Disorder Of Immune Regulation Research Articles

T Cell Depleted Haploidentical Hematopoietic Stem Cell Transplantation for Patients with Familial Hemophagocytic Lymphohistiocytosis Who Do Not Have Matched Family Donors: Experience in Oman.

Familial hemophagocytic lymphohistiocytosis (FHLH) is a potentially fatal disorder of immune regulation. Management includes chemotherapy followed by hematopoietic stem cell transplantation (HSCT). T cell depleted (TCD)-haploidentical HSCT could be an option for those patients who do not have HLA matching family donor. The objective of this study was to report on the outcome of TCD-haploidentical HSCT in patients with FHLH who underwent transplantation at Sultan Qaboos University Hospital (SQUH). This is a retrospective report on 12 patients with FHLH who received TCD- haploidentical HSCT at SQUH between August 2010 and December 2018. Epidemiologic characteristics and details on the transplantation procedures and complications were collected from patients' electronic records. Twelve patients with FHLH received TCD-haploidentical HSCT after a myeloablative conditioning regimen composed of treosulfan/thiotepa/fludarabine/anti-thymocyte globulin and rituximab. The mean age at transplantation was 11.67 ± 8 months. All patients had Perforin gene mutations, except 1 patient who had an UNC-13D mutation. Most patients received TCRαβ+/CD19+ depleted grafts for faster immune reconstitution. Seven patients (58.3%) have been cured with a mean follow-up duration of 3.44 years. Four patients died of multiorgan failure secondary to gram-negative sepsis. One patient had primary graft failure, and 2 patients had mild graft-versus-host disease. Two patients had Pneumocystis carinii pneumonia, 2 had adenoviremia, and 9 patients had cytomegalovirus (CMV) viremia. Among patients with CMV viremia, 2 had evidence of disease (retinitis, enteritis). All patients with CMV viremia were treated successfully with foscarnet pre-engraftment and ganciclovir postengraftment, respectively. TCD-haploidentical HSCT could be a viable option for patients with FHLH who do not have HLA matching family donors. Infectious complications are the leading cause of death in that setting. CMV viremia was the most frequently encountered infectious complication.

Progranulin Mediates Proinflammatory Responses in Systemic Lupus Erythematosus: Implications for the Pathogenesis of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by the disorders of immune regulation but its pathogenesis is poorly understood. Progranulin (PGRN) is an immunomodulatory protein that is upregulated in SLE patients. However, the factors involved in regulating the pathogenesis of SLE by PGRN are largely unknown. We sought to investigate the role and molecular mechanisms of PGRN in SLE to develop a novel therapeutic target. We used an animal model of SLE that was induced in PGRN-deficient and normal wild type (WT) mice using pristane. PGRN concentrations were measured in SLE and the impact of PGRN deficiency was examined by measuring tissue injury and immune responses of T cells (Th1, Th2, Th17, and Treg) and B cells. SLE patients and mice showed elevated PGRN levels. Compared with WT SLE mice, inflammatory cell infiltration, tissue edema, and necrosis were alleviated in PGRN-/- SLE mice and the levels of serum chemistry markers of tissue damage and the presence of anti-double-stranded DNA and anti-ribosomal protein P0 antibodies were all significantly decreased. We further discovered that PGRN deficiency could disturb the immune responses of T cell (Th1, Th2, Th17, and Treg) and B cell responses, leading to the decrease of inflammatory cytokines including interferon-γ and interleukin-17A and increased levels of regulatory B cells. PGRN plays a proinflammatory role in the development of SLE partially through promoting the production of autoantibodies and enhancing Th1 and Th17 cell responses. This may provide new therapeutic options for patients with SLE.

