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.

Ruxolitinib Pharmacokinetics and Pharmacodynamics in Children with Acute and Chronic Graft-versus-Host Disease.

We evaluated the pharmacokinetics (PK) of oral ruxolitinib in children with steroid-refractory acute graft-versus-host disease (aGVHD) (age <12 years) and chronic GVHD (cGVHD) (age ≤18 years) using our published pediatric dosing. PK sampling was performed before and 2 hours after ruxolitinib administration in patients with established cGVHD. More extensive PK analyses were performed in patients with newly diagnosed aGVHD or cGVHD before and .5, 1, 2, 4, and 6 hours after ruxolitinib administration in patients weighing >10 kg and before, 3+, and 6+ hours in children weighing <10 kg. pSTAT1, pSTAT3, and pSTAT5 expression levels were measured on CD4+ and CD8+ T cells before and 2 hours after ruxolitinib administration as a pharmacodynamic marker of JAK/STAT inhibition. Thirteen patients were prospectively enrolled, including 8 with existing cGVHD (age 0 to ≤18 years), 4 with new-onset steroid-refractory aGVHD (age 0 to <12 years) and 1 with newly diagnosed steroid-refractory cGVHD. Great variability in PK was seen. Mean oral clearance (CL/F) was 7.76 ± 4.09 L/h (range, 3.1 to 15.3 L/h). The average elimination half-life was 2.32 ± 1.0 hours. Mean ruxolitinib clearance was higher in children age <2 years versus those age >2 years (12.1 ± 3.0 L/h versus 5.7 ± 2.8 L/h; P = .005) and was reduced with concurrent treatment with azoles and azithromycin. We saw a variable reduction in pSTAT1/3/5 expression on T cells at time of peak ruxolitinib absorption (2 hours after dosing). Children <10 kg had lower ruxolitinib exposure, possibly due to inherent increased drug clearance or variability in dosing methods, leading to decreased drug absorption.

Quercetin ameliorates XIAP deficiency-associated hyperinflammation.

XIAP (X-linked inhibitor of apoptosis) deficiency is a rare inborn error of immunity. XIAP deficiency causes hyperinflammatory disease manifestations due to dysregulated TNF (tumor necrosis factor)-receptor signaling and NLRP3 (NOD- [nucleotide-binding oligomerization domain], LRR- [leucine-rich repeat] and pyrin domain-containing protein 3) inflammasome function. Safe and effective long-term treatments are needed and are especially important to help prevent the need for high-risk allogeneic hematopoietic cell transplantation. Here we evaluated inflammasome inhibitors as potential therapeutics with a focus on the natural flavonoid antioxidant quercetin. Bone marrow (BM)-derived macrophages were derived from XIAP-deficient or wild-type (WT) mice. Human monocytes were obtained from control or XIAP-deficient patients. Cells were stimulated with TLR (Toll-like receptor) agonists or TNF-α ± inhibitors or quercetin. For in vivo lipopolysaccharide (LPS) challenge experiments, XIAP-deficient or WT mice were fed mouse chow ± supplemental quercetin (50 mg/kg per day exposure) for 7 days followed by a challenge with 10 ng/kg LPS. IL-1ß (interleukin-1ß) and IL-18 were measured by ELISA (enzyme-linked immunosorbent assay). In murine studies, quercetin prevented IL-1ß secretion from XIAP knockout cells following TLR agonists or TNF-α stimulation (P < .05) and strongly reduced constitutive production of IL-18 by both WT and XIAP-deficient cells (P < .05). At 4 hours after in vivo LPS challenge, blood levels of IL-1ß and IL-18 were significantly decreased in mice that had received quercetin-supplemented chow (P < .05). In experiments using human cells, quercetin greatly reduced IL-1ß secretion by monocytes following TNF-α stimulation (P < .05). Our data suggest that quercetin may be an effective natural therapeutic for the prevention of XIAP deficiency-associated hyperinflammation. Clinical trials, including careful pharmacokinetic and pharmacodynamic studies to ensure that effective levels of quercetin can be obtained, are warranted.

