Familial Hemophagocytic Lymphohistiocytosis Research Articles

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.

Pituitary involvement in familial hemophagocytic lymphohistiocytosis type 5 associated with homozygous mutation in the syntaxin-binding protein-2 gene (STXBP2)

Pituitary involvement in familial hemophagocytic lymphohistiocytosis type 5 associated with homozygous mutation in the syntaxin-binding protein-2 gene (STXBP2)

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

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. 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. 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. 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.

Early hematopoietic cell transplantation for familial hemophagocytic lymphohistiocytosis in a regional treatment network in Japan.

Familial hemophagocytic lymphohistiocytosis (FHLH) is a fatal hyperinflammation syndrome arising from the genetic defect of perforin-mediated cytolysis. Curative hematopoietic cell transplantation (HCT) is needed before development of central nervous system (CNS) disease. We studied treatment outcomes of 13 patients (FHLH2 n = 11, FHLH3 n = 2) consecutively diagnosed from 2011 to 2022 by flow cytometric screening for non-myeloablative HCT in a regional treatment network in Kyushu, Japan. One patient with a novel PRF1 variant escaped screening, but all patients with FHLH2 reached diagnosis and 8 of them received HCT until 3 and 9months of age, respectively. The earliest HCT was conducted 65days after birth. Three pretransplant deaths occurred in newborns with liver failure at diagnosis. Ten posttransplant patients have remained disease-free, 7 of whom had no neurological involvement. Time from first etoposide infusion to HCT was shorter in patients without CNS disease or bleeding than in patients with those factors (median [range] days: 62 [50-81] vs. 122 [89-209], p = 0.016). Six of 9 unrelated patients had a PRF1 c.1090_1091delCT variant. These results suggest that the critical times to start etoposide and HCT are within 3months after birth and during etoposide control, respectively. Newbornscreening may increase the percentage of disease-free survivors without complications.

Germline defects of familial hemophagocytic lymphohistiocytosis-related genes presenting as adult-onset peripheral T-cell lymphoma.

Germline mutations in genes involved in perforin-granzyme-mediated cytotoxicity such as PRF1, UNC13D, STX11, and STXBP2 were known to cause familial hemophagocytic lymphohistiocytosis (FHL). In this study, we reported a unique group of 3 patients with germline mutations of UNC13D and STX11 genes and presented as adult-onset peripheral T-cell lymphoma (PTCL) with cytotoxic T-cell phenotype and atypical lymphoma presentations. CD107a degranulation assay and NK-cell activity analysis demonstrated impaired cytotoxic function of the NK/T-cells of the patients with FHL-related mutations. Gene expression profile study revealed that up-regulated genes of the cytotoxic T-cells were enriched in autoimmune-related pathways. It was possible that impaired cytotoxic lymphocyte-mediated immune surveillance and autoantigen stimulation may both participate in PTCL oncogenesis. Germline defects of FLH-related genes may represent a novel predisposing factor for PTCLs.

Familial Hemophagocytic Lymphohistiocytosis Due to PRF1 Mutation Triggered by Enterovirus.

Familial Hemophagocytic Lymphohistiocytosis Due to PRF1 Mutation Triggered by Enterovirus.

Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus-based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells efficiently cured lethal hyperinflammation in a mouse model of Epstein-Barr virus-triggered FHL2, a subtype caused by perforin-1 (Prf1) deficiency. Furthermore, repair of PRF1 and Munc13-4 (UNC13D)-whose deficiency causes the FHL subtype FHL3-in mutant memory T cells from two critically ill patients with FHL restored T cell cytotoxicity. These results provide a starting point for the treatment of genetic T cell immune dysregulation syndromes with repaired autologous T cells.

