Key Points• Syntaxin-11-deficient mice as the first animal model for human familial hemophagocytic lymphohistiocytosis type 4 (FHL4).• T-cell exhaustion limits HLH progression in syntaxin-11-deficient mice. IntroductionCytolytic activity is a key function of cytotoxic T lymphocytes (CTLs) and NK cells to eliminate infected and malignant cells. Thereby, cytotoxicity is mediated by polarized exocytosis of lytic granules at the immunological synapse. Lytic granules are secretory lysosomes containing effector molecules, such as perforin and granzymes. On interaction with target cells, CTLs undergo polarization, characterized by Ca 2ϩ mobilization, reorientation of the microtubule organization center, and directed movement of lytic granules. The docking-priming-fusion steps of lytic granules are tightly controlled and result in the release of effector molecules at the immunological synapse. Synaptogamin-like proteins, members of the Rab and the Sec1/Munc18 protein families, as well as SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are involved in this process. SNARE proteins are helical transmembrane molecules expressed on the vesicle and target membranes. On association, they build a trans-SNARE complex, which leads to the fusion of docked lipid bilayers. 1 Syntaxin-11 (Stx11) is an atypical member of the Q-SNARE family and expressed in various cells of the immune system. 2-8 Stx11 associates with Vti1b, 7,9 SNAP23, 10 and syntaxin binding protein 2 (STXBP2/Munc18-2), 11,12 regulating the fusion of lytic granules with the plasma membrane at the immunological synapse. Consistent with this, CTLs and NK cells from patients with mutated STX11 gene or with experimentally reduced Stx11 expression levels displayed defects in degranulation and cytolytic activities. 5,6 In addition, it was described recently that Stx11 plays a role in platelet exocytosis. 13 Patients with mutations in the STX11 gene develop hemophagocytic lymphohistiocytosis (HLH), a lifethreatening disorder of severe hyperinflammation resulting from immune dysregulation. 4,6,14 HLH is associated with defects in components of the cytolytic machinery of T and NK cells [15][16][17][18] and characterized by inflammatory processes in various tissues as a result of infiltrating, hyperactive T cells, NK cells and macrophages, accompanied by massive cytokine production (IFN-␥, TNF-␣, . 19 Because of this loss in immune There is an Inside Blood commentary on this article in this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From homeostasis, patients present with prolonged fever, hepatosplenomegaly, neurologic manifestations, hypercytokinemia, and hemophagocytosis. The diagnostic criteria for HLH according to the Histiocyt...

Key Points Hermansky-Pudlak syndrome type 2 confers a moderate risk for hemophagocytic lymphohistiocytosis.

Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease of hyperinflammation resulting from immune dysregulation due to inherited defects in the cytolytic machinery of natural killer and T cells. In humans, mutations in seven genes encoding proteins involved in cytolytic effector functions have so far been identified that predispose to HLH. However, although most affected patients develop HLH eventually, disease onset and severity are highly variable. Due to the genetic heterogeneity and variable time and nature of disease triggers, the immunological basis of these variations in HLH progression is incompletely understood. Several murine models of primary HLH have been established allowing to study HLH pathogenesis under more defined conditions. Here we directly compare the clinical HLH phenotype in six HLH-prone mouse strains with defects in the granule-dependent cytotoxic pathway. A severity gradient of HLH manifestations could be identified that is defined by the genetically determined residual lytic activity of cytotoxic T lymphocytes (CTL) and their ability to control lymphocytic choriomeningitis virus, which was used as a trigger for disease induction. Importantly, analysis of cohorts of HLH patients with severe bi-allelic mutations in the corresponding genes yielded a similar severity gradient in human HLH as reflected by the age at disease onset. Our findings define HLH as a threshold disease determined by subtle differences in the residual lytic activity of CTL.

