Search

Antibody production by the B cell compartment is a crucial part of the adaptive immune response. Dysregulated antibody production in the form of autoantibodies can cause autoimmune disease. To date, B‐cell depletion with anti‐CD20 antibodies is commonly applied in autoimmunity, but pre‐existing plasma cells are not eliminated in this way. Alternative ways of more selective inhibition of antibody production would add to the treatment of these autoimmune diseases. To explore novel therapeutic targets in signaling pathways essential for plasmablast formation and/or immunoglobulin production, we performed a compound screen of almost 200 protein kinase inhibitors in a robust B‐cell differentiation culture system. This study yielded 35 small cell‐permeable compounds with a reproducible inhibitory effect on B‐cell activation and plasmablast formation, among which was the clinically applied mammalian target of rapamycin (mTOR) inhibitor rapamycin. Two additional compounds targeting the phosphoinositide 3‐kinase‐AKT‐mTOR pathway (BKM120 and WYE‐354) did not affect proliferation and plasmablast formation, but specifically reduced the immunoglobulin production. With this compound screen we successfully applied a method to investigate therapeutic targets for B‐cell differentiation and identified compounds in the phosphoinositide 3‐kinase‐AKT‐mTOR pathway that could specifically inhibit immunoglobulin production only. These drugs may well be explored to be of value in current B‐cell‐depleting treatment regimens in autoimmune disorders., Through a large compound screen involving 192 inhibitors, the functional effects on B‐cell activation, differentiation, and antibody production were investigated. This highlighted multiple compounds, of which drugs targeting the phosphoinositide 3‐kinase‐AKT‐mTOR‐axis were further validated. These drugs could complement current B‐cell‐depleting treatment regimens in autoantibody‐mediated autoimmune disorders .

We report the natural history, clinical manifestations, genetics, and immunohematological findings in 14 patients from 11 families with ARPC1B deficiency, delineating the spectrum of the disease that appears progressive and challenging to manage clinically.

Contains fulltext : 229571.pdf (Publisher’s version ) (Closed access) BACKGROUND: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes. OBJECTIVE: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations. METHODS: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling. RESULTS: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents. CONCLUSIONS: We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future.

Contains fulltext : 169755.pdf (Publisher’s version ) (Open Access) EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal dysplasia syndrome, and to gain insight into the pathogenesis of the disorder, we analyzed the localization of EXTL3 in fibroblasts derived from affected individuals and determined glycosaminoglycan concentrations in these cells as well as in urine and blood. We observed abnormal glycosaminoglycan concentrations and increased concentrations of the non-sulfated chondroitin disaccharide D0a0 and the disaccharide D0a4 in serum and urine of all analyzed affected individuals. In summary, we show that biallelic mutations in EXTL3 disturb glycosaminoglycan synthesis and thus lead to a recognizable syndrome characterized by variable expression of skeletal, neurological, and immunological abnormalities.

Career situation of first and presenting author Post-doctoral fellow. Introduction The pivotal role of B cells in the pathogenesis of autoimmune diseases such as ANCA-associated vasculitis (AAV) is well-established and further substantiated by beneficial therapeutic effects of rituximab (anti-CD20 B cell targeted therapy). However, this results in prolonged B cell depletion while long-lived plasma cells are not targeted. Thus, there is a need for novel therapeutics targeting the B-cell lineage in AAV. NF-κB signalling pathways that act downstream of various B cell surface receptors, including the B cell receptor, CD40, BAFFR and TLRs, are crucially involved in B cell responses and may be suitable as novel targets. Objectives To identify whether inhibition of NF-κB signalling by novel pharmacological inhibitors is effective in targeting B cell responses in general and more specifically blocks (auto)antibody production and plasmablast differentiation in B cells from AAV patients. Methods PBMC and sorted B cells from AAV patients and healthy donors were cultured with T cell-dependent (anti-IgM+anti CD40+IL-21) and T cell-independent (CpG+IL-2) stimuli. NF-κB signalling was targeted in these cultures by small molecule inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signalling) and Inhibitor of κB kinase β (IKKβ, canonical NF-κB signalling). Downstream NF-κB signalling and nuclear NF-κB translocation was determined by Western blot and confocal imaging. Effects on B cell proliferation and differentiation were determined by CFSE dilution assays and flow cytometric analysis of B cell markers. (Auto)antibody production was measured by ELISA. Results In B cells of AAV patients and healthy donors, targeting of NIK and IKKβ effectively inhibited downstream non-canonical or canonical NF-κB signalling, respectively. In a B cell stimulation assay, NIK and IKKβ inhibition significantly reduced T cell-dependent (anti-IgM+anti-CD40+IL-21) and T cell-independent (CpG+IL-2) B cell proliferation. In addition, B cell differentiation towards plasmablasts (CD27++/CD38+) and functional antibody production was attenuated by both NIK and IKKβ inhibitors. Interestingly, the effects of NIK inhibition appeared to be B cell-specific as T cell proliferation was largely unaffected. Conclusions These data demonstrate that inhibition of NF-κB signalling in AAV B cells results in the modulation of various B cell responses. Ongoing studies will indicate whether targeting of NF-κB signalling in B cells may be an effective novel treatment modality for AAV. Disclosure of Interest J. P. Van Hamburg: None declared, P. Tuijnenburg: None declared, B. Helder: None declared, L. S. van Keep: None declared, L. A. Rutten: None declared, K. Wesenhagen: None declared, P. Kucharzewska Grant/research support from: small molecule inhibitors, M. H. Jansen: None declared, A. Al-Soudi: None declared, P. L. Klarenbeek: None declared, H. K. Olsson Grant/research support from: small molecule inhibitors, N. de Vries: None declared, T. W. Kuijpers: None declared, S. W. Tas: None declared.

