In vivo single-cell high-dimensional mass cytometry analysis to track the interaction between Klebsiella pneumoniae and myeloid cells.

In vivo single-cell approaches have transformed our understanding of the immune populations in tissues.  Mass cytometry (CyTOF), that combines the resolution of mass spectrometry with the ability to conduct multiplexed measurements of cell molecules at the single cell resolution, has enabled to resolve the diversity of immune cell subsets, and their heterogeneous functionality. Here we assess the feasibility of taking CyTOF one step further to immuno profile cells while tracking their interaction with bacteria, a method we term Bac-CyTOF. We focus on the pathogen Klebsiella pneumoniae interrogating the pneumonia mouse model. Using Bac-CyTOF, we unveil the atlas of immune cells of mice infected with a K. pneumoniae virulent strain. The atlas is characterized by a decrease in the populations of alveolar macrophages and monocyte-derived macrophages, whereas neutrophils, and inflammatory monocytes are characterized by an increase in the subpopulations expressing markers characteristic of less active cells such as the immune checkpoint PD-L1. These cells are those with associated bacteria. We show that the type VI secretion system (T6SS) contributes to shape the lung immune landscape. The T6SS governs the interaction with monocytes/macrophages by shifting Klebsiella from alveolar macrophages to interstitial macrophages and limiting the infection of inflammatory monocytes. Lack of T6SS results in cells expressing markers of active cells, and a decrease in the subpopulations expressing PD-L1. By infecting with Klebsiella, and Acinetobacter baumannii strains cleared by mice, we uncover that a heightened recruitment of neutrophils, and relative high levels of alveolar macrophages and eosinophils and the recruitment of a characteristic subpopulation of neutrophils are features of infected mice clearing infections. Lastly, we leverage Bac-CyTOF-generated knowledge platform to investigate the role of the DNA sensor STING in Klebsiella infections. sting-/- infected mice present features of mice clearing the infection including the reduced levels of PD-L1. STING absence facilitates Klebsiella clearance. ### Competing Interest Statement The authors have declared no competing interest.