Detection of a novel gross deletion in the UNC13D gene ends the diagnostic odyssey for a family with familial hemophagocytic lymphohistiocytosis 3

BackgroundFamilial hemophagocytic lymphohistiocytosis (FHL) is an immunological disorder characterized by overactivation of macrophages and T lymphocytes. This autosomal recessive condition has been characterized into multiple types depending on the genetic etiology. FHL type 3 is associated with bi-allelic pathogenic variants in the UNC13D gene.Case presentationWe present a 12-year diagnostic odyssey for a family with FHL that signifies the advances of FHL genetic testing in a clinical genetic diagnostic laboratory setting. We describe the first case of a large UNC13D gross deletion in trans to a nonsense variant in a family with FHL3, which may have been mediated by Alu elements within introns 12 and 25 of the UNC13D gene.ConclusionsThis case highlights the importance of re-evaluating past genetic testing for a patient and family as test technology evolves in order to end a diagnostic odyssey.

Deep Proteomic Analysis Reveals Shared Terminal Mechanisms for Familial Hemophagocytic Lymphohistiocytosis and Lymphoma-Associated Hyperinflammation

Background: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome increasingly observed in patients with hematologic malignancies (HM-HLH). Familial HLH (FHL) is seen in young children and is typically caused by mutations affecting the perforin pathway of lymphocyte cytotoxic function, leading to excessive CD8+ T cell activation and interferon-gamma production. Though HM-HLH has clinical similarities to FHL, little is known regarding the pathophysiology of HM-HLH. We previously developed the ‘Optimized HLH Inflammatory (OHI) index,‘ combining soluble CD25 and ferritin elevations, to improve recognition of hyperinflammation and prognostication in HM patients (Zoref-Lorenz et al., Blood; 2022). Based on these findings, we hypothesize that OHI+ HM's share similar immune pathophysiology with FHL and aim to validate this hypothesis using an unbiased proteomic approach. Methods: We conducted an unbiased proteomic analysis of samples from Schneider Children's, Meir, Sheba, and Beilinson Medical Centers. Our cohort included FHL patients, healthy pediatric controls, and patients with HMs, classified as HM-OHI+(sCD25>3,900 U/mL and ferritin>1,000 ng/mL) or HM-OHI-. SomaLogic proteomics platform, an aptamer-based method, assessed 7,596 serum proteins representing 6,412 genes. Unsupervised clustering techniques (primary component analysis and unbiased hierarchical clustering) were used to group patient results. Gene set enrichment analysis (GSEA) of the hallmark set was used to identify enriched biologic pathways in HM-OHI+ and FHL. Differential expression analysis (Student t-test) identified differentially expressed proteins (DEPs, q<0.1, p<0.05) between FHL vs. healthy controls and HM-OHI+ vs. HM-OHI-. Venn analysis revealed overlapping DEPs between FHL and OHI+ HM-HLH patients. GSEA with hallmark pathways evaluated the overlapping genes in each pairwise comparison within the entire sets and the intersecting genes. Spearman's correlation analysis examined the relationship between fold changes of proteins and normalized enrichment scores of hallmark GSEA comparisons within intersecting proteins and genes of FHL and HM-OHI+. Logistic regression created an equation for the linear curve of these correlations. Results: We analyzed 42 patients, including 14 HM-OHI+ and 14 HM-OHI- patients, matched by malignancy type, 7 FHL patients, and age-matched controls. Unsupervised hierarchical clustering (Figure 1A) showed that HM-OHI+ patients clustered with FHL patients, while healthy controls and HM-OHI- patients clustered together. GSEA analysis identified common enriched biological pathways between HM-OHI+ and FHL: interferon-γ response, IL6 JAK STAT3 signaling, IL2 STAT5 signaling, and oxidative phosphorylation (Normalized enrichment scores of 1.82:1.92; 1.76:2.23; 1.76:1.76 and 1.68:1.78 respectively for HM-OHI+ and FHL). In pairwise statistical comparisons, 841 differential proteins were shared between FHL and HM-OHI+. HM-OHI+ and FHL exhibited highly correlated fold changes (R=0.97, p<0.0001, Y= 1.362 X-0.2597, Figure 1B), with highly significant elevated proteins in HM-OHI+ vs. HM-OHI- that are key in the mechanism of FHL: T cell activation (GRZB, FC-1.6 q=0.01; GRZH: FC-1.5, q= 0.002), IFN-γ response (CXCL10, FC-2.1,q=0.001), and macrophage activation (CD14: FC- 1.6, q=0.001; CSF1:FC- 2.1, 1<0.0001; IL1RL: FC- 1.8,q=0.0009). The intersecting genes in the GSEA analysis revealed a high correlation of the biological pathways (R=0.94, p<0.0001, Y=1.282X+0), with the IFN-γ response, the known driver of FHL being the most significantly over-enriched pathway in both pairwise comparisons (q<0.25). These findings indicate shared pathophysiology mechanisms between HM-OHI+ and FHL. We validated key proteins using ELISA as an orthogonal method. Conclusions: This study establishes a common downstream mechanism of hyperinflammation in hematologic malignancies and FHL, closely linked to the IFN-γ response, and validates the OHI index as a diagnostic tool for HM-HLH. Understanding the shared pathophysiology between HM-OHI+ and FHL sheds light on the poor prognosis of HM-associated hyperinflammation and emphasizes the need to study the upstream mechanisms of HM-HLH to define targets for improved therapeutic approaches.

