PB1170 - High dimensional morphometric analysis of circulating blood cells reveals eosinophils as the predominant H3Cit+ granulocyte population

Background: Coronavirus disease 19 (COVID-19) represents a perfect storm for immunothrombosis leading to severe disease, with release of neutrophil extracellular traps (NETs) being an important contributor now frequently described. NET formation is an active process involving drastic cell biological changes including chromatin decondensation, nuclear membrane disintegration, cytoskeletal rearrangement, and translocation of granule proteins into the nucleus. The histone modification of citrullination promotes this process.

Aims: We aimed to characterize early activation toward extracellular trap formation in circulating granulocytes using high dimensional mass cytometric analysis.

Methods: We designed a custom panel of 42 antibodies to stain peripheral blood collected from COVID-19 patients in ICU at UZ Leuven during the COVID-19 Delta-wave. This panel comprised cell-specific epitopes, surface activation markers, and intracellular staining for NET- associated cell biological changes, including histone citrullination (H3Cit). Samples were run using cytometry by time-of-flight (CyTOF) on a Helios mass cytometer and analyzed using FlowJo software with FlowSOM clustering and tSNE visualisation. Granulocytes were gated as CD11b-intermediate, CD66b-positive cells for further analysis. Eosinophils were distinguished from neutrophils as CD16-negative, CD49d-positive cells. Relative expression of H3Cit as well as morphological changes related to cytoskeletal rearrangement or chromatin decondensation was analyzed.

Results: Nonbiased clustering analysis identified 18 distinct populations of granulocytes, of which 3 were consistent with eosinophil markers. A single population of citrullinated histone- positive cells was also in COVID-19 patients, with higher H3Cit expression than any of the neutrophil populations. This population was also consistent with eosinophils, and also indicated complexation with platelets by CD61-positivity. However, morphometric analysis using scatterbody targets failed to identify cell biological changes common to NET formation in neutrophils, including upon ex vivo stimulation. Challenges in this technique included long incubation times and low cell recovery.

Conclusion(s): High dimensional profiling of circulating blood cells identified eosinophils as the predominant H3Cit+ population in COVID-19 and upon ex vivo stimulation.