Targeting Immune Dysregulation in Childhood Evans Syndrome

Background: Evans Syndrome (ES) describes the simultaneous or sequential occurrence of 2 or more autoimmune cytopenias - most often autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP), but also autoimmune neutropenia (AIN) - which are often refractory to therapy and chronic in nature. Historically, ES has been attributed to idiopathic autoantibody production, but recent advances in our understanding of this disease have revealed associations with more well-described underlying disorders of immune regulation when properly investigated. These include autoimmune lymphoproliferative syndrome (ALPS) and more novel immune dysregulation disorders such as Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) defects, among the more historically well-known systemic autoimmune diseases (such as systemic lupus erythematosus [SLE]) and common variable immunodeficiency (CVID). Importantly, recent data shows improved outcomes among ES patients in whom underlying immune dysregulation is identified and for whom appropriately targeted immunomodulating treatments are then utilized. Despite this, the majority of underlying and potentially targetable pathologies driving ES in children remain undiagnosed; and treatment strategies are therefore empiric, resulting in sub-optimal outcomes among this population of patients.Aims: We aim to better describe the diagnostic rates, treatment strategies, and clinical outcomes among a large cohort of pediatric patients with this rare and poorly studied disease, in order to identify barriers to appropriate and timely diagnostic investigation, to highlight areas for future research, and to better inform clinical practices.Methods: We completed a retrospective chart review including pediatric ES patients followed at 3 large tertiary centers over a recent 6-year period (2012 - 2018). All patients meeting eligibility criteria (those aged 6 months - 21 years, with 2 documented autoimmune cytopenias as defined by current international expert committee standards, and not having undergone solid organ or stem cell transplantation) were reviewed in accordance with IRB-approved protocols. Chi-squared test or Fisher's exact test were utilized to compare nonparametric categorical data. Mann-Whitney U-test was used to compare nonparametric continuous data. A Kruskal-Wallis one way ANOVA test was used to compare ranks between more than two categories. Statistical analyses were performed using SPSS Statistics 24 (IBM, Armonk, NY). A Bonferroni correction was applied to correct for multiple comparisons. A p<0.05 was defined as statistically significant.Results: 48 eligible patients with ES were identified, with length of follow-up ranging from 0 to 12 years (median = 2.7 years). Underlying immune dysregulation was identified in 46% of patients - 12 with systemic autoimmune disease, 4 with ALPS, 3 with CVID, and 3 with other disorders of immune regulation (n=22). Notably, 100% of pediatric ES patients in whom a definitive immune dysregulation disorder was identified required ≥ 3 therapies (mean follow-up = 3.7 years), vs. only 58% of ES patients in whom no unifying immune diagnosis was made (mean follow-up = 3.2 years) [p=0.008]. Furthermore, second- and third-line immunomodulatory agents (including mycophenolate, hydroxychloroquine, 6-mercaptopurine, belimumab, ofatumumab, abatacept, rituximab, sirolimus, cyclosporine, azathioprine, danazol, and methotrexate) were utilized at a significantly higher rate among those with underlying immune dysregulation identified (82%) vs. those without (31%) [p=0.003]; and ≥3 immunomodulatory agents were required significantly more often in the group with underlying immune dysregulation identified (32%) vs. those without (0%) [p=0.016]. Among those in whom a unifying diagnosis was made, difference in time to diagnosis among groups was statistically significant (p=0.036), with the time to diagnosis for systemic autoimmune disease shorter than that for other immune dysregulation disorders. Overall, ES preceded identification of an underlying immune dysregulation disorder by an average of 3.7 years.Conclusion: A thorough investigative approach is necessary in pediatric ES patients, given the importance of identifying the more global immune dysregulation which would allow for targeted treatment strategies and lead to improved overall long-term outcomes among this population of patients. [Display omitted] DisclosuresLambert:Bayer: Membership on an entity's Board of Directors or advisory committees; Summus: Consultancy; Rigel: Consultancy; Sysmex: Consultancy; Shionogi: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Educational Concepts in Medicine: Consultancy; CSL: Consultancy. Grace:Agios Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy. Despotovic:Novartis: Research Funding; AmGen: Research Funding; Sanofi: Consultancy.

DISORDERS OF THE IMMUNOREACTIVITY AS A MARKER OF PROFESSIONAL RISK TO THE HEALTH OF WORKERS IN THE PRODUCTION OF VINYL CHLORIDE

Production of vinyl chloride (VC) is one of the largest world production of volume organic chemical intermediates, consumption of which, according to estimates from 2015 to 2020 will grow. Currently, the VC production involving more than 2 million people, the risk of formation of occupational and industrial-caused diseases might be significant. The purpose of this study was to evaluate the status of the immune reactivity of the organism of workers of different professional and experienced groups in the modern VC production to justify informative immunological criteria of the occupational health risk. There were performed the laboratory and immunological study of 77 men working in the VC production. The paper considers the regularities of formation of compensatory-adaptive immune responses in working in the VC production depending on the intensity and duration of exposure to adverse production factors. There is a more pronounced imbalance in the immune system workers of the main occupations (operatives, technicians) with long (10 or more years) experience. It characterized by a decrease in IL-2, IL-4, IgM and increased the production of IL-8, INF-α, TNF-α. The engineering staff experiencing less stress by toxicants, showed the compensatorily increased production of IL-1β and decrease of IgA and also unidirectional, but less pronounced changes in the content of IL-8, INF-γ. There is the relationship between the development of disorders in the immune system of workers in various occupational groups and the level of occupational risk for these specialties. Substantiated informative immunological parameters for the early detection of functional disorders of immune regulation underlying the formation of various diseases are necessary for the timely implementation of effective prevention programmes. Thus, the state of the immune reactivity of the body reflects the level of the adaptation potential of employees of various professions and experienced groups and may be an integrated assessment of the risk of violations of occupational health. The implementation of methods of immunodiagnostics will as predict and identify the disease at the early stages of its development that can optimize the resistance of the organism involved in the VC production as reducing the incidence.

