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MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO)., Necroptosis is a regulated form of inflammatory cell death driven by activated MLKL. Here, the authors identify a mutation in the brace region that confers constitutive activation, leading to lethal inflammation in homozygous mutant mice and providing insight into human mutations in this region.

Common variable immunodeficiency (CVID) is a disease characterized by increased susceptibility to infections, hypogammaglobulinemia, and immune dysregulation. Although CVID is thought to be a disorder of the peripheral B-cell compartment, in 25% of patients, early B-cell development in the bone marrow is impaired. Because poor B-cell reconstitution after hematopoietic stem cell transplantation has been observed, we hypothesized that in some patients the bone marrow environment is not permissive to B-cell development. Studying the differentiation dynamics of bone marrow-derived CD34+ cells into immature B cells in vitro allowed us to distinguish patients with B-cell intrinsic defects and patients with a nonpermissive bone marrow environment. In the former, immature B cells did not develop and in the latter CD34+ cells differentiated into immature cells in vitro, but less efficiently in vivo. In a further group of patients, the uncommitted precursors were unable to support the constant development of B cells in vitro, indicating a possible low frequency or exhaustion of the precursor population. Hematopoietic stem cell transplantation would result in normal B-cell repopulation in case of intrinsic B-cell defect, but in defective B-cell repopulation in a nonpermissive environment. Our study points to the importance of the bone marrow niche in the pathogenesis of CVID.

ObjectivesANCA-associated vasculitides (AAV) affect small- and medium-sized blood vessels. In active disease, vessel wall infiltrates are mainly composed of monocytes and macrophages. Immune checkpoint molecules are crucial for the maintenance of self-tolerance and the prevention of autoimmune diseases. After checkpoint inhibitor therapy, the development of autoimmune vasculitis has been observed. However, defects of immune checkpoint molecules in AAV patients have not been identified yet.MethodsMonocytes and monocyte-derived macrophages from AAV patients and healthy age-matched controls were tested for surface expression of immunoinhibitory checkpoint programmed cell death ligand-1 (PD-L1). Using in vitro co-culture approaches, the effect of monocyte PD-L1 expression on CD4+ T cell activation and proliferation was tested.ResultsMonocytes from AAV patients displayed lower PD-L1 expression and a defective PD-L1 presentation upon activation, an effect that was correlated with disease activity. Lower PD-L1 expression was due to increased lysosomal degradation of PD-L1 in AAV monocytes. We identified a reduced expression of CMTM6, a protein protecting PD-L1 from lysosomal breakdown, as the underlying molecular defect. PD-L1low AAV monocytes showed increased stimulatory capacity and induced T cell activation and proliferation. Inhibiting lysosomal function corrected this phenotype by increasing PD-L1, thus normalizing the pro-stimulatory behavior of AAV monocytes.ConclusionsThis study identifies a defect of the immunoinhibitory checkpoint PD-L1 in monocytes from patients with AAV. Low expression of CMTM6 results in enhanced lysosomal degradation of PD-L1, thus providing insufficient negative signaling to T cells. Correcting this defect by targeting lysosomal function may represent a novel strategy to treat AAV.

SUMMARYWe have isolated a mouse strain with a single missense mutation in the gene encoding MLKL, the essential effector of necroptotic cell death. The resulting substitution lies within the two-helix ‘brace’ and confers constitutive, RIPK3 independent, killing activity to MLKL. Mice homozygous forMlklD139Vdevelop lethal inflammation within days of birth, implicating the salivary glands and pericardium as hotspots for necroptosis and inflammatory infiltration. The normal development ofMlklD139Vhomozygotes until birth, and the absence of any overt phenotype in heterozygotes provides importantin vivoprecedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common humanMLKLpolymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, CRMO.

