Understanding the Cellular Ecology of Mtb Granulomas Using Single-Cell Sequencing

Abstract

Infection with Mycobacterium tuberculosis (Mtb) results in the formation of pulmonary granulomas, which are complex structures comprised of variable mixtures of stromal, parenchymal and immune cells. While Mtb granulomas within an individual have variable ability to control bacterial infection, we lack a complete understanding of the immunologic basis for bacterial control. Here, we develop and apply novel methods for high-throughput single-cell mRNA Sequencing (scRNA-Seq) to explore the relationship between the cellular composition of Mtb granulomas and their ability to control bacterial replication. We initially report the development, optimization and application of Seq-Well, a portable, low-cost platform for high-throughput single-cell mRNA sequencing designed for use in BSL3 facilities. We further developed an optimized protocol that significantly improves the per-cell information content of scRNA-Seq data generated with Seq-Well. Using this improved technique, we went on to construct an atlas of multiple inflammatory skin conditions including acne, alopecia, granuloma annulare, leprosy, and psoriasis. We next used Seq-Well to understand the relationship between the cellular and molecular features of Mtb granulomas and bacterial control and performed scRNA-Seq on a total of 26 nonhuman primate granulomas from 4 cynomolgus macaques. In low-burden lesions, we observe an overall expansion of T cells, particularly a group of T1-T17 T cells, while in high-burden lesions, there is an expansion of mast and plasma cells. Using longitudinal PET-CT imaging to track the development of granulomas throughout the course of infection, we observe that lesions that form later in infection are better able to control bacterial infection following the onset of adaptive immunity. Collectively, these data reveal a nuanced relationship between cellular composition, timing of granuloma formation and bacterial control. Finally, we profile bronchoalveolar lavage of rhesus macaques following intravenous-BCG vaccination (IV-BCG), which results in robust protection against subsequent Mtb challenge. Here, we find a correlated expression program that includes genes associated with Th1 and Th17 effector function following IV-BCG vaccination. In summary, we have developed and deployed a novel technique or scRNA-Seq to explore immunologic control in Mtb infection. Our data reveal a high resolution image of T-cell mediated control in the setting of Mtb infection and protective vaccination.