Haploidentical STEM-Cell Transplantation for Children with ACUTE Leukemia and NON-Malignant Disorders, Using ΑΒ + T Cell /CD19 + B-Cell Depletion, a Single Center Experience

Background: αβ+ T cells/CD19+ B cells depleted haploidentical Hematopoietic stem cell transplantation (αβ+ TCR/ CD19+ depleted haplo-HSCT) is increasingly used in children with acute leukemia (AL) and many non-malignant disorders (NMD) such as severe combined immune deficiency (SCID), inborn errors of immunity (IEI), bone marrow failure (BMF, acquired/congenital) and familial Hemophagocytic lymphohistiocytosis (HLH), in need of a transplant and not having a matched related donor. Within this heterogenous group of patients, it is important to try to define introductory requirements for successful outcomes, and to identify risk factors for transplant related complications. Methods: Data was collected retrospectively regarding children age <21 years with AL or NMD who underwent αβ+ TCR/ CD19+ depleted haplo-HSCT at Sheba Medical Center between the years 2012-2021. Results: Seventy-four children underwent αβ+ TCR/ CD19+ depleted haplo-HSCT, for AL and NMD. Acute Leukemia subgroup Thirty-eight children had AL of them 34 patients engrafted successfully. The median time for neutrophil and platelet engraftment was 10 (range 9-17) and 13 (range 10-22) days, respectively. We observed 5-years overall survival (OS) and event free survival of 51% and 42%, respectively for the whole cohort. There were no cases of grade 3-4 acute graft versus host disease and only two patients had chronic graft versus host disease. EFS was higher in patients with graft composition of γδ+ T cells greater than the median value (MV) of 9.5X10^6 cells/kg than in patients who recieved cell dose lower than MV (54% vs 26%, P=0.04). Non-malignant Disorders Thirty-six children had NMD. Twenty-six of them (72%) engrafted and ten did not (did not achieved neutrophil count above 500 cells/uL at day 30). Engraftment kinetics was similar to AL subgroup. Secondary rejection was observed in two patients. Conditioning regimen varied within this subgroup. For patients with BMF, busulfan or treosulfan/fludarabine was administrated. All patients with HLH conditioned with treosulfan/fludarabine/thiotepa regimen. Patients with SCID, either did not recieved conditioning or received fludarabine/thiotepa/melphalan or treosulfan/fludarabin conditioning. For patients with IEI the protocol was based on treosulfan/fludarabin/thiotepa. Full donor chimerism was observed at day 30 in all patients who engrafted. Five-year OS was 56% for the whole cohort. OS was higher for patients without active infection at the day of transplant compared to patients with active infection (91.7% vs 33%, respectively, p=6.4X10-4). Mortality was high with thirteen patients dying from infection and one from veno-occlusive disease. Cumulative incidence of aGVHD was 30% with the majority were no greater than grade 2 and for cGVHD was 19%. Conclusions: αβ+/CD19+ haploidentical Hematopoietic stem cell transplantation can offer long-term survival for children with acute leukemia and NMD. For children with AL, we observed a presumed protective role for higher content of γδ+ T cell in the graft which need to be evaluate prospectively in a larger cohort. There were no cases of severe GVHD and the rate of grade 1-2 GVHD was low. For children with NMD αβ+ TCR/CD19+ depleted haplo-HSCT is feasible and effective. Children with active infections at day 0 had poorer outcomes whereas children without an active infection had excellent outcomes. Thus, patients who have a clear indication for transplant should be evaluated for transplant as early as possible.

