Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Gene editing of hematopoietic stem cells restores T-cell response in familial hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. OBJECTIVE: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). METHODS: We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection. RESULTS: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. CONCLUSIONS: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.

Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A).

Introduction: Inborn errors of immunity (IEI) are characterized by a dysfunction of the immune system leading to increased susceptibility to infections, impaired immune regulation and cancer. We present a unique consanguineous family with a history of Hodgkin lymphoma, impaired EBV control and a late onset hemophagocytic lymphohistiocytosis (HLH). Methods and results: Overall, family members presented with variable impairment of NK cell and cytotoxic T cell degranulation and cytotoxicity. Exome sequencing identified homozygous variants in RAB27A, FBP1 (Fructose-1,6-bisphosphatase 1) and ACAD9 (Acyl-CoA dehydrogenase family member 9). Variants in RAB27A lead to Griscelli syndrome type 2, hypopigmentation and HLH predisposition. Discussion: Lymphoma is frequently seen in patients with hypomorphic mutations of genes predisposing to HLH. We hypothesize that the variants in FBP1 and ACAD9 might aggravate the clinical and immune phenotype, influence serial killing and lytic granule polarization by CD8 T cells. Understanding of the interplay between the multiple variants identified by whole exome sequencing (WES) is essential for correct interpretation of the immune phenotype and important for critical treatment decisions.

IFNγ binding to extracellular matrix prevents fatal systemic toxicity.

Interferon-γ (IFNγ) is an important mediator of cellular immune responses, but high systemic levels of this cytokine are associated with immunopathology. IFNγ binds to its receptor (IFNγR) and to extracellular matrix (ECM) via four positively charged C-terminal amino acids (KRKR), the ECM-binding domain (EBD). Across evolution, IFNγ is not well conserved, but the EBD is highly conserved, suggesting a critical function. Here, we show that IFNγ lacking the EBD (IFNγΔKRKR) does not bind to ECM but still binds to the IFNγR and retains bioactivity. Overexpression of IFNγΔKRKR in tumors reduced local ECM binding, increased systemic levels and induced sickness behavior, weight loss and toxicity. To analyze the function of the EBD during infection, we generated IFNγΔKRKR mice lacking the EBD by using CRISPR-Cas9. Infection with lymphocytic choriomeningitis virus resulted in higher systemic IFNγΔKRKR levels, enhanced sickness behavior, weight loss and fatal toxicity. We conclude that local retention of IFNγ is a pivotal mechanism to protect the organism from systemic toxicity during prolonged immune stimulation.

Adoptive T cell therapy cures mice from active hemophagocytic lymphohistiocytosis (HLH).

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.

Definition and validation of serum biomarkers for optimal differentiation of hyperferritinaemic cytokine storm conditions in children: a retrospective cohort study.