Efficacy of Moderately Dosed Etoposide in Macrophage Activation Syndrome-Hemophagocytic Lymphohistiocytosis.

OBJECTIVE: Macrophage activation syndrome (MAS) constitutes 1 subtype of the hyperinflammatory syndrome hemophagocytic lymphohistiocytosis (HLH), and the term MAS-HLH was recently proposed for HLH with underlying autoimmune/autoinflammatory conditions. The mortality of MAS-HLH has been estimated at 5-10%. Here we report our experiences with moderately dosed etoposide in severe MAS-HLH; the objective was to effectively reduce severe hyperinflammatory activity with limited side effects. METHODS: In addition to conventional antiinflammatory treatment, moderately dosed etoposide was administered to 7 children affected by rapidly progressing MAS-HLH with central nervous system (n = 5) and/or pulmonary (n = 5) involvement. Three had underlying systemic juvenile idiopathic arthritis (sJIA), 2 had atypical sJIA (no arthritis at diagnosis), and 2 had systemic lupus erythematosus. We performed lymphocyte cytotoxicity analyses in all 7 and genetic analyses in 6. RESULTS: All children promptly responded to moderately dosed etoposide (50-100 mg/m2 once weekly), added to conventional MAS-HLH treatment that was considered insufficient. The mean accumulated etoposide dose was 671 mg/m2 (range 300-1050 mg/m2) as compared to 1500 mg/m2 recommended in the first 8 weeks of the HLH-94/HLH-2004 protocols. One child developed neutropenic fever and another neutropenic sepsis (neutrophils 0.3 × 109/L at therapy onset). Five of 7 children had low percentages (< 5%) of circulating natural killer (NK) cells prior to or in association with diagnosis; NK cell activity was pathologically low in 2 of 5 children studied. Disease-causing variants in HLH-associated genes were not found. All children were alive at latest follow-up (2-9 yrs after onset); neurological symptoms had normalized in 4 of 5 affected children. CONCLUSION: Moderately dosed etoposide may be beneficial in severe and/or refractory MAS-HLH.

RhoG deficiency abrogates cytotoxicity of human lymphocytes and causes hemophagocytic lymphohistiocytosis.

Exocytosis of cytotoxic granules (CG) by lymphocytes is required for the elimination of infected and malignant cells. Impairments in this process underly a group of diseases with dramatic hyperferritinemic inflammation termed hemophagocytic lymphohistiocytosis (HLH). Although genetic and functional studies of HLH have identified proteins controlling distinct steps of CG exocytosis, the molecular mechanisms that spatiotemporally coordinate CG release remain partially elusive. We studied a patient exhibiting characteristic clinical features of HLH associated with markedly impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell exocytosis functions, who beared biallelic deleterious mutations in the gene encoding the small GTPase RhoG. Experimental ablation of RHOG in a model cell line and primary CTLs from healthy individuals uncovered a hitherto unappreciated role of RhoG in retaining CGs in the vicinity of the plasma membrane (PM), a fundamental prerequisite for CG exocytotic release. We discovered that RhoG engages in a protein-protein interaction with Munc13-4, an exocytosis protein essential for CG fusion with the PM. We show that this interaction is critical for docking of Munc13-4+ CGs to the PM and subsequent membrane fusion and release of CG content. Thus, our study illuminates RhoG as a novel essential regulator of human lymphocyte cytotoxicity and provides the molecular pathomechanism behind the identified here and previously unreported genetically determined form of HLH.

Clinical and laboratory signs of haemophagocytic lymphohistiocytosis associated with pandemic influenza A (H1N1) infection in patients needing extracorporeal membrane oxygenation: A retrospective observational study.