Genetic Disorders of Immune Tolerance: The Flip Side of Immune Deficiency Where Autoimmunity Trumps Infection

Primary Immune Regulatory Disorders (PIRD) are group of congenital Immunodeficiencies that present primarily with autoimmunity and less frequently with infections. The paradigm for these disorders is the syndrome of Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX), a disorder caused by a lack of functional regulatory T cells due defects in the lineage-specifying transcription factor, FOXP3. We have collected and evaluated a large cohort of patients with clinical manifestations suggestive of IPEX and have found FOXP3 mutations in more than 90 patients. This has informed our further investigation into the structure and function of the FOXP3 transcription factor. In patients that have IPEX-like clinical features but lack FOXP3 mutations, we have identified defects in several other genes now associated with an IPEX-like clinical phenotype, including germline STAT1 and STAT3 gain-of-function (GOF) mutations, CTLA4 haploinsufficiency, LRBA deficiency and others. We have shown that within this cohort, all have IPEX-like clinical features but there are some clinical and laboratory features that can help with differentiating between the different genetic syndromes. To determine the best approach to treatment of these disorders, we have also collected data on the international experience with bone marrow transplant for some of these disorders including IPEX, CTLA4 haploinsufficiency, STAT1-GOF, and STAT3-GOF disease. We have demonstrated that bone marrow transplant can be curative for each of these disorders but that patients with autosomal dominant gain-of-function mutations in STAT1 and STAT3 may pose unique challenges for transplant that will be discussed. These data will be presented along with preliminary data suggesting non-transplant approaches to management using targeted therapies. DisclosuresTorgerson:CSL Behring: Consultancy, Research Funding; Shire Bioopharmaceuticals: Consultancy, Research Funding; UCB Biopharma: Consultancy; ADMA Biosciences: Consultancy.

Immune response regulatory disorders and redox homeostasis in pregnant women with isoimmune conflict

Immune response regulatory disorders and redox homeostasis in pregnant women with isoimmune conflict

Modeling primary immune regulatory disorders: Ferrari or Yugo?

Despite about 75 million years of evolutionary separation, mice have proven to be remarkably informative for understanding the human immune system. Many immune signaling and developmental pathways are nearly identical between mouse and man, and some pathways, such as NK cell sensing of self, display clear evolutionary convergence. In this issue, Burnett et al. and Gamez-Diaz et al. independently describe the phenotype of Lipopolysaccharide Responsive Beige-like Anchor (LRBA) deficient mice. LRBA-deficient humans have a striking and severe syndrome of varied autoimmunity, lymphoproliferation, gut inflammation and immune deficiency.1 In contrast, these two groups report that LRBA-deficient mice are generally quite well. Despite aging and/or challenge with infectious organisms, these animals display none of the pathologies seen in humans. Of note, LRBA knockout mice have decreased CTLA4 protein levels, as is seen in LRBA-deficient patients, and is thought to underlie disease development. Both groups also noted other subtle immune abnormalities not correlated in patients to date: a decrease of the innate-like B1 B cells, and evidence of increased IgA production. These abnormalities are not predicted by any known function of LRBA, but may provide additional clues to disease pathogenesis.

Elevated serum concentrations of β-2-microglobulin are often found at the time of diagnosis of hemophagocytic lymphohistiocytosis in adults with lymphoid and myeloid malignancies

Background: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune regulation. In patients over 60 years of age, HLH associated with hematological malignancies (hM-HLH) is the most prevalent. β-2-Microglobulin (B2M) plays an important role in antigen presentation and immunological regulation. Elevated B2M levels reflect T-cell activation. Objective: The aim of this study was to determine serum B2M concentrations in adults with hM-HLH and to interpret its significance in the context of overall survival (OS). Patients and methods: Serum B2M concentration was determined in 31 adults aged 22–84 years at the time of hM-HLH diagnosis. Lymphoid malignancy was diagnosed in 22 patients and myeloid malignancy in 9 patients. Results: The serum concentration of B2M was elevated in 100% of the examined patients. Mean and median serum B2M concentrations were 5.3 and 4.2mg/L, respectively (range 2–17mg/L). We have not found any significant differences in terms of the studied serum B2M concentrations between patients with T/NK-cell lymphomas, B-cell lymphomas, and myeloid malignancies. The outcome of HLH was poor in vast majority of patients with the median OS for the entire group of 46 days. Conclusions: Elevated serum B2M level is a frequent finding at the time of hM-HLH diagnosis in adults. It seems to be a useful indicator of HLH for its early detection and evaluation afterward, as well as for immediate therapeutic intervention. Further prospective studies answering the question whether serum B2M can be used as a prognostic factor in hM-HLH would be of interest.