BACKGROUND: Cytokine storm syndromes are life-threatening complications that can occur in children with rheumatic conditions (macrophage activation syndrome [MAS]), inherited cytotoxicity defects (ie, primary haemophagocytic lymphohistiocytosis [HLH]), or as a result of infection or malignancies (ie, secondary HLH). To adequately steer treatment, an early and clear discrimination of these entities is essential. We aimed to define and validate serum biomarker profiles that can differentiate between primary HLH, secondary HLH (predominantly infection-associated), and MAS associated with systemic juvenile idiopathic arthritis (systemic JIA-MAS). METHODS: In this multicentre, retrospective, cohort study, serum samples from patients (0-18 years) with a clinical diagnosis of primary HLH, secondary HLH, or systemic JIA-MAS were analysed by immunoassays for 55 cytokines and chemokines. Serum samples were collected from patients treated at seven clinical centres in Europe and North America. 15 serum biomarkers were validated using an independent commercial assay, and the diagnostic accuracy of the best performing biomarkers was tested in an independent validation cohort. FINDINGS: Serum samples were collected between Dec 7, 2010, and Jan 26, 2018. In the discovery cohort of 43 patients (24 girls and 19 boys) multi-marker analyses revealed distinct serum biomarker profiles associated with primary or secondary HLH versus systemic JIA-MAS. Ten biomarkers were identified that were differentially elevated in either HLH or systemic JIA-MAS and distinguished between these clinical entities, six of which were tested in an independent validation cohort of 79 patients (34 girls and 45 boys). Serum concentrations of S100A12 and interleukin-18, as well as ratios of both S100A12 and IL-18 with chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 were identified as the most promising candidates for differential diagnostics. INTERPRETATION: At initial presentation, when it is unclear whether a patient with excessive hyperferritinaemic inflammation has primary HLH, infection-associated secondary HLH, or MAS, high serum concentrations of S100A12 indicate an initial differential diagnosis of systemic JIA-MAS, thus helping to guide subsequent treatment decisions. We therefore suggest the inclusion of serum S100A12 and IL-18 in the diagnostic investigations for hyperferritinaemic syndromes; however, the definition and introduction of universially applicable cutoff values are still required. FUNDING: German Research Foundation, the Center for Interdisciplinary Clinical Research at University Hospital Muenster, the EU's Horizon 2020 research and innovation programme, and the Deutsche Kinderkrebsstiftung.

Griscelli Syndrome Type 2 Sine Albinism: Unraveling Differential RAB27A Effector Engagement.

Griscelli syndrome type 2 (GS-2) is an inborn error of immunity characterized by partial albinism and episodes of hemophagocytic lymphohistiocytosis (HLH). It is caused by RAB27A mutations that encode RAB27A, a member of the Rab GTPase family. RAB27A is expressed in many tissues and regulates vesicular transport and organelle dynamics. Occasionally, GS-2 patients with RAB27A mutation display normal pigmentation. The study of such variants provides the opportunity to map distinct binding sites for tissue-specific effectors on RAB27A. Here we present a new case of GS-2 without albinism (GS-2 sine albinism) caused by a novel missense mutation (Val143Ala) in the RAB27A and characterize its functional cellular consequences. Using pertinent animal cell lines, the Val143Ala mutation impairs both the RAB27A-SLP2-A interaction and RAB27A-MUNC13-4 interaction, but it does not affect the RAB27A-melanophilin (MLPH)/SLAC2-A interaction that is crucial for skin and hair pigmentation. We conclude that disruption of the RAB27A-MUNC13-4 interaction in cytotoxic lymphocytes leads to the HLH predisposition of the GS-2 patient with the Val143Ala mutation. Finally, we include a review of GS-2 sine albinism cases reported in the literature, summarizing their genetic and clinical characteristics.

NK1.1+ innate lymphoid cells in salivary glands inhibit establishment of tissue-resident memory CD8+ T cells in mice.

NK1.1+ cells found in salivary glands (SG) represent a unique cell population of innate lymphoid cells (ILC) with characteristics of both conventional NK cells and ILC1. Here, we demonstrate that these NK1.1+  cells limit the accumulation and differentiation of virus-specific tissue-resident memory CD8+ T cells (TRM  cells) in SG of mice infected with lymphocytic choriomeningitis virus (LCMV). The negative regulation of LCMV-specific CD8+ TRM  cells by NK1.1+  cells in SG is independent of NKG2D, NKp46, TRAIL, and perforin. Moreover, analysis of NKp46iCre+ Eomesfl/fl mice revealed that Eomes-dependent conventional NK cells are dispensable for negative regulation. Since the SG are prone to autoimmune reactions, regulation of TRM  cells by tissue-resident ILC may be particularly important to prevent immunopathology in this organ.

Modeling MyD88 Deficiency In Vitro Provides New Insights in Its Function.

