Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity

Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45⁺HLA-DR^-CD123⁺). Through CyTOF and fluorescence-activated cell sorting (FACS), we classified these four PBG subpopulations as (I) CD16^lowFcεRI^highCD244^high (88.5 ± 1.2%), (II) CD16^highFcεRI^highCD244^high (9.1 ± 0.4%), (III) CD16^lowFcεRI^lowCD244^low (2.3 ± 1.3), and (IV) CD16^highFcεRI^lowCD244^low (0.4 ± 0.1%). Prospective isolation confirmed basophilic-morphology of PBG I-III, but neutrophilic-morphology of PBG IV. Functional interrogation via IgE-crosslinking or IL-3 stimulation demonstrated that PBG I-II had significant increases in CD203c expression, whereas PBG III-IV remained unchanged compared with media-alone conditions. Thus, PBG III-IV could serve roles in non-IgE-mediated immunity. Our findings offer new perspectives in human basophil heterogeneity and the varying functional potential of these new subsets in health and disease.