Incidence and Outcomes of Central Nervous System Hemophagocytic Lymphohistiocytosis Relapse after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation

Hemophagocytic lymphohistiocytosis (HLH) is an immune regulatory disorder that commonly presents with central nervous system (CNS) involvement. The only cure for genetic HLH is hematopoietic stem cell transplantation (HSCT), typically treated with reduced-intensity conditioning (RIC) regimens. We sought to estimate the incidence of CNS relapse after RIC HSCT, determine risk factors, and evaluate outcomes. We performed a retrospective chart review of 94 consecutive children and young adults with primary HLH who received RIC HSCT. CNS relapse within 1 year after transplantation was diagnosed by review of clinical symptoms, cerebral spinal fluid (CSF), and radiologic findings. Four (4.25%) patients developed symptoms of possible CNS HLH after HSCT and 3 patients were diagnosed. Eight patients underwent screening lumbar puncture because of history of active CNS disease at the onset of the conditioning regimen and 4 had evidence of continued disease. The overall incidence of CNS relapse and continued CNS disease after RIC HSCT was 8%. All patients with CNS disease after HSCT responded to CNS-directed therapy. Whole blood donor chimerism at the time of CNS relapse was low at 1% to 34%, but it remained high at 88% to 100% for patients with continued CNS disease. Overall survival for patients with CNS relapse was 50%, compared with 75% for patients without CNS disease (P = .079). Our data suggest that a low level of donor chimerism or active CNS disease at the time of transplantation increase the risk of CNS HLH after HSCT. Surveillance CSF evaluation after allogeneic RIC HSCT should be considered in patients with risk factors and CNS-directed treatment should be initiated if appropriate.

Familial haemophagocytosis lymphohisticytosis type 3: A case report

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare autosomal recessive disorder of immune regulation. Here, we report on a fatal case of type 3 FHL (FHL3) in a 45-day-old boy. Clinically, the infant presented with fever and hepatosplenomegaly. Biology showed pancytopenia, elevated ferritin, and decreased fibrinogen. Images of hemophagocytosis were found at the bone morrow examination. The diagnosis of FHL type 3 was made by the identification of homozygous mutation in the Munc13-4 gene (UNC13D) located in exon 20: 1822 del 12bp (V608fs). This mutation was previously observed in a Tunisian and in Moroccan families.

Comparison of Th1/Th2 cytokine profiles between primary and secondary haemophagocytic lymphohistiocytosis.

BackgroundHaemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune regulation, and HLH patients with mutations in genes including PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK were reported to be primary HLH. Due to the different treatment options, the differentiation between primary and secondary HLH is critical. Our previous studies have showed that a Th1/Th2 cytokine profile is diagnostic for HLH, yet the cytokine profiles between primary and secondary HLH have not been compared. The aim of the study was to test whether the Th1/Th2 cytokine profile could be used as a tool to differentiate between primary and secondary HLH.MethodsA total of 45 hospitalized Chinese children with HLH during the period of February 2010 through September 2012 were enrolled in the study. Fifty healthy children were enrolled as controls. Primary HLH related genes were sequenced using genomic DNA samples. The Th1/Th2 cytokine levels including interferon-γ (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10, IL-6, IL-4 and IL-2 were quantitatively determined by cytometric bead assay techniques.ResultsPrimary HLH group (n = 4) included one patient with biallelic heterozygous mutations in PRF1 gene, and three patients with hemizygous mutation in SH2D1A gene. Based on the available genetic data, the other 41 patients were classified into the secondary HLH group. When compared the cytokine levels between the two groups, IL-4 level in primary-HLH was significantly lower than that in secondary HLH (P = 0.025), while IFN-γ level in primary HLH had a tendency of statistically lower than that in secondary HLH (P = 0.051). Area under receiver operating characteristic (ROC) curves of IL-4 and IFN-γ, IL-10, TNF-α, IL-2, and IL-6 levels were 0.841, 0.799, 0.506, 0.494, 0.457, and 0.250, respectively. ROC curves showed that 1.7 pg/ml of IL-4 had sensitivity and specificity for differentiation between primary and secondary HLH as 70.7 and 100.0 %, while 433.9 pg/ml of IFN-γ had sensitivity and specificity as 51.2 and 100.0 %, respectively.ConclusionsHLH patients with lower IL-4 and IFN-γ levels have higher possibility to be primary HLH. The cytokine profile may be used as an additional tool for the quick differential diagnosis between primary and secondary HLH.Electronic supplementary materialThe online version of this article (doi:10.1186/s13052-016-0262-7) contains supplementary material, which is available to authorized users.

T-cell depleted haploidentical hematopoietic stem cell transplantation is the optimal graft source for patients with familial hemophagocytic lymphohistiocytosis who do not have matched family donors.

