A patient-based murine model recapitulates human STAT3 gain-of-function syndrome.

STAT3 gain-of-function (GOF) variants results in a heterogeneous clinical syndrome characterized by early onset immunodeficiency, multi-organ autoimmunity, and lymphoproliferation. While 191 documented cases with STAT3 GOF variants have been reported, the impact of individual variants on immune regulation and the broad clinical spectrum remains unclear. We developed a Stat3p.L387R mouse model, mirroring a variant identified in a family exhibiting common STAT3 GOF symptoms, and rare phenotypes including pulmonary hypertension and retinal vasculitis. In vitro experiments revealed increased STAT3 phosphorylation, nuclear migration, and DNA binding of the variant. Our Stat3p.L387R model displayed similar traits from previous Stat3GOF strains, such as splenomegaly and lymphadenopathy. Notably, Stat3p.L387R/+ mice exhibited heightened embryonic lethality compared to prior Stat3GOF/+ models and ocular abnormalities were observed. This research underscores the variant-specific pathology in Stat3p.L387R/+ mice, highlighting the ability to recapitulate human STAT3 GOF syndrome in patient-specific transgenic murine models. Additionally, such models could facilitate tailored treatment development.

The Association of Serum Immunoglobulins with Risk of Cardiovascular Disease and Mortality: the Rotterdam Study.

PURPOSE: Inflammation is implicated in cardiovascular disease (CVD), but the association of total serum immunoglobulin (Ig) A, G, and M with CVD across the whole spectrum of atherosclerosis in community-dwelling elderly is unknown. METHODS: This study was embedded in the Rotterdam Study, an ongoing population-based cohort study. We performed Cox regression for the associations of Igs with incident atherosclerotic CVD (ACVD; composite of myocardial infarction, revascularization, and stroke), cardiovascular mortality, and all-cause mortality, and multinomial logistic regression for the association between Igs and coronary artery calcification (CAC) scores. We adjusted for age, sex, lifestyle, and cardiovascular risk factors and presented results per standard deviation increase. RESULTS: We included 8767 participants (median age 62.2 years, 57% women). Higher IgG was associated with an increased ACVD risk (hazard ratio [HR]: 1.08; 95% confidence interval [95% CI]: 1.01-1.15). Higher IgA and IgG were associated with an increased cardiovascular mortality risk, mainly within Ig reference ranges, and with an increased all-cause mortality risk, although less marked. Higher IgA was associated with severe atherosclerosis, i.e., CAC score > 400 (odds ratio: 1.29; 95% CI: 1.03-1.62), while for higher IgG a trend was seen with severe atherosclerosis. CONCLUSION: In middle-aged and older individuals from the general population, higher serum IgA and IgG, but not IgM, are associated with CVD, cardiovascular mortality, and severe atherosclerosis, particularly within Ig reference ranges and independent of serum C-reactive protein. Future studies are needed to elucidate potential causality of the reported associations.

High-titer convalescent plasma plus nirmatrelvir/ritonavir treatment for non-resolving COVID-19 in six immunocompromised patients.

OBJECTIVES: Immunocompromised patients have an increased risk of severe or prolonged COVID-19. Currently available drugs are registered to treat COVID-19 during the first 5 to 7 days after symptom onset. Data on the effectivity in immunocompromised patients with chronic non-resolving COVID-19 are urgently needed. Here, we report the outcome of patients treated with nirmatrelvir/ritonavir together with high-titer convalescent plasma (CP) in six immunocompromised patients with non-resolving COVID-19. METHODS: Immunocompromised patients with persisting COVID-19 (positive PCR with Ct values <30 for ≥20 days) received off-label therapy with nirmatrelvir/ritonavir. It was combined with CP containing BA.5 neutralizing titers of ≥1/640 whenever available. Follow-up was done by PCR and sequencing on nasopharyngeal swabs on a weekly basis until viral genome was undetectable consecutively. RESULTS: Five immunocompromised patients were treated with high-titer CP and 5 days of nirmatrelvir/ritonavir. One patient received nirmatrelvir/ritonavir monotherapy. Median duration of SARS-CoV-2 PCR positivity was 70 (range 20-231) days before nirmatrelvir/ritonavir treatment. In four patients receiving combination therapy, no viral genome of SARS-CoV-2 was detected on day 7 and 14 after treatment while the patient receiving nirmatrelvir/ritonavir monotherapy, the day 7 Ct value increased to 34 and viral genome was undetectable thereafter. Treatment was unsuccessful in one patient. In this patient, sequencing after nirmatrelvir/ritonavir treatment did not show protease gene mutations. CONCLUSIONS: In immunocompromised patients with non-resolving COVID-19, the combination of nirmatrelvir/ritonavir and CP may be an effective treatment. Larger prospective studies are needed to confirm these preliminary results and should compare different treatment durations.