Preclinical Assessment for Gene Addition Strategy in Familial Hemophagocytic Lymphohistiocytosis Related to Munc 13-4 Deficiency

Introduction: Familial Hemophagocytic Lymphohistiocytosis (FHL) comprises a group of rare genetic disorders with defect cytotoxicity in T and NK cells predisposing to severe hyperinflammation with the clinical hallmark of the Hemophagocytic LymphoHistiocytosis (HLH) syndrome. The uncontrolled proliferation of activated T-CD8 lymphocytes and macrophages infiltrates lymphoid tissues, bone marrow, and multiple organs including the brain. Thus, if untreated full-scale FHL is usually fatal. FHL3 is due to mutations in the UNC13D gene coding for Munc 13-4 protein, one of the components of the perforin-dependent cytotoxicity apparatus. This condition accounts for 30% to 35 % of all genetic cases of HLH. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, remains difficult even when a compatible donor is available because of the patients' critical inflammatory background. In this context, gene therapy could be a promising therapeutic option, especially for those patients without HLA-compatible donor. Method: The feasibility and efficacy of gene addition strategy for FHL3 have been previously demonstrated in mouse models. We then translated this approach to human cells using a third-generation self-inactivated lentivirus vector pseudo-typed with a conventional vesicular stomatitis virus-G (VSVG) expressing a codon-optimized UNC13D gene under the transcriptional control of EF1 alpha (LV-EF1a-UNC13D). HSPC or CD3+ T-cells were transduced with a GMP-like-grade LV-EF1a-UNC13D lentiviral vector. We studied the restoration of cytotoxic function. Results: Our GMP transduction protocol led to efficient transcription of optimized mRNA codon and expression of Munc 13-4 protein as evidenced by RT qPCR and western blot, respectively. Interestingly, we show restored cytotoxicity activity after CD34+ differentiation toward CD3+ T-cells and NK cells. The vector copy number is compatible with a clinical approach. In vivo experiments are ongoing to validate the absence of genotoxicity in this setting. Discussion conclusion: Our results support the feasibility and efficacy of UNCD13D gene transfer into hematopoietic stem and progenitor cells or T cells from Munc 13-4 deficient patients. Alternatively, gene therapy of HSPCs could be combined with transduced autologous T-cells infusion. The first attempts regarding this option are encouraging. UNC13D gene addition strategy could dramatically improve the care of FHL3 patients lacking an HLA-genoidentical donor.

Complementary HLH susceptibility factors converge on CD8 T-cell hyperactivation

AbstractHemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes. Familial HLH is caused by genetic impairment of granule-mediated cytotoxicity (eg, perforin deficiency). MAS is linked to excess activity of the inflammasome-activated cytokine interleukin-18 (IL-18). Though individually tolerated, mice with dual susceptibility (Prf1⁻/⁻Il18tg; DS) succumb to spontaneous, lethal hyperinflammation. We hypothesized that understanding how these susceptibility factors synergize would uncover key pathomechanisms in the activation, function, and persistence of hyperactivated CD8 T cells. In IL-18 transgenic (Il18tg) mice, IL-18 effects on CD8 T cells drove MAS after a viral (lymphocytic choriomeningitis virus), but not innate (toll like receptor 9), trigger. In vitro, CD8 T cells also required T-cell receptor (TCR) stimulation to fully respond to IL-18. IL-18 induced but perforin deficiency impaired immunoregulatory restimulation-induced cell death (RICD). Paralleling hyperinflammation, DS mice displayed massive postthymic oligoclonal CD8 T-cell hyperactivation in their spleens, livers, and bone marrow as early as 3 weeks. These cells increased proliferation and interferon gamma production, which contrasted with increased expression of receptors and transcription factors associated with exhaustion. Broad-spectrum antibiotics and antiretrovirals failed to ameliorate the disease. Attempting to genetically “fix” TCR antigen-specificity instead demonstrated the persistence of spontaneous HLH and hyperactivation, chiefly on T cells that had evaded TCR fixation. Thus, drivers of HLH may preferentially act on CD8 T cells: IL-18 amplifies activation and demand for RICD, whereas perforin supplies critical immunoregulation. Together, these factors promote a terminal CD8 T-cell activation state, combining features of exhaustion and effector function. Therefore, susceptibility to hyperinflammation may converge on a unique, unrelenting, and antigen-dependent state of CD8 T-cell hyperactivation.