Genetic studies are identifying an increasing number of monogenic causes of Common Variable Immunodeficiency (CVID). Pathogenic variants in the C-terminus of NFKB2 have been identified in the subset of CVID patients whose immunodeficiency is associated with ectodermal dysplasia and central adrenal insufficiency. We describe 2 unrelated CVID pedigrees with 4 cases of pathogenic stop gain variants (c.1903C > T) in the ankyrin repeat domain (ARD) of NF-κB2, leading to a premature truncation of the protein at p.Arg635Term (R635X). By immunophenotyping and functional ex vivo B- and T-cell experiments we characterized the variant by reduced class-switched memory B-cell counts and immature plasmablasts, unable to produce IgG and IgA. Features of a poor proliferative T-cell response and reduced expansion of CD4 + CXCR5 + T cells was only observed in the two clinically affected index cases without any clear clinical correlate. In conclusion, pathogenic stop variants in the ARD of NFKB2 can cause ‘infection-only’ CVID with an abnormal B-cell phenotype and a variable clinical penetrance.

Primary immunodeficiency (PID) is characterised by recurrent and often life-threatening infections, autoimmunity and cancer, and it presents major diagnostic and therapeutic challenges. Although the most severe forms present in early childhood, the majority of patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent, and up to 10% develop lymphoid malignancies1–3. Consequently, in sporadic PID genetic diagnosis is difficult and the role of genetics is not well defined. We addressed these challenges by performing whole genome sequencing (WGS) of a large PID cohort of 1,318 participants. Analysis of coding regions of 886 index cases found disease-causing mutations in known monogenic PID genes in 10.3%, while a Bayesian approach (BeviMed4) identified multiple potential new candidate genes, including IVNS1ABP. Exploration of the non-coding genome revealed deletions in regulatory regions which contribute to disease causation. Finally, a genome-wide association study (GWAS) identified PID-associated loci and uncovered evidence for co-localisation of, and interplay between, novel high penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to variable penetrance and phenotypic complexity in PID. Thus, a cohort-based WGS approach to PID diagnosis can increase diagnostic yield while deepening our understanding of the key pathways influencing human immune responsiveness.

