Infection dose shapes granuloma phenotypes in a humanized mouse model of tuberculosis

Tuberculosis (TB) remains a leading cause of infectious disease mortality worldwide, yet current murine models fail to fully recapitulate human granuloma biology. Here, we utilized hCD34 + NCG humanized mice (humouse) to investigate how infection dose shapes granuloma formation and disease progression following aerosol infection with Mtb CDC1551. Humouse were infected with either low-dose (10³ CFU) or high-dose (10⁶ CFU) Mtb and compared with conventional C57BL/6 mice. High-dose infection induced significant weight loss, severe pulmonary pathology, and the formation of compact, highly cellular granulomas containing multinucleated giant cells, closely resembling human TB lesions. In contrast, low-dose infection produced sparse organized granulomas with reduced pathology and delayed immune recruitment. Granuloma phenotypes were independent of reconstitution efficiency, supporting the robustness of the model. Progressive disease was associated with depletion of hCD8 + T cells and hNKT cells, mirroring immune signatures observed in human TB. Low-dose infection promoted distinct immunoregulatory responses characterized by pulmonary MIP-3α, IL-17A, and IL-15 signaling together with systemic IL-10 induction. IHC and RNAscope analyses revealed dose-dependent macrophage accumulation and actively replicating bacilli within granulomatous lesions. Collectively, these findings demonstrate that infection dose is a major determinant of granuloma heterogeneity in the humouse model.