Objective: Rheumatic diseases with involvement of the central nervous system (RDwCNS) may mimic multiple sclerosis (MS). Inversely, up to 60% of MS-patients have antinuclear autoantibodies (ANAs) and may be misdiagnosed as RDwCNS. The detection of antibodies against extractable nuclear antigens (ENA) and oligoclonal bands (OCB) are established valuable diagnostic tools in the differential diagnosis of RDwCNS and MS. The MRZ-reaction (MRZR) is defined by three antibody indices (AIs) against neurotropic viruses and is frequently positive in MS. To investigate the added value of MRZR combined with testing for antibodies against ENAs and OCB detection to distinguish RDwCNS from ANA positive MS.Methods: MRZR was evaluated in RDwCNS (n = 40) and 68 ANA positive MS-patients. Two stringency levels, MRZR-1 and MRZR-2 (at least one respectively two of three AIs positive) were applied. Autoantibody testing included ANA plus ENA and anti-dsDNA antibodies, antiphospholipid antibodies, and anti-neutrophil cytoplasmic antibodies.Results: Most of the RDwCNS patients (n = 32; 80%) suffered from systemic lupus erythematosus. Within the RDwCNS group 20% had a positive MRZR-1 and 8.5% a positive MRZR-2 compared to 80.9 and 60%, respectively within the MS-group (p < 0.0001 for both comparisons). Oligoclonal bands were found in 28.6% of the RDwCNS patients and 94.3% of the MS-patients (p < 0.0001). Conversely, autoantibodies to specific nuclear antigens or phospholipids were found more frequently in RDwCNS. A positive MRZR in conjunction with the absence of ENA autoantibodies distinguished MS from RDwCNS with high specificity (97.5%).Conclusions: We suggest combining MRZR, OCBs, and specific autoantibody diagnostics to differentiate RDwCNS from MS.

Background:ALPS patients show impaired generation of humoral memory for T independent antigens whereas they generate memory for self-antigens due to impaired FAS-dependent removal of autoreactive germinal center B cells. It is known that FAS signaling via caspase activation results in cell apoptosis. However, FAS ligation may also initiate or modulate non-apoptotic signaling as shown for example by its ability to activate NF-κB. Recent data implicate a regulatory role of FAS in the modulation of mTOR signaling in ALPS double-negative T cells. Moreover, a recently described C194V FAS mutation disturbs its post-translational modification leading to impaired apoptosis induction while non-apoptotic signalling is still intact. Consequently, C194V FAS protects from the autoimmune phenotype in the murine ALPS system. This supports the view that FAS may prevent autoimmunity with other mechanisms than inducing apoptosis.Objectives:We hypothesize that FAS mutations impair this modulatory signaling, leading to hyper-activation of B cells. Therefore we aim to investigate non apoptotic FAS signaling in B cells derived from healthy individuals and ALPS patients.Methods:We studied resting and activated B cells in ALPS patients in presence or absence of FAS ligand by flow cytometry analysing relevant molecules to the CD40 signaling pathway. We used mass cytometry to perform functional phenotyping of B cells isolated from secondary lymphoid organs. Proteomic studies were performed to identify potential signaling circuits and RNA sequencing to study the consequences of FAS signaling on B cell fate.Results:In CD40L activated B cells, FAS signaling results in specific modulation of the mTOR signaling pathway. This modulation is absent in ALPS derived B cells. In line with these data germinal center B cells and plasmablast from secondary lymphoid organs of ALPS patients show hyperactive mTOR signaling pathway. Proteomic studies identify a circuit that links FAS to the phosphatase PTEN via DAXX and the deubiquitinase USP7.Conclusion:We describe a new role of FAS in the regulation of B cell activation. Defects in FAS signaling in ALPS contribute to dysregulation of the mTOR signaling pathway and disturbed B cell development.Disclosure of Interests:None declared

