Marie Ouachée-Chardin

Munc13-4 Is Essential for Cytolytic Granules Fusion and Is Mutated in a Form of Familial Hemophagocytic Lymphohistiocytosis (FHL3)

Secretion of cytolytic granules content at the immunological synapse is a highly regulated process essential for lymphocyte cytotoxicity. This process requires the rapid transfer of perforin containing lytic granules to the target cell interface, followed by their docking and fusion with the plasma membrane. Defective cytotoxicity characterizes a genetically heterogeneous condition named familial hemophagocytic lymphohistiocytosis (FHL), which can be associated with perforin deficiency. The locus of a perforin (+) FHL subtype (FHL3), observed in 10 patients, was mapped to 17q25. This region contains hMunc13-4, a member of the Munc13 family of proteins involved in vesicle priming function. HMunc13-4 mutations were shown to cause FHL3. HMunc13-4 deficiency results in defective cytolytic granule exocytosis, despite polarization of the secretory granules and docking with the plasma membrane. Expressed tagged hMunc13-4 localizes with cytotoxic granules at the immunological synapse. HMunc13-4 is therefore essential for the priming step of cytolytic granules secretion preceding vesicle membrane fusion.

Autoantibodies against IL-17A, IL-17F, and IL-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I

Most patients with autoimmune polyendocrine syndrome type I (APS-I) display chronic mucocutaneous candidiasis (CMC). We hypothesized that this CMC might result from autoimmunity to interleukin (IL)-17 cytokines. We found high titers of autoantibodies (auto-Abs) against IL-17A, IL-17F, and/or IL-22 in the sera of all 33 patients tested, as detected by multiplex particle-based flow cytometry. The auto-Abs against IL-17A, IL-17F, and IL-22 were specific in the five patients tested, as shown by Western blotting. The auto-Abs against IL-17A were neutralizing in the only patient tested, as shown by bioassays of IL-17A activity. None of the 37 healthy controls and none of the 103 patients with other autoimmune disorders tested had such auto-Abs. None of the patients with APS-I had auto-Abs against cytokines previously shown to cause other well-defined clinical syndromes in other patients (IL-6, interferon [IFN]-γ, or granulocyte/macrophage colony-stimulating factor) or against other cytokines (IL-1β, IL-10, IL-12, IL-18, IL-21, IL-23, IL-26, IFN-β, tumor necrosis factor [α], or transforming growth factor β). These findings suggest that auto-Abs against IL-17A, IL-17F, and IL-22 may cause CMC in patients with APS-I.

Clinical similarities and differences of patients with X-linked lymphoproliferative syndrome type 1 (XLP-1/SAP-deficiency) versus type 2 (XLP-2/XIAP-deficiency)

X-linked lymphoproliferative syndromes (XLP) are primary immunodeficiencies characterized by a particular vulnerability toward Epstein-Barr virus infection, frequently resulting in hemophagocytic lymphohistiocytosis (HLH). XLP type 1 (XLP-1) is caused by mutations in the gene SH2D1A (also named SAP), whereas mutations in the gene XIAP underlie XLP type 2 (XLP-2). Here, a comparison of the clinical phenotypes associated with XLP-1 and XLP-2 was performed in cohorts of 33 and 30 patients, respectively. HLH (XLP-1, 55%; XLP-2, 76%) and hypogammaglobulinemia (XLP-1, 67%; XLP-2, 33%) occurred in both groups. Epstein-Barr virus infection in XLP-1 and XLP-2 was the common trigger of HLH (XLP-1, 92%; XLP-2, 83%). Survival rates and mean ages at the first HLH episode did not differ for both groups, but HLH was more severe with lethal outcome in XLP-1 (XLP-1, 61%; XLP-2, 23%). Although only XLP-1 patients developed lymphomas (30%), XLP-2 patients (17%) had chronic hemorrhagic colitis as documented by histopathology. Recurrent splenomegaly often associated with cytopenia and fever was preferentially observed in XLP-2 (XLP-1, 7%; XLP-2, 87%) and probably represents minimal forms of HLH as documented by histopathology. This first phenotypic comparison of XLP subtypes should help to improve the diagnosis and the care of patients with XLP conditions.