Abstract Familial hemophagocytic lymphohistiocytosis (HLH) is a fatal disorder of immune regulation. When managed appropriately with systemic chemotherapy, cure can be achieved by subsequent allogeneic hematopoietic stem cell transplantation (HSCT). Although the best results are obtained with matched-family donors (MFD), alternative donor transplants may be necessary when such donors are not available. As there is urgency to proceed with curative therapy, T-cell depleted haploidentical HSCT could be a viable option as a parent or other haploidentical family member would be available instantly. Between August 2010 and September 2015 we performed haploidentical HSCT for six patients with HLH in this institution. These patients did not have MFDs, and in our center it was logistically easier to perform an haploidentical HSCT rather than to pursue with an unrelated donor or cord blood search from the international registries. The median age at transplant was 6 months (5–31 months) and all had perforin gene mutations. The first two patients had CD3/CD19 depleted procedures while the rest underwent TCR alpha/beta and CD19 depletion for faster immune reconstitution. All six patients engrafted following the transplant with minimal graft versus hot disease. Four patients are cured from the disease with a median follow-up of 3 years (7mths to 5 years). Two patients (siblings) who were transplanted in partial remission expired 2 months following transplant from multi-organ failure after successful engraftment. In the presence of an appropriate transplant laboratory facility, T-cell depleted haploidentical HSCT should be considered as the graft of choice for patients with HLH who do not have HLA-matched family donors.

Quercitrin ameliorates the development of systemic lupus erythematosus-like disease in a chronic graft-versus-host murine model.

Systemic lupus erythematosus (SLE) is a serious disorder of immune regulation characterized by overproduction of autoantibodies, lupus nephritis, CD4+ T cell aberrant activation, and immune complex-mediated inflammation. The chronic graft vs. host disease (cGVHD) mouse model is a well-established model of SLE. Quercitrin is a natural compound found in Tartary buckwheat with a potential anti-inflammatory effect that is used to treat heart and vascular conditions. In our previous study, we determined that quercitrin is an immunosuppressant with beneficial effects in mouse models of immune diseases. We hypothesized that quercitrin could prevent lupus nephritis in the cGVHD mouse model by decreasing the production of autoantibodies and inflammatory cytokines, and reducing immune cell activation. cGVHD was induced by injecting DBA/2 spleen cells into the tail vein of BDF1 mice. The cGVHD mice exhibited significant proteinuria, which is a marker of nephritis. Quercitrin decreased the number of serum antibodies, CD4+ T cell activation, as well as the expression levels of T-bet, GATA-3, and selected cytokines. Moreover, quercitrin treatment decreased the expression of inflammatory genes and cytokines in the kidney, as well as in peritoneal macrophages. In addition, quercitrin inhibited LPS-induced cytokines as well as the phosphorylation of ERK, p38 MAPK, and JNK in Raw264.7 cells. Overall, quercitrin ameliorated the symptoms of lupus nephritis in the cGVHD mouse model, which may be due to the inhibition of CD4 T cell activation and anti-inflammatory effects on macrophages.

Treatment of Langerhans cell histiocytosis: role of BRAF/MAPK inhibition

AbstractLangerhans cell histiocytosis (LCH) is a clonally derived neoplasm with a highly variable clinical course. Although LCH was once considered a disorder of immune regulation, the identification of activating mutations in the proto-oncogene BRAF-V600E in ∼50%-60% of cases and MEK and ERK phosphorylation in 100% of examined cases, has changed the definition of LCH to a dendritic cell neoplasm with a strong inflammatory component. Current international LCH trials are focused on further improving the outcome of high-risk multisystem LCH patients, by decreasing the reactivation rate, optimizing early salvage regimens, and preventing late sequelae. Anecdotal responses to vemurafenib, a BRAF-V600E inhibitor, have been reported in a few cases of LCH and Erdheim–Chester disease. However, the development of resistance, as well as the potential risks of cutaneous and pancreatic cancers in patients with BRAF-V600E-mutated melanoma treated with single inhibitors, suggest the need for prospective trials with BRAF inhibitors, alone or in combination with other inhibitors of this pathway, for patients with refractory or multiply-relapsed LCH. The recent discovery of somatic mutations in ARAF and in MAP2K1, which lead to activation of the RAS-RAF-MEK –ERK pathway in the setting of wild-type BRAF, as well as the finding that activating mutation in MAP2K1 are relatively insensitive to MEK inhibitors, suggest that a more detailed understanding of this pathway in LCH may be necessary for the development of more effective targeted therapies.

Treatment of Langerhans cell histiocytosis: role of BRAF/MAPK inhibition.

