Mucosal BCG reshapes lung immunity and boosts TB defense in mice

Tuberculosis remains a global challenge, and the only licensed vaccine, Bacille Calmette–Guérin (BCG), gives inconsistent protection against disease. Work in non-human primates has suggested that how BCG is given can matter a lot — for example, intravenous BCG can protect better than the usual intradermal route — but it has been hard to see exactly how different routes reshape lung immunity at the level of individual cells and their spatial organization. Mice are the main system for detailed mechanistic work, yet many studies have focused on one major lung cell type, alveolar macrophages, leaving interstitial macrophages largely unexplored in the vaccination context. To fill that gap, a team led by Aaron James Forde set out to map the cellular and spatial immune landscape in mouse lungs after vaccination. They compared three delivery routes and used approaches that look at both which cells are present and where they sit within lung tissue, aiming to connect route of vaccination to specific immune changes tied to protection.

To define how vaccination route shapes lung immunity, the investigators compared intratracheal (IT), intravenous (IV), and subcutaneous (SC) delivery of Bacille Calmette–Guérin (BCG) in mice. They used flow cytometry to profile immune cell populations, single-cell RNA sequencing to read gene programs in individual cells, and spatial transcriptomics to map those programs back into lung tissue. The study evaluated macrophage responses in particular because lung macrophages are key effectors during Mycobacterium tuberculosis infection and because interstitial macrophage responses to vaccination had been largely unexplored. Across these single-cell and spatial tools, mucosal IT delivery stood out: it uniquely reprograms interstitial macrophages, and it organizes immune cells into spatially distinct hubs that include CD4 T cells. Importantly, the IT route provided superior protection against infectious challenge compared with the other routes tested in the mouse model.

These results point to a previously underappreciated role for interstitial macrophages (IM) as mediators of vaccine-induced protection in the lung. By combining flow cytometry, single-cell RNA sequencing, and spatial transcriptomics, the study shows that not all vaccination routes are equivalent: intratracheal mucosal delivery can both change macrophage states and arrange immune cells into localized hubs with CD4 T cells, changes that correlate with better protection in mice. For researchers and vaccine developers, this work offers a framework for thinking about how to optimize vaccines against respiratory pathogens: targeting mucosal immunity and specifically engaging IM could be a strategic direction. The findings also reinforce the value of high-resolution cellular and spatial methods in revealing which cell types and tissue arrangements underlie protective immune responses.