Klaus Warnatz

Homozygous loss of ICOS is associated with adult-onset common variable immunodeficiency

No genetic defect is known to cause common variable immunodeficiency (CVID), a heterogeneous human disorder leading to adult-onset panhypogammaglobulinemia. In a search for CVID candidate proteins, we found four of 32 patients to lack ICOS, the “inducible costimulator” on activated T cells, due to an inherited homozygous deletion in the ICOS gene. T cells from these individuals were normal with regard to subset distribution, activation, cytokine production and proliferation. In contrast, naive, switched and memory B cells were reduced. The phenotype of human ICOS deficiency, which differs in key aspects from that of the ICOS−/− mouse, suggests a critical involvement of ICOS in T cell help for late B cell differentiation, class-switching and memory B cell generation.

A homozygous CARD9 mutation in a family with susceptibility to fungal infections.

BACKGROUND Chronic mucocutaneous candidiasis may be manifested as a primary immunodeficiency characterized by persistent or recurrent infections of the mucosa or the skin with candida species. Most cases are sporadic, but both autosomal dominant inheritance and autosomal recessive inheritance have been described. METHODS We performed genetic studies in 36 members of a large, consanguineous five-generation family, in which 4 members had recurrent fungal infections and an additional 3 members died during adolescence, 2 after invasive infection of the brain with candida species. All 36 family members were enrolled in the study, and 22 had blood samples taken for DNA analysis. Homozygosity mapping was used to locate the mutated gene. In the 4 affected family members (patients) and the 18 unaffected members we sequenced CARD9, the gene encoding the caspase recruitment domain-containing protein 9, carried out T-cell phenotyping, and performed functional studies, with the use of either leukocytes from the patients or a reconstituted murine model of the genetic defect. RESULTS We found linkage (lod score, 3.6) to a genomic interval on chromosome 9q, including CARD9. All four patients had a homozygous point mutation in CARD9, resulting in a premature termination codon (Q295X). Healthy family members had wild-type expression of the CARD9 protein; the four patients lacked wild-type expression, which was associated with low numbers of Th17 cells (helper T cells producing interleukin-17). Functional studies based on genetic reconstitution of myeloid cells from Card9(-/-) mice showed that the Q295X mutation impairs innate signaling from the antifungal pattern-recognition receptor dectin-1. CONCLUSIONS An autosomal recessive form of susceptibility to chronic mucocutaneous candidiasis is associated with homozygous mutations in CARD9.

ICOS Deficiency Is Associated with a Severe Reduction of CXCR5+CD4 Germinal Center Th Cells

ICOS is expressed on activated T cells and particularly on CXCR5+ follicular Th cells in germinal centers (GC). Its deletion leads to a profound deficiency in memory B cell formation and switched Ab response in humans. Here, we show that in ICOS-deficient patients the generation of GCs is severely disturbed, and the numbers of circulating CXCR5+CD45RO+ memory CD4 T cells are significantly reduced, indicating an essential role of ICOS in the differentiation of CXCR5+CD4 T cells. The GC-specific CD57+CXCR5+ subpopulation is virtually absent. In ICOS−/− mice, the decrease of circulating CXCR5+CD4 T cells reflects the reduction of CXCR5+ follicular Th cells in lymph nodes and spleen. Therefore, in concurrence with the absence of CXCR5+ T cells in the blood of CD40L-deficient patients, these data support the hypothesis that circulating CD57+CXCR5+ T cells are GC derived and thus may serve as a surrogate marker for the presence of functional GCs in humans.

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.

