PMC6289061_01
Clinical presentation of VZV disease in GATA2-haploinsufficient patient (A) An erytematous, vesicular skin rash on the back of patient 1 with GATA2-haploinsufficiency, 4 days after the onset of VZV disease. (B) Targetoid lesions on the left arm of the same patient, 7 days after the onset of illness. (C) A chest X-ray 8 days after the onset of illness, showing diffuse alveolar involvement with a miliary pattern of pulmonary opacities. (D) Histograms showing perforin expression in CD56+CD16+ lymphocytes from patient 1 (gray), patient 2 (black), an individual with heterozygous A91V PRF1 mutation (green), a healthy control (blue) and isotype control (red dotted line) as assessed by intracellular flow cytometry.
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F1
SAP and XIAP expression in patient 2 were normal (Figure 1D).
PMC6289061_02
Clinical presentation of VZV disease in GATA2-haploinsufficient patient (A) An erytematous, vesicular skin rash on the back of patient 1 with GATA2-haploinsufficiency, 4 days after the onset of VZV disease. (B) Targetoid lesions on the left arm of the same patient, 7 days after the onset of illness. (C) A chest X-ray 8 days after the onset of illness, showing diffuse alveolar involvement with a miliary pattern of pulmonary opacities. (D) Histograms showing perforin expression in CD56+CD16+ lymphocytes from patient 1 (gray), patient 2 (black), an individual with heterozygous A91V PRF1 mutation (green), a healthy control (blue) and isotype control (red dotted line) as assessed by intracellular flow cytometry.
fimmu-09-02766-g0001.jpg
PMC6289061_02_fimmu-09-02766-g0001.jpg
F1
SAP and XIAP expression in patient 2 were normal (Figure 1D).
High-dimensional mass cytometry for the analysis of immune populations in GATA2-haploinsufficient patients PBMCs of 3 patients (patients 2-4) and 3 healthy controls were stained with 27 heavy-metal labeled antibodies (Supplementary Table 2) and acquired on a CYTOF2 mass cytometer. (A) Cells identified as CD45+ were exported, downsampled to 900 events and the combined dataset (5,400 cells) subjected to dimensionality reduction using the t-SNE algorithm. Density plot of total (left panel), control (middle panel) and patient (right panel) cells based on tSNE analysis. The manually gated XYZ population consist of closely grouped cells present mainly in the patients and not belonging to any immune subpopulation defined by manual annotation. (B) Color-coded manual gates are plotted by density for the combined dataset. (C) Density plots of the different subpopulations in patients and controls with the fraction out of all CD45+ cells indicated. (D) Histograms of markers with the largest expression discrepancies between patients and controls in the XYZ population and among monocytes based on visual analysis. (E) Percentages of XYZ cells from the CyTOF data, out of all CD45+ plotted for patient 2 (HLH), the 2 additional patients with germline GATA2-mutations (patient 3 and 4) and the 3 healthy controls. (F) Timecourse of TCRgammadelta+ cells (percentage of lymphocytes) measured by conventional flow cytometry in GATA2-haploinsufficient patients 1 to 4.
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F2
To visualize the lymphocyte pattern differences in peripheral blood samples from these three patients and three healthy controls, we used t-Disturbed Stochastic Neighbor Embedding (tSNE) to visualize mass cytometry datasets (Figures 2A,B), using the antibody panel shown in Supplementary Table 2. All patients with GATA2-haploinsufficiency had markedly reduced peripheral blood B cells, NK cells and monocytes (Figure 2C). The patients furthermore displayed an exceedingly high frequency of a CD3+ CD56+ CD16- CD8- CD4- cell population ("XYZ-population" in Figures 2A,D,E). Although largely different in ratios, this and the monocyte population retained the same phenotype as the ones present in healthy controls (Figure 2D). TCRgammadelta+ or NKT lymphocytes. This was confirmed by flow cytometry analysis of samples collected at different time points using a TCRgammadelta specific antibody (Figure 2F). On the contrary, TCRgammadelta+ cells in patient 1 and patient 3 remained below 5% of lymphocytes during the period of monitoring (between the age of 8 and 10 years for patient 1, and 22 and 25 years for patient 3), raising the possibility that populations other than TCRgammadelta+ T cells are expanded in GATA2-haploinsufficiency (Figure 2F).