Altered leukocyte subsets and immune proteome indicate proinflammatory mechanisms in mastocytosis.

BACKGROUND: Indolent systemic mastocytosis (ISM) is characterized by pathologic accumulation of mast cells. The mechanism behind its phenotypic heterogeneity is not well understood. Interaction of mast cells with other immune cells might cause systemic inflammation and thereby associated symptoms. OBJECTIVE: We investigated peripheral leukocyte compartments and serum immune proteome in ISM. METHODS: Peripheral blood leukocyte phenotyping using flow cytometry in a cohort of 18 adults with ISM and 12 healthy controls. Targeted proteomics was performed to measure 169 proteins associated with inflammation on serum of another 20 ISM patients and 20 healthy controls. RESULTS: Proportions of plasmacytoid dendritic cells and monocytes were significantly decreased while TH2 cells were increased in peripheral blood of ISM patients. Furthermore, a shift from naive to memory T cells was observed. Hierarchical clustering of the serum proteome revealed 2 distinct subgroups within ISM patients. In subgroup A (n = 8), 62 proteins were significantly overexpressed, whereas those of subgroup B (n = 12) were comparable to healthy controls. Patients in subgroup A displayed upregulated signaling pathways downstream of Toll-like receptor 4, TNF-α, and IFN-γ. Fatigue was more often present in subgroup A compared to B (75% vs 33% respectively, P = .06). CONCLUSIONS: Altered distribution of leukocyte subsets and a proinflammatory proteome were observed in subsequent 2 cohorts of ISM patients. We hypothesize that neoplastic mast cells recruit and activate plasmacytoid dendritic cells, monocytes, and T cells, leading to a vicious cycle of inflammation.

Immunogenicity of the mRNA-1273 COVID-19 vaccine in adult patients with inborn errors of immunity.

BACKGROUND: Patients with inborn errors of immunity (IEI) are at increased risk of severe coronavirus disease-2019 (COVID-19). Effective vaccination against COVID-19 is therefore of great importance in this group, but little is known about the immunogenicity of COVID-19 vaccines in these patients. OBJECTIVES: We sought to study humoral and cellular immune responses after mRNA-1273 COVID-19 vaccination in adult patients with IEI. METHODS: In a prospective, controlled, multicenter study, 505 patients with IEI (common variable immunodeficiency [CVID], isolated or undefined antibody deficiencies, X-linked agammaglobulinemia, combined B- and T-cell immunodeficiency, phagocyte defects) and 192 controls were included. All participants received 2 doses of the mRNA-1273 COVID-19 vaccine. Levels of severe acute respiratory syndrome coronavirus-2-specific binding antibodies, neutralizing antibodies, and T-cell responses were assessed at baseline, 28 days after first vaccination, and 28 days after second vaccination. RESULTS: Seroconversion rates in patients with clinically mild antibody deficiencies and phagocyte defects were similar to those in healthy controls, but seroconversion rates in patients with more severe IEI, such as CVID and combined B- and T-cell immunodeficiency, were lower. Binding antibody titers correlated well to the presence of neutralizing antibodies. T-cell responses were comparable to those in controls in all IEI cohorts, with the exception of patients with CVID. The presence of noninfectious complications and the use of immunosuppressive drugs in patients with CVID were negatively correlated with the antibody response. CONCLUSIONS: COVID-19 vaccination with mRNA-1273 was immunogenic in mild antibody deficiencies and phagocyte defects and in most patients with combined B- and T-cell immunodeficiency and CVID. Lowest response was detected in patients with X-linked agammaglobulinemia and in patients with CVID with noninfectious complications. The assessment of longevity of immune responses in these vulnerable patient groups will guide decision making for additional vaccinations.

