History of Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a severe cytokine storm syndrome (CSS), which until the turn of the century, was barely known but is now receiving increased attention. The history of HLH dates back to 1939 when it was first described in adults, to be followed in 1952 by the first description of its primary, familial form in children. Secondary forms of HLH are far more frequent and occur with infections, malignancies, metabolic diseases, iatrogenic immune suppression, and autoinflammatory/autoimmune diseases. Identification of the genetic defects leading to the defective function of natural killer (NK) cells and cytotoxic T cells as well as the corresponding mouse models have revolutionized our understanding of HLH and of immune function. Diagnosis relies on clinical and laboratory criteria; functional and genetic tests can help separate primary from secondary forms. Treatment with immunochemotherapy and hematopoietic stem cell transplantation has considerably improved survival in children with primary HLH, a formerly uniformly fatal disease.

Genetics of Acquired Cytokine Storm Syndromes : Secondary HLH Genetics.

Secondary hemophagocytic lymphohistiocytosis (sHLH) has historically been defined as a cytokine storm syndrome (CSS) occurring in the setting of triggers leading to strong and dysregulated immunological activation, without known genetic predilection. However, recent studies have suggested that existing underlying genetic factors may synergize with particular diseases and/or environmental triggers (including infection, autoimmune/autoinflammatory disorder, certain biologic therapies, or malignant transformation), leading to sHLH. With the recent advances in genetic testing technology, more patients are examined for genetic variations in primary HLH (pHLH)-associated genes, including through whole exome and whole genome sequencing. This expanding genetic and genomic evidence has revealed HLH as a more complex phenomenon, resulting from specific immune challenges in patients with a susceptible genetic background. Rather than a simple, binary definition of pHLH and sHLH, HLH represents a spectrum of diseases, from a severe complication of common infections (EBV, influenza) to early onset familial diseases that can only be cured by transplantation.

Genetics of Macrophage Activation Syndrome in Systemic Juvenile Idiopathic Arthritis.

Macrophage activation syndrome (MAS) is a life-threatening episode of hyperinflammation driven by excessive activation and expansion of T cells (mainly CD8) and hemophagocytic macrophages producing proinflammatory cytokines. MAS has been reported in association with almost every rheumatic disease, but it is by far most common in systemic juvenile idiopathic arthritis (SJIA). Clinically, MAS is similar to familial or primary hemophagocytic lymphohistiocytosis (pHLH), a group of rare autosomal recessive disorders linked to various genetic defects all affecting the perforin-mediated cytolytic pathway employed by NK cells and cytotoxic CD8 T lymphocytes. Decreased cytolytic activity in pHLH patients leads to prolonged survival of target cells associated with increased production of proinflammatory cytokines that overstimulate macrophages. The resulting cytokine storm is believed to be responsible for the frequently fatal multiorgan system failure seen in MAS. Whole exome sequencing as well as targeted sequencing of pHLH-associated genes in patients with SJIA-associated MAS demonstrated increased "burden" of rare protein-altering variants affecting the cytolytic pathway compared to healthy controls, suggesting that as in pHLH, genetic variability in the cytolytic pathway contributes to MAS predisposition. Functional studies of some of the novel variants have shown that even in a heterozygous state, their presence partially reduces cytolytic activity that may lead to increased cytokine production.

Murine Models of Familial Cytokine Storm Syndromes.

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disease caused by mutations in effectors and regulators of cytotoxicity in cytotoxic T cells (CTL) and natural killer (NK) cells. The complexity of the immune system means that in vivo models are needed to efficiently study diseases like HLH. Mice with defects in the genes known to cause primary HLH (pHLH) are available. However, these mice only develop the characteristic features of HLH after the induction of an immune response (typically through infection with lymphocytic choriomeningitis virus). Nevertheless, murine models have been invaluable for understanding the mechanisms that lead to HLH. For example, the cytotoxic machinery (e.g., the transport of cytotoxic vesicles and the release of granzymes and perforin after membrane fusion) was first characterized in the mouse. Experiments in murine models of pHLH have emphasized the importance of cytotoxic cells, antigen-presenting cells (APC), and cytokines in hyperinflammatory positive feedback loops (e.g., cytokine storms). This knowledge has facilitated the development of treatments for human HLH, some of which are now being tested in the clinic.

IL-1 Family Blockade in Cytokine Storm Syndromes.

