Lung mucosal and systemic responses at single-cell resolution in an aerosolised Mycobacterium bovis BCG human challenge model

The development of an effective vaccine against tuberculosis (TB) is hindered by the limited understanding of the dynamics of cellular responses to Mycobacterium tuberculosis infection in humans. Here, in our aerosolised Mycobacterium bovis BCG human challenge study, we used single-cell and bulk immune profiling of bronchoalveolar lavage and blood samples to temporally resolve the dynamics of immune responses in BCG-naïve individuals. We observed a rapid change in cellular composition and gene expression in both the lung mucosa and blood. TB-specific gene signatures were observed on days 2 and 7 following aerosol challenge. T cell receptor (TCR) clones that expanded in the blood seven days post-challenge (BCG-expanded TCR clones) persisted in the lung mucosa for up to 56 days post-challenge, with most of these detectable in the blood prior to challenge. Pre-existing BCG-expanded TCR clones were more likely to be activated CD4⁺ T cells and were more enriched in the lung mucosa than newly expanded TCR clones. Only a small proportion of mycobacterial antigen-reactive TCR clones in the blood, detected by the activation-induced marker assay, were enriched in the lung mucosa. Several public BCG-expanded TCR clones across individuals were identified in the lung mucosa and shown to be reactive to mycobacterial antigens using artificial antigen-presenting cells and reporter T cells. Overall, our study provides crucial insights into the dynamics of human mucosal and systemic immunity following aerosolised BCG challenge, informing the design of more effective TB vaccines.