OP0314 DOCK8 MUTATIONS IN COVID-19 AND MIS-C CYTOKINE STORM SYNDROME

Background:We recently identified DOCK8 as a novel gene associated with cytokine storm syndrome (CSS)1. Heterozygous missense mutations in DOCK8 diminish NK cell lytic function and contribute to increased pro-inflammatory cytokine production (CSS). CSS is a potential complication of COVID-19 with severe consequences2. Children are at risk of a SARS-CoV-2 post-infectious CSS, multisystem inflammatory syndrome in children (MIS-C)3. Host genetic factors associated with COVID-19 CSS and MIS-C CSS are unknown.Objectives:The goals are to identify and functionally study rare mutations in DOCK8 in patients with SARS-CoV-2 COVID-19 and MIS-C.Methods:To date, 16 adult patients enrolled in a COVID-19 CSS clinical trial at UAB had whole genome sequencing. Four (25%) had rare heterozygous DOCK8 mutations (3 missense, 1 intronic). A COVID-19 CSS adult patient in Seattle also had a DOCK8 missense mutation. In addition, DOCK8 missense mutations were identified in five children (UAB & Northwell) hospitalized with MIS-C. DOCK8 mutations, or wild-type (WT) sequence controls, were introduced into human NK-92 cells by FOAMY virus transduction. WT and mutant DOCK8-expressing NK-92 cells were incubated with K562 target cells and compared for cytolysis and degranulation (CD107a).Results:One COVID-19 patient DOCK8 mutation (Gly523Arg) reduced NK cell degranulation by 30% and cytolysis by 23% (n=3) (Figure 1). Similar studies of 3 MIS-C patients with DOCK8 missense mutations (Arg899Trp, Ala2Thr, Pro687Leu) revealed up to 31% reduced NK cell degranulation and 48% reduction in cytolysis by 3 distinct mutations (n=3). Two-way ANOVA analysis revealed statistically significant (p<0.05) differences in NK cell degranulation and lysis for four unique DOCK8 mutations.Conclusion:Heterozygous DOCK8 missense mutations may contribute to severe COVID-19 and MIS-C CSS by partial dominant-negative effects yielding decreased NK cell cytolysis.References:[1]Schulert GS, Cron RQ. The genetics of macrophage activation syndrome. Genes Immun 2020:21:169-181.[2] Cron RQ, Chatham WW. The rheumatologist’s role in COVID-19. J Rheumatol 2020:47:639-642.[3]Reiff D, Mannion ML, Samuy N, Scalici P, Cron RQ. Distinguishing active pediatric COVID-19 from MIS-C. Pediatr Rheumatol Online J, in press.Disclosure of Interests:Randy Cron Consultant of: SOBI, Novartis, Pfizer, Sironax, Grant/research support from: SOBI, Mingce Zhang: None declared, Remy Cron: None declared, Devin Absher: None declared, John Bridges: None declared, Amanda Schnell: None declared, Pavan Bhatraju: None declared, Anshul Vagrecha: None declared, Shannon Lozinsky: None declared, Suchitra Acharya: None declared, Carolyn Levy: None declared, Winn Chatham Grant/research support from: SOBI.

Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.

New Insights Into Biomphalysin Gene Family Diversification in the Vector Snail Biomphalaria glabrata

Aerolysins initially characterized as virulence factors in bacteria are increasingly found in massive genome and transcriptome sequencing data from metazoans. Horizontal gene transfer has been demonstrated as the main way of aerolysin-related toxins acquisition in metazoans. However, only few studies have focused on their potential biological functions in such organisms. Herein, we present an extensive characterization of a multigene family encoding aerolysins - named biomphalysin - in Biomphalaria glabrata snail, the intermediate host of the trematode Schistosoma mansoni. Our results highlight that duplication and domestication of an acquired bacterial toxin gene in the snail genome result in the acquisition of a novel and diversified toxin family. Twenty-three biomphalysin genes were identified. All are expressed and exhibited a tissue-specific expression pattern. An in silico structural analysis was performed to highlight the central role played by two distinct domains i) a large lobe involved in the lytic function of these snail toxins which constrained their evolution and ii) a small lobe which is structurally variable between biomphalysin toxins and that matched to various functional domains involved in moiety recognition of targets cells. A functional approach suggests that the repertoire of biomphalysins that bind to pathogens, depends on the type of pathogen encountered. These results underline a neo-and sub-functionalization of the biomphalysin toxins, which have the potential to increase the range of effectors in the snail’s immune arsenal.

Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior

Cytotoxic immune cells are endowed with a high degree of heterogeneity in their lytic function, but how this heterogeneity is generated is still an open question. We therefore investigated if human CD8+ T cells could segregate their lytic components during telophase, using imaging flow cytometry, confocal microscopy and live cell imaging. We show that CD107a+-intracellular vesicles, perforin and granzyme B unevenly segregate in a constant fraction of telophasic cells during each division round. Mathematical modeling posits that unequal lytic molecule inheritance by daughter cells results from the random distribution of lytic granules on the two sides of the cleavage furrow. Finally, we establish that the level of lytic compartment in individual CTL dictates CTL killing capacity. Together, our results show the stochastic asymmetric distribution of effector molecules in dividing CD8+ T cells. They propose uneven mitotic repartition of pre-packaged lytic components as a mechanism generating non-hereditary functional heterogeneity in CTL.

Evolution along the parasitism-mutualism continuum determines the genetic repertoire of prophages

Integrated into their bacterial hosts’ genomes, prophage sequences exhibit a wide diversity of length and gene content, from highly degraded cryptic sequences to intact, functional prophages that retain a full complement of lytic-function genes. We apply three approaches—bioinformatics, analytical modelling and computational simulation—to understand the diverse gene content of prophages. In the bioinformatics work, we examine the distributions of over 50,000 annotated prophage genes identified in 1384 prophage sequences, comparing the gene repertoires of intact and incomplete prophages. These data indicate that genes involved in the replication, packaging, and release of phage particles have been preferentially lost in incomplete prophages, while tail fiber, transposase and integrase genes are significantly enriched. Consistent with these results, our mathematical and computational approaches predict that genes involved in phage lytic function are preferentially lost, resulting in shorter prophages that often retain genes that benefit the host. Informed by these models, we offer novel hypotheses for the enrichment of integrase and transposase genes in cryptic prophages. Overall, we demonstrate that functional and cryptic prophages represent a diversity of genetic sequences that evolve along a parasitism-mutualism continuum.

Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior

Cytotoxic immune cells are endowed with a high degree of heterogeneity in their lytic function, but how this heterogeneity is generated is still an open question. We therefore investigated if human CD8+ T cells could segregate their lytic components during telophase, using imaging flow cytometry, confocal microscopy and live cell imaging. We show that CD107a+-intracellular vesicles, perforin and granzyme B unevenly segregate in a constant fraction of telophasic cells during each division round. Mathematical modeling posits that unequal lytic molecule inheritance by daughter cells results from the random distribution of lytic granules on the two sides of the cleavage furrow. Finally, we establish that the level of lytic compartment in individual CTL dictates CTL killing capacity. Together, our results show the stochastic asymmetric distribution of effector molecules in dividing CD8+ T cells. They propose uneven mitotic repartition of pre-packaged lytic components as a mechanism generating non-hereditary functional heterogeneity in CTL.

Human CD8+ T-cells Require Glycolysis to Elicit Effector Function

Targeting human T-cell metabolism for modulating immune function requires an understanding of macronutrient utilization. Using metabolic inhibition during activation of human naïve CD8+ T-cells, we demonstrate blocking glycolysis or mitochondrial respiration prevents T-cell proliferation. However, after activation and differentiation, the metabolic program changes. Inhibition of glycolysis abolished cytotoxic T-lymphocyte (CTL) activity, whereas mitochondrial inhibition had no effect on CTL lytic function. Studies with uniformly labeled 13C-glucose confirmed CTL convert the majority of glucose to lactate. The role of glycolysis in CTL function was assessed using NOD models of Type 1 diabetes (T1D). Treatment of NOD models with a glycolysis inhibitor resulted in reduced and delayed T1D incidence and significantly preserved β-cell mass. We conclude glycolysis and mitochondrial ATP production are essential for efficient T-cell activation, but only glycolysis is essential for CTL lytic function. These data suggest targeting glycolysis in CTLs is a promising pathway to prevent T-cell-mediated autoimmunity.