key: cord-0306362-0mllx6x0 authors: Shanthikumar, Shivanthan; Ranganathan, Sarath C.; Saffery, Richard; Neeland, Melanie R. title: Application of high dimensional flow cytometry and unsupervised analysis to define the immune cell landscape of early childhood respiratory and blood compartments date: 2021-03-22 journal: bioRxiv DOI: 10.1101/2021.03.21.436363 sha: 72d0ec41e46b20e434c441168523010f76e7796b doc_id: 306362 cord_uid: 0mllx6x0 The cellular landscape of the paediatric respiratory system remains largely uncharacterised and as a result, the mechanisms of highly prevalent childhood respiratory diseases remain poorly understood. A major limitation in defining mechanisms of disease has been the availability of tissue samples collected in early life, as well as technologies that permit deep immune analysis from limited sample volumes. In this work, we developed new experimental methods and applied unsupervised analytical tools to profile the local (bronchoalveolar lavage) and systemic (whole blood) immune response in childhood respiratory disease. We quantified and comprehensively phenotyped immune cell populations across blood and lung compartments in young children (under 6 years of age), showed that inflammatory cells in the BAL express higher levels of activation and migration markers relative to their systemic counterparts, and applied new analytical tools to reveal novel tissue-resident macrophage and infiltrating monocyte populations in the paediatric lung. To our knowledge, this is the first description of the use of these methods for paediatric respiratory samples. Combined with matched analysis of the systemic immune cell profile, the application of these pipelines will increase our understanding of childhood lung disease with potential to identify clinically relevant disease biomarkers. Whilst the first to report detailed immune cell frequencies from childhood lung samples, there 76 were several limitations of this work, including use of cryopreserved samples (resulting in 77 incomplete phenotyping of granulocytes), as well as limited unsupervised analyses. In the present study, we sought to provide advanced experimental and analytical methods for at 300 x g for 10 mins at 4°C. Supernatant was discarded and the cell pellet resuspended in 106 PBS for viability staining using near infra-red viability dye according to manufacturers' 107 instructions. Following blood collection, 100 µl of EDTA whole blood was aliquoted for flow 108 cytometry analysis and lysed with 1mL of red cell lysis buffer for 10 mins at room temperature. Cells were washed with 1mL PBS and centrifuged at 400 x g for 5 mins. Following another 110 wash, cells were resuspended in PBS for viability staining using near infra-red viability dye 111 according to manufacturers' instructions. For both BAL and whole blood samples, the viability 112 dye reaction was stopped by the addition of FACS buffer (2% heat-inactivated FCS in 2mM 113 EDTA) and cells were centrifuged at 400 x g for 5 mins. Cells were then resuspended in human Table 1) . 137 Matched BAL and blood samples from 11 children with CF aged between 1-5 years were used 138 in this study (Table 1) Figures 3-4) . As we generated flow cytometry data on matched lung and blood samples from every 204 individual, we next explored the relationship between immune cell profiles within and across 205 the two compartments. As can be seen in Figure 1G , granulocytes from BAL expressed higher 206 levels of CD66b, CD11b and CD15 relative to the equivalent granulocyte population in whole 207 blood. Similarly, CD16 + monocytes from BAL expressed high levels of CD206, whilst the 208 CD16 + monocytes in blood do not express this marker ( Figure 1G ). The heatmap in Figure 1H There are several limitations related to these data that must be acknowledged. 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