Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)1,2, conferring a predisposition to life-threatening COVID-19 pneumonia3. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52LOF/IκBδGOF). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52LOF/IκBδLOF) or gain-of-function of p52 (hereafter, p52GOF/IκBδLOF). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.

Combined Immunodeficiency Caused by a Novel Nonsense Mutation in LCK.

Mutations affecting T-cell receptor (TCR) signaling typically cause combined immunodeficiency (CID) due to varying degrees of disturbed T-cell homeostasis and differentiation. Here, we describe two cousins with CID due to a novel nonsense mutation in LCK and investigate the effect of this novel nonsense mutation on TCR signaling, T-cell function, and differentiation. Patients underwent clinical, genetic, and immunological investigations. The effect was addressed in primary cells and LCK-deficient T-cell lines after expression of mutated LCK. RESULTS: Both patients primarily presented with infections in early infancy. The LCK mutation led to reduced expression of a truncated LCK protein lacking a substantial part of the kinase domain and two critical regulatory tyrosine residues. T cells were oligoclonal, and especially naïve CD4 and CD8 T-cell counts were reduced, but regulatory and memory including circulating follicular helper T cells were less severely affected. A diagnostic hallmark of this immunodeficiency is the reduced surface expression of CD4. Despite severely impaired TCR signaling mTOR activation was partially preserved in patients' T cells. LCK-deficient T-cell lines reconstituted with mutant LCK corroborated partially preserved signaling. Despite detectable differentiation of memory and effector T cells, their function was severely disturbed. NK cell cytotoxicity was unaffected. Residual TCR signaling in LCK deficiency allows for reduced, but detectable T-cell differentiation, while T-cell function is severely disturbed. Our findings expand the previous report on one single patient on the central role of LCK in human T-cell development and function.

Interferon-Driven Immune Dysregulation in Common Variable Immunodeficiency-Associated Villous Atrophy and Norovirus Infection.

PURPOSE: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. METHODS: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. RESULTS: VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. CONCLUSIONS: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.

Naive- and Memory-like CD21low B Cell Subsets Share Core Phenotypic and Signaling Characteristics in Systemic Autoimmune Disorders.

An expansion of CD21low B cells has been described in a variety of diseases associated with persistent immune stimulation as in chronic infection, immunodeficiency, or autoimmunity. Different developmental stages of CD21low B cells have been highlighted in specific diseases; however, a systematic comparison of distribution, phenotype, and signaling capacity of these populations has not yet been performed to delineate the pivotal character of this unusual B cell population. Screening of more than 200 patients with autoimmune disease demonstrated that the prevalence of patients with expanded CD21low B cells varies between diseases. The expansion was frequent in patients with systemic lupus erythematosus, in which it correlated to relative B cell lymphopenia and duration of disease. Different proportions of distinct developmental stages of CD21low B cells co-occur in nearly all patients with autoimmune disease. Although in most patients, naive-like and CD27- switched memory B cells were the most prominent CD21low subpopulations, there was no detectable association of the pattern with the underlying disease. Despite their distinct developmental stage, all CD21low B cells share a common core phenotype including the increased expression of inhibitory receptors, associated with an elevated constitutive phosphorylation of proximal signaling molecules downstream of the BCR but impaired Ca2+ mobilization and NF-κB activation after BCR stimulation. Further, this was accompanied by impaired upregulation of CD69, although CD86 upregulation was preserved. Beyond maturation-associated differences, the common core characteristics of all CD21low B cell populations suggests either a common ancestry or a shared sustained imprint by the environment they originated in.

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.

Genomic Spectrum and Phenotypic Heterogeneity of Human IL-21 Receptor Deficiency.

Biallelic inactivating mutations in IL21R causes a combined immunodeficiency that is often complicated by cryptosporidium infections. While eight IL-21R-deficient patients have been reported previously, the natural course, immune characteristics of disease, and response to hematopoietic stem cell transplantation (HSCT) remain to be comprehensively examined. In our study, we have collected clinical histories of 13 patients with IL-21R deficiency from eight families across seven centers worldwide, including five novel patients identified by exome or NGS panel sequencing. Eight unique mutations in IL21R were identified in these patients, including two novel mutations. Median age at disease onset was 2.5 years (0.5-7 years). The main clinical manifestations were recurrent bacterial (84.6%), fungal (46.2%), and viral (38.5%) infections; cryptosporidiosis-associated cholangitis (46.2%); and asthma (23.1%). Inflammatory skin diseases (15.3%) and recurrent anaphylaxis (7.9%) constitute novel phenotypes of this combined immunodeficiency. Most patients exhibited hypogammaglobulinemia and reduced proportions of memory B cells, circulating T follicular helper cells, MAIT cells and terminally differentiated NK cells. However, IgE levels were elevated in 50% of IL-21R-deficient patients. Overall survival following HSCT (6 patients, mean follow-up 1.8 year) was 33.3%, with pre-existing organ damage constituting a negative prognostic factor. Mortality of non-transplanted patients (n = 7) was 57.1%. Our detailed analysis of the largest cohort of IL-21R-deficient patients to date provides in-depth clinical, immunological and immunophenotypic features of these patients, thereby establishing critical non-redundant functions of IL-21/IL-21R signaling in lymphocyte differentiation, humoral immunity and host defense against infection, and mechanisms of disease pathogenesis due to IL-21R deficiency. Outcome following HSCT depends on prior chronic infections and organ damage, which should thus be considered as early as possible following molecular diagnosis.

