AbstractCD8⁺T cells contribute to tuberculosis (TB) infection control by inducing death of infected macrophages. Mycobacterium tuberculosis (Mtb) infection is associated with increased PD-1/PD-L1 expression and alternative activation of macrophages. We aimed to study the role of PD-1 pathway and macrophage polarization on Mtb-specific CD8⁺T cell-induced macrophage death. We observed that both PD-L1 on CD14⁺ cells and PD-1 on CD8⁺T cells were highly expressed at the site of infection in pleurisy TB patients’ effusion samples (PEMC). Moreover, a significant increase in CD8⁺T cells’ Mtb-specific degranulation from TB-PEMC vs. TB-PBMC was observed, which correlated with PD-1 and PDL-1 expression. In an in vitro model, M1 macrophages were more susceptible to Mtb-specific CD8⁺T cells’ cytotoxicity compared to M2a macrophages and involved the transfer of cytolytic effector molecules from CD8⁺T lymphocytes to target cells. Additionally, PD-L1 blocking significantly increased the in vitro Ag-specific CD8⁺T cell cytotoxicity against IFN-γ-activated macrophages but had no effect over cytotoxicity on IL-4 or IL-10-activated macrophages. Interestingly, PD-L1 blocking enhanced Mtb-specific CD8⁺ T cell killing of CD14⁺ cells from human tuberculous pleural effusion samples. Our data indicate that PD-1/PD-L1 pathway modulates antigen-specific cytotoxicity against M1 targets in-vitro and encourage the exploration of checkpoint blockade as new adjuvant for TB therapies.