Interleukin-1 is a prototypic proinflammatory cytokine that is elevated in cytokine storm syndromes (CSSs), such as secondary hemophagocytic lymphohistiocytosis (sHLH) and macrophage activation syndrome (MAS). IL-1 has many pleotropic and redundant roles in both innate and adaptive immune responses. Blockade of IL-1 with recombinant human interleukin-1 receptor antagonist has shown efficacy in treating CSS. Recently, an IL-1 family member, IL-18, has been demonstrated to be contributory to CSS in autoinflammatory conditions, such as in inflammasomopathies (e.g., NLRC4 mutations). Anecdotally, recombinant IL-18 binding protein can be of benefit in treating IL-18-driven CSS. Lastly, another IL-1 family member, IL-33, has been postulated to contribute to CSS in an animal model of disease. Targeting of IL-1 and related cytokines holds promise in treating a variety of CSS.

Genetics of Primary Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) constitutes a rare, potentially life-threatening hyperinflammatory immune dysregulation syndrome that can present with a variety of clinical signs and symptoms, including fever, hepatosplenomegaly, and abnormal laboratory and immunological findings such as cytopenias, hyperferritinemia, hypofibrinogenemia, hypertriglyceridemia, elevated blood levels of soluble CD25 (interleukin (IL)-2 receptor α-chain), or diminished natural killer (NK)-cell cytotoxicity (reviewed in detail in Chapter 11 of this book). While HLH can be triggered by an inciting event (e.g., infections), certain monogenic causes have been associated with a significantly elevated risk of development of HLH, or recurrence of HLH in patients who have recovered from their disease episode. These monogenic predisposition syndromes are variably referred to as "familial" (FHL) or "primary" HLH (henceforth referred to as "pHLH") and are the focus of this chapter. Conversely, secondary HLH (sHLH) often occurs in the absence of monogenic etiologies that are commonly associated with pHLH and can be triggered by infections, malignancies, or rheumatological diseases; these triggers and the genetics associated with sHLH are discussed in more detail in other chapters in this book.

CD8+ T Cell Biology in Cytokine Storm Syndromes.

Familial forms of hemophagocytic lymphohistiocytosis (HLH) are caused by loss-of-function mutations in genes encoding perforin as well as those required for release of perforin-containing cytotoxic granule constituent. Perforin is expressed by subsets of CD8+ T cells and NK cells, representing lymphocytes that share mechanism of target cell killing yet display distinct modes of target cell recognition. Here, we highlight recent findings concerning the genetics of familial HLH that implicate CD8+ T cells in the pathogenesis of HLH and discuss mechanistic insights from animal models as well as patients that reveal how CD8+ T cells may contribute to or drive disease, at least in part through release of IFN-γ. Intriguingly, CD8+ T cells and NK cells may act differentially in severe hyperinflammatory diseases such as HLH. We also discuss how CD8+ T cells may promote or drive pathology in other cytokine release syndromes (CSS). Moreover, we review the molecular mechanisms underpinning CD8+ T cell-mediated lymphocyte cytotoxicity, key to the development of familial HLH. Together, recent insights to the pathophysiology of CSS in general and HLH in particular are providing promising new therapeutic targets.

Familial Hemophagocytic Lymphohistiocytosis Screening in Neonatal Sepsis.

OBJECTIVE: Neonatal sepsis and familial hemophagocytic lymphohistiocytosis (fHLH) have similar clinical and laboratory symptoms and the possibility of overlooking fHLH diagnosis is high in newborns with sepsis. History of consanguineous marriage and/or sibling death, hepatomegaly/splenomegaly, and hyperferritinemia (>500 ng/mL) are likely to support fHLH in newborns with sepsis. Therefore, in newborns with sepsis in whom at least 2 of these 3 criteria were detected, genetic variants was investigated for the definitive diagnosed of fHLH. According to the results of genetic examination, we investigated whether these criteria supporting fHLH could be used as a screening test in fHLH. MATERIALS AND METHODS: fHLH-associated genetic variants were investigated in 22 patients diagnosed with neonatal sepsis who fulfilled at least 2 of the following criteria (1) history of consanguineous marriage and/or sibling death, (2) hepatomegaly/splenomegaly, and (3) hyperferritinemia (>500 ng/mL). RESULTS: Heterozygous variants were determined in 6 patients (27.2%): 3 STXBP2 , 1 STX11 , 1 UNC13D , and 1 PRF1 . Polymorphisms associated with the clinical symptoms and signs of HLH were determined in 5 patients (22.7%): 4 UNC13D , 1 PRF1 . Two patients were in the heterozygous variants and polymorphism associated with the clinical symptoms and signs of HLH groups. In 12 patients, benign polymorphisms were detected in STXBP2 and UNC13D genes. No change in fHLH associated genes were found in 1 patient. CONCLUSION: Some variants and/or polymorphisms identified in our patients have been previously reported in patients with HLH. Therefore, we recommend further investigation of fHLH in patients with neonatal sepsis who fulfill at least 2 of the above 3 criteria.

