Sara Ciullini Mannurita

Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation–polyendocrinopathy–enteropathy–X-linked–like syndrome

BACKGROUND Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

Immune deficiencies, infection, and systemic immune disordersDominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation–polyendocrinopathy–enteropathy–X-linked–like syndrome

BACKGROUND Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

The spectrum of autoantibodies in IPEX syndrome is broad and includes anti-mitochondrial autoantibodies

IPEX syndrome is a congenital disorder of immune regulation caused by mutations in the FOXP3 gene, which is required for the suppressive function of naturally arising CD4 + CD25 + regulatory T cells. In this case series we evaluated serum samples from 12 patients with IPEX syndrome for the presence of common autoantibodies associated with a broad range of autoimmune disorders. We note that 75% of patients (9/12) had 1 or more autoantibodies, an incidence far above the cumulative rate observed in the general population. The range of autoantibodies differed between patients and there was no predominant autoantibody or pattern of autoantibodies present in this cohort. Surprisingly, one patient had high-titer anti-mitochondrial antibodies (AMA) typically associated with primary biliary cirrhosis (PBC) although the patient had no signs of cholestasis. PBC is a well-characterized autoimmune disease that occurs primarily in women and includes the serological hallmarks of serum AMA and elevated IgM which were both present in this patient. PBC is virtually absent in children with the exception of one reported child with interleukin 2 receptor α (CD25) deficiency which is associated with an IPEX-like regulatory T cell dysfunction. Based on the present data and the available literature we suggest a direct role for CD4 + CD25 + regulatory T cells in restraining B cell autoantibody production and that defects in regulatory T cells may be crucial to the development of PBC.

Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity.

Cell-surface CD25 expression is critical for maintaining immune function and homeostasis. As in few reported cases, CD25 deficiency manifests with severe autoimmune enteritis and viral infections. To dissect the underlying immunological mechanisms driving these symptoms, we analyzed the regulatory and effector T cell functions in a CD25 deficient patient harboring a novel IL2RA mutation. Pronounced lymphoproliferation, mainly of the CD8+ T cells, was detected together with an increase in T cell activation markers and elevated serum cytokines. However, Ag-specific responses were impaired in vivo and in vitro. Activated CD8+STAT5+ T cells with lytic potential infiltrated the skin, even though FOXP3+ Tregs were present and maintained a higher capacity to respond to IL-2 compared to other T-cell subsets. Thus, the complex pathogenesis of CD25 deficiency provides invaluable insight into the role of IL2/IL-2RA-dependent regulation in autoimmunity and inflammatory diseases.

Single centre experience of haematopoietic SCT for patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

Single centre experience of haematopoietic SCT for patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

CACP syndrome: identification of five novel mutations and of the first case of UPD in the largest European cohort

Camptodactyly-Arthropathy-Coxa vara-Pericarditis (CACP) syndrome is a rare autosomal recessive disorder caused by mutations in PRG4 gene that encodes for proteoglycan 4, a mucin-like glycoprotein that is the major lubricant for joints and tendon surfaces. The molecular studies reported so far have described the identification of 15 mutations associated with this syndrome and the majority of them were found in families of Arabian origin. Here we report the molecular investigation of the largest European cohort that comprises 13 patients, and allowed the identification of 5 novel mutations and of the first case of CACP syndrome resulting from uniparental disomy of chromosome 1.

Novel molecular defects associated with very early-onset inflammatory bowel

Purpose of review Immune dysregulation disorders present with common clinical features of multiorgan autoimmunity. Gastrointestinal involvement is the hallmark of an impaired immune homeostasis. This review will give an overview on the novel phenotypes, highlighting the major points that will help to enable early diagnosis and treatment. Recent findings The rapid progress on DNA sequencing technologies have led to the identification of monogenic defects that adversely impact the control of immune homeostasis. Lymphocytes may be present but dysfunctional, allowing for the development of excessive autoreactivity and resultant autoimmune disease. Regulatory T cells (Tregs) play an essential role in enforcing immune tolerance. Here we illustrate disorders caused by impairment of mechanisms ensuring Tregs function (Tregs related) in which autoimmunity is a hallmark of the clinical disease presentation and other disorders, affecting molecules more broadly involved in immune responses and indirectly causing immune dysregulation (Tregs unrelated). Clinical presentation is sometime mischievous and often symptoms are analogous in different diseases and can mislead diagnosis. Summary The increasing comprehension of immunological concepts behind immune dysregulation diseases will allow better and in some cases possibly even targeted treatment. A genetic diagnosis therefore becomes important information in this group of patients, especially as some patients might require hematopoietic stem cell transplantation.

