CD203c distinguishes the erythroid and mast cell-basophil differentiation trajectories among human FcεRI+ bone marrow progenitors.

To the Editor,

IgE molecules that bind their specific antigen crosslink FcεRI receptors present on mast cells and basophils. Downstream signaling results in cell activation and subsequent release of diverse compounds that exhibit potential to trigger allergic symptoms. Although mature FcεRI+ cells have been extensively studied, less is known about the FcεRI+ progenitors and their differentiation capacity.1 Here, we analyzed the FcεRI+ progenitor population from human bone marrow with multicolor flow cytometry and fate assays. The results revealed distinct subpopulations of FcεRI+ progenitors, all showing capacity to form mast cells and basophil‐like cells but not granulocytes or monocytes. The CD203c− subsets displayed erythroid potential, whereas the CD203c+ subset did not, altogether providing early evidence for a common mast cell‐basophil‐erythroid differentiation trajectory in human, distinct from the granulocyte‐monocyte axis.

The CD34+ CD117+ FcεRI+ phenotype identifies the human mast cell progenitor population in blood.2 Other characteristics include expression of the IL‐3 receptor and the absence of CD45RA, positioning the cells among common myeloid progenitors (CMPs) when analyzing the progenitors with flow cytometry.2, 3 In contrast to blood, we recently demonstrated that CMPsFcεRI+ in bone marrow do not exclusively form CD117hi mast cells.4 This observation warranted further characterization of the bone marrow CMPsFcεRI+ phenotype and cell‐forming potential.

Morphologic assessment following cell sorting and May‐Grünwald Giemsa stain revealed that the CMPFcεRI+ population was heterogenous (Figure 1A,C; see Methods S1 for materials and methods). Some cells exhibited a blast‐like phenotype with little cytoplasm, whereas other displayed numerous metachromatic granules (Figure 1C). The cell heterogeneity prompted us to design a multicolor flow cytometry panel that further characterizes the progenitors. Analyzing the CD203c and integrin β7 expression patterns revealed subpopulations of CMPFcεRI+ cells (Figure S1). Three CMPFcεRI+ subpopulations—CD203c+, integrin β7+ CD203c−, and integrin β7− CD203c− cells—exhibited distinct protein expression profiles and were studied further (Figure 1B,D). These three populations, along with CMPsFcεRI− and granulocyte/monocyte progenitors (GMPs), were sorted and cultured to investigate their cell‐forming potential (Figure 2A). The five bone marrow progenitor populations were first cultured with IL‐3 and IL‐6. These conditions support mast cell progenitors from peripheral blood to form CD117hi FcεRI+ mast cells.3 We analyzed the cultured bone marrow cells with a flow cytometry panel that distinguished three subsets of FcεRI+ cells separated based on the CD117 expression, CD235a+ erythroid cells, and CD14+ or CD15+ granulocyte‐monocyte output (Figure 2B visualizes the gating strategy). The CMPFcεRI+ largely maintained their FcεRI+ phenotype during culture (Figure 2C). None of the three CMPFcεRI+ subpopulations produced pure populations of CD117hi mast cells or CD117− basophil‐like cells, but instead constituted a mix of cells with variable CD117 expression (Figure 2F‐H).

Figure 1

Bone marrow CMPsFcεRI+ comprises three distinct progenitor populations. A, Flow cytometry analysis of human bone marrow cells. B, CD203c and integrin β7 distinguishes three distinct CMPFcεRI+ subpopulations. C, May‐Grünwald Giemsa staining of CMPsFcεRI+. D, Surface expression analysis of CD203c+ (green), Iβ7+ CD203c− (blue), and Iβ7− CD203c− (orange) CMPFcεRI+ compared with CMPsFcεRI− (red), and GMPs (purple). Negative controls (gray) represent internal control populations from the sample that are negative for the marker of interest. One representative bone marrow sample is shown