BACKGROUND: Severe pandemic influenza has been associated with the hyperinflammatory condition secondary haemophagocytic lymphohistiocytosis (HLH). OBJECTIVES: To determine the frequency, degree, character and possible cause of influenza-associated HLH in critically ill patients with severe acute respiratory distress syndrome due to influenza A (H1N1) infection requiring extracorporeal membrane oxygenation (ECMO) support at our hospital. DESIGN: A retrospective observational study. PATIENTS AND SETTING: Medical data were retrieved retrospectively from 11 consenting patients of thirteen adults infected with pandemic influenza A (H1N1) 2009 requiring ECMO between July 2009 and January 2010 at the ECMO Centre of Karolinska University Hospital, Stockholm, Sweden. All patients were evaluated for HLH using HLH-2004 criteria and HScore. RESULTS: Eleven patients (median age 31 years) were included in the study and all survived. All patients showed signs of multiple organ dysfunction and pronounced inflammation, more severe in the four patients with HLH who had significantly higher peak serum concentrations of ferritin (P = 0.024), alkaline phosphatase (P = 0.012) and gamma-glutamyl transferase (P = 0.024), lower concentration of albumin (P = 0.0086) and more frequently hepatomegaly (P = 0.048). Abnormal lymphocyte cytotoxicity (lytic units <10) and a low proportion of natural killer (NK) cells were observed in three of four patients with HLH. Notably, we found a significant inverse correlation between serum ferritin concentration and NK cell and cytotoxic T lymphocyte percentages (rs = -0.74, P = 0.0013 and rs = -0.79, P = 0.0025, respectively). One HLH patient received HLH-directed cytotoxic therapy, another intravenous immunoglobulin and the other two no specific HLH-directed therapy. CONCLUSION: Critically ill patients, including healthy young adults, with pandemic influenza may develop HLH and should be monitored for signs of hyperinflammation and increasing organ dysfunction, and evaluated promptly for HLH because HLH-directed therapy may then be beneficial. The association of low NK percentages with hyperferritinaemia may suggest a role for reduced NK cell numbers, possibly also cytotoxic T lymphocytes, and subsequently reduced lymphocyte cytotoxicity, in the pathogenesis of hyperinflammation and secondary HLH.

Diagnostic challenges for a novel SH2D1A mutation associated with X-linked lymphoproliferative disease.

Mutations in SH2D1A, encoding the intracellular adaptor signaling lymphocyte activation molecule associated protein (SAP), are associated with X-linked lymphoproliferative disease type 1 (XLP1). We identified a novel hemizygous SH2D1A c.49G > A (p.E17K) variant in a 21-year-old patient with fatal Epstein-Barr virus infection-associated hemophagocytic lymphohistiocytosis. Cellular and biochemical assays revealed normal expression of the SAP variant protein, yet binding to phosphorylated CD244 receptor was reduced by >95%. Three healthy brothers carried the SH2D1A c.49G > A variant. Thus, data suggest that this variant represents a pathogenic mutation, but with variable expressivity. Importantly, our results highlight challenges in the clinical interpretation of SH2D1A variants and caution in using functional flow cytometry assays for the diagnosis of XLP1.

A Rare Case of Activated Phosphoinositide 3-Kinase Delta Syndrome (APDS) Presenting With Hemophagocytosis Complicated With Hodgkin Lymphoma.

Gain of function mutations in the p110δ catalytic subunit of the phosphatidylinositol-3-OH kinase (PIK3CD) classified as activated phosphoinositide 3-kinase delta syndrome (APDS) are the cause of a primary immunodeficiency characterized by recurrent sinopulmonary infections, and lymphoproliferation. Previously, autoimmunity and Epstein-Barr virus-related B-cell lymphoma have been documented for patients with APDS; here, we present a case that extends the picture, as the patient shows the full diagnostic criteria of hemophagocytic lymphohistiocytosis at 6 months of age. He experienced Hodgkin lymphoma as a 2.5-year-old baby. Next-generation sequencing returned a de novo heterozygous missense variant in PIK3CD (LRG_191t1: c.3061G>A; p.Glu1021Lys), confirming the primary immunodeficiency. After 2 courses of ifosfamide, cisplatin, and etoposide combined with brentuximab, the patient successfully underwent allogeneic hematopoietic stem cell transplantation from his HLA full matched sister, and he has been well for 18 months after that. The hematologist treating Hodgkin lymphoma and/or hemophagocytic lymphohistiocytosis should be vigilant about the possible underlying immune deficiency, and they should consider APDS in their differential diagnosis.