Background:Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare form of systemic vasculitis, which is characterized by bronchial asthma, hypereosinophilia, and systemic vasculitis. B-lymphocytes play a key role in EGPA as producers of IgE and anti-neutrophil cytoplasmic antibodies (ANCAs). Indeed, the neutrophils that are targeted by these antibodies are widely described as the mechanism of endothelial damage in this disease. On the other side, the therapeutic response to rituximab in EGPA patients provides evidence for a role of B-cells in the pathogenesis of EGPA. Therefore characterizing B cell subpopulations may help in understanding the disease and the treatment.Objectives:To characterize the peripheral B cell compartment in patients with EGPA and to analyze the in vivo potential of B lymphocytes to class-switch to IgE and to assess in vitro the differentiation potential of naïve B cells of EGPA patients into IgE-secreting plasmablasts.Methods:Clinical characteristics of the patients, including organ involvement and treatment regimen were evaluated. Laboratory work-up included ANCA-status, eosinophils, IgE, IgG, IgA, IgM, and peripheral CD19+B-cell count. For immunophenotyping isolated PBMCs were stained with monoclonal or polyclonal antibodies and B cells were classified into: naïve, marginal zone, class-switched memory B cells, unconventional memory B cells, transitional and plasmablasts. Furthermore, the expression of IgG+ and subclasses IgG1-4, IgA+, IgE+B cells, BAFFR and TACI was quantified. For in vitro differentiation assays magnetically isolated B lymphocytes from EGPA patients and age-matched healthy controls were stimulated with CD40L, IL-21 and IL-4. Starting the culture with equal number of B cells, the absolute number of plasmablasts, and IgE class switched cells after 9 days was determined by counting the events in the CD27highCD38high gate or the IgG/A/D-IgE+gate by flow cytometry. IgE secretion in the supernatant was measured by ELISA. JAK-STAT signalling pathway was analyzed in response to IL-4 and IL-21 stimulation and phosphorylation of STAT5 and 6 measured by flow cytometry.Results:34 patients with EGPA diagnosed according to ACR and CHC-criteria were included into the study. Ten of these patients were analysed separately because they received rituximab therapy. Peripheral B cell numbers in EGPA patients were markedly diminished. B cell subpopulation phenotyping showed in average 57.9% naïve B cells, 12.5 % marginal zone like B cells and 19.2% switched memory B cells. Plasmablasts constituted in average 1.15% of the peripheral B cell compartment, transitional B cells 2.0%. Interestingly, the expression of BAFF receptor and TACI in the memory B cell subset was significantly decreased in EGPA patients when compared with healthy donors. In vitro assays of isolated B cells from EGPA patients demonstrated an increased proportion of IgE-class-switched B cells after 9 days of culture under IL4 stimulation compared with controls. However, no differences were observed in the phosphorylation of STAT5 and STAT6 after stimulation with IL-4 or IL-21.Conclusion:In the EGPA-patients we observed markedly diminished B-cells despite of normal lymphocyte counts. B cells showed a reduced expression of BAFF-R and TACI. Class switch to IgE is enhanced in EGPA patients.Disclosure of Interests:None declared

The maintenance of B cell homeostasis requires a tight control of B cell generation, survival, activation, and maturation. In lymphocytes upon activation, increased sensitivity to apoptotic signals helps controlling differentiation and proliferation. The death receptor Fas is important in this context because genetic Fas mutations in humans lead to an autoimmune lymphoproliferative syndrome that is similar to lymphoproliferation observed in Fas-deficient mice. In contrast, the physiological role of TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs) in humans has been poorly studied so far. Indeed, most studies have focused on tumor cell lines and on mouse models whose results are difficult to transpose to primary human B cells. In the present work, the expression of apoptosis-inducing TRAIL-R1 and TRAIL-R2 and of the decoy receptors TRAIL-R3 and TRAIL-R4 was systematically studied in all developmental stages of peripheral B cells isolated from the blood and secondary lymphoid organs. Expression of TRAIL-Rs is modulated along development, with highest levels observed in germinal center B cells. In addition, T-dependent and T-independent signals elicited induction of TRAIL-Rs with distinct kinetics, which differed among B cell subpopulations: switched memory cells rapidly upregulated TRAIL-R1 and -2 upon activation while naïve B cells only reached similar expression levels at later time points in culture. Increased expression of TRAIL-R1 and -2 coincided with a caspase-3-dependent sensitivity to TRAIL-induced apoptosis in activated B cells but not in freshly isolated resting B cells. Finally, both TRAIL-R1 and TRAIL-R2 could signal actively and both contributed to TRAIL-induced apoptosis. In conclusion, this study provides a systematic analysis of the expression of TRAIL-Rs in human primary B cells and of their capacity to signal and induce apoptosis. This dataset forms a basis to further study and understand the dysregulation of TRAIL-Rs and TRAIL expression observed in autoimmune diseases. Additionally, it will be important to foresee potential bystander immunomodulation when TRAIL-R agonists are used in cancer treatment.