Hemophagocytic lymphohistiocytosis associated with sodium valproate

Hemophagocytic lymphohistiocytosis(HLH) is known to have numerous causes, such as chronic inflammation, infection, malignancy, drug use, and primary or familial HLH. HLH triggered by valproate (VPA) has rarely been reported in the literature. Here we describe a pediatric patient with HLH induced by VPA. A 5-years-old girl presented to our center with recurrent fever accompanied by diffuse generalized rash after 2 weeks of taking oral VPA. Physical examination revealed hepatosplenomegaly; laboratory findings showed bicytopenia (hemoglobin and platelet), hemophagocytic cells on the bone marrow smear, hypofibrinogenaemia and hypertriglyceridaemia, and a high ferritin level. She was diagnosed to have HLH associated with VPA. She was treated with intravenous immunoglobulin, glucocorticoid and withdrawal of the sodium valproate, and she completely recovered. In conclusion, VPA can trigger HLH, a potentially fatal condition.

The opposing effects of two gene defects in STX11 and SLP76 on the disease in a patient with an inborn error of immunity

Inborn errors of immunity are mostly monogenic. However, disease phenotype and outcome may be modified by the coexistence of a second gene defect. We sought to identify the genetic basis of the disease in a patient who experienced bleeding episodes, pancytopenia, hepatosplenomegaly, and recurrent pneumonia that resulted in death. Genetic analysis was done using next-generation sequencing. Protein expression and phosphorylation were determined by immunoblotting. T-cell proliferation and F-actin levels were studied by flow cytometry. The patient harbored 2 homozygous deletions in STX11 (c.369_370del, c.374_376del; p.V124fs60∗) previously associated with familial hemophagocytic lymphohistiocytosis and a novel homozygous missense variant in SLP76 (c.767C>T; p.T256I) that resulted in an approximately 85% decrease in SLP76 levels and absent T-cell proliferation. The patient's heterozygous family members showed an approximately 50% decrease in SLP76 levels but normal immune function. SLP76-deficient J14 Jurkat cells did not express SLP76 and had decreased extracellular signal-regulated kinase signaling, basal F-actin levels, and polymerization following T-cell receptor stimulation. Reconstitution of J14 cells with T256I mutant SLP76 resulted in low protein expression and abnormal extracellular signal-regulated kinase phosphorylation and F-actin polymerization after T-cell receptor activation compared with normal expression and J14 function when wild-type SLP76 was introduced. The hypomorphic mutation in SLP76 tones down the hyperinflammation due to STX11 deletion, resulting in a combined immunodeficiency that overshadows the hemophagocytic lymphohistiocytosis phenotype. To our knowledge, this study represents the first report of the opposing effects of 2 gene defects on the disease in a patient with an inborn error of immunity.

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

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. 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). 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. 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. 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.