A degranulation assay using Vγ9Vδ2 T cells for the rapid diagnosis of familial hemophagocytic syndromes.

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder characterized by uncontrolled lymphocyte and macrophage activation and a subsequent cytokine storm. The timely initiation of immunosuppressive treatment is crucial for survival. Methods: Here, we harnessed Vγ9Vδ2 T cell degranulation to develop a novel functional assay for the diagnosis of HLH. We compared the novel assay with the conventional natural killer (NK) cell stimulation method in terms of efficiency, specificity, and reliability. Our analysis involved 213 samples from 182 individuals, including 23 samples from 12 patients with degranulation deficiency (10 individuals with UNC13D deficiency, 1 with STXBP2 deficiency, and 1 with RAB27A deficiency). Results: While both tests exhibited 100% sensitivity, the Vγ9Vδ2 T cell degranulation assay showed a superior specificity of 86.2% (n=70) compared to the NK cell degranulation assay, which achieved 78.9% specificity (n=213). The Vγ9Vδ2 T cell degranulation assay offered simpler technical requirements and reduced labor intensity, leading to decreased susceptibility to errors with faster processing times. Discussion: This efficiency stemmed from the sole requirement of dissolving (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) powder, contrasting with the intricate maintenance of K562 cells necessary for the NK cell degranulation assay. With its diminished susceptibility to errors, we anticipate that the assay will require fewer repetitions of analysis, rendering it particularly well-suited for testing infants. Conclusion: The Vγ9Vδ2 T cell degranulation assay is a user-friendly, efficient diagnostic tool for HLH. It offers greater specificity, reliability, and practicality than established methods. We believe that our present findings will facilitate the prompt, accurate diagnosis of HLH and thus enable rapid treatment and better patient outcomes.

Efficacy of T-cell assays for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis.

ABSTRACT: Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder associated with autosomal recessive variants in genes required for perforin-mediated lymphocyte cytotoxicity. A rapid diagnosis is crucial for successful treatment. Although defective cytotoxic T lymphocyte (CTL) function causes pathogenesis, quantification of natural killer (NK)-cell exocytosis triggered by K562 target cells currently represents a standard diagnostic procedure for primary HLH. We have prospectively evaluated different lymphocyte exocytosis assays in 213 patients referred for evaluation for suspected HLH and related hyperinflammatory syndromes. A total of 138 patients received a molecular diagnosis consistent with primary HLH. Assessment of Fc receptor-triggered NK-cell and T-cell receptor (TCR)-triggered CTL exocytosis displayed higher sensitivity and improved specificity for the diagnosis of primary HLH than routine K562 cell-based assays, with these assays combined providing a sensitivity of 100% and specificity of 98.3%. By comparison, NK-cell exocytosis after K562 target cell stimulation displayed a higher interindividual variability, in part explained by differences in NK-cell differentiation or large functional reductions after shipment. We thus recommend combined analysis of TCR-triggered CTL and Fc receptor-triggered NK-cell exocytosis for the diagnosis of patients with suspected familial HLH or atypical manifestations of congenital defects in lymphocyte exocytosis.

[Clinical and genetic mutation characteristics of patients with primary hemophagocytic lymphohistiocytosis and their impact on prognosis].

