View Abstract

A1435 - Population Analysis of BAL Cells by Using Mass Cytometry in a Subject Undergoing SBP-AG
Author Block: H. Moon1, S. Kim1, S. J. Ackerman1, V. Natarajan2, G. Park1; 1University of Illinois at Chicago, Chicago, IL, United States, 2Dept of Pharmacology, University of Illinois at Chicago, Chicago, IL, United States.
Rationale: Diverse immune cells are involved in asthma pathogenesis. Upon airway challenge with allergen, a flux of immune and inflammatory cells is actively recruited into the airspaces. Flow cytometry is limited in its ability to analyze this complex cell population. Mass cytometry is a new method based on mass spectrometry using heavy metal-conjugated antibodies to phenotypic biomarkers followed by time-of-flight mass spectrometry. This method overcomes the limitations of flow cytometry in terms of overlapping fluorescent signals, allowing for greater definition of cell populations using an increased number of phenotypic markers. Methods: We exploited the state-of-the-art mass cytometry technique to analyze cell populations in paired BAL samples (before and 48 hours after allergen challenge) from an asthmatic subject who underwent subsegmental bronchoprovocation with allergen (SBP-AG). Total BAL cells were collected and immunostained with a panel of 21 different heavy metal-conjugated antibodies. Samples were analyzed on a CyToF2 (Fluidigm, San Francisco, CA) in the Research Resource Center of the University of Illinois at Chicago. The data were analyzed using SPADE and viSNE (Cytobank, Santa Clara, CA) software packages for unsupervised and density-dependent analysis without input from investigators. Results: A significant shift in airway inflammatory cell populations was observed in BAL fluid following allergen challenge in the SBP-AG protocol. Using the 21 phenotypic markers, we clearly identified T cells, B cells, resident alveolar macrophages, eosinophils, dendritic cells, monocyte-derived alveolar macrophages, and newly recruited macrophages. Furthermore, we could define new clusters of sub-populations within each cell type. For example, classical alveolar macrophages could be further subdivided into classical and atypical alveolar macrophages depending on their expression pattern of surface biomarkers. Even in single sub-population, different phenotypic groups of cells could be distinguished. In addition, we identified a unique cell population co-expressing distinct phenotype markers of two different cell types. These cells have never been reported using flow cytometry because they would likely have been excluded from analysis by the gating process. Conclusion: Mass cytometry is a powerful new tool for analyzing complex inflammatory cell populations in allergic airway responses in the lung, and to identify novel subpopulations and cellular phenotypes. Using this technique, we identified novel cell populations in BAL fluid samples of human allergic asthmatics who underwent the SBP-AG protocol.