Impaired polysaccharide responsiveness without agammaglobulinaemia in three patients with hypomorphic mutations in Bruton Tyrosine Kinase-No detection by newborn screening for primary immunodeficiencies.

Hypomorphic mutations in the gene encoding Bruton tyrosine kinase (BTK) may result in milder phenotypes and delayed diagnosis of B-cell related immunodeficiencies due to residual BTK function. Newborn screening for kappa-deleting-recombination-excision circles (KRECs) reliably identifies classical X-linked agammaglobulinaemia (XLA) patients with profound B-cell lymphopenia at birth but has not been evaluated in patients with residual BTK function. We aimed to evaluate clinical findings, BTK function and KREC copy numbers in three patients with BTK mutations presenting with impaired polysaccharide responsiveness without agammaglobulinaemia. One patient had an invasive pneumococcal infection at the age of 4 years. All three patients (two brothers) had visible tonsils, normal to slightly decreased immunoglobulin G levels, undetectable pneumococcal antibodies despite pneumococcal conjugate vaccinations, no antibody response after a diagnostic polysaccharide vaccination as well as profound B-cell lymphopenia with residual B-cell differentiation. BTK mutations were identified by Sanger sequencing. BTK staining and phosphorylation assays were performed on peripheral B cells. KREC copy numbers were determined from dried blood spots obtained within the first week of life as well as once at the age of 8, 6 and 3 years, respectively. BTK staining showed residual protein expression. Also, residual BTK activity could be demonstrated. KREC copy numbers from dried blood spots were above the threshold set for detection of patients with profound B-cell lymphopenia. Male patients with impaired polysaccharide responsiveness should be evaluated for B-cell lymphopenia followed by BTK analyses irrespective of immunoglobulin levels or tonsil size.

High SYK Expression Drives Constitutive Activation of CD21low B Cells.

Human CD21low B cells present with an activated phenotype and accumulate in distinct disorders connected with chronic immune stimulation. Signaling studies had revealed an increased basal phosphorylation of spleen tyrosine kinase (SYK) and phospholipase Cγ2. Additional BCR stimulation of these constitutively active cells, however, led to reduced activation of these signaling molecules and subsequently NF-κB and Ca2+ activation. In this article, we demonstrate that high SYK expression is a common feature of CD21low B cells independent of the underlying disorder, and that this high expression is sufficient to drive constitutive phosphorylation of SYK and its immediate targets Bruton's tyrosine kinase and phospholipase Cγ2. Inhibition of SYK activity eliminated features of the constitutive activation in these cells and partly restored BCR signaling. High SYK expression is especially induced by CpG or CD40L in combination with IL-21, but not BCR stimulation, suggesting the importance of the immune-stimulatory context for the induction of this B cell phenotype. In summary, high SYK expression is a common feature of human CD21low B cells and presumably results from chronic activation in inflammatory environments present in a subgroup of patients with heterogeneous disorders like chronic infection, autoimmunity, and immunodeficiency. High SYK expression by itself drives the constitutive activation observed in these B cells, which in turn may contribute to the hyporesponsiveness upon BCR stimulation. Given the high prevalence of autoreactive clones among CD21low B cells in autoimmune disorders, the dominant role of SYK in CD21low B cells may provide a new option for therapeutic interventions in patients with expanded CD21low B cells and humoral autoimmunity.

The TH1 phenotype of follicular helper T cells indicates an IFN-γ-associated immune dysregulation in patients with CD21low common variable immunodeficiency.