Immunotherapy of familial hemophagocytic lymphohistiocytosis with antithymocyte globulins: a single-center retrospective report of 38 patients.

OBJECTIVES. Familial hemophagocytic lymphohistiocytosis is a genetically determined condition that is characterized by unremitting CD8 T lymphocyte and macrophage activation and leads to death in the absence of therapy. On the basis of the immunologic pathophysiology of familial hemophagocytic lymphohistiocytosis, we propose a therapy with a combination of antithymocyte globulins with corticosteroids, cyclosporin A, and intrathecal injections of methotrexate. METHODS. We retrospectively analyzed the outcome of antithymocyte globulin–based therapy that was performed in 38 consecutive patients who had familial hemophagocytic lymphohistiocytosis and were treated in a single center between 1991 and 2005. Overall, they received 45 courses of antithymocyte globulin (5–10 mg/kg per day for 5 days). RESULTS. This regimen was associated with infections after 10 of 45 courses of antithymocyte globulin. There were 6 events after 11 antithymocyte globulin courses given as second-line therapy against 4 after 34 antithymocyte globulin courses in patients who were treated primarily with antithymocyte globulin. Antithymocyte globulin administration led to rapid and complete response of familial hemophagocytic lymphohistiocytosis in 73% of cases, partial response in 24%, and no response only once. When hematopoietic stem cell transplantation was performed early after complete or partial response induction, it led to a high rate of cure, in 16 of 19 cases. Overall survival was 21 of 38 with 4 toxic deaths. CONCLUSION. Antithymocyte globulin based immunotherapy of familial hemophagocytic lymphohistiocytosis is efficient and carries an acceptable toxicity when used as a first treatment of familial hemophagocytic lymphohistiocytosis.

Functional consequences of perforin gene mutations in 22 patients with familial haemophagocytic lymphohistiocytosis

Summary. Familial haemophagocytic lymphohistiocytosis (FHL), an inherited form of haemophagocytic lymphohistiocytosis (HLH) syndrome, is characterized by the overwhelming activation of T lymphocytes and macrophages invariably leading to death in the absence of treatment. FHL is a heterogeneous autosomal recessive disorder, with one known causative gene which codes for perforin, a cytotoxic effector protein. In this study, we have characterized the genotype and phenotype of 14 unrelated families with perforin deficiency. Four new missense mutations of the perforin gene were identified. In every case, perforin gene mutations led to undetectable intracellular perforin expression within cytotoxic cells, while some residual T‐cell cytotoxic activity could be associated with certain missense mutations. Clinical and biological analyses did not differentiate between patients with nonsense or missense mutations, although age at diagnosis, which tended to be similar within members of the same family, was delayed in patients from two families belonging to the second group. In one case, consequences of perforin deficiency, diagnosed at birth, could be assessed prior to onset of clinical manifestations. No evidence for T‐cell activation could be shown, suggesting that an exogenous event is required to trigger the disease manifestation. Control assessment of perforin expression and cytotoxic assays by lymphocytes from young children led to the conclusion that perforin content of natural killer cells could be a reliable diagnostic test at any age. Altogether, these data enabled a better characterization of perforin deficiency and its consequences, and defined reliable diagnostic tools.