Patients with Chromosome 11q Deletions Are Characterized by Inborn Errors of Immunity Involving both B and T Lymphocytes.

Disorders of the long arm of chromosome 11 (11q) are rare and involve various chromosomal regions. Patients with 11q disorders, including Jacobsen syndrome, often present with a susceptibility for bacterial and prolonged viral and fungal infections partially explained by hypogammaglobulinemia. Additional T lymphocyte or granular neutrophil dysfunction may also be present. In order to evaluate infectious burden and immunological function in patients with 11q disorders, we studied a cohort of 14 patients with 11q deletions and duplications. Clinically, 12 patients exhibited prolonged and repetitive respiratory tract infections, frequently requiring (prophylactic) antibiotic treatment (n = 7), ear-tube placement (n = 9), or use of inhalers (n = 5). Complicated varicella infections (n = 5), chronic eczema (n = 6), warts (n = 2), and chronic fungal infections (n = 4) were reported. Six patients were on immunoglobulin replacement therapy. We observed a high prevalence of low B lymphocyte counts (n = 8), decreased T lymphocyte counts (n = 5) and abnormal T lymphocyte function (n = 12). Granulocyte function was abnormal in 29% without a clinical phenotype. Immunodeficiency was found in patients with terminal and interstitial 11q deletions and in one patient with terminal 11q duplication. Genetically, FLI1 and ETS1 are seen as causative for the immunodeficiency, but these genes were deleted nor duplicated in 4 of our 14 patients. Alternative candidate genes on 11q may have a role in immune dysregulation. In conclusion, we present evidence that inborn errors of immunity are present in patients with 11q disorders leading to clinically relevant infections. Therefore, broad immunological screening and necessary treatment is of importance in this patient group.

Novel RAB27A Variant Associated with Late-Onset Hemophagocytic Lymphohistiocytosis Alters Effector Protein Binding.

Autosomal recessive mutations in RAB27A are associated with Griscelli syndrome type 2 (GS2), characterized by hypopigmentation and development of early-onset, potentially fatal hemophagocytic lymphohistiocytosis (HLH). We describe a 35-year old male who presented with recurrent fever, was diagnosed with Epstein-Barr virus-driven chronic lymphoproliferation, fulfilled clinical HLH criteria, and who carried a novel homozygous RAB27A c.551G > A p.(R184Q) variant. We aimed to evaluate the contribution of the identified RAB27A variant in regard to the clinical phenotype as well as cellular and biochemical function. The patient displayed normal pigmentation as well as RAB27A expression in blood-derived cells. However, patient NK and CD8+ T cell exocytosis was low. Ectopic expression of the RAB27A p.R184Q variant rescued melanosome distribution in mouse Rab27a-deficient melanocytes, but failed to increase exocytosis upon reconstitution of human RAB27A-deficient CD8+ T cells. Mechanistically, the RAB27A p.R184Q variant displayed reduced binding to SLP2A but augmented binding to MUNC13-4, two key effector proteins in immune cells. MUNC13-4 binding was particularly strong to an inactive RAB27A p.T23N/p.R184Q double mutant. RAB27A p.R184Q was expressed and could facilitate melanosome trafficking, but did not support lymphocyte exocytosis. The HLH-associated RAB27A variant increased Munc13-4 binding, potentially representing a novel mode of impairing RAB27A function selectively in hematopoietic cells.

Clinical course of COVID-19 infections in patients with Behçet's disease in The Netherlands.