Inherited defects in MyD88 and IRAK4, two regulators in Toll-like receptor (TLR) signaling, are clinically highly relevant, but still incompletely understood. MyD88- and IRAK4-deficient patients are exceedingly susceptible to a narrow spectrum of pathogens, with ∼50% lethality in the first years of life. To better understand the underlying molecular and cellular characteristics that determine disease progression, we aimed at modeling the cellular response to pathogens in vitro. To this end, we determined the immunophenotype of monocytes and macrophages derived from MyD88- and IRAK4-deficient patients. We recognized that macrophages derived from both patients were particularly poorly activated by streptococci, indicating that both signaling intermediates are essential for the immune response to facultative pathogens. To characterize this defect in more detail, we generated induced pluripotent stem cells (iPSCs) of fibroblasts derived from an MyD88-deficient patient. The underlying genetic defect was corrected using Sleeping Beauty transposon vectors encoding either the long (L) or the short (S) MYD88 isoform, respectively. Macrophages derived from these iPSC lines (iMacs) expressed typical macrophage markers, stably produced either MyD88 isoform, and showed robust phagocytic activity. Notably, iMacs expressing MyD88-L, but not MyD88-S, exhibited similar responses to external stimuli, including cytokine release patterns, as compared to genetically normal iMacs. Thus, the two MyD88 isoforms assume distinct functions in signaling. In conclusion, iPSC technology, in combination with efficient myeloid differentiation protocols, provides a valuable and inexhaustible source of macrophages, which can be used for disease modeling. Moreover, iPSC-derived macrophages may eventually aid in stabilizing MyD88-deficient patients during pyogenic infections.

Clinical and Immunological Phenotype of Patients With Primary Immunodeficiency Due to Damaging Mutations in NFKB2.

Non-canonical NF-κB-pathway signaling is integral in immunoregulation. Heterozygous mutations in NFKB2 have recently been established as a molecular cause of common variable immunodeficiency (CVID) and DAVID-syndrome, a rare condition combining deficiency of anterior pituitary hormone with CVID. Here, we investigate 15 previously unreported patients with primary immunodeficiency (PID) from eleven unrelated families with heterozygous NFKB2-mutations including eight patients with the common p.Arg853* nonsense mutation and five patients harboring unique novel C-terminal truncating mutations. In addition, we describe the clinical phenotype of two patients with proximal truncating mutations. Cohort analysis extended to all 35 previously published NFKB2-cases revealed occurrence of early-onset PID in 46/50 patients (mean age of onset 5.9 years, median 4.0 years). ACTH-deficiency occurred in 44%. Three mutation carriers have deceased, four developed malignancies. Only two mutation carriers were clinically asymptomatic. In contrast to typical CVID, most patients suffered from early-onset and severe disease manifestations, including clinical signs of T cell dysfunction e.g., chronic-viral or opportunistic infections. In addition, 80% of patients suffered from (predominately T cell mediated) autoimmune (AI) phenomena (alopecia > various lymphocytic organ-infiltration > diarrhea > arthritis > AI-cytopenia). Unlike in other forms of CVID, auto-antibodies or lymphoproliferation were not common hallmarks of disease. Immunophenotyping showed largely normal or even increased quantities of naïve and memory CD4+ or CD8+ T-cells and normal T-cell proliferation. NK-cell number and function were also normal. In contrast, impaired B-cell differentiation and hypogammaglobinemia were consistent features of NFKB2-associated disease. In addition, an array of lymphocyte subpopulations, such as regulatory T cell, Th17-, cTFH-, NKT-, and MAIT-cell numbers were decreased. We conclude that heterozygous damaging mutations in NFKB2 represent a distinct PID entity exceeding the usual clinical spectrum of CVID. Impairment of the non-canonical NF-κB pathways affects function and differentiation of numerous lymphocyte-subpopulations and thus causes a heterogeneous, more severe form of PID phenotype with early-onset. Further characteristic features are multifaceted, primarily T cell-mediated autoimmunity, such as alopecia, lymphocytic organ infiltration, and in addition frequently ACTH-deficiency.