Introduction The pivotal role of B cells in the pathogenesis autoimmune diseases such as ANCA-associated vasculitis (AAV) is well-established and further substantiated by beneficial therapeutic effects of rituximab (anti-CD20 B cell targeted therapy). However, this results in prolonged B cell depletion while long-lived plasma cells are not targeted. Thus there is a need for novel therapeutics targeting cells in the B-cell lineage in AAV. Novel targets might be encountered in the NF-κB signalling pathway, which acts downstream of various B cell surface receptors, including the B cell antigen receptor, CD40, BAFFR and TLRs, and is crucially involved in B cell responses. Objectives To identify whether inhibition of NF-κB signalling by novel pharmacological inhibitors is effective in targeting B cell responses in general and more specifically blocks (auto)antibody production and plasmablast differentiation in B cells from AAV patients. Methods PBMC and sorted B cells from AAV patients and healthy donors were cultured with T cell-dependent (anti-IgM +anti CD40+IL-21) and T cell-independent (CpG +IL-2) stimuli. NF-κB signalling was targeted in these cultures by small molecule inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signalling) and IKKβ (canonical NF-κB signalling). Downstream NF-κB signalling and nuclear NF-κB translocation was determined by Western blot and confocal imaging. Effects on B cell proliferation and differentiation were determined by CFSE dilution assays and flow cytometric analysis of B cell markers. (Auto)antibody production was measured by ELISA. Results In B cells, targeting of NIK and IKKβ effectively inhibited non-canonical or canonical NF-κB signalling, respectively. In a B cell stimulation assay, NIK and IKKβ inhibition significantly reduced T cell-dependent (anti-IgM +anti CD40+IL-21) and T cell-independent (CpG +IL-2) B cell proliferation, plasmablast differentiation (CD27++/CD38+), and antibody production. The effects of NIK inhibition appeared to be B cell-specific as T cell proliferation was largely unaffected. Currently, studies are ongoing to investigate the effect of IKKβ inhibition on B cell responses and to explore the effects of targeting NF-κB signalling in AAV B cells. Conclusions These data demonstrate that inhibition of NF-κB signalling in B cells results in the modulation of various B cell responses. Ongoing studies will indicate whether targeting of NF-κB signalling in B cells may be an effective novel treatment modality for AAV. Disclosure of interest None declared

After exiting the thymus, Foxp3(+) regulatory T (Treg) cells undergo further differentiation in the periphery, resulting in the generation of mature, fully suppressive effector (e) Treg cells in a process dependent on TCR signaling and the transcription factor IRF4. Here, we show that tumor necrosis factor receptor superfamily (TNFRSF) signaling plays a crucial role in the development and maintenance of eTreg cells. TNFRSF signaling activated the NF-kappa B transcription factor RelA, which was required to maintain eTreg cells in lymphoid and non-lymphoid tissues, including ROR gamma t(+) Treg cells in the small intestine. In response to TNFRSF signaling, RelA regulated basic cellular processes, including cell survival and proliferation, but was dispensable for IRF4 expression or DNAbinding, indicating that both pathways operated independently. Importantly, mutations in the RelA binding partner NF-kappa B1 compromised eTreg cells in humans, suggesting that the TNFRSF-NF-kappa B axis was required in a non-redundant manner to maintain eTreg cells in mice and humans

Background The genetic cause of primary immunodeficiency disease (PID) carries prognostic information. Objective We conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource–Rare Diseases cohort. Methods In the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense, and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n = 390) in the cohort. Amino acid substitutions predicted to be pathogenic were assessed by means of analysis of structural protein data. Immunophenotyping, immunoblotting, and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype cosegregation analyses. Results Both sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B-lymphocyte differentiation. Detailed assessment of B-lymphocyte numbers, phenotype, and function identifies the presence of an increased CD21low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases. Conclusion We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells., Graphical abstract

We describe here the case of a boy who presented with pulmonary infections, feeding difficulties due to velopharyngeal insufficiency and gastroesophageal reflux, myopathy, and hypotonia soon after birth. Later, he was also found to have an elevated immunoglobulin (Ig) E and mild eczema and was diagnosed with inflammatory bowel disease. Further immunological screening at the age of 7 years showed low B and NK cell numbers but normal CD4(+) and CD8(+) T cells and notably, normal numbers of CD4(+) regulatory T (Treg) cells. Serum IgG, IgA, and IgM were low to normal, but he had a deficient response to a pneumococcal polysaccharide vaccine and thus a humoral immunodeficiency. To our surprise, whole exome sequencing revealed a mutation in forkhead box protein 3 (FOXP3), encoding an essential transcription factor for the development and function of Treg cells. This classical mutation is associated with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Further in vitro studies indeed showed defective function of Treg cells despite normal FOXP3 protein expression and nuclear localization. The boy underwent hematopoietic stem cell transplantation at 11 years of age and despite the temporary development of diabetes while on prednisone is now doing much better, IgE levels have declined, and his fatigue has improved. This case illustrates that a classical pathogenic mutation in FOXP3 can lead to a clinical phenotype where the diagnosis of IPEX syndrome was never considered because of the lack of diabetes and the presence of only mild eczema, in addition to the normal Treg cell numbers and FOXP3 expression