While in patients with rheumatoid arthritis B-cell repopulation starts within 9 months after rituximab (RTX) therapy, a delayed B-cell repopulation was reported in some RTX-treated patients with ANCA-associated vasculitides (AAV). To date, the frequency of AAV patients with impaired peripheral B-cell regeneration and the mechanisms leading to the constricted regenerative capacity are unknown. We analyzed the B-cell repopulation kinetic in 37 AAV patients treated with RTX followed by maintenance immunosuppressants. We report on serum concentrations of the B-cell-activating factor BAFF, immunoglobulins and B-cell subpopulations in patients that relapsed after RTX. B-cells were re-detectable in only one patient within 9 months after RTX. In 14 patients (41%), B-cell repopulation started later, after a mean observation time of 21 months. Only seven of these patients had detectable B-cells within the first year after RTX. Twenty patients (59%) had no B-cell reconstitution within the observation period. BAFF was increased in RTX-treated AAV patients compared to healthy controls and correlated inversely with peripheral B-cell numbers, IgG- and IgA concentrations. Immunoglobulin concentrations declined significantly after RTX and the IgG concentration correlated with B-cell numbers. Thirteen patients relapsed after RTX. Relapses occurred exclusively either after B-cell reconstitution had started or were accompanied by rising ANCA titres. In relapsed patients, the B-lymphocyte compartment consisted mainly of switched memory B-cells. Our data indicate that RTX treatment can induce secondary immunodeficiency in AAV, with hypogammaglobulinemia and long-lasting B-lymphopenia. Further studies are needed to define the pathophysiology of the impaired B-cell development in RTX-treated AAV patients.

B-cells are pivotal to the pathogenesis of rheumatoid arthritis and tofacitinib, a JAK inhibitor, is effective and safe in its treatment. Tofacitinib interferes with signal transduction via cytokine receptors using the common γ-chain. Despite extensive data on T-lymphocytes, the impact of tofacitinib on B-lymphocytes is poorly understood. In this study we assessed the effect of tofacitinib on B-lymphocyte differentiation and function. Tofacitinib treatment strongly impaired in vitro plasmablast development, immunoglobulin secretion and induction of B-cell fate determining transcription factors, Blimp - 1 , Xbp - 1 , and IRF - 4 , in naive B-cells. Interestingly, class switch and activation-induced cytidine deaminase ( AICDA ) induction was only slightly reduced in activated naive B-cells. The effect of tofacitinib on plasmablast formation, immunoglobulin secretion and proliferation was less profound, when peripheral blood B-cells, including not only naive but also memory B-cells, were stimulated. In line with these in vitro results, the relative distribution of B-cell populations remained stable in tofacitinib treated patients. Nevertheless, a temporary increase in absolute B-cell numbers was observed 6–8 weeks after start of treatment. In addition, B-cells isolated from tofacitinib treated patients responded rapidly to in vitro activation. We demonstrate that tofacitinib has a direct impact on human naive B-lymphocytes, independently from its effect on T-lymphocytes, by impairing their development into plasmablasts and immunoglobulin secretion. The major effect of tofacitinib on naive B-lymphocyte development points to the potential inability of tofacitinib-treated patients to respond to novel antigens, and suggests planning vaccination strategies prior to tofacitinib treatment.

PubMedID: 25724123 Background Mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency (CID) also classified as autosomal recessive (AR) hyper-IgE syndrome (HIES). Recognizing patients with CID/HIES is of clinical importance because of the difference in prognosis and management. Objectives We sought to define the clinical features that distinguish DOCK8 deficiency from other forms of HIES and CIDs, study the mutational spectrum of DOCK8 deficiency, and report on the frequency of specific clinical findings. Methods Eighty-two patients from 60 families with CID and the phenotype of AR-HIES with (64 patients) and without (18 patients) DOCK8 mutations were studied. Support vector machines were used to compare clinical data from 35 patients with DOCK8 deficiency with those from 10 patients with AR-HIES without a DOCK8 mutation and 64 patients with signal transducer and activator of transcription 3 (STAT3) mutations. Results DOCK8-deficient patients had median IgE levels of 5201 IU, high eosinophil levels of usually at least 800/µL (92% of patients), and low IgM levels (62%). About 20% of patients were lymphopenic, mainly because of low CD4+ and CD8+ T-cell counts. Fewer than half of the patients tested produced normal specific antibody responses to recall antigens. Bacterial (84%), viral (78%), and fungal (70%) infections were frequently observed. Skin abscesses (60%) and allergies (73%) were common clinical problems. In contrast to STAT3 deficiency, there were few pneumatoceles, bone fractures, and teething problems. Mortality was high (34%). A combination of 5 clinical features was helpful in distinguishing patients with DOCK8 mutations from those with STAT3 mutations. Conclusions DOCK8 deficiency is likely in patients with severe viral infections, allergies, and/or low IgM levels who have a diagnosis of HIES plus hypereosinophilia and upper respiratory tract infections in the absence of parenchymal lung abnormalities, retained primary teeth, and minimal trauma fractures. © 2015 American Academy of Allergy, Asthma & Immunology. National Institutes of Health: R01AI065617 National Institutes of Health: R21AI087627

