ABSTRACT
Purpose: Cytokine storm syndrome (CSS), also known as hemophagocytic lymphohistiocytosis (HLH), is a life-threatening condition presenting with fever and shock-like multi-organ dysfunction (MOD). Familial CSS/HLH results from homozygous defects in genes critical to perforin-mediated cytolysis by cytolytic lymphocytes. 30-40% of CSS cohorts possess heterozygous defects in familial CSS/HLH genes resulting in decreased cytolysis, prolonged interaction with antigen presenting cells, and subsequent increased pro-inflammatory cytokines resulting in MOD. Since NK cell dysfunction is common in CSS, there are likely other genes contributing to CSS via decreased cytolysis. Methods: CSS patients at UAB, and children with SARS-CoV-2 multisystem inflammatory syndrome in children (MIS-C) at Northwell were screened for mutations via genome sequencing or a commercial immunodeficiency panel. Seven patients (3 with MIS-C) had mutations in the guanine nucleotide exchange factor DOCK8 critical to NK cell function. DOCK8 mutations, or wild-type (WT) sequence controls, were introduced into human NK-92 cells by FOAMY virus (FV) transduction. FV-transduced WT and mutant DOCK8-expressing NK-92 cells were incubatedwith K562 target cells and compared for cytolytic activity, degranulation (CD107a), and cytokine [interferon-g (IFNg), tumor necrosis factor (TNF)] production by flow cytometry. Results: Four patients had rare heterozygous missense DOCK8 mutations, and 2 had the same DOCK8 polymorphism (Asp63Asn, 12% of population). One splice acceptor variant (c.54-1G>T, 0.03%) disrupted RNA splicing by exon-trapping. One novel (Ala261Val) DOCK8 mutant decreased NK cell lytic activity (n = 3, decreased -50% versusWT, p = 0.007) and degranulation by >50% (n = 3, p = 0.013). During incubation with K562 targets, NK cells expressing the novel DOCK8 mutant increased IFNg and TNF expression by >200% (p = 0.019 and p = 0.003, respectively). The DOCK8 polymorphism decreased lysis and degranulation to a lesser degree. Conclusion: Heterozygous mutations in DOCK8, a novel CSS/HLHassociated gene, contribute to CSS pathology through a partial dominant-negative or hypomorphic effect resulting in decreased cytolysis and increased pro-inflammatory cytokine production.