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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.

This study investigated the hypothesis that high-resolution MRI can reveal mural inflammatory changes of the superficial temporal artery in giant cell arteritis (GCA).MRI of the temporal artery of 20 patients with suspected GCA was performed on a 1.5-T scanner using a dedicated eight-element phased-array head coil. Contrast-enhanced multislice T1-weighted spin-echo sequences were acquired perpendicular to the orientation of the vessel, with a submillimeter spatial resolution of 0.2 x 0.3 mm and a slice thickness of 3 mm. Mural thickness and lumen diameter of the temporal artery were measured, and mural contrast enhancement was graded on a four-point scale by two radiologists. For all patients, the MRI results were compared with the findings of clinical examination and laboratory tests. In addition, biopsy samples of the temporal artery were taken from 16 of these patients.MRI sharply demonstrated the superficial temporal artery, allowing an evaluation of its lumen and wall. Seventeen patients were GCA-positive according to criteria of the American College of Rheumatology. Of these 17, 16 had true-positive MRI findings and one had false-negative MRI findings. The 3 patients who were GCA-negative according to the American College of Rheumatology criteria had true-negative MRI findings. The mean thickness of the vessel wall and the lumen diameter were 0.88 +/- 0.23 mm and 0.78 +/- 0.29 mm, respectively, in GCA-positive patients and 0.57 +/- 0.25 mm and 0.7 +/- 0.1 mm, respectively, in GCA-negative patients.High-resolution contrast-enhanced MRI of the temporal artery allowed visualization of the temporal artery and evaluation of possible inflammation of the vessel wall. Our initial results with this noninvasive technique agreed well with histologic results and with the clinical criteria of the American College of Rheumatology.

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

Objectives 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. Methods 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. Results 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). Conclusions 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.

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.