Gastrointestinal manifestations are frequent in patients with common variable immunodeficiency (CVID), and some of the patients present with celiac-like features. Diagnosing celiac disease (CD) in CVID however is challenging, as autoantibody detection and histopathology of the small intestine cannot reliably discriminate between classic CD and a celiac-like disease in these individuals. For the development of classic gluten-sensitive CD a certain HLA haplotype involving the loci DQA1* and DQB1* and encoding two different HLA DQ heterodimers is the prerequisite. We aimed to determine the frequency of these haplotypes in CVID patients with suspected CD. Furthermore, we report on autoimmune manifestations and the lymphocyte phenotype in these patients. By retrospective analysis data on gastrointestinal symptoms, diet, concurrent autoimmune diseases, and routine laboratory values were collected. CVID patients were classified according to their B-cell phenotype. Expression of HLA-DQA1* and HLA-DQB1* alleles were determined by genetic analysis. Twenty out of 250 CVID patients presented with a clinical phenotype resembling celiac disease. Four (20 %) out of these CVID patients carried the CD-associated HLA DQ2.5 or DQ8 heterodimer, while HLA DQ2.5 was present in 100 % of a CD control cohort. Gluten-free diet (GFD) resulted in a clinical and histological response in two out of four patients with HLA high-risk alleles for CD. The response could not be assessed in the remaining two patients, as these patients did not adhere sufficiently long to GFD. The percentage of autoimmune manifestations other than CD was high (50 %) in CVID patients presenting with a CD-like enteropathy, and most of these patients had an expansion of B-cells with low expression of CD21 (CD21low B-cells). In CVID patients with suspected celiac disease typing of the HLA loci DQA1 and DQB1 can help to identify those that have a genetic susceptibility for CD. In CVID patients with a celiac-like phenotype but negative for CD-associated HLA-DQ markers, an autoimmune enteropathy (AIE) as part of an extended autoimmune dysregulation needs to be considered. This has important implications for further diagnostics and therapy of these patients.

The TNF family member protein BAFF/BLyS is essential for B cell survival and plays an important role in regulating class switch recombination as well as in the selection of autoreactive B cells. In humans, increased concentrations of soluble BAFF are found in different pathological conditions, which may be as diverse as autoimmune diseases, B cell malignancies, and primary Ab deficiencies (PAD). Because the mechanisms that regulate BAFF levels are not well understood, we newly developed a set of mAbs against human BAFF to study the parameters that determine the concentrations of soluble BAFF in circulation. Patients with PAD, including severe functional B cell defects such as BTK, BAFF-R, or TACI deficiency, were found to have higher BAFF levels than asplenic individuals, patients after anti-CD20 B cell depletion, chronic lymphocytic leukemia patients, or healthy donors. In a comparable manner, mice constitutively expressing human BAFF were found to have higher concentrations of BAFF in the absence than in the presence of B cells. Therefore, our data strongly suggest that BAFF steady-state concentrations mainly depend on the number of B cells as well as on the expression of BAFF-binding receptors. Because most patients with PAD have high levels of circulating BAFF, the increase in BAFF concentrations cannot compensate defects in B cell development and function.

Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21low B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients’ B cells, including normal phospholipase Cγ2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21low B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21low B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy.

