Neonatal hemophagocytic lymphohistiocytosis (HLH) is a medical emergency that can be associated with significant morbidity and mortality. Often these patients present with familial HLH (f-HLH), which is caused by gene mutations interfering with the cytolytic pathway of cytotoxic T-lymphocytes (CTLs) and natural killer cells. Here we describe a male newborn who met the HLH diagnostic criteria, presented with profound cholestasis, and carried a maternally inherited heterozygous mutation in syntaxin-binding protein-2 [ STXBP2 , c.568C>T (p.Arg190Cys)] in addition to a severe pathogenic variant in glucose 6-phosphate dehydrogenase [ G6PD , hemizygous c.1153T>C (Cys385Arg)]. Although mutations in STXBP2 gene are associated with f-HLH type 5, the clinical and biological relevance of the p.Arg190Cys mutation identified in this patient was uncertain. To assess its role in disease pathogenesis, we performed functional assays and biochemical and microscopic studies. We found that p.Arg190Cys mutation did not alter the expression or subcellular localization of STXBP2 or STX11, neither impaired the STXBP2/STX11 interaction. In contrast, forced expression of the mutated protein into normal CTLs strongly inhibited degranulation and reduced the cytolytic activity outcompeting the effect of endogenous wild-type STXBP2. Interestingly, arginine 190 is located in a structurally conserved region of STXBP2 where other f-HLH-5 mutations have been identified. Collectively, data strongly suggest that STXBP2-R190C is a deleterious variant that may act in a dominant-negative manner by probably stabilizing non-productive interactions between STXBP2/STX11 complex and other still unknown factors such as the membrane surface or Munc13-4 protein and thus impairing the release of cytolytic granules. In addition to the contribution of STXBP2-R190C to f-HLH, the accompanied G6PD mutation may have compounded the clinical symptoms; however, the extent by which G6PD deficiency has contributed to HLH in our patient remains unclear.
Cytotoxic T‐lymphocytes (CTLs) regulate the immune response by recognizing and destroying cancerous or virus‐infected cells through the polarized release of apoptosis‐inducing proteins at the contact area known as the immunological synapse (IS). The synaptic membrane is a heterogenous region where protein complexes rearrange on the cell surface. Within the membrane are phospholipids that assemble to relay information from extracellular receptors to induce global cellular changes. Diacylglycerol (DAG) is a key secondary messenger of T‐cell receptor (TCR) activation which induces microtubule‐organizing center polarization towards the IS and signaling via MAPK and NFkB pathways. Abnormal lipid homeostasis at the synapse is linked to autoimmune disorders. However, our understanding on how cells regulate dynamic transfer of lipids from one cellular compartment to another is still scant. Evidence points to the role of lipid transport proteins (LTPs) in the regulation of lipid homeostasis without the need for membrane fusion. Extended‐Synaptotagmins (E‐Syts) are a group of LTPs tethering the endoplasmic reticulum (ER) to plasma membrane (PM), thus controlling membrane lipid compositions by transporting DAG down its concentration gradient. Herein, we tested the hypothesis that E‐Syts play a role in the modulation of diacylglycerol at the synaptic membrane, ER‐PM morphology and signaling dynamics by performing the following experiments: 1) assessing the role of E‐Syts in regulating the effector functions of human lymphocytes by measuring cellular cytotoxicity, degranulation, TCR signaling via phospho‐protein analysis and intracellular Ca2+ levels; 2) testing the role of E‐Syts in DAG distribution at the IS via stimulated emission depletion (STED) microscopy. Our results indicate that E‐Syts down‐modulate human lymphocyte activation as their knock‐down leads to enhanced TCR signaling, degranulation, and cytokine production measured by flow cytometry. Using GFP‐based bioprobes for DAG and tagged E‐Syt constructs, we observed proximal localization of E‐Syts with signaling protein LAT and CD4 via super‐resolution microscopy. Accordingly, E‐Syt1 knockout Jurkat cells at resting state express localized DAG accumulation similar to activated WT Jurkat cells. Our results provide, for the first time, an understanding of spatio‐temporal distribution as well as the role of lipid regulators at membrane junction sites during IS formation. We believe that E‐Syts play an essential role in maintenance of synaptic lipids via regulation of non‐vesicular lipid transport, whose loss might contribute to enhanced activity of CTLs at resting conditions. Collectively, our results suggest that E‐Syts are critical components to orchestrate lipid distribution during synapse formation. Future experiments will be required to shed light on additional steady state regulators of synaptic membrane lipids, highlighting the importance of membrane crosstalk in the setting of CTLs.
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