OBJECTIVES: The aim of this study is to investigate the cumulative incidence and the severity of COVID-19 infections in patients with Behçet's disease. METHODS: A retrospective cohort study of patients with Behçet's disease was conducted. We obtained the data systematically from electronic patient files and through telephone interviews between February 2020 and May 1, 2021. Main outcomes were COVID-19 infection, disease duration, hospitalisation, intensive care admission and mortality. Secondary outcome was adherence to quarantine measures as recommended by the government. RESULTS: 185 Behçet's disease patients were included (mean age 42.2 years, 54% female); 58% of the patients were receiving colchicine, 30% anti-TNFα, 16% azathioprine and 8% systemic steroids. 30 patients (16.2%) were positive for COVID-19. Within our cohort, the cumulative incidence of COVID-19 was therefore 16.2% (95% CI 11.2-22.3%), which is significantly increased when compared to the general Dutch population (8.7% (95% CI 8.72-8.73%)) (p < 0.001). Four out of 30 (13%) patients were admitted to the hospital. There was no COVID-19 related mortality observed. Patients adhered to government measures; except in the period between the 1st of June and the 28th of September, this cohort received more visitors than in period 1 and 3. CONCLUSIONS: In this cohort, Behçet's disease patients have a higher risk for COVID-19 infection, without an increase of virus-related mortality. The course of COVID-19 disease in this cohort is relatively mild, with a lower admission rate than expected of patients using immunosuppressive medication.

Blood biomarkers recommended for diagnosing and monitoring IgG4-related disease. Considerations from the ERN ReCONNET and collaborating partners.

Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a chronic, clinically heterogenous fibroinflammatory condition, characterised by an accumulation of IgG4 secreting plasma cells in affected tissues and associated with increased serum IgG4 concentrations. Despite a growing recognition of the disease among clinicians from different specialties worldwide, its indolent nature, lack of a single diagnostic test and ability to mimic other malignant, infective and inflammatory conditions, makes the diagnosis challenging. As treatment options evolve, biomarkers correlating with disease activity, predicting prognosis and response to treatment are deemed required. A multidisciplinary panel of experts from the European Reference Network for Rare and Complex Connective tissue diseases (ERN ReCONNET) and affiliated international partners have performed a narrative literature search and reviewed the current evidence of biomarkers in IgG4-RD, including immunoglobulins, cytokines, chemokines and other soluble immune mediators, and cellular components of the immune system. The aim of this paper is to provide useful information for clinicians as to the utility of biomarkers for diagnosing and monitoring IgG4-RD in clinical routine and sets out recommendations for clinical decision making.

Did variants in inborn errors of immunity genes contribute to the extinction of Neanderthals?

BACKGROUND: Neanderthals were a species of archaic humans that became extinct around 40,000 years ago. Modern humans have inherited 1-6% of Neanderthal DNA as a result of interbreeding. These inherited Neanderthal genes have paradoxical influences, while some can provide protection to viral infections, some others are associated with autoimmune/auto-inflammatory diseases. OBJECTIVE: We aim to investigate whether genetic variants with strong detrimental effects on the function of the immune system could have potentially contributed to the extinction of the Neanderthal population. METHODS: We used the publically available genome information from an Altai Neanderthal and filtered for potentially damaging variants present in genes associated with inborn errors of immunity (IEI) and checked whether these variants were present in the genomes of the Denisovan, Vindija and Chagyrskaya Neanderthals. RESULTS: We identified 24 homozygous variants and 15 heterozygous variants in IEI-related genes in the Altai Neanderthal. Two homozygous variants in the UNC13D gene and one variant in the MOGS gene were present in all archaic genomes. Defects in the UNC13D gene are known to cause a severe and often fatal disease called hemophagocytic lymphohistiocystosis (HLH). One of these variants p.(N943S) has been reported in patients with HLH. Variants in MOGS are associated with glycosylation defects in the immune system affecting the susceptibility for infections. CONCLUSIONS: Although the exact functional impact of these three variants needs further elucidation, we speculate that they could have resulted in an increased susceptibility to severe diseases and may have contributed to the extinction of Neanderthals after exposure to specific infections.