Background Common variable immunodeficiency (CVID) comprises a heterogeneous group of primary antibody deficiencies with complex clinical and immunological phenotypes. The recent discovery that some CVID patients show monogenic defects in the genes encoding ICOS, TACI or CD19 prompted us to investigate several functional candidate genes in individuals with CVID. Results The exonic, protein coding regions of the genes encoding: APRIL, BCMA, IL10, IL10Rα, IL10Rβ, IL21, IL21R, and CCL18, were analyzed primarily in familial CVID cases, who showed evidence of genetic linkage to the respective candidate gene loci and CVID families with a recessive pattern of inheritance. Two novel SNPs were identified in exon 5 and exon 8 of the IL21R gene, which segregated with the disease phenotype in one CVID family. Eleven additional SNPs in the genes encoding BCMA, APRIL, IL10, IL10Rα, IL21 and IL21R were observed at similar frequencies as in healthy donors. Conclusion We were unable to identify obvious disease causing mutations in the protein coding regions of the analyzed genes in the studied cohort.

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3' untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

CD20-specific antibodies show remarkable therapeutic efficacy in B-cell malignancy and autoimmune diseases, but due to the occurrence of a significant treatment resistance, a critical, unmet need for improving B-cell-depleting approaches remains. A CD20 transcript variant (D393-CD20) appears to be associated with Rituximab treatment resistance in malignant B cells, but is lacking in patients with autoimmune dermatoses as shown by Gamonet et al. While CD20-specific factors certainly play a major role in the pathogenesis of Rituximab treatment resistance, the D393-CD2 transcript may greatly facilitate the development of clinical markers for monitoring the therapy in B-cell malignancies and (auto)antibody-mediated diseases. Its association with systemic autoimmune diseases should be therefore explored in further studies.

Background The dynamics of B cell reconstitution in relapsing or refractory ANCA-associated vasculitides (AAV) treated with rituximab (RTX) followed by standard immunosuppressants are not clear. While in patients with rheumatoid arthritis B cell repopulation generally starts 6 to 9 months after RTX therapy, an impaired early B-lymphocyte development resulting in a protracted peripheral B cell repopulation was reported recently in some AAV patients after B cell depletion with RTX. Objectives This is of clinical importance as the relapse rates in AAV after discontinuation of RTX therapy are high, and there are no data on safety and efficacy of RTX retreatment in persistently B cell depleted patients. Furthermore, there are no defined parameters that could guide RTX retreatment. To date the frequency of AAV patients with impaired peripheral B cell regeneration and the mechanism leading to the constricted regenerative capacity of the B cell compartment are unknown. Methods We analysed in a single center cohort comprising 125 patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) the B cell repopulation kinetics in 37 patients treated for relapsing or refractory disease with RTX followed by maintenance immunosuppressants in most patients. Furthermore, we report on serum concentrations of the B cell-activating factor of the TNF family (BAFF, BLyS) and immunoglobulin concentrations as well as on peripheral B cell subpopulations in a subgroup of patients that relapsed after RTX treatment. Results B cells were re-detectable in only one patient within the 9 months period after RTX treatment. After a mean observation time of 21 months 14 patients (41%) had started repopulating the peripheral B cell compartment, with 7 of the 14 patients having re-detectable B cells within the first year after RTX treatment. In 20 patients (59%) no repopulation occurred within the observation period. Eight of these patients were observed for more than 24 months without a starting B cell repopulation. BAFF concentrations in serum of AAV patients after RTX treatment were increased compared to healthy controls and correlated inversely with peripheral B cell numbers, IgG- and IgA concentrations. Serum immunoglobulin concentrations declined significantly after RTX treatment, and serum IgG concentrations correlated with peripheral B cell numbers after RTX therapy. 13 patients relapsed after RTX treatment. All of the 13 relapses either occurred after beginning B cell reconstitution or were accompanied by rising ANCA titres. In patients that relapsed after RTX the B lymphocyte compartment consisted mainly of switched memory B cells. Conclusions In summary, in our cohort of GPA and MPA patients treated with RTX followed by a standard maintenance immunosuppressive therapy a high proportion of patients had a long-lasting B cell depletion. Retreatment with RTX in long-term B cell depleted patients warrants close monitoring for adverse events and especially infectious complications and monitoring of serum immunoglobulin. Relapses after RTX treatment either occurred after beginning B cell reconstitution or were accompanied by rising ANCA titres. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4213