Langerhans cell histiocytosis (LCH) is a clonally derived neoplasm with a highly variable clinical course. Although LCH was once considered a disorder of immune regulation, the identification of activating mutations in the proto-oncogene BRAF-V600E in ~50%-60% of cases and MEK and ERK phosphorylation in 100% of examined cases, has changed the definition of LCH to a dendritic cell neoplasm with a strong inflammatory component. Current international LCH trials are focused on further improving the outcome of high-risk multisystem LCH patients, by decreasing the reactivation rate, optimizing early salvage regimens, and preventing late sequelae. Anecdotal responses to vemurafenib, a BRAF-V600E inhibitor, have been reported in a few cases of LCH and Erdheim-Chester disease. However, the development of resistance, as well as the potential risks of cutaneous and pancreatic cancers in patients with BRAF-V600E-mutated melanoma treated with single inhibitors, suggest the need for prospective trials with BRAF inhibitors, alone or in combination with other inhibitors of this pathway, for patients with refractory or multiply-relapsed LCH. The recent discovery of somatic mutations in ARAF and in MAP2K1, which lead to activation of the RAS-RAF-MEK -ERK pathway in the setting of wild-type BRAF, as well as the finding that activating mutation in MAP2K1 are relatively insensitive to MEK inhibitors, suggest that a more detailed understanding of this pathway in LCH may be necessary for the development of more effective targeted therapies.

A Novel Targeted Approach to the Treatment of Hemophagocytic Lymphohistiocytosis (HLH) with an Anti-Interferon Gamma (IFNγ) Monoclonal Antibody (mAb), NI-0501: First Results from a Pilot Phase 2 Study in Children with Primary HLH

Introduction Primary HLH (pHLH) is a rare immune regulatory disorder invariably lethal if untreated. It is driven by pathologic immune activation, leading to the development of fever, splenomegaly, cytopenias and coagulopathy, which ultimately may cause multi-organ failure (MOF) and death. Based on data from murine models of primary and secondary HLH (sHLH), and observational studies in patients with HLH, the high production of IFNγ is thought to be a critical factor driving development of the disease. Immune-chemotherapy, primarily etoposide-based regimens, is at present the only pharmacological approach able to control HLH and bring patients to curative allogeneic hematopoietic stem cell transplant (allo-HSCT). In spite of recent attempts to further intensify treatment regimens, mortality and morbidity remain high, in part due to drug-related toxicities.NI-0501 is a fully human, high affinity, anti-IFNγ mAb that binds to and neutralizes human IFNγ, offering a novel and targeted approach for the control of HLH.Methods An open-label Phase 2 study has been conducted in United States and Europe to evaluate safety and efficacy of NI-0501 in children with either confirmed or suspected pHLH. NI-0501 was administered at initial dose of 1 mg/kg every 3 days, with possible dose increase guided by PK data and/or clinical response in each patient, on initial background dexamethasone dose of 5-10 mg/m2. Treatment duration ranged from 4 to 8 weeks. Ability to move to allo-HSCT, relevant HLH disease parameters, and 8-week survival were assessed.Study Population A total of 13 patients were enrolled: 8F/5M, median age 1.0y (range 2.5mo-13y). Twelve patients received NI-0501 as a second line treatment after having received conventional therapy and either reactivating, obtaining unsatisfactory response, or being intolerant to therapy. One patient was treated with NI-0501 in first line. Nine patients carried a known HLH genetic defect (3 FHL2, 2 FHL3, 2 GS-2, 1 XLP1, 1 XLP2). The majority of patients were at the severe end of HLH spectrum, in compromised general condition (2 patients in ICU) and carrying significant toxicities from previous HLH treatments. At baseline ferritin was elevated in 12/13 patients and sCD25 in 8, cytopenias were present in 10 patients, splenomegaly in 8, hypofibrinogenemia and hypertriglyceridemia in 9. Liver and CNS involvement were present in 7 and 3 patients, respectively.Results Overall, NI-0501 treatment significantly improved parameters of HLH disease activity (Fig. 1), and 9 of 13 patients achieved a satisfactory response. Seven patients have proceeded to allo-HSCT. Allo-HSCT is planned for two patients with good HLH control upon identification of an appropriate donor. In one patient (who achieved disease control with first line NI-0501) allo-HSCT is not yet planned given the absence of a causative HLH gene mutation. Eleven of 13 patients were alive at 8 weeks. Two patients died of HLH/MOF at day 53 and 36, respectively. CNS signs and symptoms resolved in the 2 evaluable patients. Greater than 50% reduction of dexamethasone dose was possible in 50% of patients during the first 4 weeks of NI-0501 treatment. IFNγ neutralization was demonstrated by a sharp decrease in CXCL9, a chemokine exquisitely IFNγ-induced. CXCL9 levels tightly correlated with IFNγ production, estimated by measuring IFNγ bound to NI-0501 (Fig. 2), suggesting CXCL9 as a potential new biomarker for HLH. NI-0501 was well tolerated and no safety concern was identified. None of the infections known to be favored by IFNγ neutralization were reported, and no infections occurred in patients who had not previously received chemotherapy. Seven patients reported at least one SAE, all assessed by the DMC as not related to NI-0501 administration. No unexpected events attributable to “off target” effects of NI-0501 (myelotoxicity, hemodynamic alterations) were observed.Conclusions Neutralization of IFNγ by NI-0501 offers an innovative targeted and potentially less toxic approach to HLH management. The results of this study show that NI-0501 is a safe and effective therapeutic option in patients with pHLH who have demonstrated unsatisfactory response, or are intolerant, to conventional therapy. Furthermore, therapy with NI-0501 was not associated with any of the typical short- or long-term toxicities reported for etoposide-based regimens. Assessment of NI-0501 as first line treatment for pHLH is ongoing. [Display omitted] [Display omitted] Disclosures:Jordan:Novimmune: Consultancy. Allen:Roche: Consultancy , Other: unpaid ; NovImmune: Consultancy , Other: unpaid. De Benedetti:Novimmune: Consultancy. Grom:Novartis: Consultancy , Research Funding ; Novimmune: Consultancy. Ballabio:Novimmune SA: Employment. Ferlin:Novimmune SA: Employment. De Min:Novimmune SA: Employment.

