Project Summary/Abstract Understanding and eliminating TB depends on understanding the host and pathogen dynamics in granulomas, where the outcomes of M. tuberculosis infection are determined. Despite substantial efforts, the cellular composition, spatial interactions, and mechanisms that determine outcomes such as pathologic granuloma necrosis are poorly understood. One explanation for the limited understanding of granulomas is that most studies are performed in a single experimental system in isolation. We have formed a consortium to perform and integrate studies of TB granulomas in three systems: humans, rhesus macaques, and new strains of mice. In these three systems, we will address several major questions in TB granuloma biology: what is the extent of diversity in cell composition in TB granulomas? What regulates cell trafficking and spatial interactions in TB granulomas? What are the roles of type I interferons (TIIFN) and interferon gamma (IFNg) in regulating and determining cell trafficking, differentiation, spatial relationships, and activation states in TB granulomas? What are the pathogen and host determinants of necrosis, a pathologic outcome, in TB granulomas? Our primary mode for investigating these and other questions will be high-parameter multiplex immunostaining of TB granulomas from the three species, as this will provide essential insight into the spatial relationships between immune cell subsets. Multiplex immunostaining will be complemented by studies of live imaging of cell dynamics in rhesus macaque granulomas and by strategically-timed transfers of labeled cells in rhesus macaques and diverse strains of mice. The value of the multiplex immunostaining studies will be further enhanced by deep characterization of dissociated cell populations at the single-cell level, using high parameter flow cytometry and single-cell transcriptomics. A uniquely important contribution of our proposed project is the computational integration of data from humans, rhesus macaques, and new strains of mice. By integrating multispecies data, we will identify the features of TB granulomas that are common to all three species, and we will identify factors that are unique to granulomas in each species, to guide further modifications to improve the utility of studies in animal models. Integration of the data from the three species will allow studies in rhesus macaques to be compared with those of human samples and new strains of mice; this will improve the understanding and interpretation of human pathology studies, and enhance the value of studies that can take advantage of using the unique advantages of mice for mechanistic studies. Together, our studies will advance the knowledge and understanding of TB biology, and guide development of interventions to improve treatment outcomes, limit lung damage, and improve lung tissue repair in people suffering from TB.

Project Narrative Granulomas are the sites where M. tuberculosis and host cells interact, yet despite the importance of granulomas in TB, they are poorly understood. In this project, we employ the strengths of studies in 3 species, including humans, rhesus macaques, and new strains of mice, to understand the cell composition, spatial interactions, movements, and functional states in TB granulomas.