Spatial and temporal immune landscapes of non-necrotic granulomas in two murine tuberculosis models with divergent disease outcomes

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major health issue worldwide. Granulomas are characteristic lesions of TB, functioning both as host defense structures and as sites where bacteria persist. To explore immune features associated with bacterial control versus disease progression, we examined the cellular composition and spatial arrangement of non-necrotic granulomas in two mouse models with distinct immune responses and outcomes: Balb/c mice, which partially control infection, and C3HeB/FeJ mice, which develop high bacterial loads and extensive pathology. Using multiplex immunohistochemistry, digital pathology, and spatial analysis, we measured immune cell densities, phenotypic diversity, and cell-to-cell spatial relationships, including median nearest-neighbor distances within granulomas over time. Balb/c granulomas showed higher cell density, greater immune diversity, and increased interactions between CD4+ T cells and macrophages, all of which are markers of effective IFN gamma-dependent macrophage activation. These mice also exhibited early accumulation of Foxp3+ regulatory T cells and IL-10+ cells, followed by controlled infiltration of CD8+ T cells and Ly6G+ neutrophils, correlating with a reduction in lung bacterial burden. Conversely, C3HeB/FeJ granulomas were larger but less dense, dominated early on by Ly6G+ neutrophils, and revealed increased median nearest-neighbor distances between CD4+ T cells and macrophages, despite ongoing immune cell presence and bacterial loads. Our comparison of non-necrotic granulomas across both models indicates that granuloma size alone does not determine bacterial control. Instead, differences in immune cell composition, spatial arrangement, and specific cell interactions distinguish granulomas in mice without evident disease progression from those showing disease progression. These results suggest that spatial immune architecture is a crucial factor influencing the outcome of TB disease.