In Vitro Evidence for Immune-Modulatory Properties of Non-Digestible Oligosaccharides: Direct Effect on Human Monocyte Derived Dendritic Cells.

Infant formulas containing non-digestible oligosaccharides (NDO) similar to the composition in breast milk or a combination of lactic acid bacteria (LAB) and NDO have been shown to harbor preventive effects towards immune-regulatory disorders. The aim of this study was to investigate the immune-modulatory potential of non-digestible short chain galacto- and long chain fructo-oligosaccharides (scGOS/lcFOS) mimicking the natural distribution of oligosaccharides in human breast milk in presence or absence of certain LAB strains in human monocyte derived dendritic cells (MoDC). Immature human MoDC prepared from peripheral blood of healthy non-atopic volunteers were screened in vitro after stimulation with specific scGOS/lcFOS in presence or absence of LAB. IL-10 and IL-12p70 release was analyzed after 24 hours in cell-free supernatants by enzyme-linked immunosorbent assay (ELISA). A luminex-based assay was conducted to assess further cytokine and chemokine release by MoDC. To investigate the resulting T cell response, stimulated MoDC were co-incubated with naïve T cells in allogeneic stimulation assays and intracellular Foxp3 expression, as well as immune-suppressive capacity was determined. Oligosaccharides did not induce relevant amounts of IL-12p70 production, but did promote IL-10 release by MoDC. Furthermore, scGOS/lcFOS mixtures exerted a significant enhancing effect on LAB induced IL-10 secretion by MoDC while no increase in IL-12p70 production was observed. Blocking toll like receptor (TLR)4 abrogated the increase in IL-10 in both the direct stimulation and the LAB stimulation of MoDC, suggesting that scGOS/lcFOS act via TLR4. Finally, scGOS/lcFOS-treated MoDC were shown to upregulate the number of functional suppressive Foxp3 positive T cells following allogeneic stimulation. Our results indicate anti-inflammatory and direct, microbiota independent, immune-modulatory properties of scGOS/lcFOS mixtures on human MoDC suggesting a possible induction of regulatory T cells (Tregs). The tested combinations of LAB and scGOS/lcFOS might represent a useful dietary ingredient for the maintenance of intestinal homeostasis via the induction of Tregs.

Real-Time Quantitative Polymerase Chain Reaction for Apolipoprotein E genotyping

Background : Apolipoprotein E (ApoE) is an important substance responsible for the lipoprotein metabolism and thetransportation of lipid and known to play an important role in the body's physiological and pathological processes. Recently,ApoE genotyping test has been applied to the field of diagnostic methods for predicting high risk of Alzheimer's disease,cardiovascular diseases, as well as new diseases such as immune regulation disorders. Therefore, a faster and more accurateApoE genotyping test is required for the prevention of related disease and the general public health.Methods : We evaluated 220 serum specimens using two kinds of assays: one-stop Real-Q ApoE genotyping (BioSewoom,Seoul, Korea) and Seeplex ApoE ACE genotyping (Seegene, Seoul, Korea). The results were finally confirmed using sequencingas the reference. Statistical analysis was performed for the concordance rates between each assay and sequencing and thesensitivity and specificity were calculated with their 95% confidence intervals.Results : Sequencing results of 220 DNA samples showed 1 case of E2/E2, 139 of E3/E3, 9 of E4/E4, 17 of E2/E3, 4 ofE2/E4, 50 of E3/E4. The concordance rate between One-step Real-Q ApoE genotyping and sequencing was 100% (220/220),and that between Seeplex ApoE ACE genotyping and sequencing was 99.5% (219/220). Both sensitivity and specificity of OnestepReal-Q ApoE genotyping for every genotypes was 100%. The sensitivities and specificities of Seeplex ApoE ACEgenotyping were almost 100%, with two exception: 99.3% sensitivity in E3/E3 and 99.5% specificity in E2/E2.Conclusions : The performances of One-step Real-Q ApoE genotyping and Seeplex ApoE ACE genotyping were comparableoverall. However, the former showed better sensitivity and specificity comparable with those of sequencing. Hence, One-stepApoE genotyping could be a faster and more accurate option for ApoE genotyping.