Figure 2

Cell fate assays reveal the cell‐forming potential of the CMPFcεRI+ populations. A, Schematic diagram indicating the cell fate assay methodology. B, Gating strategy of the cultured cells. Cells cultured for 5‐6 d with the myeloerythroid‐promoting cytokines IL‐3, IL‐6, SCF, EPO, and GM‐CSF are shown. Percentages of (C) FcεRI+, (D) CD14+ or CD15+, and (E) CD235a+ (Glycophorin A+) cells after 5‐6 d of culture. Percentages of (F) CD117hi FcεRI+ mast cells, (G) CD117+ FcεRI+ precursors, and (H) CD117− FcεRI+ basophils after 5‐6 d of culture. Panels C‐H display cells cultured with IL‐3 and IL‐6 (left) or the myeloerythroid‐promoting cytokines IL‐3, IL‐6, SCF, EPO, GM‐CSF (right). The cell fate assay was performed from 3‐4 donors per population and condition as indicated

The three CMPFcεRI+ populations did not display granulocyte‐monocyte potential (Figure 2D). By contrast, culture of CMPsFcεRI− and GMPs resulted in substantial granulocyte‐monocyte output. The CMPFcεRI− population included progenitors with potential to upregulate FcεRI expression, suggesting that this population contains precursors of CMPsFcεRI+. GMPs did not produce FcεRI+ cells, indicating that this population lacks mast cell and basophil‐forming capacity (Figure 2C). Bühring et al previously reported that the CD34+ CD203c+ progenitors exhibit mast cell‐forming and high basophil‐forming potential, agreeing with our results that the CD203c+ subset of the CMPsFcεRI+ form these cell types.5 However, the CD34+ CD203c+ progenitors cultured in the study by Bühring et al exhibit residual granulocyte‐monocyte‐forming potential and were proposed to contain multipotent progenitors.5 We sorted and cultured CD203c+ cells from the CMPFcεRI+ fraction, constituting cells that likely are more differentiated than CD34+ CD203c+ progenitors in general, which could explain the observation that CD203c+ CMPFcεRI+ cells lack granulocyte‐monocyte potential.

No or few erythroid cells developed from any of the starting populations in the IL‐3 and IL‐6 conditions (Figure 2E), which is in agreement with lack of sufficient stimulus for erythroid development. Through a combined single‐cell RNA sequencing and cell culture‐based approach, Tusi et al6 recently identified progenitors with combined erythroid and mast cell‐basophil output in mouse bone marrow. Hence, we investigate whether the CMPFcεRI+ populations exhibited erythroid potential. We cultured the CMPsFcεRI+ with the myeloerythroid‐promoting cytokines IL‐3, IL‐6, SCF, EPO, and GM‐CSF. Cell culture assays in these conditions revealed that all CMPFcεRI+ subpopulations still maintained FcεRI+ cells (Figure 2B‐C). Notably, we observed clear erythroid output when culturing the two CD203c− subpopulations (Figure 2E). A population of FcεRI− CD117hi cells was also present in cultures of the CD203c− subpopulations, cells with dim CD45 expression that likely constituted erythroid precursors (Figure S2A‐B).

We cannot exclude that unipotent erythroid progenitors contaminated the CMPFcεRI+ sort gate. However, performing single‐cell culture experiments to resolve this question is complicated by the poor proliferation capacity of human mast cell progenitors,2 making it difficult to identify and characterize mixed colonies. Though, it is worth to point out that erythrocytes were not present in cultures derived from the CD203c+ subpopulation across cell fate assays of 4 donors, suggesting that CD203c upregulation is associated with loss of erythroid potential.

Progressive loss of proliferation capacity with mast cell differentiation may explain the relatively low frequency of mast cells derived from CD203c+ compared with integrin β7+ CD203c− CMPFcεRI+ cells in IL‐3 and IL‐6 conditions.

Resolving the differentiation trajectories from hematopoietic stem cells to FcεRI+ mast cells and basophils could significantly improve our understanding of, for example, IgE‐mediated allergic diseases as well as the mast cell‐driven disease systemic mastocytosis. The frequency of putative bone marrow mast cell progenitors was recently demonstrated to be elevated in systemic mastocytosis patients compared with healthy subjects.7 Thus, the establishment of flow cytometry gating strategies for the identification of progenitors with basophil and mast cell‐forming capacity can help us to understand why mature cells accumulate in disease. Taken together, the results presented here provide early evidence that the mast cell and basophil differentiation trajectories are closely linked to the erythropoiesis in human. Further studies on the topic are warranted, and fate assays of individual cells coupled with single‐cell transcriptomics represent a promising way forward.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

Click here for additional data file.

Click here for additional data file.