A CD57+ CTL Degranulation Assay Effectively Identifies Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients
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Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is a genetic disorder that results in immune dysregulation. It requires prompt and accurate diagnosis. A natural killer (NK) cell degranulation assay is often used to screen for FHL3 patients. However, we recently encountered two cases of late-onset FHL3 carrying novel UNC13D missense mutations: in these cases, the degranulation assays using freshly isolated and interleukin (IL)-2-activated NK cells yielded contradictory results. Since the defective degranulation of CD57+ cytotoxic T lymphocytes (CTLs) in these cases was helpful for making the diagnosis, we assessed whether the CD57+ CTL degranulation assay more effectively identified FHL3 patients than the NK cell assays.
Forty additional patients with hemophagocytic lymphohistiocytosis were prospectively screened for FHL3 by measuring the perforin expression in NK cells and the expression of Munc13-4, syntaxin-11, and Munc18-2 in platelets and by performing NK cell and CTL degranulation assays. The results were confirmed by genetic analysis.
The freshly isolated NK cell degranulation assay detected FHL3 patients with high sensitivity (100%) but low specificity (71%). The IL-2-stimulated NK cell assay had improved specificity, but 3 out of the 31 non-FHL3 patients still showed degranulation below the threshold level. The CD57+ CTL degranulation assay identified FHL3 patients with high sensitivity and specificity (both 100%).
The CD57+ CTL degranulation assay more effectively identified FHL3 patients than the NK cell-based assays.
KeywordsFamilial hemophagocytic lymphohistiocytosis type 3 lysosomal degranulation defect functional screening assay UNC13D
The authors are grateful to all of the participating patients, their families, and the referring physicians for their generous cooperation.
Contribution: T.Y., R.N., and T.H. designed the research; M.I., N.N., H.T., and E.I. treated patients 1 and 2; M.H., S.S., H.S., E.H., K.I., and T.K. performed the degranulation and protein expression assays; H.O. and O.O. performed the genetic analyses; R.S. and H.H. prepared the anti-Munc13-4 and anti-Syntaxin11 antibodies; M.H., T.Y., K.I., T.K., R.N., S.M., and T.H. analyzed and discussed the results; T.Y. and S.M. performed the statistical analysis; and M.H. and T.Y. wrote the paper.
Compliance with Ethical Standards
Informed consent was obtained from the patients and their parents in accordance with the institutional review board of Kyoto University Hospital and the Declaration of Helsinki.
Conflict of Interest
The authors declare that they have no competing interests.
This work was supported by JSPS KAKENHI (Grant Numbers 26461582 and 25670475) and by grants from the “Research on Measures for Intractable Diseases” Project: matching fund subsidy from the Japanese Ministry of Health, Labor, and Welfare.
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