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

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.