Immunotherapy becomes a promising line of treatment for breast cancer (BC) however, its success rate is still limited.MethodsThe study was designed to optimize the condition for producing an effective dendritic cell (DCs) based immunotherapy by using DCs and T lymphocytes together with tumor-infiltrating lymphocytes (TILs) and tumor-infiltrating DCs (TIDCs), treated with anti-PD1 and anti-CTLA4 monoclonal antibodies. This mixture of immune cells was co-cultured with autologous breast cancer cells (BCCs) isolated from 26 BC females.ResultsThere was a significant upregulation of CD86 and CD83 on DCs (p= 0.001 and 0.017, respectively), similarly upregulation of CD8, CD4 and CD103 on T cells (p= 0.031, 0.027, and 0.011, respectively). While there was a significant downregulation of FOXP3 and combined CD25.CD8 expression on regulatory T cells (p= 0.014 for both). Increased CD8/Foxp3 ratio (p< 0.001) was also observed. CD133, CD34 and CD44 were downregulated on BCCs (p= 0.01, 0.021, and 0.015, respectively). There was a significant increase in interferon-γ (IFN-γ,p< 0.001), lactate dehydrogenase (LDH,p= 0.02), and a significant decrease in vascular endothelial growth factor (VEGF,p< 0.001) protein levels. Gene expression of FOXP3 and Programmed cell death ligand 1 (PDL-1) were downregulated in BCCs (p< 0.001, for both), similarly cytotoxic T lymphocyte antigen-4 (CTLA4,p= 0.02), Programmed cell death 1 (PD-1,p< 0.001) and FOXP3 (p< 0.001) were significantly downregulated in T cells.ConclusionEx-vivoactivation of immune cells (DCs, T cells, TIDCs, and TILs) with immune checkpoint inhibitors could produce a potent and effective BC immunotherapy. However, these data should be validated on an experimental animal model to be transferred to the clinical setting.