237. Genetic Therapy for Perforin Deficiency Associated Hemophagocytic Lymphohistiocytosis Requires High Level Expression of the Perforin Gene for Adequate Correction

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal immune regulatory disorder, whereby heightened immune activation following viral infections causes hypercytokinemia, pancytopenia and end-organ damage. The majority of familial HLH is caused by Perforin-1 (prf1) gene mutations. Perforin is expressed in CD8+ T cells and natural killer (NK) cells and is critical for lymphocyte cytotoxicity. Currently, immunosuppressive therapy followed by allogeneic hematopoietic stem cell (HSC) transplant is the only curative option for HLH, but is restricted by availability of matched donors and a high (20-50%) transplant related mortality. We posited that perforin gene transfer into autologous HSC may be curative, have lower mortality, and not be limited by donor availability. We recently reported significant, but partial, correction of HLH symptoms in prf1-/- mice using lentivirus vectors (LV) expressing perforin from a ubiquitous phosphoglycerate kinase (PGK) promoter or a tissue specific perforin gene promoter (PRF). We hypothesized that negative selection of high perforin expressing HSC and the perforin gene dosage delivered by the LV contributes to the partial correction. We redesigned and developed a series of LV and compared prf1 expression from the PGK/PRF promoters to that from the MND promoter/enhancer; additionally we restricted perforin expression from PGK and MND in HSC with 4 repeats of miR126 (4T) target sequence, a miRNA specifically expressed in HSCs but not in lymphocytes, thus, circumventing the negative selection of high perforin expressing HSC. Improved perforin expression and cytotoxicity was observed with the ‘HSC-restricted’ MND4TLV in the KHYG1 human NK cell line, and in prf1-/- P14 mice, transgenic for a T-cell receptor that recognizes a lymphocytic choriomeningitis virus (LCMV) glycoprotein. These LV were compared in prf1-/- mice that were challenged with LCMV following transplant of gene-modified HSC. High gene marked NK chimerism was achieved: 87.6±1.7%, 83.1±2.4% and 58.1±4.6% with the MND4T, PGK4T and PRF0T LV. Following LCMV challenge, γ-IFN levels in MND4T, PRF0T and PGK4T mice were 1.7±0.3, 1.5±0.5 and 5.9±2.1 ng/ml, respectively, compared to 1.7±0.5 ng/ml in prf1-/- mice receiving WT HSC. Similarly, the degree of anemia in WT and MND4T mice was comparable 15 days after LCMV challenge: hemoglobins declined by 4.3±0.3, 3.7±0.3, 5.6±0.3 and 5.8±0.7 g/dL in WT, MND4T, PGK4T and PRF0T mice, respectively. Most importantly, 66.7% of the mice in the MND4T group survived the LCMV challenge, while only 10% and no mice survived in PGK4T and PRF0T groups, suggesting that perforin expression from PGK4T and PRF0T were insufficient to prevent fatal HLH in the majority of animals. Overall, we show that perforin deficient HLH requires a robust perforin expression in cytotoxic T and NK cells for adequate correction of the disease phenotype.

Interstitial lung disease with multiple microgranulomas in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease that is characterized by susceptibility to bacterial and fungal infections. CGD patients also suffer from immune regulatory disorders, such as CGD-associated bowel inflammation with granuloma, which could be caused by excessive inflammation without demonstrable infection. We investigated the clinical manifestation of interstitial lung disease (ILD) resulting from excessive inflammation in X-linked CGD patients. Pulmonary CT images and testing of serum KL-6 levels were performed to assess ILD in the patients. For this study, patients with pulmonary lesions due to demonstrable infections were excluded from among ILD patients. Among 33 CGD patients, four developed ILD; they had increased reticulo-nodular opacities on CT images and elevated serum KL-6 levels. Histopathological examinations revealed multiple homogeneous microgranulomas in the lesions of inflammatory cell infiltration. Mononuclear cells obtained from their pulmonary lesions produced higher amounts of inflammatory cytokines than the peripheral blood mononuclear cells of CGD patients, suggesting that the only infiltrating cells in the pulmonary lesions were activated and produced large amounts of inflammatory cytokines in ILD patients. Interestingly, an anti-inflammatory drug, such as a corticosteroid or thalidomide, but not anti-bacterial or anti-fungal drugs, improved CT image findings and reduced their KL-6 levels. CGD patients' daily exposures to inhaled antigens may induce excessive reactions with the production of inflammatory cytokines leading to the development of ILD with multiple microgranulomas, which could be due to an inadequate production of reactive oxygen species in CGD.