Objective: To investigate the clinical and genetic mutation characteristics of patients with primary hemophagocytic lymphohistiocytosis (HLH) and their impact on prognosis. Methods: Sixty-three primary HLH patients with complete medical records admitted and diagnosed at Beijing Friendship Hospital of Capital Medical University from January 2013 to December 2022 were selected. The patients' clinical and laboratory features, genetic and rapid immunological indicator characteristics, treatment outcomes and prognosis were retrospectively analyzed. Follow-up was up to June 30, 2023, with a median follow-up time [M (Q1, Q3)] of 47 (21, 76) months. Overall survival was analyzed using Kaplan-Meier survival curve, and prognostic factors were analyzed using Cox proportional hazards regression model. Results: Sixty-three primary HLH patients included 35 males and 28 females, with a median age [M (Q1, Q3)] of 17 (7, 27) years. Clinical manifestations at the initial diagnosis mainly included fever (93.7%, 59/63), splenomegaly (87.3%, 55/63), hemophagocytosis (65.1%, 41/63), hepatomegaly (52.4%, 33/63) and central nervous system (CNS) involvement (38.1%, 24/63). A total of 39 patients (61.9%) were diagnosed with EB virus (EBV) infection at initial diagnosis.PRF1 and UNC13D gene mutations were the most common mutations, and the highest frequency mutation site in the PRF1 gene was c.1349C>T, and that of UNC13D gene was c.2588G>A. A total of 76.2% (48/63) of patients had reduced activity of natural killer (NK) cells. Cytotoxic cell degranulation function was impaired or absent in 52.7% (29/55) of patients, of which 79.2% (19/24) of patients with primary HLH with defects in degranulation-related genes had impaired degranulation function. The 1-year and 3-year overall survival rates were 74.8% and 66.7%, respectively. Cox multivariate analysis suggested that peripheral blood EBV≥10 000 copies/ml (HR=3.523, 95%CI: 1.418-8.757, P=0.007) was the risk factor for prognosis. Conclusions: The main clinical manifestations of primary HLH patients at the initial diagnosis include fever, splenomegaly, hemophagocytosis, hepatomegaly, and CNS involvement. PRF1 and UNC13D are the most commonly mutated genes. High copy number EBV infection in peripheral blood is the risk factor for prognosis.

Allogeneic hematopoietic stem cell transplant for familial hemophagocytic lymphohistiocytosis: a case report and literature review.

Objectives: This study aims to discuss the clinical manifestations and treatment of Familial hemophagocytic lymphohistiocytosis (FHL) caused by a mutation in the UNC13D gene. Methods: A 6-year-old female child presented with unexplained febricity, splenomegaly, pancytopenia, hemophagocytic lymphohistiocytosis in bone marrow, decreased NK cell activity, soluble CD25 levels > 44000ng/ml. Genetic sequencing revealed a mutation in the UNC13D gene. Additionally, the patient experienced intermittent fever with seizures characterized by involuntary twitching of the left upper limb. Head magnetic resonance imaging (MRI) showed white matter lesions. Results: According to the HLH-2004 diagnostic criteria revised by the International Society of Histiocytosis the patient was diagnosed with FHL. Despite receiving HLH-2004 treatment, the disease relapsed. However, after a salvage allogeneic Hematopoietic Stem Cell Transplant (HSCT), febricity, abnormal blood cells, and neurological symptoms significantly improved. Conclusions: Prompt performance of allogeneic HSCT is crucial upon diagnosis of FHL, especially when neurological involvement is present.

HLH and Recurrent EBV Lymphoma as the presenting manifestation of MAGT1 Deficiency: A Systematic Review of the Expanding Disease Spectrum.

Magnesium transporter 1 (MAGT1) gene loss-of-function variants lead to X-linked MAGT1 deficiency with increased susceptibility to EBV infection and N-glycosylation defect (XMEN), a condition with a variety of clinical and immunological effects. In addition, MAGT1 deficiency has been classified as a congenital disorder of glycosylation (CDG) due to its unique role in glycosylation of multiple substrates including NKG2D, necessary for viral protection. Due to the predisposition for EBV, this etiology has been linked with hemophagocytic lymphohistiocytosis (HLH), however only limited literature exists. Here we present a complex case with HLH and EBV-driven classic Hodgkin lymphoma (cHL) as the presenting manifestation of underlying immune defect. However, the patient's underlying immunodeficiency was not identified until his second recurrence of Hodgkin disease, recurrent episodes of Herpes Zoster, and after he had undergone autologous hematopoietic stem cell transplant (HSCT) for refractory Hodgkin lymphoma. This rare presentation of HLH and recurrent lymphomas without some of the classical immune deficiency manifestations of MAGT1 deficiency led us to review the literature for similar presentations and to report the evolving spectrum of disease in published literature. Our systematic review showcased that MAGT1 predisposes to multiple viruses (including EBV) and adds risk of viral-driven neoplasia. The roles of MAGT1 in the immune system and glycosylation were highlighted through the multiple organ dysfunction showcased by the previously validated Immune Deficiency and Dysregulation Activity (IDDA2.1) score and CDG-specific Nijmegen Pediatric CDG Rating Scale (NPCRS) score for the patient cohort in the systematic review.

Serum proteomics reveals hemophagocytic lymphohistiocytosis-like phenotype in a subset of patients with multisystem inflammatory syndrome in children.