Background B-lymphocytes are involved in the pathogenesis of ANCA-associated vasculitides (AAVs), but the knowledge about alterations in peripheral blood B-lymphocytes of AAV patients and the changes in B-lymphocyte subsets after immunosuppressive therapy is sparse. Alterations in B-cell subpopulations can shed light on AAVs pathogenesis and may guide treatment protocols. Immunosuppressive drugs like cyclophosphamide, azathioprine, and methotrexate, which constitute the prerequisite of a successful AAV therapy, may exert their beneficial effects by their influence on peripheral B-cell homeostasis and function. Using surface staining for CD19, CD20, CD27, IgM, IgD, CD38, and CD21, transitional B-cells, marginal zone B-cells, naive B-cells, class-switched memory B-cells (smBs), and plasmablasts can be identified. Objectives To assess the effect of immunosuppressive therapies on peripheral B cell homeostasis and to investigate B cell repopulation after therapy with rituximab in patients with AAV. Methods Staining and flow cytometric analysisof B cells was performed using a FACS Canto II. 5000 to 10000 B-cells were analyzed. Results The B-cell compartment was analysed in 61 AAV patients. After immunosuppressive treatment B-lymphocytes and marginal zone B-cells (MZB) were persistently lowered. Low transitional B-cells (TR) indicated an impaired early B-lymphocyte development. B-lymphocytes and MZB were even decreased in patients with stable disease without immunosuppressive therapy. In untreated AAV patients the functionally exhausted CD21low-B-cells was expanded indicating an ongoing immunostimulatory process. Different from other autoimmune diseases like SLE plasmablasts were only slightly increased, showed no correlation to disease activity, and were efficiently targeted by immunosuppression. B-lymphocytes, MZB, TR, and plasmablasts correlated with immunoglobulin concentrations. Seven patients were analysed for B-cell repopulation after RTX treatment. B-cell repopulation was analysed by measuring total B-cell numbers and transitional B-cells. Time from RTX to B-cell analysis ranged from 7 to 42 months in these patients. In 3 patients B-cell numbers were below 0.2% impeding a further detection of CD19 + CD38 + IgM high transitional B-cells because of paucity of events in the CD19 gate. In two of these patients time from RTX application to B-cell analysis was less than 12 months, but in one patient the absence of B-cells was observed 42 months after RTX therapy indicating a long-lasting effect on the B-cell compartment. In a further patient 0.17% transitional B-cells were measured, indicating an insufficient B-cell regeneration. Only two patients had moderately increased percentages of transitional B-cells, indicating a beginning recovery of the B-cell compartment 18 and 19 months after RTX, but absolute numbers of transitional B-cells were still low in these patients. Conclusions Immunosuppression (IS) disturbs the B-cell compartment in AAV. Marginal zone B-cells (MZB) and transitional B-cells (TR) are decreased. Low MZB correlate with IgM levels. TR are reduced in AAV after immunosuppressive therapy, indicating an impaired early B-cell development. The reoccurrence of TR in AAV patients after RTX treatment is delayed. CD21 low B cells are expanded in AAVs. Disclosure of Interest None Declared

We have investigated the molecular etiology of autosomal recessive congenital neutropenia in a pedigree of 15 members with four affected children. Combining genome wide linkage analysis and a transcriptional profiling approach, we identified a homozygous mutation in the 3′-UTR of p14 (MAPBPIP), a recently identified adaptor molecule controlling late endosomal signaling by the MP1-MAPK complex. The mutation segregated with the disease, and neutrophils from all patients showed decreased RNA and protein levels of p14. To further assess the significance of this point mutation, we designed firefly luciferase reporter plasmids carrying either wildtype or mutated 3′-UTRs and cotransfected these constructs together with renilla luciferase expression plasmids into CHO cells. The specific luciferase activity was consistently decreased in the mutated constructs, suggesting that the mutated 3′-UTR is associated with decreased RNA levels in a heterologous system. Mechanistically, we excluded aberrant splicing and aberrant polyadenylation as a cause for decreased RNA levels. However, an increased ratio of unprocessed to processed nuclear RNA in transfected cells suggests a defect in RNA processing. Decreased levels of p14 lead to an abnormal configuration of lysosomes in granulocytes as shown by transmission electron microscopy. Whereas phagocytosis capacity is normal, killing of pathogenic bacteria is reduced in p14-deficient granulocytes. This defect is reversed upon retroviral p14 gene transfer into hematopoietic stem cells and subsequent differentiation into neutrophils in vitro. In summary, we provide evidence that decreased levels of p14 cause neutropenia. Furthermore, our report extends the small list of hereditary diseases associated with 3′-UTR mutations.