PMC6438155_01
NK cell degranulation assay results in FA patients: (A) Samples were analyzed by flow cytometry, gating on lymphocytes by forward/side scatter. CD107a expression was analyzed on natural killer (NK) cells (CD56+CD3-) (B) Degranulation assay results from a healthy control are shown (representative plot) and from the patients with FA included in this study. The percentage of CD107a+ NK cells is indicated on each plot. In the healthy controls the CD107a expression on NK cells increased significantly after Ca-I and PMA stimulation. However, in FA patients CD107a expression on stimulated NK cells was significantly lower than the healthy controls, indicating defective granule release mechanism.
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F1
However, all patients had abnormal GRA on the NK cells (<15%; reference range 28 +- 8%) (p < 0.0001) (Figure 1) and had NK cell cytotoxicity lower than compared to the healthy controls (Table 2) (p < 0.0001; Figure 2; Supplementary Figure 3).
NK cell cytotoxicity in FA patients (A) Comparison of NK cell cytotoxicity in healthy control (median of all healthy controls processed) and FA patients at different E:T ratio (50:1, 100:1, and 200:1) FA patients had significantly low NK cell cytotoxicity compared to the healthy controls. (B) Comparison of NK cell cytotoxicity in healthy controls and FA patients at E:T ratio 200:1. Box-and-whiskers graph. The box extends from the minimum to the maximum and the line at the middle is the median. Mann-Whitney U test was used to evaluate differences (***P < 0.01).
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F2
Perforin expression on NK cells in all these patients was within the reference range (>65%; reference range 72 +- 2%) (Supplementary Figure 2). However, all patients had abnormal GRA on the NK cells (<15%; reference range 28 +- 8%) (p < 0.0001) (Figure 1) and had NK cell cytotoxicity lower than compared to the healthy controls (Table 2) (p < 0.0001; Figure 2; Supplementary Figure 3).
Comparison of serum cytokine levels Box-and-whiskers graph and the line at the middle is the median. **P < 0.05, ***P < 0.01. (A) FA patients (n = 5), HLH patients with abnormal CD107a expression on stimulated NK cells (GRA) (n = 13) and healthy controls (n = 12). Mann-Whitney U test was used to evaluate differences between cytokine concentrations in different groups. (B) FA patients with mild and severe cytopenia. Unpaired t-test was used to evaluate differences between cytokine concentrations in different groups.
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F3
However, all patients had abnormal GRA on the NK cells (<15%; reference range 28 +- 8%) (p < 0.0001) (Figure 1) and had NK cell cytotoxicity lower than compared to the healthy controls (Table 2) (p < 0.0001; Figure 2; Supplementary Figure 3). IFN-gamma, IL-6, IL-7, IL-10, GM-CSF, MCP-1, MIP-1alpha, MIP-1beta, and IP-10 levels (Figure 3A). In FHL patients, the defect in granule release mechanism leads to an inefficient clearing of the infecting cells, causing hypercytokinemia. Interestingly, when FA patients with and without cytopenia were compared, it was observed that patients with cytopenia had significantly elevated IL-10 levels but reduced MIP-1beta levels (Figure 3B).