Children with Multisystem Inflammatory Syndrome in Children (MIS-C) can present with thrombocytopenia, which is a key feature of hemophagocytic lymphohistiocytosis (HLH). We hypothesized that thrombocytopenic MIS-C patients have more features of HLH. Clinical characteristics and routine laboratory parameters were collected from 228 MIS-C patients, of whom 85 (37%) were thrombocytopenic. Thrombocytopenic patients had increased ferritin levels; reduced leukocyte subsets; and elevated levels of ASAT and ALAT. Soluble IL-2RA was higher in thrombocytopenic children than in non-thrombocytopenic children. T-cell activation, TNF-alpha and IFN-gamma signaling markers were inversely correlated with thrombocyte levels, consistent with a more pronounced cytokine storm syndrome. Thrombocytopenia was not associated with severity of MIS-C and no pathogenic variants were identified in HLH-related genes. This suggests that thrombocytopenia in MIS-C is not a feature of a more severe disease phenotype, but the consequence of a distinct hyperinflammatory immunopathological process in a subset of children.

Atypical familial hemophagocytic lymphohistiocytosis type 3 in children: A report of cases and literature review.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is caused by UNC13D variants. The clinical manifestations of FHL3 are highly diverse and complex. Some patients exhibit atypical or incomplete phenotypes, making accurate diagnosis difficult. Our study aimed to broaden the understanding of the atypical FHL3 clinical spectrum. METHODS: In our study, we analyzed in detail the clinical features of four Chinese patients with UNC13D variants. Additionally, we conducted a comprehensive review of the existing literature on previously reported atypical manifestations and summarized the findings. RESULTS: Two of our patients presented with muscle involvement, while the other two had hematological involvement; none of them met the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH). However, protein expression and functional analysis ultimately confirmed diagnostic criteria for FHL3 in all patients. From the literature we reviewed, many atypical FHL3 patients had neurological involvement, especially isolated neurological manifestations. At the same time, arthritis and hypogammaglobulinemia were also prone to occur. CONCLUSION: Our study highlights that the expression of the Munc13-4 protein may not fully indicate the pathogenicity of UNC13D variants, whereas CD107a analysis could be more sensitive for disease diagnosis. These findings contribute to a broader understanding of the FHL3 clinical spectrum and may offer new insights into the underlying pathogenesis of UNC13D variants. It is crucial to prioritize the timely and accurate diagnosis of atypical patients, as they may often be overlooked among individuals with rheumatic or hematological diseases.

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 Monotherapy for a Child With HAVCR2 Gene Mutation Associated Subcutaneous Panniculitis-like T-cell Lymphoma: A Case Report.

BACKGROUND: The occurrence of hemophagocytic lymphohistiocytosis (HLH) in patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL) may be due to HAVCR2 gene mutation, leading to T-cell immunoglobulin and mucin domain-containing molecule 3 deficiency, T-cell and macrophage activation, and proinflammatory cytokine production. OBSERVATION: We report a patient with SPTCL and HLH for whom ruxolitinib, used as a novel treatment, showed notable therapeutic effects. CONCLUSIONS: Remission of both HAVCR2 mutation-induced high inflammatory characteristics and significant symptoms post-ruxolitinib administration suggested that patients with SPTCL and HLH may not represent typical lymphoma cases. Ruxolitinib, with its relatively low toxic side effects, can provide favorable outcomes.

Successful Second CBT for Graft Failure After First CBT for Adult-Onset Familial Hemophagocytic Lymphohistiocytosis Type 3: A Case Report.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare inherited autosomal recessive immune deficiency that usually manifests during infancy or early childhood, rarely occurring in adults. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for FHL. However, optimal conditioning regimens for adult-onset FHL have not yet been established. Herein, we report a case of adult-onset FHL. A 37-year-old man presented with fever, liver dysfunction, and pancytopenia, which improved temporarily with corticosteroid therapy. However, he later developed encephalitis and myelitis. Genetic analysis revealed rare variants of UNC13D (c.2367+1 g>a and c.2588 g>a), which were compound heterozygous pathogenic mutations. FHL type 3 was diagnosed, and treatment based on the hemophagocytic lymphohistiocytosis (HLH) 1994 protocol was initiated. The patient underwent cord blood transplantation (CBT) with myeloablative conditioning using fludarabine, melphalan, and total-body irradiation (TBI), which resulted in graft rejection. The patient was successfully rescued by a second CBT following reduced-intensity conditioning with fludarabine, cyclophosphamide, and TBI. Although graft failure is an important complication especially in CBT, it could be managed by appropriate treatment, and that cord blood would be a promising alternative source with the advantages of rapidity and avoidance of related donors with a high risk of harboring the same genetic mutation.