk or inoperable breast cancer (BC) tumors, yet its success rate falls below 50%, highlighting the need for better strategies. T cell-based therapies, involving ex vivo expansion of patients' T lymphocytes, have emerged as a promising alternative, particularly for chemotherapy-resistant tumors, but their efficacy is often limited. Building on our previous findings that the antigen-presenting molecule, HLA-DR, is crucial in tumor infiltrating cytotoxic T lymphocytes (CTLs) for a favorable response to neoadjuvant chemotherapy, we further investigated the role of HLA-DR-expressing CTLs in anti-tumor responses and evaluated strategies to amplify these cells.

To elucidate the role of HLA-DR in improving CTL-mediated tumor elimination, we conducted in vitro and in vivo experiments using zebrafish xenograft models. Then, aiming at increasing the population of HLA-DR-expressing CTLs, we optimized an ex vivo stimulation protocol using cells isolated from peripheral blood. Additionally, we tested the potential of different immune agents to boost their cytotoxicity against BC cells. This study included 209 BC patients, from whom both blood and tumor biopsy samples were collected, and 32 healthy donors who provided blood samples. These samples were used across all in vitro assays, including CTLs expansion, functional evaluation, and screening experiments.

Our findings revealed that only CTLs expressing HLA-DR effectively eliminate tumor cells. Moreover, CD4⁺ T cells were identified as essential drivers of CTLs activation and cytotoxicity. Indeed, blocking HLA-DR or depleting CD4+ T cells impaired CTLs function, underscoring the importance of this interaction for therapeutic success. Notably, our study revealed that short-term stimulation increased HLA-DR expression while preserving CTLs functionality, whereas prolonged expansion diminished their effectiveness, underscoring the importance of prioritizing cell quality over quantity. Furthermore, we demonstrated that PD-1 blockade with nivolumab further upregulated HLA-DR levels, amplifying CTL-mediated tumor cell killing.

Overall, our results emphasize the critical role of HLA-DR in T cell-based therapies and highlight the synergistic benefits of ex-vivo PD-1 treatment along with short-term stimulation in enhancing CTLs efficacy. These insights pave the way for improved immunotherapeutic strategies for chemotherapy-resistant BC.