Hypoxia restricts the generation of BCG-responsive polycytotoxic T cells.

The Bacillus Calmette-Guérin (BCG) vaccine protects infants from severe tuberculosis by eliciting cellular immune responses, particularly through the activation of CD8+ cytotoxic T lymphocytes. Given that hypoxia is a defining feature of the pulmonary microenvironment during Mycobacterium tuberculosis infection, especially within granulomatous lesions, we investigated how limited oxygen availability influences the expansion and effector functions of BCG-induced cytotoxic T cells. Mycobacteria-specific short-term T-cell lines were generated and restimulated with BCG-infected macrophages under normoxic and hypoxic conditions. To analyze the immune response, Boolean gating was applied to flow cytometry data to compare the frequencies of cytotoxic T-cell subsets within BCG-responsive CD8+ T-cell population. Under normoxia, BCG-responsive CD8+ T cells exhibited elevated expression of perforin, granulysin, and granzyme B, with a significant enrichment of polycytotoxic cells co-expressing all three molecules. Hypoxia differentially modulated these markers: perforin expression remained unchanged, granulysin was substantially increased, whereas granzyme B declined, resulting in a net reduction of polycytotoxic T cells. These findings demonstrate that oxygen availability shapes the functional profile of vaccine-induced cytotoxic T cells. Considering the hypoxic nature of inflamed and infected lung tissue, our results highlight the importance of evaluating vaccine efficacy in physiologically relevant microenvironments and may inform strategies to enhance polycytotoxic T-cell responses for improved tuberculosis protection.