Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity

Abstract

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.

Keywords: Immunology; Systems Biology.

Graphical abstract

Figure 1

Mass Cytometry Analysis Reveals Four Phenotypically Basophilic Granulocyte Subpopulations Identified by Traditional CD45⁺HLA-DR⁻CD123⁺ Gating (A) Gating strategy plots for total phenotypically basophilic granulocytes (PBGs, CD45⁺HLA-DR^-CD123⁺), eosinophils (CD45⁺HLA-DR⁻CD123⁻CD14⁻CD66b⁺CD193⁺Siglec8⁺), and neutrophils (CD45⁺HLA-DR^-CD123⁻CD14⁻CD66b⁺CD16⁺) using classic lineage markers. (B) Implementation of Leiden clustering algorithm using the following markers: CD123, CD16, FcεRI, CD244, CD53, CD305, and CD193. (C) Four donors' mean median expression (arcsinh with cofactor 5 transformation) across clusters identified by the Leiden algorithm, hierarchically clustered by protein expression and by Leiden clusters. (D) Normalized single cell expression across of the following proteins: CD123, CD16, FcεRI, and CD244. (E) CD16, FcεRI, and CD244 used to manually draw gates in biaxial plots to distinguish the four PBG subpopulations. (F) The four-gated PBG subpopulations superimposed on the original tSNE plot coordinates. See also Figures S1 and S2.

Figure 2

Characterization of Four Phenotypically Basophilic Granulocyte Subpopulations across Donors (A) Four donors' mean median expression (arcsinh with cofactor 5 transformation) for CD123, CD16, FcεRI, and CD244 shown across basophil subpopulations. (B) Plot indicating mean abundance and standard error of each cluster in the “classic” basophil gate across female and male blood donors. There were not any statistically significant differences between female and male basophil subpopulation abundances. See also Table S2.

Figure 3

Three Out of Four Phenotypically Basophilic Granulocyte Subpopulations Are Morphologically Basophils (A) The CD45⁺HLA-DR⁻CD123⁺ gate traditionally assigned as basophils was analyzed for CD16 and split into high and low CD16 levels. These two CD16 sub-gates were then analyzed for CD244 and FcεRI levels. Subpopulations were identified as follows: subpopulation I as CD16^lowCD244^highFcεRI^high, subpopulation II as CD16^highCD244^highFcεRI^high, subpopulation III as CD16^lowCD244^lowFcεRI^low, and subpopulation IV as CD16^highCD244^lowFcεRI^low. (B) Cytocentrifugation, Wright-Giemsa staining, and light microscopy revealed that basophil subpopulations I, II, and III appeared morphologically to be basophils, but cluster IV showed typical neutrophil morphology. Colored frames correspond to subpopulation colors in Figure 2.

Figure 4

Phenotypic Profile of “Basophil” Subpopulations Compared with Traditional Granulocyte Populations A heatmap summary of median protein marker levels detected across eosinophils (E), total PBG (CD45⁺HLA-DR⁻CD123⁺) (B), PBG subpopulations (I, II, III, and IV), and classical neutrophils (Nϕ). Protein markers were organized by their use as clustering markers and by their known expression (Table 1) across traditional granulocyte subpopulations. Marker medians were transformed by calculating the difference of inverse hyperbolic sine (arcsinh) and normalized by individual markers. Columns indicate the five donors that were compared across granulocyte populations.

Figure 5

Functionality of Phenotypically Basophilic Granulocyte Subpopulations Compared with Traditional Granulocyte Subpopulations The CD203c basophil activation marker (A) was used to examine the potential for activation across PBG subpopulations (I, II, III, and IV) compared with total PBGs, neutrophils (Nϕ), and eosinophils (E). Stimulations were carried out in response to anti-IgE (red) or IL-3 (blue) stimulation compared with control RPMI media (open circles). Protein markers CD16 (B), CD244 (C), and FcεRI (D), used to delineate the four PBG subpopulations, were also analyzed to determine their modulation upon anti-IgE or IL-3 stimulus. Significant findings are denoted by ∗, ∗∗, and ∗∗∗∗ where p < 0.05, <0.01, and <0.0001 compared with values for RPMI controls. See also Figure S3.

Figure 6

Profile of Phenotypically Basophilic Granulocyte Subpopulations in Healthy and CML Samples, across Compartments A heatmap summary of median marker levels detected in PBG subpopulations compared with bulk PBG basophils and neutrophils. Markers include granule specific proteins important in basophil functional profiles. Marker medians were first transformed by calculating the difference of inverse hyperbolic sine (arcsinh), followed by normalization by individual markers. (A) Total PBG basophils, the four identified subpopulations (I, II, III, and IV), and neutrophils (Nϕ) were profiled in paired samples of peripheral blood (PB) and bone marrow (BM) in healthy donors and compared with bone marrow from donors with CML. (B) Plot indicating abundance of each subpopulation in the total PBG gate across peripheral blood, bone marrow, and bone marrow of CML donors.