Chronically Activated T-cells Retain Their Inflammatory Properties in Common Variable Immunodeficiency.

PURPOSE: Immune dysregulation complications cause significant morbidity and mortality in common variable immunodeficiency (CVID), but the underlying pathophysiology is poorly understood. While CVID is primarily considered a B-cell defect, resulting in the characteristic hypogammaglobulinemia, T-cells may also contribute to immune dysregulation complications. Here, we aim to further characterize T-cell activation and regulation in CVID with immune dysregulation (CVIDid). METHODS: Flow cytometry was performed to investigate T-cell differentiation, activation and intracellular cytokine production, negative regulators of immune activation, regulatory T-cells (Treg), and homing markers in 12 healthy controls, 12 CVID patients with infections only (CVIDio), and 20 CVIDid patients. RESULTS: Both CD4 + and CD8 + T-cells in CVIDid showed an increased activation profile (HLA-DR + , Ki67 + , IFNγ +) when compared to CVIDio, with concomitant upregulation of negative regulators of immune activation PD1, LAG3, CTLA4, and TIGIT. PD1 + and LAG3 + subpopulations contained equal or increased frequencies of cells with the capacity to produce IFNγ, Ki67, and/or GzmB. The expression of PD1 correlated with serum levels of CXCL9, 10, and 11. Treg frequencies were normal to high in CVIDid, but CVIDid Tregs had reduced CTLA-4 expression, especially on CD27 + effector Tregs. Increased migratory capacity to inflamed and mucosal tissue was also observed in CVIDid T-cells. CONCLUSION: CVIDid was characterized by chronic activation of peripheral T-cells with preserved inflammatory potential rather than functional exhaustion, and increased tissue migratory capacity. While Treg numbers were normal in CVIDid Tregs, low levels of CTLA-4 indicate possible Treg dysfunction. Combined studies of T-cell dysfunction and circulating inflammatory proteins may direct future treatment strategies.

Targeted Proteomics Reveals Inflammatory Pathways that Classify Immune Dysregulation in Common Variable Immunodeficiency.

Patients with common variable immunodeficiency (CVID) can develop immune dysregulation complications such as autoimmunity, lymphoproliferation, enteritis, and malignancy, which cause significant morbidity and mortality. We aimed to (i) assess the potential of serum proteomics in stratifying patients with immune dysregulation using two independent cohorts and (ii) identify cytokine and chemokine signaling pathways that underlie immune dysregulation in CVID. A panel of 180 markers was measured in two multicenter CVID cohorts using Olink Protein Extension Assay technology. A classification algorithm was trained to distinguish CVID with immune dysregulation (CVIDid, n = 14) from CVID with infections only (CVIDio, n = 16) in the training cohort, and validated on a second testing cohort (CVIDid n = 23, CVIDio n = 24). Differential expression in both cohorts was used to determine relevant signaling pathways. An elastic net classifier using MILR1, LILRB4, IL10, IL12RB1, and CD83 could discriminate between CVIDid and CVIDio patients with a sensitivity of 0.83, specificity of 0.75, and area under the curve of 0.73 in an independent testing cohort. Activated pathways (fold change > 1.5, FDR-adjusted p < 0.05) in CVIDid included Th1 and Th17-associated signaling, as well as IL10 and other immune regulatory markers (LAG3, TNFRSF9, CD83). Targeted serum proteomics provided an accurate and reproducible tool to discriminate between patients with CVIDid and CVIDio. Cytokine profiles provided insight into activation of Th1 and Th17 pathways and indicate a possible role for chronic inflammation and exhaustion in immune dysregulation. These findings serve as a first step towards the development of biomarkers for immune dysregulation in CVID.

Immunoglobulin Replacement Therapy Versus Antibiotic Prophylaxis as Treatment for Incomplete Primary Antibody Deficiency.