Biallelic mutations in the human lipopolysaccharide responsive beige-like anchor (LRBA) gene lead to a primary immunodeficiency known as LRBA deficiency, characterized by a broad range of clinical manifestations including autoimmunity, organomegaly, hypogammaglobulinemia and recurrent infections. Considering the phenotypic heterogeneity in patients and the severity of the disease, our aim was to assess the role of LRBA in immune cells and to understand the underlying pathomechanisms through the study of a Lrba knockout (Lrba) mouse model. LRBA-deficient mice did not show severe clinical or immunological signs of disease, either at steady state under specific-pathogen-free conditions, after vaccination with T-dependent and T-independent antigens, or in the context of acute infections with lymphocytic choriomeningitis virus (LCMV) or Salmonella Typhimurium. Although Lrba mice were able to produce normal serum immunoglobulin M (IgM) and IgG and to mount a specific immune response after immunization, they showed elevated serum and secretory basal IgA levels. LRBA was dispensable for B- and T-cell development, as well as for in vitro B-cell proliferation, survival, isotype switching and plasmablast differentiation. Interestingly, Lrba mice displayed decreased cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) expression by regulatory T cells and activated conventional CD4 and CD8 T lymphocytes, reduced frequency of peritoneal B-1a cells along with diminished interleukin-10 production and increased percentages of T follicular helper cells in Peyer's patches, but without developing overt signs of autoimmunity. Our findings expand the role of LRBA in immune regulatory mechanisms previously reported in patients, and suggest a novel role in IgA production that is crucial for the protection of mucosal surfaces and gut-associated immune tolerance.

Familial hemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory syndrome affecting patients with genetic cytotoxicity defects. Perforin‐deficient (PKO) mice recapitulate the full clinical picture of FHL after infection with lymphocytic choriomeningitis virus (LCMV). Hyperactivated CD8+ T cells and IFN‐γ have been identified as the key drivers of FHL and represent targets for therapeutic interventions. However, the response of patients is variable. This could be due to trigger‐dependent differences in pathogenesis, which is difficult to address in FHL patients, since the trigger frequently escapes detection. We established an alternative FHL model using intravenous infection of PKO mice with murine CMV (MCMV)Smith. PKO mice developed acute FHL after both infections and fulfilled HLH diagnostic criteria accompanied by excessive IFN‐γ production by disease‐inducing T cells, that enrich in the BM. However, direct comparison of the two infection models disclosed trigger‐dependence of FHL progression and revealed a higher contribution of CD4 T cells and NK cells to IFN‐γ production after MCMV infection. Importantly, therapeutic intervention by IFN‐γ neutralization or CD8+ T‐cell depletion had less benefit in MCMV‐triggered FHL compared to LCMV‐triggered FHL, likely due to MCMV‐induced cytopathology. Thus, the context of the specific triggering viral infection can impact the success of targeted immunotherapeutic HLH control.

The two T-box transcription factors T-bet and Eomesodermin (Eomes) are important regulators of cytotoxic lymphocytes (CTLs), such as activated CD8 T cells, which are essential in the fight against intracellular pathogens and tumors. Both transcription factors share a great degree of homology based on sequence analysis and as a result exert partial functional redundancy during viral infection. However, the actual degree of redundancy between T-bet and Eomes remains a matter of debate and is further confounded by their distinct spatiotemporal expression pattern in activated CD8 T cells. To directly investigate the functional overlap of these transcription factors, we generated a new mouse model in which Eomes expression is under the transcriptional control of the endogenous Tbx21 (encoding for Tbet) locus. Applying this model, we demonstrate that the induction of Eomes in lieu of T-bet cannot rescue T-bet deficiency in CD8 T cells during acute lymphocytic choriomeningitis virus (LCMV) infection. We found that the expression of Eomes instead of T-bet was not sufficient for early cell expansion or effector cell differentiation. Finally, we show that imposed expression of Eomes after acute viral infection promotes some features of exhaustion but must act in concert with other factors during chronic viral infection to establish all hallmarks of exhaustion. In summary, our results clearly underline the importance of T-bet in guiding canonical CTL development during acute viral infections.

Primary hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome caused by impaired lymphocyte cytotoxicity. First‐line therapeutic regimens directed against activated immune cells or secreted cytokines show limited efficacy since they do not target the underlying immunological problem: defective lymphocyte cytotoxicity causing prolonged immune stimulation. A potential rescue strategy would be the adoptive transfer of ex vivo gene‐corrected autologous T cells. However, transfusion of cytotoxicity‐competent T cells under conditions of hyperinflammation may cause more harm than benefit. As a proof‐of‐concept for adoptive T cell therapy (ATCT) under hyperinflammatory conditions, we transferred syngeneic, cytotoxicity‐competent T cells into mice with virally triggered active primary HLH. ATCT with functional syngeneic trigger‐specific T cells cured Jinx mice from active HLH without life‐threatening side effects and protected Perforin‐deficient mice from lethal HLH progression by reconstituting cytotoxicity. Cured mice were protected long‐term from HLH relapses. A threshold frequency of transferred T cells with functional differentiation was identified as a predictive biomarker for long‐term survival. This study is the first proof‐of‐concept for ATCT in active HLH.

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