B-cell activating factor receptor deficiency is associated with an adult-onset antibody deficiency syndrome in humans

B-cell survival depends on signals induced by B-cell activating factor (BAFF) binding to its receptor (BAFF-R). In mice, mutations in BAFF or BAFF-R cause B-cell lymphopenia and antibody deficiency. Analyzing BAFF-R expression and BAFF-binding to B cells in common variable immunodeficiency (CVID) patients, we identified two siblings carrying a homozygous deletion in the BAFF-R gene. Removing most of the BAFF-R transmembrane part, the deletion precludes BAFF-R expression. Without BAFF-R, B-cell development is arrested at the stage of transitional B cells and the numbers of all subsequent B-cell stages are severely reduced. Both siblings have lower IgG and IgM serum levels but, unlike most CVID patients, normal IgA concentrations. They also did not mount a T-independent immune response against pneumococcal cell wall polysaccharides but only one BAFF-R-deficient sibling developed recurrent infections. Therefore, deletion of the BAFF-R gene in humans causes a characteristic immunological phenotype but it does not necessarily lead to a clinically manifest immunodeficiency.

Autologous Hematopoietic Stem Cell Transplantation vs Intravenous Pulse Cyclophosphamide in Diffuse Cutaneous Systemic Sclerosis: A Randomized Clinical Trial

IMPORTANCE High-dose immunosuppressive therapy and autologous hematopoietic stem cell transplantation (HSCT) have shown efficacy in systemic sclerosis in phase 1 and small phase 2 trials. OBJECTIVE To compare efficacy and safety of HSCT vs 12 successive monthly intravenous pulses of cyclophosphamide. DESIGN, SETTING, AND PARTICIPANTS The Autologous Stem Cell Transplantation International Scleroderma (ASTIS) trial, a phase 3, multicenter, randomized (1:1), open-label, parallel-group, clinical trial conducted in 10 countries at 29 centers with access to a European Group for Blood and Marrow Transplantation-registered transplant facility. From March 2001 to October 2009, 156 patients with early diffuse cutaneous systemic sclerosis were recruited and followed up until October 31, 2013. INTERVENTIONS HSCT vs intravenous pulse cyclophosphamide. MAIN OUTCOMES AND MEASURES The primary end point was event-free survival, defined as time from randomization until the occurrence of death or persistent major organ failure. RESULTS A total of 156 patients were randomly assigned to receive HSCT (n = 79) or cyclophosphamide (n = 77). During a median follow-up of 5.8 years, 53 events occurred: 22 in the HSCT group (19 deaths and 3 irreversible organ failures) and 31 in the control group (23 deaths and 8 irreversible organ failures). During the first year, there were more events in the HSCT group (13 events [16.5%], including 8 treatment-related deaths) than in the control group (8 events [10.4%], with no treatment-related deaths). At 2 years, 14 events (17.7%) had occurred cumulatively in the HSCT group vs 14 events (18.2%) in the control group; at 4 years, 15 events (19%) had occurred cumulatively in the HSCT group vs 20 events (26%) in the control group. Time-varying hazard ratios (modeled with treatment × time interaction) for event-free survival were 0.35 (95% CI, 0.16-0.74) at 2 years and 0.34 (95% CI, 0.16-0.74) at 4 years. CONCLUSIONS AND RELEVANCE Among patients with early diffuse cutaneous systemic sclerosis, HSCT was associated with increased treatment-related mortality in the first year after treatment. However, HCST conferred a significant long-term event-free survival benefit. TRIAL REGISTRATION isrctn.org Identifier: ISRCTN54371254.

Clinical picture and treatment of 2212 patients with common variable immunodeficiency

BACKGROUND Common variable immunodeficiency (CVID) is an antibody deficiency with an equal sex distribution and a high variability in clinical presentation. The main features include respiratory tract infections and their associated complications, enteropathy, autoimmunity, and lymphoproliferative disorders. OBJECTIVE This study analyzes the clinical presentation, association between clinical features, and differences and effects of immunoglobulin treatment in Europe. METHODS Data on 2212 patients with CVID from 28 medical centers contributing to the European Society for Immunodeficiencies Database were analyzed retrospectively. RESULTS Early disease onset (<10 years) was very frequent in our cohort (33.7%), especially in male subjects (39.8%). Male subjects with early-onset CVID were more prone to pneumonia and less prone to other complications suggesting a distinct disease entity. The diagnostic delay of CVID ranges between 4 and 5 years in many countries and is particularly high in subjects with early-onset CVID. Enteropathy, autoimmunity, granulomas, and splenomegaly formed a set of interrelated features, whereas bronchiectasis was not associated with any other clinical feature. Patient survival in this cohort was associated with age at onset and age at diagnosis only. There were different treatment strategies in Europe, with considerable differences in immunoglobulin dosing, ranging from 130 up to 750 mg/kg/mo. Patients with very low trough levels of less than 4 g/L had poor clinical outcomes, whereas higher trough levels were associated with a reduced frequency of serious bacterial infections. CONCLUSION Patients with CVID are being managed differently throughout Europe, affecting various outcome measures. Clinically, CVID is a truly variable antibody deficiency syndrome.

Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells.

The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21low B cells are polyclonal, unmutated IgM+IgD+ B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21low B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21low B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21low B cells represent a human innate-like B cell population.

15-Deoxyspergualin in Patients with Refractory ANCA-Associated Systemic Vasculitis: A Six-Month Open-Label Trial to Evaluate Safety and Efficacy

The combination of cyclophosphamide (CYC) and oral corticosteroids is effective in the majority of patients with antineutrophil cytoplasmic antibody-associated vasculitis (AASV), but it carries substantial risk of drug-related morbidity and mortality. New regimens are desired, especially in refractory cases. The immunosuppressant 15-deoxyspergualin (DSG) is effective in experimental autoimmune disease and transplantation as well as in acute kidney transplant rejection in humans. To assess the efficacy and safety of DSG, an open label multicenter trial was conducted in patients with AASV who were either unresponsive or had contraindications for standard immunosuppressants. Included were 19 cases of Wegener granulomatosis and one case of microscopic polyangiitis. Nine of them had received CYC shortly before study entry without apparent therapeutic success. DSG (0.5 mg/kg per d) was given for 2 to 3 wk until the WBC count dropped to 3000/ micro l followed by a rest until at least a WBC of 4000/ micro l was reached again. This was repeated up to six cycles. During the study, no other immunosuppressants besides steroids were allowed. Disease improvement during treatment with DSG was achieved in 70% of cases (six cases of complete remission; eight cases of partial remission). Leucopenia occurred in each patient in a regular pattern during the cycles and was transient without exception. No mortality or septicemia was observed. Mild to moderate infections mainly in the respiratory tract were observed but resolved under adequate treatment without sequel. It is concluded that treatment with DSG is successful in patients with refractory Wegener granulomatosis under careful monitoring of WBC count.

Genetic CD21 deficiency is associated with hypogammaglobulinemia

BACKGROUND Complement receptor 2 (CR2/CD21) is part of the B-cell coreceptor and expressed by mature B cells and follicular dendritic cells. CD21 is a receptor for C3d-opsonized immune complexes and enhances antigen-specific B-cell responses. OBJECTIVE Genetic inactivation of the murine CR2 locus results in impaired humoral immune responses. Here we report the first case of a genetic CD21 deficiency in human subjects. METHODS CD21 protein expression was analyzed by means of flow cytometry and Western blotting. CD21 transcripts were quantified by using real-time PCR. The CD21 gene was sequenced. Wild-type and mutant CD21 cDNA expression was studied after transfection of 293T cells. Binding of EBV-gp350 or C3d-containing immune complexes and induction of calcium flux in CD21-deficient B cells were analyzed by means of flow cytometry. Antibody responses to protein and polysaccharide vaccines were measured. RESULTS A 28-year-old man presented with recurrent infections, reduced class-switched memory B cells, and hypogammaglobulinemia. CD21 receptor expression was undetectable. Binding of C3d-containing immune complexes and EBV-gp350 to B cells was severely reduced. Sequence analysis revealed a compound heterozygous deleterious mutation in the CD21 gene. Functional studies with anti-immunoglobulin- and C3d-containing immune complexes showed a complete loss of costimulatory activity of C3d in enhancing suboptimal B-cell receptor stimulation. Vaccination responses to protein antigens were normal, but the response to pneumococcal polysaccharide vaccination was moderately impaired. CONCLUSIONS Genetic CD21 deficiency adds to the molecular defects observed in human subjects with hypogammaglobulinemia.