Is an infectious trigger always required for primary hemophagocytic lymphohistiocytosis? Lessons from in utero and neonatal disease.

In this report, we evaluate the hypothesis that hemophagocytic lymphohistiocytosis in patients with defects of lymphocyte cytotoxicity is usually triggered by infections. We show that in the majority of patients, extensive virus PCR panels performed in addition to routine microbiological investigations remain negative and summarize 25 patients with onset of hemophagocytic lymphohistiocytosis in utero or within the first 10 days of life, in none of which an associated bacterial or viral infection was reported. These observations, even though preliminary, invite to consider a key role of lymphocyte cytotoxicity in controlling T-cell homeostasis also in the absence of apparent infectious stimuli.

CD57 identifies T cells with functional senescence before terminal differentiation and relative telomere shortening in patients with activated PI3 kinase delta syndrome.

Premature T-cell immunosenescence with CD57+ CD8+ T-cell accumulation has been linked to immunodeficiency and autoimmunity in primary immunodeficiencies including activated PI3 kinase delta syndrome (APDS). To address whether CD57 marks the typical senescent T-cell population seen in adult individuals or identifies a distinct population in APDS, we compared CD57+ CD8+ T cells from mostly pediatric APDS patients to those of healthy adults with similarly prominent senescent T cells. CD57+ CD8+ T cells from APDS patients were less differentiated with more CD27+ CD28+ effector memory T cells showing increased PD1 and Eomesodermin expression. In addition, transition of naïve to CD57+ CD8+ T cells was not associated with the characteristic telomere shortening. Nevertheless, they showed the increased interferon-gamma secretion, enhanced degranulation and reduced in vitro proliferation typical of senescent CD57+ CD8+ T cells. Thus, hyperactive PI3 kinase signaling favors premature accumulation of a CD57+ CD8+ T-cell population, which shows most functional features of typical senescent T cells, but is different in terms of differentiation and relative telomere shortening. Initial observations indicate that this specific differentiation state may offer the opportunity to revert premature T-cell immunosenescence and its potential contribution to inflammation and immunodeficiency in APDS.

CD59 deficiency presenting as polyneuropathy and Moyamoya syndrome with endothelial abnormalities of small brain vessels.

CD59 is involved in lymphocyte signal transduction and regulates complement-mediated cell lysis by inhibiting the membrane attack complex. In the cases reported so far, congenital isolated CD59 deficiency was associated with recurrent episodes of hemolytic anemia, peripheral neuropathy, and strokes. Here, we report on a patient from a consanguineous Turkish family, who had a first episode of hemolytic anemia at one month of age and presented at 14 months with acute Guillain-Barré syndrome (GBS). The child suffered repeated infection-triggered relapses leading to the diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP). Although partly steroid-responsive, the polyneuropathy failed to be stabilized by a number of immunosuppressive agents. At the age of 6 years, he developed acute hemiparesis and showed progressive stenosis of proximal cerebral arteries, evolving into Moyamoya syndrome (MMS) with recurrent infarctions leading to death at 8 years of age. Post-mortem genetic analysis revealed a pathogenic p.(Asp49Valfs*31) mutation in CD59. Re-analysis of brain biopsy specimens showed absent CD59 expression and severe endothelial damage. Whereas strokes are a known feature of CD59 deficiency, MMS has not previously been described in this condition. Therefore, we conclude that in MMS combined with hemolysis or neuropathy CD59 deficiency should be considered. Establishing the diagnosis and targeted therapy with eculizumab might have prevented the lethal course in our patient.

Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4T337S mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4T337S-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4T337S intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ-induced genes. Assessing cellular and tissue expression, classical inflammasome components such as Il1b, Nlrp3, and Mefv predominated in neutrophils, whereas Nlrc4 and Il18 were distinctly epithelial. Demonstrating the importance of free IL-18, Il18 transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4T337S mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis.

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with "full" HLH and 79/111 patients with "incomplete/atypical" HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.