KIT signaling is dispensable for human mast cell progenitor development

Abstract

Human hematopoietic progenitors are generally assumed to require stem cell factor (SCF) and KIT signaling during differentiation for the formation of mast cells. Imatinib treatment, which inhibits KIT signaling, depletes mast cells in vivo. Furthermore, the absence of SCF or imatinib treatment prevents progenitors from developing into mast cells in vitro. However, these observations do not mean that mast cell progenitors require SCF and KIT signaling throughout differentiation. Here, we demonstrate that circulating mast cell progenitors are present in patients undergoing imatinib treatment. In addition, we show that mast cell progenitors from peripheral blood survive, mature, and proliferate without SCF and KIT signaling in vitro. Contrary to the prevailing consensus, our results show that SCF and KIT signaling are dispensable for early mast cell development.

Figure 1.

Imatinib treatment does not affect the frequency of blood mast cell progenitors in vivo. (A) Peripheral blood mast cell progenitors and mast cells were gated as SSC^loLin⁻CD45^intCD34⁺CD117^int/hiFcεRI⁺ cells and CD45⁺CD14⁻CD34⁻CD117^hiFcεRI^hi cells, respectively. The percentages indicate the frequency of the gated cells normalized to the number of CD45⁺ singlet cells. The frequencies of mast cell progenitors (B) and mast cells (C) from the CD45⁺ cell population in healthy controls (n = 6), patients with CML (n = 5), patients with CML under treatment with imatinib (n = 5), patients with GIST under treatment with imatinib (n = 5), and patients with systemic mastocytosis (SM) carrying the D816V KIT mutation (n = 9) are shown. The lines represent the geometric means. The triangles and squares represent the same patients before and during imatinib treatment. One-way ANOVA with Tukey’s multiple comparisons test on log-transformed data. *Adjusted P < .05; **adjusted P < .01. FSC, forward scatter; SSC, side scatter.

Figure 2.

SCF is dispensable for mast cell progenitor survival in vitro. CD34⁺ progenitors were purified from buffy coats from healthy donors and analyzed with flow cytometry. (A-B) The representative gating strategy and the quantification of CD34⁺CD117^int/hiFcεRI⁺ mast cell progenitors are shown (n = 9). The representative example in panel A is indicated with an open circle. The line in panel B represents the geometric mean. (C-G) CD34-enriched cells were cultured and analyzed by flow cytometry. (D) The fraction of CD117^hiFcεRI⁺ pre–mast cells from panel C was normalized to the combined IL-3 and IL-6 condition for each buffy coat, and the results from 3 buffy coats were pooled. The bars represent the means ± standard error of the mean (SEM). (E) CD34⁺ progenitors were cultured with IL-3 and IL-6, and the fractions of CD34-expressing cells out of the CD117^hiFcεRI⁺ mast cell population (days 3-7) are shown. The CD34 expression of CD117^int/hiFcεRI⁺ cells is shown as the day 0 control. The bars represent the means ± SEM of 3 buffy coats. (F) The CD117 expression of CD34⁺ cells was analyzed on day 3. The red histogram indicates the cells cultured without cytokines. The blue line indicates the cells cultured with IL-3 and IL-6. The dashed line indicates the cells cultured with IL-3, IL-6, and SCF. One representative experiment out of 3 is shown. (G) The CD117 expression on CD34⁺ cells at day 3, shown in panel F, was quantified by calculation of the median fluorescence intensity. The expression level of CD117 is shown as a percentage of the IL-3 and IL-6 condition. The bars represent the means ± SEM of 3 buffy coats. (H-J) CD34⁺ cells were enriched from buffy coats, cultured, and analyzed by flow cytometry. The medium was supplemented with polyclonal goat IgG or anti-SCF neutralizing antibodies as indicated. (J) The integrin β7 expression was analyzed by flow cytometry before and after culture with IL-3, IL-6, and anti-SCF. The bars in panels I and J represent the means ± SEM of 3 buffy coats. Live singlet cells are shown in the flow cytometric graphs. The statistical analyses in panels D, E, G, and I were performed using 1-way ANOVA with Tukey’s multiple comparisons test. **Adjusted P < .01; ****adjusted P < .0001; ns = not significant. The unpaired 2-tailed Student t test was used for the statistical analysis in panel J. ****P < .0001.

Figure 3.

KIT signaling is dispensable for mast cell progenitor survival. (A-C) CD34⁺ progenitors were enriched from buffy coats, cultured for 5 days, and analyzed by flow cytometry. The medium was supplemented with imatinib dissolved in dimethyl sulfoxide where indicated. Vehicle indicates that the medium was supplemented with dimethyl sulfoxide only. (B) The fraction of CD117^hiFcεRI⁺ pre–mast cells from panel A was normalized to the combined IL-3, IL-6, and vehicle condition for each buffy coat (n = 3). The bars represent the means ± SEM. (C) The CD117 expression on CD34⁺ cells from day 5 was quantified by calculation of the median fluorescence intensity. The expression level of CD117 is shown as a percentage of the IL-3, IL-6, and vehicle condition. The bars represent the means ± SEM of 3 buffy coats. Live singlet cells are shown in the flow cytometric graphs. All statistical analyses were performed using 1-way ANOVA with Tukey’s multiple comparisons test. *Adjusted P < .05; **adjusted P < .01; ***adjusted P < .001; ****adjusted P < .0001.

