Natural Killer Cell Degranulation Defect: A Cause for Impaired NK-Cell Cytotoxicity and Hyperinflammation in Fanconi Anemia Patients

Abstract

Fanconi anemia (FA) is a rare inherited syndrome characterized by progressive bone marrow failure (BMF), abnormal skin pigmentation, short stature, and increased cancer risk. BMF in FA is multifactorial and largely results from the death of hematopoietic stem cells due to genomic instability. Also, inflammatory pathology in FA has been previously reported, however the mechanism is still not clear. In literature, decreased NK-cell count and/or impaired NK-cell activity, along with other immunological abnormalities have been described in FA-patients (1). However, to the best of our knowledge, this is the first report showing a defective degranulation mechanism leading to abnormal NK-cell cytotoxicity in FA-patients, which may explain the development of a hyperinflammatory response in these patients. This may predispose some patients to develop Hemophagocytic lymphohistiocytosis (HLH) which manifests with prolonged fever, progressive cytopenias and organomegaly. Early diagnosis and initiation of immunosuppressive therapy in these patients will help to better manage these patients. We also propose FA genes to be listed as a cause of familial HLH.

Keywords: Familial Hemophagocytic lymphohistiocytosis (FHL); Fanconi anemia; HLH-targeted therapy; NK cell cytotoxicity; NK cell degranulation defect.

Figure 1

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.

Figure 2

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).

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.