Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors

Abstract

Mast cell accumulation is a hallmark of a number of diseases, including allergic asthma and systemic mastocytosis. Immunoglobulin E-mediated crosslinking of the FcεRI receptors causes mast cell activation and contributes to disease pathogenesis. The mast cell lineage is one of the least studied among the hematopoietic cell lineages, and controversies remain about whether FcεRI expression appears during the mast cell progenitor stage or during terminal mast cell maturation. Here, we used single-cell transcriptomics analysis to reveal a temporal association between the appearance of FcεRI and the mast cell gene signature in CD34+ hematopoietic progenitors in adult peripheral blood. In agreement with these data, the FcεRI+ hematopoietic progenitors formed morphologically, phenotypically, and functionally mature mast cells in long-term culture assays. Single-cell transcriptomics analysis further revealed the expression patterns of prospective cytokine receptors regulating development of mast cell progenitors. Culture assays showed that interleukin-3 (IL-3) and IL-5 promoted disparate effects on progenitor cell proliferation and survival, respectively, whereas IL-33 caused robust FcεRI downregulation. Taken together, we showed that FcεRI expression appears at the progenitor stage of mast cell differentiation in peripheral blood. We also showed that external stimuli regulate FcεRI expression of mast cell progenitors, providing a possible explanation for the variable FcεRI expression levels during mast cell development.

Graphical abstract

Figure 1

Expression of FcεRI subunit genes in hematopoietic progenitors is associated with a mast cell gene signature. (A) UMAP visualization of the single-cell transcriptomes of 6874 Lin⁻ c-Kit⁺ peripheral blood cells. The colors indicate Leiden clusters. Expression patterns of lineage genes used to annotate the landscape are provided in supplemental Figure 1. (B) Gene expression levels of mast cell genes and FcεRI subunit genes. (C) Gene expression of CD34. (D) Post-sort flow cytometry analysis of Lin⁻ c-Kit⁺ cells. The red contour plot shows back-gated FcεRI⁺ cells. One buffy coat was used to produce the data presented in this figure.

Figure 2

Single-cell transcriptomics reveals a temporal association between FcεRI and the mast cell gene signature. (A) Representation of the cells in the first data set relative to the cells in the prospective second data set. (B) Experimental approach to hashtag a cell subset with a PE-oligonucleotide barcode. (C) Post-sort flow cytometry analysis of pooled cells, constituting Lin⁻ CD34⁺ c-Kit⁺ cells without and Lin⁻ CD34⁺ c-Kit⁺ FcεRI⁺ cells with the PE-oligonucleotide hashtag. (D) UMAP visualization of the entire Lin⁻ CD34⁺ c-Kit⁺ data set (4063 cells). The colors indicate Leiden clusters. Panels E and G and supplemental Figure 3 provide landscape annotation. Population A (Pop A) consisted of 2 main clusters, Pop A1 and Pop A2. (E) Gene expression levels of mast cell genes and FcεRI subunit genes. (F) Histogram showing the distribution of the PE-oligonucleotide in the single-cell transcriptomics data, resembling the flow cytometry data in panel C. Gray bins represent ambiguous events. (G) Plots showing the PE⁻ Lin⁻ CD34⁺ c-Kit⁺ cells and PE⁺ Lin⁻ CD34⁺ c-Kit⁺ FcεRI⁺ cells (population A). (H) Visualization of highly dynamic genes along the mast cell differentiation trajectory. Cells are ordered according to cluster and pseudotime. (I) Visualization of dynamic genes that positively correlate with rank ordering, also referred to as Pop A signature genes. (J) Plot showing the temporal upregulation of mast cell genes and FcεRI subunit genes. The blue to yellow color gradients in panels H and I specify low to high gene expression. One buffy coat, independent of the buffy coat used for Figure 1, was used to produce the data presented in panels C to J. Ab, antibody; Ery, erythroid; Ly/Mono/DC, lymphocyte/monocyte/dendritic cell; MNCs, mononuclear cells; MPP, multipotent progenitor; Neu, neutrophil; scRNA-seq, single-cell RNA-sequencing.