High frequency of GATA2 mutations in patients with mild chronic neutropenia evolving to MonoMac syndrome, myelodysplasia, and acute myeloid leukemia

UNLABELLED Congenital neutropenia is a group of genetic disorders that involve chronic neutropenia and susceptibility to infections. These neutropenias may be isolated or associated with immunologic defects or extra-hematopoietic manifestations. Complications may occur as infectious diseases, but also less frequently as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Recently, the transcription factor GATA2 has been identified as a new predisposing gene for familial AML/MDS. In the present study, we describe the initial identification by exome sequencing of a GATA2 R396Q mutation in a family with a history of chronic mild neutropenia evolving to AML and/or MDS. The subsequent analysis of the French Severe Chronic Neutropenia Registry allowed the identification of 6 additional pedigrees and 10 patients with 6 different and not previously reportedGATA2 mutations (R204X, E224X, R330X, A372T, M388V, and a complete deletion of the GATA2 locus). The frequent evolution to MDS and AML in these patients reveals the importance of screening GATA2 in chronic neutropenia associated with monocytopenia because of the frequent hematopoietic transformation, variable clinical expression at onset, and the need for aggressive therapy in patients with poor clinical outcome. KEY POINTS Mutations of key transcription factor in myeloid malignancies.

Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype

Since their discovery in patients with autosomal dominant (AD) chronic mucocutaneous candidiasis (CMC) in 2011, heterozygous STAT1 gain-of-function (GOF) mutations have increasingly been identified worldwide. The clinical spectrum associated with them needed to be delineated. We enrolled 274 patients from 167 kindreds originating from 40 countries from 5 continents. Demographic data, clinical features, immunological parameters, treatment, and outcome were recorded. The median age of the 274 patients was 22 years (range, 1-71 years); 98% of them had CMC, with a median age at onset of 1 year (range, 0-24 years). Patients often displayed bacterial (74%) infections, mostly because of Staphylococcus aureus (36%), including the respiratory tract and the skin in 47% and 28% of patients, respectively, and viral (38%) infections, mostly because of Herpesviridae (83%) and affecting the skin in 32% of patients. Invasive fungal infections (10%), mostly caused by Candida spp. (29%), and mycobacterial disease (6%) caused by Mycobacterium tuberculosis, environmental mycobacteria, or Bacille Calmette-Guérin vaccines were less common. Many patients had autoimmune manifestations (37%), including hypothyroidism (22%), type 1 diabetes (4%), blood cytopenia (4%), and systemic lupus erythematosus (2%). Invasive infections (25%), cerebral aneurysms (6%), and cancers (6%) were the strongest predictors of poor outcome. CMC persisted in 39% of the 202 patients receiving prolonged antifungal treatment. Circulating interleukin-17A-producing T-cell count was low for most (82%) but not all of the patients tested. STAT1 GOF mutations underlie AD CMC, as well as an unexpectedly wide range of other clinical features, including not only a variety of infectious and autoimmune diseases, but also cerebral aneurysms and carcinomas that confer a poor prognosis.

Human leucocyte antigen‐identical haematopoietic stem cell transplantation in major histocompatiblity complex class II immunodeficiency: reduced survival correlates with an increased incidence of acute graft‐versus‐host disease and pre‐existing viral infections

Major histocompatibility complex class II deficiency, a rare autosomal recessive primary immunodeficiency, is caused by the defective expression of human leucocyte antigen (HLA) class II molecules due to mutated trans‐acting elements of any one of four regulatory genes (CIITA, RFXANK, RFX5, RFXAP). The impaired CD4 T‐cell differentiation and antigen presentation in the periphery results in a severe defect of cellular and humoral response consistent with severe recurrent infections, leading to a poor prognosis. Currently, allogeneic haematopoietic stem cell transplantation (HSCT) is the only curative approach, but the overall cure rate is lower than in other immunodeficiencies. We report a single centre experience of 17 HSCTs with 15 HLA‐identical donors between 1981 and 2004. Eight patients survived, while the occurrence of acute graft‐versus‐host disease (GVHD) was 50%. This study aimed to identify potential risk factors for GVHD and outcome within pre‐HSCT complications related to the immunodeficiency. Five of seven patients with pre‐existing viral infections developed acute GVHD ≥ grade II, of whom four died. Two of seven patients without detectable pre‐existing viral infection developed GVHD ≥ grade II, and one died. The difference was significant (P < 0·05). A plausible link with other factors potentially associated with the development of GVHD could not be found. We suggest that the reduced survival after HLA‐identical HSCT may be caused by the high incidence of pre‐existing viral infections and associated with the onset of severe acute GVHD.