Different Clinical Presentation of 3 Children With Familial Hemophagocytic Lymphohistiocytosis With 2 Novel Mutations.

Although familial hemophagocytic lymphohistiocytosis (FHL) generally manifest with a combination of unremitting fever, hepatosplenomegaly, and pancytopenia; unusual presentations should also be taken into account. Herein, we present 3 FHL cases with 2 novel mutations with different initial presentations. The first patient bearing a homozygous truncation mutation in UNC13D (c.2650C>T.p.Gln884Ter) presented with central nervous system involvement and skin rash. The patient responded to the HLH-2004 protocol, and allogenic hematopoietic stem cell transplantation was performed from her healthy sister. The second and third patients with homozygous splice site mutation (c.430-1G>A) in STXBP2 were siblings who presented at birth with fevers, elevated aspartate aminotransferase, alanine aminotransferase, and hyperferritinemia but did not fulfill FHL criteria. The last 2 infants died despite intervention. Hematologists should be vigilant about the different presentation of FHL in children.

Elevated ferritin and soluble CD25 in critically ill patients are associated with parameters of (hyper) inflammation and lymphocyte cytotoxicity.

BACKGROUND: Critically ill patients may develop a potentially fatal hyperinflammatory condition known as secondary (acquired) hemophagocytic lymphohistiocytosis (sHLH), the cause of which is unclear. We evaluated serum ferritin and soluble CD25 (sCD25) in critically ill patients, and their association with other parameters of inflammation and critical illness. Moreover, aiming to better understand the pathogenesis of sHLH, we also evaluated lymphocyte cytotoxicity parameters and correlations with the inflammatory markers ferritin and sCD25. METHODS: In a prospective observational study, 32 patients with ferritin ≥500 µg/L (24 with sepsis) were studied on admission to an intensive care unit (ICU) with regard to ferritin and corresponding clinical and laboratory features including sCD25, and detailed lymphocyte cytotoxicity and genetic analyses whenever possible. RESULTS: Critically ill patients had elevated, positively correlated levels of serum ferritin and sCD25 (rs=0.465, P=0.008); both associated with other risk factors of poor outcome in critically ill, such as thrombocytopenia (rs=-0.534, P=0.002 and rs=-0.421, P=0.018, respectively), and sCD25 with hypoalbuminemia (rs=-0.678, P<0.001) and life support treatments (rs=0.479, P=0.006). Interestingly, ferritin levels were inversely associated with natural killer (NK)-cell cytotoxicity (rs=-0.462, P=0.047) and degranulation (rs=-0.504, P=0.030). Moreover, of four patients with abnormally low cytotoxicity, three (75%) had <5% circulating NK-cells. CONCLUSIONS: Our study suggests that hyperferritinemia and sCD25 correlate with other laboratory parameters indicative of severe hyperinflammation and organ dysfunction in critically ill ICU-patients, indicating their value in identifying hyperinflammatory critically ill patients for early intervention. Furthermore, it suggests that hyperferritinemia and hyperinflammation may partly be associated with a low percentage circulating NK-cells, and hence, the associated low lymphocyte cytotoxicity.

Fatal Central Nervous System Lymphocytic Vasculitis after Treatment for Burkitt Lymphoma in a Patient with a SH2D1A Mutation.

Very rarely, patients with X-linked lymphoproliferative syndrome type 1 present central nervous system vasculitis. We report a patient carrying a SH2D1A mutation that, after treatment for lymphoma developed fatal central nervous system vasculitis. He lacked signs of ongoing Epstein-Barr virus infection. We propose that impaired T cell homeostasis caused by SAP deficiency facilitates aberrant CD8 T cell activation against vascular antigens promoting clinical manifestations.