BACKGROUND: Patients with an IgG subclass deficiency (IgSD) ± specific polysaccharide antibody deficiency (SPAD) often present with recurrent infections. Previous retrospective studies have shown that prophylactic antibiotics (PA) and immunoglobulin replacement therapy (IRT) can both be effective in preventing these infections; however, this has not been confirmed in a prospective study. OBJECTIVE: To compare the efficacy of PA and IRT in a randomized crossover trial. METHODS: A total of 64 patients (55 adults and 9 children) were randomized (2:2) between two treatment arms. Treatment arm A began with 12 months of PA, and treatment arm B began with 12 months of IRT. After a 3-month bridging period with cotrimoxazole, the treatment was switched to 12 months of IRT and PA, respectively. The efficacy (measured by the incidence of infections) and proportion of related adverse events in the two arms were compared. RESULTS: The overall efficacy of the two regimens did not differ (p = 0.58, two-sided Wilcoxon signed-rank test). A smaller proportion of patients suffered a related adverse event while using PA (26.8% vs. 60.3%, p < 0.0003, chi-squared test). Patients with persistent infections while using PA suffered fewer infections per year after switching to IRT (2.63 vs. 0.64, p < 0.01). CONCLUSION: We found comparable efficacy of IRT and PA in patients with IgSD ± SPAD. Patients with persistent infections during treatment with PA had less infections after switching to IRT. CLINICAL IMPLICATION: Given the costs and associated side-effects of IRT, it should be reserved for patients with persistent infections despite treatment with PA.

Cost and impact of early diagnosis in primary immunodeficiency disease: A literature review.

BACKGROUND: New, innovative, costly diagnostic methods for patients with primary immunodeficiencies (PID) demand upfront insight into their potential cost savings and added value for individual patients. As such, high quality, comparable economic evaluations are of utmost importance to enable informed decisions. The objective of this review was therefore to create an extensive overview of current costing studies and potential cost savings of early diagnosis in primary immunodeficiency disease. METHODS: A literature search in PubMed was conducted and studies involving any form of costing study in the field of PIDs were included. Of the included studies, study characteristics, cost parameters and benefits of early diagnosis were extracted and outlined in separate tables. RESULTS: Twenty two studies met the inclusion criteria and were included in the review. The papers were categorized according to their subject: neonatal screening for severe combined immunodeficiency (SCID), Ig replacement therapies and studies reporting on costs of general or specific PIDs. Within and between these groups variability in reported costing characteristics was observed. In studies that reported cost savings pre- and post-diagnosis, cost savings ranged from 6500 to 108,463 USD of total costs per patient. CONCLUSION: This literature review shows that, regardless of what aspect of PIDs has been studied, in nearly all cases early diagnosis reduces health care consumption and leads to better health outcomes for patients with PIDs. We found considerable variability in costing characteristics of economic evaluations of PID patients, which hampers the comparability of outcomes. More effort is needed to create uniformity and define cost parameters in economic evaluations in the field of PIDs, facilitating further prospective research to extensively assess the benefits of early diagnosis.

Precursor B-cell development in bone marrow of Good syndrome patients.

Good syndrome is an immunodeficiency presenting with thymoma, hypogammaglobulinemia and almost absent B cells. To investigate the origin of the B-cell lymphopenia in these patients, we studied B cell differentiation in the bone marrow of Good syndrome patients. We found very low numbers of precursor B cells in bone marrow of Good syndrome patients and a differentiation arrest after the pro-B-cell stage; this is different from other agammaglobulinemia patients with a defect in pre B-cell receptor signaling.

ALPK1 missense pathogenic variant in five families leads to ROSAH syndrome, an ocular multisystem autosomal dominant disorder.