Quantitative and Qualitative CD4 T Cell Immune Responses Related to Adenovirus DNAemia in Hematopoietic Stem Cell Transplantation

The nature of adenovirus (AdV)-specific T cells that could best predict the capacity of immunocompromised host to fight AdV is unclear. To this aim, 47 pediatric patients were enrolled for at least 3 months either at allogeneic bone marrow transplantation (BMT) (23 genoidentical, 18 unrelated of which 9 were 10/10 and 9 were 9/10 HLA-matched) or at unrelated cord blood transplantation (n = 6). Enumeration of AdV-specific CD4 T cells secreting cytokines (flow cytometry) and proliferative responses to AdV ((3)HT-incorporation) were compared to AdV-DNAemia. A total of 44/47 patients did not evidence AdV-DNAemia. Thirty-two of 44 (73%) developed CD4-mediated interferon-gamma (IFN-γ) responses to AdV (median 0.36 CD4/μL of blood) since the first month post-HSCT (n = 11: 8 genoidentical and 3 unrelated) or the third month (n = 21 additional patients). At 3 months, both incidence and level intensities of AdV-specific CD4 appeared similar in genoidentical and unrelated BMT (70% and 80%; 0.36 and 0.21 CD4/μL, respectively) and not statistically different from age-matched controls (76%; 1.35 CD4/μL), whereas cord blood transplanted patients exhibited similar incidence but higher level intensities (67%; 1.49 CD4/μL). Polyfunctional (IL2 + IFN-γ) and proliferative responses appeared later, after the third month. Three of 4 9/10 HLA-matched unrelated HSCT that did not develop immunity to AdV presented chemotherapy-resistant AdV-DNAemia at 3 to 5 months post-hematopoietic stem cell transplantation (HSCT). Two were successfully treated with AdV-specific CTL infusion. Monitoring, since month 1 post-HSCT, of IFN-γ-secreting AdV-specific CD4 appears suitable for early detection of at-risk patients especially in 9/10 HLA-matched unrelated HSCT and preferable to monitoring of more delayed IL2- and proliferative responses.

Contribution of HLA-A/B/C/DRB1/DQB1 Common Haplotypes to Donor Search Outcome in Unrelated Hematopoietic Stem Cell Transplantation

In unrelated hematopoietic stem cell transplantation (HSCT), the prediction of donor search outcome at the time of search initiation is of great value for the physicians to delineate the strategy of patient care. The probability of finding an unrelated donor is high for patients who carry at least 1 of the 10 most common HLA haplotypes in Caucasians. As only 10% to 20% patients respond to this criterion, here we aimed at finding additional common haplotypes to improve the prediction of a successful search. HLA broad HLA-A/B/DRB1 haplotypes that were observed with frequencies ≥0.19% in patient families of European origin and that split into ≤2 predominant 4-digit HLA-A/B/C/DRB1/DQB1 haplotypes were considered as common. Carriage of at least 1 of those in 168 patients of various geographic areas with no family donor was confronted to the chance of finding ≥9/10 HLA-matched unrelated donors. Fifty common 4-digit haplotypes were identified. A higher (P < 5 × 10(-6)) chance of finding a suitable donor was found for 55 of 170 (32%) recipients that carried at least 1 of these common haplotypes. Up to now, estimates classified patients into ≥3 groups of probability with ≥1 intermediate group of poor utility for the clinicians. Considering carriage of these common haplotypes together with the frequencies of alleles and of B/C and DRB1/DQB1 associations, which are carried by patient HLA haplotypes, we could classify the patients into 2 groups of probability with a 98% and 26% chance of finding a donor, respectively. Prediction of search outcome could be improved by including the 50 most common HLA haplotypes in the current approaches.