Figure 4.

Mast cell progenitors mature in the absence of SCF. (A) The CD34-enriched progenitors were purified from buffy coats and cultured with IL-3 and IL-6. The FSC and SSC parameters of the primary mast cell progenitors (CD117^int/hiFcεRI⁺ cells, left) and the cultured pre–mast cells (CD117^hiFcεRI⁺ cells, right) are shown in black height curves. The gray height curves indicate unstimulated CD34⁺ cells. One representative buffy coat out of 3 is shown. (B) The mean fluorescence intensities of the FSC and SSC parameters were calculated for each population described in panel A. The ratio between the CD117^int/hiFcεRI⁺ cells and the unstimulated CD34⁺ cells was then calculated for each time point independently, as the photomultiplier tube voltages of those parameters were adjusted between day 0 and 5 to visualize the cells on scale. The results were pooled from 3 buffy coats. The bars represent the means ± SEM. Unpaired 2-tailed Student t tests. **P < .01; ***P < .001. (C) CD117⁺ cells were enriched from buffy coats, and CD34⁺CD117^int/hiFcεRI⁺ cells were isolated with FACS. The isolated CD34⁺CD117^int/hiFcεRI⁺ cells were stained with May-Grünwald Giemsa. (D) Sorted CD34⁺CD117^int/hiFcεRI⁺ cells were cultured with IL-3 and IL-6 for 5 days. The cultured cells were analyzed with May-Grünwald Giemsa staining, an enzyme cytochemical staining for trypsinlike activity as a measure of tryptase, and flow cytometry. Live singlet cells are shown in the flow cytometric graph. (E) Sorted CD34⁺CD117^int/hiFcεRI⁺ cells were first cultured with IL-3 and IL-6 for 5 days and then cultured with SCF and IL-6 for an additional 5 to 7 days. The cultured cells were analyzed with May-Grünwald Giemsa staining and an enzyme cytochemical staining for trypsinlike activity as a measure of tryptase. (F) The largest cell diameter of the primary mast cell progenitors, pre–mast cells, and mast cells was measured using ImageJ (NIH, Bethesda, MD). Each dot in the graph corresponds to 1 cell. The means ± SEM are shown. One-way ANOVA with Tukey’s multiple comparisons test. ***Adjusted P < .001; ****adjusted P < .0001. Images were captured using the Eclipse E400 microscope, the Digital Camera DXM1200, and ACT-1 software (Nikon, Tokyo, Japan). The width of the May-Grünwald Giemsa photos corresponds to 28 μm. The width of the enzyme cytochemical staining photos corresponds to 35 μm. The cultures at day 5 and days 10 to 12 were performed for 2 buffy coats with similar results. The day 0 May-Grünwald Giemsa staining was performed for 1 buffy coat.

Figure 5.

Mast cell progenitors proliferate in the absence of SCF. CD34-enriched cells purified from buffy coats were stained with CellTrace Far Red. (A) The cells were cultured with IL-3 and IL-6 for 5 days and analyzed by flow cytometry. Live singlets were gated. The red-filled histogram represents unstimulated CD34⁺ cells, the blue line indicates CD117^hiFcεRI⁺ pre–mast cells, and the dotted light green line indicates cells that did not fall into the CD117^hiFcεRI⁺ pre–mast cell gate. (B) Cell proliferation was analyzed using the proliferation platform in FlowJo. The model was adjusted to 8 peaks. The non–pre-mast-cell population was used as a positive control for cell division, and the cell generation gates were calculated accordingly. The undivided peak was set according to the unstimulated sample, which was stored at 2°C–8°C throughout the culture period. The division index corresponds to the mean number of cell divisions undergone by the cells in the original culture. The bars represent the means ± SEM of 3 buffy coats. One-way ANOVA with Tukey’s multiple comparisons test. ****Adjusted P < .0001.

Figure 6.

IL-3 is sufficient for mast cell progenitor survival in vitro. CD34⁺ progenitors were enriched from buffy coats and cultured for 5 days with the cytokines indicated in the figure. (A) The cultured cells were analyzed with flow cytometry, and the frequency of CD117^hiFcεRI⁺ pre–mast cells as a fraction of live singlet cells was quantified. One representative experiment out of 3 is shown. (B) The frequency of CD117^hiFcεRI⁺ pre–mast cells as a fraction of live singlet cells was normalized to the combined IL-3 and IL-6 condition for each buffy coat, and the results from 3 buffy coats were pooled. The bars represent the means ± SEM of each group. One-way ANOVA with Tukey’s multiple comparisons test. ***Adjusted P < .001; ****adjusted P < .0001. (C) The IL-3 receptor (IL-3R) expression on the SSC^loCD14⁻CD34⁺CD117^int/hiFcεRI⁺ blood mast cell progenitors is shown. The red-filled histogram indicates the fluorescence minus 1 control, and the blue line indicates the sample. The analysis was performed on cells from 2 blood donors and yielded similar results.