Spatial Transcriptomics of Granuloma Microenvironments in Pulmonary Tuberculosis Resection Specimens from Kano State: Decoding Cellular Topography and Immunological Zonation

The tuberculosis granuloma is a structured multicellular immunological lesion whose spatial architecture dictates bacterial control, necrosis progression, and therapeutic drug penetration. Bulk transcriptomic analysis of TB granuloma tissue fails to resolve the spatially distinct gene expression programs operative in granuloma zones (cellular, necrotic, fibrous rim). Spatial transcriptomics enables gene expression profiling with preservation of tissue spatial coordinates, providing unprecedented resolution of granuloma immunological zonation. This dissertation presents spatial transcriptomic analysis of pulmonary TB granulomas from resection specimens of treatment-refractory patients at Aminu Kano Teaching Hospital, Kano State, Northwest Nigeria. Lung resection specimens from 15 patients with confirmed drug-resistant TB were sectioned at 10 micrometers for 10x Visium spatial gene expression library preparation. Sequencing was performed on Illumina NovaSeq, and data were analyzed using Seurat v4.3 with spatially variable gene detection by SPARK-X. Cell deconvolution was performed using RCTD with a comprehensive single-cell TB reference atlas. Multi-lineage M. tuberculosis strain diversity within individual granuloma sections was assessed by ultra-deep targeted amplicon sequencing of multi-copy genomic loci. Spatial transcriptomics identified 7 transcriptionally and spatially distinct granuloma niches, including a novel immunosuppressive LAP-TGF-beta signaling zone at the fibrous rim-cellular granuloma interface not previously described in human TB tissue. M2-polarized macrophage signatures were enriched in the peri-necrotic zone, while cytotoxic T-cell gene signatures clustered at the cellular rim. Intra-granuloma M. tuberculosis strain heterogeneity was confirmed in 40% of specimens, implicating within-granuloma evolutionary diversification as a potential treatment failure mechanism. These findings advance TB granuloma biology with original spatial resolution data from a West African clinical cohort. Keywords: spatial transcriptomics, tuberculosis granuloma, immunological zonation, drug-resistant TB, Kano State.