PURPOSE: To identify the molecular cause in five unrelated families with a distinct autosomal dominant ocular systemic disorder we called ROSAH syndrome due to clinical features of retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and migraine headache. METHODS: Independent discovery exome and genome sequencing in families 1, 2, and 3, and confirmation in families 4 and 5. Expression of wild-type messenger RNA and protein in human and mouse tissues and cell lines. Ciliary assays in fibroblasts from affected and unaffected family members. RESULTS: We found the heterozygous missense variant in the ɑ-kinase gene, ALPK1, (c.710C>T, [p.Thr237Met]), segregated with disease in all five families. All patients shared the ROSAH phenotype with additional low-grade ocular inflammation, pancytopenia, recurrent infections, and mild renal impairment in some. ALPK1 was notably expressed in retina, retinal pigment epithelium, and optic nerve, with immunofluorescence indicating localization to the basal body of the connecting cilium of the photoreceptors, and presence in the sweat glands. Immunocytofluorescence revealed expression at the centrioles and spindle poles during metaphase, and at the base of the primary cilium. Affected family member fibroblasts demonstrated defective ciliogenesis. CONCLUSION: Heterozygosity for ALPK1, p.Thr237Met leads to ROSAH syndrome, an autosomal dominant ocular systemic disorder.

Effective "activated PI3Kδ syndrome"-targeted therapy with the PI3Kδ inhibitor leniolisib.

Pathogenic gain-of-function variants in the genes encoding phosphoinositide 3-kinase δ (PI3Kδ) lead to accumulation of transitional B cells and senescent T cells, lymphadenopathy, and immune deficiency (activated PI3Kδ syndrome [APDS]). Knowing the genetic etiology of APDS afforded us the opportunity to explore PI3Kδ inhibition as a precision-medicine therapy. Here, we report in vitro and in vivo effects of inhibiting PI3Kδ in APDS. Treatment with leniolisib (CDZ173), a selective PI3Kδ inhibitor, caused dose-dependent suppression of PI3Kδ pathway hyperactivation (measured as phosphorylation of AKT/S6) in cell lines ectopically expressing APDS-causative p110δ variants and in T-cell blasts derived from patients. A clinical trial with 6 APDS patients was conducted as a 12-week, open-label, multisite, within-subject, dose-escalation study of oral leniolisib to assess safety, pharmacokinetics, and effects on lymphoproliferation and immune dysregulation. Oral leniolisib led to a dose-dependent reduction in PI3K/AKT pathway activity assessed ex vivo and improved immune dysregulation. We observed normalization of circulating transitional and naive B cells, reduction in PD-1+CD4+ and senescent CD57+CD4- T cells, and decreases in elevated serum immunoglobulin M and inflammatory markers including interferon γ, tumor necrosis factor, CXCL13, and CXCL10 with leniolisib therapy. After 12 weeks of treatment, all patients showed amelioration of lymphoproliferation with lymph node sizes and spleen volumes reduced by 39% (mean; range, 26%-57%) and 40% (mean; range, 13%-65%), respectively. Thus, leniolisib was well tolerated and improved laboratory and clinical parameters in APDS, supporting the specific inhibition of PI3Kδ as a promising new targeted therapy in APDS and other diseases characterized by overactivation of the PI3Kδ pathway. This trial was registered at www.clinicaltrials.gov as #NCT02435173.

Adalimumab provides long-lasting clinical improvement in refractory mucocutaneous Behçet's disease without formation of antidrug antibodies.

OBJECTIVES: The TNF-blocker adalimumab can be effective in Behçet's disease (BD), a multisystem auto-inflammatory disorder. Unfortunately, the therapeutic efficacy of TNF-blockers can be hampered by the formation of anti-drug antibodies. We present an observational study of adalimumab in refractory BD with measurement of anti-drug antibodies. METHODS: The effect of fortnightly 40mg adalimumab in nine patients with therapy refractory mucocutaneous, non-ocular or organ threatening BD was studied up to 60 months. Primary endpoint was a decrease in disease activity, measured by the BD Current Activity Form (BDCAF) within 6 months. Secondary endpoints included serum cytokines and the long-term formation of anti-adalimumab antibodies. RESULTS: BDCAF improved significantly in all nine patients from 5.4 (SD=1.4) to 2.4 (SD=1.4) (p=0.007) within one month up to 6 months and after prolonged follow up of 5 years. All patients could either taper or stop concomitant therapy. Symptoms of mucocutaneous lesions, erythema nodosum and joint involvement decreased or disappeared. Serum TNF-alpha levels were elevates in five patients and decreased upon treatment (p=0.017). Adalimumab was save and none of the patients experienced therapy failure or antibodies against adalimumab. CONCLUSIONS: We present an observational study on patients with BD treated with adalimumab and provide a basis for long-term use in refractory mucocutaneous BD. These findings show that adalimumab can safely be administered yielding sustainable clinical effects in refractory BD patients with mucocutaneous disease without formation of anti-adalimumab antibodies, even after long follow up.