CXCL9 inhibition does not ameliorate disease in murine models of both primary and secondary hemophagocytic lymphohistiocytosis

Hemophagocytic Lymphohistiocytosis (HLH) is a group of disorders culminating in systemic inflammation and multi-organ failure with high incidence of hepatic dysfunction. Overproduction of IFN-γ is the main immunopathological driver in this disorder. Monokine induced by IFN-γ (CXCL9) serves as a biomarker for disease activity and response to treatment in this disorder. However, very little is understood about the actual functional role of CXCL9 in pathogenesis in HLH. In the current study, we sought to determine the role of CXCL9 in pathogenesis in murine models of both Familial HLH (prf1−/−) and Toll Like Receptor (TLR) 9 repeated stimulation induced Macrophage Activation Syndrome (MAS), a form of secondary HLH. FHL and MAS were induced in both CXCL9 genetically deficient mice (cxcl9−/−) and controls as well as using AMG487, a pharmacological antagonist of the CXCL9 receptor, CXCR3. Results showed that CXCL9 genetic deficiency did not improve disease parameters or hepatitis in both models. Consistent with genetic ablation of CXCL9, inhibition of its receptor, CXCR3, by AMG487 did not show any significant effects in the FHL model. Taken together, inhibition of CXCL9-CXCR3 interaction does not ameliorate HLH physiology in general, or hepatitis as a classical target organ of disease.

Approaching hemophagocytic lymphohistiocytosis.

Hemophagocytic Lymphohistiocytosis (HLH) is a rare clinical condition characterized by sustained but ineffective immune system activation, leading to severe and systemic hyperinflammation. It may occur as a genetic or sporadic condition, often triggered by an infection. The multifaceted pathogenesis results in a wide range of non-specific signs and symptoms, hampering early recognition. Despite a great improvement in terms of survival in the last decades, a considerable proportion of patients with HLH still die from progressive disease. Thus, prompt diagnosis and treatment are crucial for survival. Faced with the complexity and the heterogeneity of syndrome, expert consultation is recommended to correctly interpret clinical, functional and genetic findings and address therapeutic decisions. Cytofluorimetric and genetic analysis should be performed in reference laboratories. Genetic analysis is mandatory to confirm familial hemophagocytic lymphohistiocytosis (FHL) and Next Generation Sequencing is increasingly adopted to extend the spectrum of genetic predisposition to HLH, though its results should be critically discussed with specialists. In this review, we critically revise the reported laboratory tools for the diagnosis of HLH, in order to outline a comprehensive and widely available workup that allows to reduce the time between the clinical suspicion of HLH and its final diagnosis.

Quantitation of Immune Synapse Duration Supports the Contribution of Perforin Haploinsufficiency to Hyperinflammation

Abstract Hyperinflammation is an acute state that occurs in a variety of infectious, inflammatory, and genetic contexts. Familial Hemophagocytic Lymphohistiocytosis (FHL) is a rare Inborn Error of Immunity resulting from biallelic disruption of perforin or other genes necessary for granule-mediated cytotoxicity. Some reports suggest that heterozygous defects in FHL genes contribute to hyperinflammation in concert with infection or rheumatic diseases. Most known causes of hyperinflammation arise from abnormal regulation of the Immune Synapse (IS) between cytotoxic CD8 T-cells and target cells. Using live imaging, we sought to assess the effects of Perforin and other putative contributors to hyperinflammation on IS duration and magnitude. We evaluated the IS between CD8 T cells from mice with different T-cell receptor signal strengths (OT1 vs P14) and various target cell types, as well as that between Prf1+/+, +/−, and −/− P14 cells and BMDCs. IS duration was calculated based on time between killer cell calcium flux and target cell Propidium Iodide uptake, and cytokine ELISA of supernatants was performed. We found that OT1, but not P14, CD8 T-cells could kill MC57 fibrosarcoma cells, but both were able to kill BMDCs. Moreover, both complete Prf1 deficiency and Prf1 haploinsufficiency prolonged the IS and increased cytokine production. Excess IL18, a hyperinflammatory susceptibility factor that synergizes with Prf1 deficiency in vivo to drive spontaneous hyperinflammation, shortened IS duration but dramatically increased IFN-γ production. Thus, showing that our in vitro IS quantitation model is sensitive to small changes that may be important contributors to pathogenic T-cell activation and life-threating hyperinflammation. Supported by grants from NIH (R01 HD098428-03-04), Children's Hospital of Philadelphia (IDF FY21-FY24), and from Histiocytosis Association (FP00039723_A1)

