Deep Learning-Based Spatial Immunoprofiling of Multiplex Immunofluorescence Images Distinguishes Tuberculosis Disease States in Diversity Outbred Mice

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis ( M . tb ), remains a major global health challenge, with approximately 10.8 million new cases and 1.25 million deaths reported in 2023. Human responses to M . tb are heterogeneous with clinical outcomes including bacterial clearance, asymptomatic latent M . tb infection, and severe fatal pulmonary TB. Here, we aim to address knowledge gaps in the organization of M . tb granulomas by identifying cell-based spatial features indicating asymptomatic lung infection. To address this gap, which cannot be directly studied in humans, we used lung sections from M . tb infected Diversity Outbred mice with acute pulmonary TB, asymptomatic M . tb infection, or chronic pulmonary TB that were stained for T cells, B cells, macrophages, and bronchiolar epithelial cells by multiplexed immunofluorescence. We first developed a new, accurate model to automatically segment lung granulomas, detect/quantify the cell types within granulomas, and extract the location of immune cells in granulomas for each disease state. Analysis of model-derived results show that lung granulomas from asymptomatic mice have a characteristic spatial profile consisting of higher CD4+ and CD8a+ T cell densities, closer B cell proximity to bronchiolar epithelium, and increased T cell-macrophage proximity in asymptomatic M . tb infection. Next, we propose a second novel approach to utilize a large language model (LLM) to independently decode complex cellular patterns within granulomas, and distinguished key immunological signatures: balanced immune expression in asymptomatic M . tb mice, dysfunctional responses with low cellularity in acute TB, and highest immune cells in chronic TB. Overall, the results from this study show that lung granulomas in asymptomatic infection are characterized by increased T cell density, increased numbers of peribronchiolar B cells, and T cells closer to macrophages as compared to acute and chronic TB. These methods and results help establish an automated pipeline to extract and analyze data from multiplexed fluorescence images and provide a foundation to better understand how granuloma architecture varies by disease state.