Expansion of blood IgG4+ B, TH2, and regulatory T cells in patients with IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition affecting various organs and has a diverse clinical presentation. Fibrosis and accumulation of IgG4+ plasma cells in tissue are hallmarks of the disease, and IgG4-RD is associated with increased IgG4 serum levels. However, disease pathogenesis is still unclear, and these cellular and molecular parameters are neither sensitive nor specific for the diagnosis of IgG4-RD. OBJECTIVE: Here we sought to develop a flow cytometric gating strategy to reliably identify blood IgG4+ B cells to study their cellular and molecular characteristics and investigate their contribution in disease pathogenesis. METHODS: Sixteen patients with histologically confirmed IgG4-RD, 11 patients with sarcoidosis, and 30 healthy subjects were included for 11-color flow cytometric analysis of peripheral blood for IgG4-expressing B cells and TH subsets. In addition, detailed analysis of activation markers and chemokine receptors was performed on IgG4-expressing B cells, and IgG4 transcripts were analyzed for somatic hypermutations. RESULTS: Cellular and molecular analyses revealed increased numbers of blood IgG4+ memory B cells in patients with IgG4-RD. These cells showed reduced expression of CD27 and CXCR5 and increased signs of antibody maturation. Furthermore, patients with IgG4-RD, but not patients with sarcoidosis, had increased numbers of circulating plasmablasts and CD21low B cells, as well as TH2 and regulatory T cells, indicating a common disease pathogenesis in patients with IgG4-RD. CONCLUSION: These results provide new insights into the dysregulated IgG4 response in patients with IgG4-RD. A specific "peripheral lymphocyte signature" observed in patients with IgG4-RD, could support diagnosis and treatment monitoring.

Hydroxychloroquine decreases human MSC-derived osteoblast differentiation and mineralization in vitro.

We recently showed that patients with primary Sjögren Syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favourable effects on BMD. To study the direct effects of HCQ on human MSC-derived osteoblast activity. Osteoblasts were cultured from human mesenchymal stromal cells (hMSCs). Cultures were treated with different HCQ doses (control, 1 and 5 µg/ml). Alkaline phosphatase activity and calcium measurements were performed to evaluate osteoblast differentiation and activity, respectively. Detailed microarray analysis was performed in 5 µg/ml HCQ-treated cells and controls followed by qPCR validation. Additional cultures were performed using the cholesterol synthesis inhibitor simvastatin (SIM) to evaluate a potential mechanism of action. We showed that HCQ inhibits both MSC-derived osteoblast differentiation and mineralization in vitro. Microarray analysis and additional PCR validation revealed a highly significant up-regulation of the cholesterol biosynthesis, lysosomal and extracellular matrix pathways in the 5 µg/ml HCQ-treated cells compared to controls. Besides, we demonstrated that 1 µM SIM also decreases MSC-derived osteoblast differentiation and mineralization compared to controls. It appears that the positive effect of HCQ on BMD cannot be explained by a stimulating effect on the MSC-derived osteoblast. The discrepancy between high BMD and decreased MSC-derived osteoblast function due to HCQ treatment might be caused by systemic factors that stimulate bone formation and/or local factors that reduce bone resorption, which is lacking in cell cultures.