Carsten Speckmann

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34+ cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification–mediated PCR (LAM-PCR) to identify clonal vector proviral integrants. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID).

Antiviral and Regulatory T Cell Immunity in a Patient with Stromal Interaction Molecule 1 Deficiency

Stromal interaction molecule 1 (STIM1) deficiency is a rare genetic disorder of store-operated calcium entry, associated with a complex syndrome including immunodeficiency and immune dysregulation. The link from the molecular defect to these clinical manifestations is incompletely understood. We report two patients with a homozygous R429C point mutation in STIM1 completely abolishing store-operated calcium entry in T cells. Immunological analysis of one patient revealed that despite the expected defect of T cell proliferation and cytokine production in vitro, significant antiviral T cell populations were generated in vivo. These T cells proliferated in response to viral Ags and showed normal antiviral cytotoxicity. However, antiviral immunity was insufficient to prevent chronic CMV and EBV infections with a possible contribution of impaired NK cell function and a lack of NKT cells. Furthermore, autoimmune cytopenia, eczema, and intermittent diarrhea suggested impaired immune regulation. FOXP3-positive regulatory T (Treg) cells were present but showed an abnormal phenotype. The suppressive function of STIM1-deficient Treg cells in vitro, however, was normal. Given these partial defects in cytotoxic and Treg cell function, impairment of other immune cell populations probably contributes more to the pathogenesis of immunodeficiency and autoimmunity in STIM1 deficiency than previously appreciated.

Outcome of hematopoietic stem cell transplantation for adenosine deaminase-deficient severe combined immunodeficiency

Deficiency of the purine salvage enzyme adenosine deaminase leads to SCID (ADA-SCID). Hematopoietic cell transplantation (HCT) can lead to a permanent cure of SCID; however, little data are available on outcome of HCT for ADA-SCID in particular. In this multicenter retrospective study, we analyzed outcome of HCT in 106 patients with ADA-SCID who received a total of 119 transplants. HCT from matched sibling and family donors (MSDs, MFDs) had significantly better overall survival (86% and 81%) in comparison with HCT from matched unrelated (66%; P < .05) and haploidentical donors (43%; P < .001). Superior overall survival was also seen in patients who received unconditioned transplants in comparison with myeloablative procedures (81% vs 54%; P < .003), although in unconditioned haploidentical donor HCT, nonengraftment was a major problem. Long-term immune recovery showed that regardless of transplant type, overall T-cell numbers were similar, although a faster rate of T-cell recovery was observed after MSD/MFD HCT. Humoral immunity and donor B-cell engraftment was achieved in nearly all evaluable surviving patients and was seen even after unconditioned HCT. These data detail for the first time the outcomes of HCT for ADA-SCID and show that, if patients survive HCT, long-term cellular and humoral immune recovery is achieved.

X-linked inhibitor of apoptosis (XIAP) deficiency: the spectrum of presenting manifestations beyond hemophagocytic lymphohistiocytosis.

X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.

Disease-causing mutations in the XIAP BIR2 domain impair NOD2-dependent immune signalling.

X‐linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro‐inflammatory signalling downstream of the nucleotide‐binding oligomerization domain containing (NOD)‐1 and ‐2 pattern recognition receptors. Mutations in XIAP cause X‐linked lymphoproliferative syndrome type‐2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2‐BIR2 mutations severely impair NOD1/2‐dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP‐deficient cell lines. XLP2‐BIR2 mutations abolish the XIAP‐RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2‐complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM‐binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2‐RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling.

Atypical familial hemophagocytic lymphohistiocytosis due to mutations in UNC13D and STXBP2 overlaps with primary immunodeficiency diseases

Background Familial hemophagocytic lymphohistiocytosis is a genetic disorder of lymphocyte cytotoxicity that usually presents in the first two years of life and has a poor prognosis unless treated by hematopoietic stem cell transplantation. Atypical courses with later onset and prolonged survival have been described, but no detailed analysis of immunological parameters associated with typical versus atypical forms of familial hemophagocytic lymphohistiocytosis has been performed. Design and Methods We analyzed disease manifestations, NK-cell and T-cell cytotoxicity and degranulation, markers of T-cell activation and B-cell differentiation as well as Natural Killer T cells in 8 patients with atypical familial hemophagocytic lymphohistiocytosis due to mutations in UNC13D and STXBP2. Results All but one patient with atypical familial hemophagocytic lymphohistiocytosis carried at least one splice-site mutation in UNC13D or STXBP2. In most patients episodes of hemophagocytic lymphohistiocytosis were preceded or followed by clinical features typically associated with immunodeficiency, such as chronic active Epstein Barr virus infection, increased susceptibility to bacterial infections, granulomatous lung or liver disease, encephalitis or lymphoma. Five of 8 patients had hypogammaglobulinemia and reduced memory B cells. Most patients had a predominance of activated CD8+ T cells and low numbers of Natural Killer T cells. When compared to patients with typical familial hemophagocytic lymphohistiocytosis, NK-cell cytotoxicity and NK-cell and CTL degranulation were impaired to a similar extent. However, in patients with an atypical course NK-cell degranulation could be partially reconstituted by interleukin-2 and cytotoxic T-cell cytotoxicity in vitro was normal. Conclusions Clinical and immunological features of atypical familial hemophagocytic lymphohistiocytosis show an important overlap to primary immunodeficiency diseases (particularly common variable immunodeficiency and X-linked lymphoproliferative syndrome) and must, therefore, be considered in a variety of clinical presentations. We show that degranulation assays are helpful screening tests for the identification of such patients.

Clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: A large patient cohort study

Background: Activated phosphoinositide 3‐kinase &dgr; syndrome (APDS) is a recently described combined immunodeficiency resulting from gain‐of‐function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3‐kinase &dgr; (PI3K&dgr;). Objective: We sought to review the clinical, immunologic, histopathologic, and radiologic features of APDS in a large genetically defined international cohort. Methods: We applied a clinical questionnaire and performed review of medical notes, radiology, histopathology, and laboratory investigations of 53 patients with APDS. Results: Recurrent sinopulmonary infections (98%) and nonneoplastic lymphoproliferation (75%) were common, often from childhood. Other significant complications included herpesvirus infections (49%), autoinflammatory disease (34%), and lymphoma (13%). Unexpectedly, neurodevelopmental delay occurred in 19% of the cohort, suggesting a role for PI3K&dgr; in the central nervous system; consistent with this, PI3K&dgr; is broadly expressed in the developing murine central nervous system. Thoracic imaging revealed high rates of mosaic attenuation (90%) and bronchiectasis (60%). Increased IgM levels (78%), IgG deficiency (43%), and CD4 lymphopenia (84%) were significant immunologic features. No immunologic marker reliably predicted clinical severity, which ranged from asymptomatic to death in early childhood. The majority of patients received immunoglobulin replacement and antibiotic prophylaxis, and 5 patients underwent hematopoietic stem cell transplantation. Five patients died from complications of APDS. Conclusion: APDS is a combined immunodeficiency with multiple clinical manifestations, many with incomplete penetrance and others with variable expressivity. The severity of complications in some patients supports consideration of hematopoietic stem cell transplantation for severe childhood disease. Clinical trials of selective PI3K&dgr; inhibitors offer new prospects for APDS treatment.

Reduced memory B cells in patients with hyper IgE syndrome

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection.

The syndrome of hemophagocytic lymphohistiocytosis in primary immunodeficiencies: implications for differential diagnosis and pathogenesis

Hemophagocytic lymphohistiocytosis is a hyperinflammatory syndrome defined by clinical and laboratory criteria. Current criteria were created to identify patients with familial hemophagocytic lmyphohistiocytosis in immediate need of immunosuppressive therapy. However, these criteria also identify patients with infection-associated hemophagocytic inflammatory states lacking genetic defects typically predisposing to hemophagocytic lymphohistiocytosis. These patients include those with primary immunodeficiencies, in whom the pathogenesis of the inflammatory syndrome may be distinctive and aggressive immunosuppression is contraindicated. To better characterize hemophagocytic inflammation associated with immunodeficiencies, we combined an international survey with a literature search and identified 63 patients with primary immunodeficiencies other than cytotoxicity defects or X-linked lymphoproliferative disorders, presenting with conditions fulfilling current criteria for hemophagocytic lymphohistiocytosis. Twelve patients had severe combined immunodeficiency with <100/μL T cells, 18 had partial T-cell deficiencies; episodes of hemophagocytic lymphohistiocytosis were mostly associated with viral infections. Twenty-two patients had chronic granulomatous disease with hemophagocytic episodes mainly associated with bacterial infections. Compared to patients with cytotoxicity defects, patients with T-cell deficiencies had lower levels of soluble CD25 and higher ferritin concentrations. Other criteria for hemophagocytoc lymphohistiocytosis were not discriminative. Thus: (i) a hemophagocytic inflammatory syndrome fulfilling criteria for hemophagocytic lymphohistiocytosis can be the initial manifestation of primary immunodeficiencies; (ii) this syndrome can develop despite severe deficiency of T and NK cells, implying that the pathophysiology is distinct and not appropriately described as “lympho”-histiocytosis in these patients; and (iii) current criteria for hemophagocytoc lymphohistiocytosis are insufficient to differentiate hemophagocytic inflammatory syndromes with different pathogeneses. This is important because of implications for therapy, in particular for protocols targeting T cells.