Defective CD107a Surface Expression Heralds Munc13-4 Defect and Discriminates between Genetic Subtypes of Familial Hemophagocytic Lymphohistiocytosis (FHL).
Authors: Aricò Maurizio , Marcenaro Stefania , Gallo Federico , Martini Stefania , Santoro Alessandra , Griffiths Gillian M. , Moretta Lorenzo , Pende Daniela 
Published: October 2019

Abstract
FHL is a rare fatal disease of early infancy characterized by hyperinflammatory sindrome, fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia, central nervous system alteration. Defective cellular cytotoxicity results in pathogen persistence, hypercytokinemia, disseminated infiltration of lymphocytes and histiocytes and hemophagocytosis. Differential diagnosis between FHL due to PRF1 (FHL2) or MUNC13–4 (FHL3) mutations, and additional unknown genetic subtypes is not easy unless mutation analysis is performed. Furthermore, some infection-associated cases of HLH (“secondary”) may mimic FHL.
We investigated natural killer (NK) cells function and phenotype in patients with FHL2 (n=5) or FHL3 (n=8). NK cells were cultured in IL-2 prior to their use in the various assays.
B-EBV cell lines and dendritic cells are suitable targets in 51Cr-release assays to reveal the FHL3 NK cell defect. Tumor targets were partially lysed by FHL3 NK cells expressing even trace amounts of Munc13–4 protein. FHL2 NK cells were completely unable to lyse target cells.
Cytokine production induced by mAb-crosslinking of triggering receptors was similar in patients and controls; under co-culture with 721.221 B-EBV cells, FHL NK cells were high producers, whereas control cells were almost ineffective. This could reflect persistence versus elimination of the source of stimulation in patients vs healthy controls, mimicking the pathophysiology of FHL. Finally, we tested cell surface CD107a expression in a degranulation assay, co-culturing 2x105 polyclonal NK cell or 24-hr IL-2 activated PBMCs with 2x105 target cells (K562, FO-1, 721.221 or P815 and anti-NKp30). CD107a expression was tested in CD56+ cell of either CD3− PBMC or polyclonal activated NK cell populations. CD107a was expressed in 11.8±7.5% cells in 6 FHL3 patients vs 48.5±11.5% in 9 controls (p<0.001). MFI was also lower in FHL3 patients vs. controls (9.3±3.1 vs. 42.6±21.9; p<0.001). Differently from FHL3, mean values of CD107a expression in 3 FHL2 patients were even higher than controls both in terms of percentage (63.3%) and MFI (86.6).
Altogether, these flow-cytometry data show that the pattern of CD107a expression represents a novel tool to identify the defect of degranulation characteristic of Munc 13–4 deficiency. Importantly, in FHL2 NK cells, whose granules lack perforin, degranulation pattern is normal. Although use of purified activated NK cells may increase discrimination power of the test, even PBMC (24-hr IL-2 activated) could be used to reveal a defect of granule exocytosis.
In addition to providing information useful for a better understanding of the pathophysiology of FHL and functional correlation with different gene mutations, our data may impact on FHL diagnosis. Combined use of surface expression of CD107a, together with intracytoplasmic staining with anti-perforin mAb, allows to promptly dissect FHL3 and FHL2 defects by the simple analysis of PBMC, so directing genetic analysis.