A Case of Familial Hemophagocytic Lymphohistiocytosis Type 4 With Involvement of the Central Nervous System Complicated With Infarct.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis (HLH) is a fatal disease affecting infants and very young children. Central nervous system involvement of HLH can cause catastrophic results. METHOD: We present a case with cranial involvement of familial HLH type 4 who showed diffuse infiltration of white matter complicated with intracranial thrombosis. A 5-year-old girl from a consanguineous couple presented with fever and pancytopenia, and was referred to our hematology unit. Examination revealed fever, lymphadenopathy, and hepatosplenomegaly. Ultrasound examination revealed hepatosplenomegaly and free intra-abdominal fluid. HLH was revealed on bone marrow aspiration biopsy. Defective natural killer and T lymphocyte cytotoxicity using degranulation tests was determined. In the genetic analysis, syntaxin gene mutation was found. On T2-weighted and T2-fluid-attenuated inversion recovery magnetic resonance imaging (MRI), diffuse hyperintense signal changes of cerebral white matter, indicating white matter demyelination, were observed. A second brain MRI showed an acute infarct involving the left temporooccipital region. Immunosuppressive therapy according to the HLH 2004 protocol was started. The infarct resolved but white matter lesions were stable on the brain MRI that was performed 1 month later. Brain MRI taken 4 months after the first examination showed stable cerebral white matter lesions, but hyperintense signal changes appeared in the cerebellar white matter and were regarded as progression. The patient died because of infection despite immunosuppressive therapy. CONCLUSIONS: Physicians managing patients with HLH must be vigilant about the possibility of central nervous system involvement including stroke.

A Newborn With Familial Hemophagocytic Lymphohistiocytosis Complicated With Transfusion Associated Graft Versus Host Disease.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by activation of cytotoxic T and natural killer (NK) cells, and macrophages related to a spectrum of hyperinflammatory disorders. The clinical findings mainly include high fever, cytopenia, splenomegaly, phagocytosis, and proliferation of histiocytes in lymphoreticular tissue. To the best of our knowledge, transfusion-associated graft versus host disease (TA-GVHD) in a 13-day old male newborn with HLH is being reported first time in the literature. The aim of this report was to emphasize the importance of blood products irradiation in the prevention of the development of graft versus host disease especially among high-risk subjects such as newborns with HLH.

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin.

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8+ Trm cells on a compartmental and functional basis. In human skin epithelia, CD8+CD49a+ Trm cells produced interferon-γ, whereas CD8+CD49a- Trm cells produced interleukin-17 (IL-17). In addition, CD8+CD49a+ Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8+CD49a+ Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8+CD49a- Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8+ Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases.

Adaptive NK cells can persist in patients with GATA2 mutation depleted of stem and progenitor cells.

Heterozygous GATA2 mutation is associated with immunodeficiency, lymphedema, and myelodysplastic syndrome. Disease presentation is variable, often coinciding with loss of circulating dendritic cells, monocytes, B cells, and natural killer (NK) cells. Nonetheless, in a proportion of patients carrying GATA2 mutation, NK cells persist. We found that peripheral blood NK cells in symptomatic patients uniformly lacked expression of the transcription factor promyelocytic leukemia zinc finger (PLZF), as well as expression of intracellular signaling proteins FcεRγ, spleen tyrosine kinase (SYK), and EWS/FLI1-Activated Transcript 2 (EAT-2) in a variegated manner. Moreover, consistent with an adaptive identity, NK cells from patients with GATA2 mutation displayed altered expression of cytotoxic granule constituents and produced interferon-γ upon Fc-receptor engagement but not following combined interleukin-12 (IL-12) and IL-18 stimulation. Canonical, PLZF-expressing NK cells were retained in asymptomatic carriers of GATA2 mutation. Developmentally, GATA-binding protein-2 (GATA-2) was expressed in hematopoietic stem cells, but not in NK-cell progenitors, CD3-CD56bright, canonical, or adaptive CD3-CD56dim NK cells. Peripheral blood NK cells from individuals with GATA2 mutation proliferated normally in vitro, whereas lineage-negative progenitors displayed impaired NK-cell differentiation. In summary, adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. Moreover, our data suggest that adaptive NK cells are more long-lived than canonical, immunoregulatory NK cells.