Cholesterol 25‐Hydroxylase Deficiency Provides an Optimal and Durable Protective T Helper 1 Immunity via Interleukin‐12–Interleukin‐2 Axis in BCG‐Vaccinated Mice

ABSTRACT Bacillus Calmette‐Guérin (BCG) remains the only licensed vaccine for tuberculosis (TB), yet its efficacy wanes over time due to insufficient durability of vaccine‐induced immunity. However, mechanisms constraining BCG vaccine‐induced dendritic cell (DC)‐mediated T cell priming are poorly understood. Cholesterol 25‐hydroxylase (CH25H) converts cholesterol into 25‐hydroxycholesterol (25‐HC) that modulates antigen‐presenting cell imprinting and innate immune responses. Here, we investigated whether CH25H regulates DC function and vaccine‐induced protective immunity against Mycobacterium tuberculosis (Mtb). We found that CH25H deficiency enhanced DC activation, characterized by increased expression of the costimulatory molecules CD80 and CD86, elevated interleukin (IL)‐12p70 production, and reduced IL‐10 secretion, resulting in augmented T cell proliferation and robust Th1 cytokine responses. Transcriptomic analyses of CH25H‐deficient and 25‐HC‐treated DCs showed that an SREBP2‐associated program may contribute to Il12a expression. BCG vaccination conferred enhanced protection in Ch25h −/− mice, including reduced Mtb burden and pulmonary inflammation. BCG‐vaccinated Ch25h −/− mice developed more durable protective immunity and increased frequencies of CD103 + CD69 + tissue‐resident memory T cells, along with sustained IL‐2‐producing T cell responses. Collectively, these findings identify CH25H as a negative regulator of DC‐mediated Th1 immunity and suggest that targeting CH25H represents a potential host‐directed immunomodulatory strategy to enhance the long‐term protective immunity of TB vaccination.