CRISPR screening for cytotoxic granule degranulation pathway in CD8 T cell

Abstract Degranulation of perforin-containing cytotoxic granules is important for NK cells and CD8 cytotoxic T lymphocytes (CTLs) to kill infected cells or tumors. Defects in this pathway cause the primary form of hemophagocytic lymphohistiocytosis (HLH), which is known as familial HLH (FHL) syndrome. Most of reported FHL causative genes encode molecules that are involved in the intracellular trafficking of cytotoxic granules and their fusion to the plasma membrane, followed by the delivery of their contents into the target cell through immunological synapses. However, other components essential for cytotoxic granule degranulation for CTLs are not well understood. To identify indispensable molecules for cytotoxic granule degranulation, we established an efficient in vitro CD8 amplification and degranulation system to perform a CRISPR-screening. To test this system, we transduced OT-I CD8 cells with a gRNA library targeting PIP 3binding proteins, which are important for cell adhesion, subcellular trafficking and T cell activation. Cells were harvested at short (Day 8) and medium-timepoints (Day 15) to evaluate the frequency of gRNAs in the degranulated (CD107+) and non-degranulated fractions. Of the top 10 hits of genes whose deletion suppressed degranulation, five were already listed in the 2022 IUIS classification of inborn error of immunity. We will use individual knockouts to evaluate how these genes affect degranulation. To expand to genome-wide screening, we have also subcloned a full library of sgRNAs into our high efficiency retroviral vector. Thus, we established an efficient assay system to perform a CRISPR-screening for CD8 T cells and can detect genes affected by primary immunodeficiencies. This work was supported by the Intramural Research Program of NIAID, NIH.

A Case of a Relapsing Remitting Macrophage Activating Syndrome After Covid-19 Vaccine In a Teenager with UNC13D Heterozygous Variant of Uncertain Significance

IntroductionMacrophage activation syndrome (MAS) is a severe hyper inflammatory condition caused by the over-activation and proliferation of T cells, NK cells and macrophages. It is often associated with complications of rheumatic/immune diseases. We present a case of a 15-year-old female who experiences recurrent episodes of MAS without any known definitive underlying etiology. Case PresentationA 15-year-old previously healthy female developed fatigue, fevers, myalgia, chest pain, splenomegaly and lymphadenopathy 10 days after receiving her first Pfizer COVID-19 vaccine. Her symptoms recurred 10 days after receiving the second dose. Her myocarditis, MIS-C, and infectious work up was negative except for positive EBV IgG. Laboratory studies revealed anemia, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia. She initially responded to decadron; however, her symptoms recurred with steroid taper. Bone marrow biopsy revealed hemophagocytosis. Whole exome sequencing (WES) revealed a heterozygous variant of uncertain significance in UNC13D c.962C>A (p.Thr321Asn). She had multiple re-admissions with significantly elevated inflammatory markers, including extremely high IL2-R, IL-18 and CXCL9. Each episode was complicated by an acute viral infection. She responds to high dose steroids, anti-IL-1, and JAK inhibitors. Nonetheless, it has been difficult to wean decadron without triggering a flare. She continues to require increasing doses of baricitinib. DiscussionMAS may be seen as a complication of rheumatic diseases, as well as inborn errors of immunity. However, none of these conditions have been diagnosed in this patient despite extensive testing, including WES. The degree of her immune dysregulation has been very severe making her disease process unpredictable and extremely difficult to control. She has frequent flares precipitated by viral infections or attempts at adjusting her immunomodulators. Weaning her medications has been challenging as she continues to require increasing doses of baricitinib and corticosteroids. The UNC13D gene is associated with autosomal recessive familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Our patient is heterozygous for an UNC13D variant of uncertain significance. Additional genetic inquiries with whole genome sequencing to help elucidate the underlying etiology of her severe condition is being conducted. We hypothesize she developed MAS due to a combination of genetic predisposition, prior EBV infection, and immune stress associated with the COVID-19 vaccine. [Display omitted] [Display omitted] [Display omitted]