Inherited Multiple Autoimmunity: Molecular and Clinical Characterization of IPEX and IPEX-Like Syndromes

describes the study design to facilitate the enrollment of patients treated in clinical settings. Study: All females who become pregnant during IgHy treatment (treated while pregnant or in the 30 days prior to conception) will be encouraged to participate. As soon as the patient becomes aware of the pregnancy, treatment with IgHy will be stopped. Subjects will receive an alternative gammaglobulin treatment and all other medical care will be performed as is standard. The overall duration of the study is approximately 6 years and there is no minimum sample size pre-specified. Data from medical records on the routine assessments during the pregnancy will be collected. In those subjects consenting to blood drawing, samples will be taken to assess antibodies against rHuPH20 every 3 months. Data will be assessed on the development of the fetus in utero, and on the growth and development of the infant for 2 years after delivery. Descriptive methods will be used to analyze and report data from this IgHy pregnancy registry. ESID-0098 Non-Interventional Post-Authorization Safety Study (PASS) on the Long-Term Safety of HYQVIA® (IGHY) K. Fielhauer, N. Nikolov, C. Hermann, A. Nagy, R.I. Schiff Bioscience, Baxter Healthcare, Vienna, Austria Bioscience, Baxter Healthcare SA, Zurich, Switzerland Bioscience, Baxter Healthcare, Westlake Village, USA Introduction: IgHy (recombinant human hyaluronidase [rHuPH20]-facilitated subcutaneous [SC] infusion of immunoglobulin [IG]) is a novel treatment that has been approved as a replacement therapy in adults (≥18 years) with primary immunodeficiency (PID) syndromes, myeloma, and chronic lymphocytic leukemia. This is a non-interventional, prospective, uncontrolled, multi-center, open label, post-authorization safety study to be conducted in the EEA to acquire additional data (including the assessment of anti-rHuPH20 antibodies) on the long-term safety of IgHy. This abstract describes the study design to facilitate the evaluation of patients treated in a

Clinical analysis of the complex world of inherited multiple autoimmunity

Continued B) In homozygous IGHG diplotypes only one of the two alternative allotypes is expressed, which result in restricted qualities of IgG molecules and B cells. IGHG gene frequencies and the allotypic IgG subclass levels, the activity of the genes, are available for children and adults of healthy Caucasians (Fig. 4). IGHG genes have impact on disease and immunotherapy. The function of IgG is related to the constant part of the heavy γ chains the Fcγ part of the molecule in parallel with the variable adaptive antibody binding site. Different effects of polysaccharide and protein vaccines, with different amounts of specific antibodies were recorded for different IGHG genes. They are also associated to different severity of bacterial and viral infections and can be used as predisposing, prognostic and sometimes carrier markers. The IgG molecules of IGHG genes are differently affected by viruses acting as FcγRs modifying the disease. Suscepetibility/resistence of various infections both bacterial and viral, allergens and tumour antigens are influenced by IGHG genes. The IGHG genes are associated with different phenotypes of allergic, autoimmune, infectious, immunodeficiency and malignt disorders. In treatments with IVIG, the levels of foreign allelic IgG subclasses are recognized and can be followed. This is the ultimate evidence of that the allelic IgG subclasses are unique. IGHG genes and allotypic IgG subclasses are predisposing or prognostic genetic markers in various infections, immunodeficiencies, different phenotypes of allergic disease, autoimmune disorders and malignant diseases. Fig.1Fig. 2Fig.3Fig. 4 29 IMMUNOPHENOTYPING OF B LYMPHOCYTES IN PATIENTS WITH COMMON VARIABLE IMMUNODEFICIENCY (CVID) F. Mazzi, G. Patuzzo, A. Vella, R. Ortolani, A. Barbieri, A. Puccetti, E. Tinazzi, G. Marchi, O.M. Codella, R. Beri, C. Lunardi Department of Medicine, Unit of Autoimmune Diseases, University of Verona, Department of Pathology and Diagnosis, Section of Immunology, University of Verona, Verona, Immunopathology, Institute G. Gaslini and University of Genova, Genova, Italy Introduction: CVID is a primary immunodeficiency characterized by failure of B lymphocytes to differentiate in plasma cells, with deficient immunoglobulin secretion. The identified genetic defects account only for a minority of cases. Objective: The importance of B cells immunophenotyping in the classification of CVID is well known. This procedure can identify some alterations on cell surface molecule expression that could explain the immunological disorder at the basis of CVID. Moreover, some immunophenotipical aspects can correlate with clinical features, severity and prognosis of the disease. Aim: We studied a cohort of 16 patients affected by CVID, to identify alterations of B cells and to find correlations with clinical features. Methods: We studied circulating B cells in patients and controls by flow-cytometry, using a specific panel of antibodies for B cells. Conclusion: We compared the population of “switched memory” IgD-CD27+B lymphocytes, used in the Friburg classification, with the population of IgM-IgDCD23-CD27+B cells, we have analysed. IgM-IgDCD23-CD27+B cells were reduced in patients compared to healthy controls and in patients they were lower than IgD-CD27+B cells. The reduction of these lymphocytes was correlated more tightly than IgDCD27+B cells to lymphoadenopathy, splenomegaly, J Clin Immunol (2012) 32 (Suppl 1):S1–S379

GUT IMMUNE RECONSTITUTION IN IPEX SYNDROME AFTER HSCT

Adoptive cell transfer (ACT) of transgenic T lymphocytes expressing tumor specific T cell receptor (TCR) is a promising therapy. However, little is known about the fate of engineered T cells and the dynamics of host immunity. We comprehensively monitored the transgenic and endogenous immune response in 8 melanoma patients over 8–10 time points in an ACT therapy of transgenic F5 MART-1 TCR T cells, using highly multiplexed clinical immune diagnostics methods (Ma, C. et al. Nature Medicine 17, 738–743 (2011)). We found that the TCR engineered T cells sustained a 2–3 week duration anti-tumor response. After day 30 a second wave endogenous multi-clonal melanoma specific T cell response emerged. The melanoma-specific T cells within the ACTwent through a process of in vivo functional changes that correlated with clinical antitumor activity. The appearance and the persistence of the second wave immune response provided indications of long term therapeutic efficacy.