Figure 3

FcεRI⁺ hematopoietic progenitors exhibit mast cell–forming potential in health and disease. (A) Experimental setup to analyze mast cell differentiation. The study used 10 ng/mL IL-3, 50 ng/mL IL-6, and 100 ng/mL stem cell factor (SCF). (B-C) Flow cytometry analysis of cultured progenitors. Filled histograms show isotype control staining. The results in panel B are quantified in Ci-ii (n = 6). (D) May-Grünwald Giemsa staining. (E) Tryptase staining (day 0, one experiment; days 7-28 from an individual experiment, culture experiment performed 6 times). Microscope: Olympus AX70 with XC10 camera. (Fi) Functional analysis of mast cells derived from hematopoietic progenitors. The CD63 gate that defines mast cell activation was set to ensure that the IgE-only group showed <1% positive events. (Fii) Quantification of activated mast cells in panel Fi (n = 3). Lines link samples from the same donor. (G) Progenitors from patients with systemic mastocytosis were sorted and cultured as indicated in panel A. Cultured FcεRI⁻ progenitors and cultured population A (Pop A) cells from each patient were analyzed by using flow cytometry on the same day (days 28-30, depending on donor). Analysis of aberrant markers was not performed. (H) Quantification of the results in panel G (n = 3). Lines link samples from the same donor. Two-tailed unpaired t tests; *P < .05, **P < .01, ****P < .0001. MCs, mast cells; ns, not significant.

Figure 4

Mast cell progenitors express transcripts of the receptors for IL-3, IL-5, EPO, and IL-33. The graphs show gene expression levels of the indicated cytokine receptors. (A) UMAP visualization of Lin⁻ c-Kit⁺ cells (data set 1). (B) UMAP visualization of Lin⁻ CD34⁺ c-Kit⁺ cells (data set 2). Ery, erythroid; MPP, multipotent progenitor; Neu, neutrophil; Pop A, population A.

Figure 5

IL-3 promotes mast cell progenitor proliferation, whereas IL-5 supports mast cell progenitor survival. (A) Gating of pre–mast cells (pre-MCs) after culture of c-Kit⁺ progenitors for 5 days. (B) Quantification of pre-MCs after culture of c-Kit⁺ progenitors in various cytokine conditions. Points in each group represent individual buffy coats. Values are normalized to the IL-3 condition of each donor. One-way analysis of variance, Dunnett's post hoc with no cytokines as control group. (Ci) The proliferative response of mast cell progenitors was analyzed by CellTrace Far Red signal in pre-MCs. The control refers to analysis of all live cells after culture without cytokines, as pre-MCs were virtually absent. (Cii) Quantification of the results in panel Ci (n = 3). (D) Analysis of the proliferative response of mast cell progenitors to various concentrations of IL-5. IL-3 served as positive control. Representative of 2 independent experiments. The study used 10 ng/mL IL-3, 20 ng/mL IL-5, 10 ng/mL GM-CSF, 1 U/mL EPO, 50 ng/mL thrombopoietin (TPO), 50 ng/mL Flt3L, and 22.5 to 100 ng/mL IL-33 unless otherwise specified. ****P < .0001. ns, not significant.

Figure 6

IL-33 downregulates FcεRI expression on mast cell progenitors (MCPs). (A) Quantification of FcεRI expression on pre–mast cells after 5 days in culture with the indicated cytokines. Points in each group represent individual buffy coats. The levels were normalized to the IL-3 condition of each donor. The study used 10 ng/mL IL-3, 20 ng/mL IL-5, 10 ng/mL GM-CSF, 1 U/mL EPO, 50 ng/mL thrombopoietin (TPO), 50 ng/mL Flt3L, and 22.5 to 100 ng/mL IL-33 unless otherwise specified. (B) Flow cytometry analysis of FACS-isolated MCPs cultured with 10 ng/mL IL-3 alone or in combination with 100 ng/mL IL-33 for 5 days. Cultures of FcεRI⁻ progenitors are shown in supplemental Figure 8. (C) Quantification of panel B (n = 3). Two-tailed one-sample t test in panels A and C, hypothetical value 1.0. ***P < .001, ****P < .0001. MFI, median fluorescent intensity.