BACKGROUND: A subgroup of patients with common variable immunodeficiency (CVID) experience immune dysregulation manifesting as autoimmunity, lymphoproliferation, and organ inflammation and thereby increasing morbidity and mortality. Therefore treatment of these complications demands a deeper comprehension of their cause and pathophysiology. OBJECTIVES: On the basis of the identification of an interferon signature in patients with CVID with secondary complications and a skewed follicular helper T-cell differentiation in defined monogenic immunodeficiencies, we sought to determine the profile of CD4 memory T cells in blood and secondary lymphatic tissues of these patients. METHODS: We quantified TH1/TH2/TH17 CD4 memory T cells in blood and lymph nodes of patients with CVID using flow cytometry, analyzed their function, and correlated all findings to the burden of immune dysregulation. RESULTS: Patients with CVID with immune dysregulation had a skewed memory CD4 T-cell differentiation toward a CXCR3+CCR6- TH1 phenotype both in blood and lymph nodes. Consistent with our phenotypic findings, we observed a higher IFN-γ production in peripheral CD4 memory T cells and lymph node-derived follicular helper T cells of patients with CVID compared with those of healthy control subjects. Increased IFN-γ production was accompanied by a poor germinal center output, an accumulation of T-box transcription factor (T-bet)+ B cells in lymph nodes, and an accumulation of T-bet+CD21low B cells in peripheral blood of affected patients. CONCLUSION: Identification of excessive IFN-γ production by blood and lymph node-derived T cells of patients with CVID with immune dysregulation will offer new therapeutic avenues for this subgroup. CD21low B cells might serve as a marker of this IFN-γ-associated dysregulation.

Haploinsufficiency of the NF-κB1 Subunit p50 in Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID), characterized by recurrent infections, is the most prevalent symptomatic antibody deficiency. In ∼90% of CVID-affected individuals, no genetic cause of the disease has been identified. In a Dutch-Australian CVID-affected family, we identified a NFKB1 heterozygous splice-donor-site mutation (c.730+4A>G), causing in-frame skipping of exon 8. NFKB1 encodes the transcription-factor precursor p105, which is processed to p50 (canonical NF-κB pathway). The altered protein bearing an internal deletion (p.Asp191_Lys244delinsGlu; p105ΔEx8) is degraded, but is not processed to p50ΔEx8. Altered NF-κB1 proteins were also undetectable in a German CVID-affected family with a heterozygous in-frame exon 9 skipping mutation (c.835+2T>G) and in a CVID-affected family from New Zealand with a heterozygous frameshift mutation (c.465dupA) in exon 7. Given that residual p105 and p50­translated from the non-mutated alleles­were normal, and altered p50 proteins were absent, we conclude that the CVID phenotype in these families is caused by NF-κB1 p50 haploinsufficiency.

T+ NK+ IL-2 Receptor γ Chain Mutation: a Challenging Diagnosis of Atypical Severe Combined Immunodeficiency.

PURPOSE: All reported patients with hypomorphic X-linked severe combined immunodeficiency (X-SCID) due to c.664C>T (p.R222C) mutations in the gene (IL2RG) encoding the common γ chain (γc) have presented with opportunistic infections within the first year of life, despite the presence of nearly normal NK and T cell numbers. Reporting five children of one extended family with hemizygous mutations in IL2RG, we explore potential diagnostic clues and extend our comprehension of the functional impact of this mutation. METHODS: Whole exome sequencing (WES); detailed immune phenotyping; cytokine-induced STAT phosphorylation; B, T, and NK cell activation; and quantification of sjTRECs in five Arab children with c.664C>T (p.R222C) IL2RG mutation. RESULTS: The mean age at clinical presentation with respiratory tract infection or diarrhea was 6.8 (range: 2-12) months. None of the children presented with opportunistic infections. Diagnostic clues were early onset in the first year of life, and a suggestive family history associated with reduced naïve CD4 T cells and absent switched memory B cells. Number and phenotype of NK cells and innate-like lymphocytes were normal. The diagnosis was made by WES and corroborated by absent STAT phosphorylation and reduced functional response after IL-2 and IL-21 stimulation. Four patients underwent successful hematopoietic stem cell transplantation. CONCLUSIONS: As early diagnosis and treatment are important, a high index of suspicion in the diagnosis of c.664C>T (p.R222C) X-SCID is needed. This requires prompt genetic testing by next generation sequencing in order to avoid unnecessary delays in the definite diagnosis since immunological work up may not be discriminating. Assays directly testing cytokine signaling or cytokine-dependent functions are helpful in confirming the functional impact of the identified hypomorphic variants.

Disturbed canonical nuclear factor of κ light chain signaling in B cells of patients with common variable immunodeficiency.

BACKGROUND: Most patients with common variable immunodeficiency (CVID) present with severely reduced switched memory B-cell counts, and some display an increase of CD21low B-cell counts (CVID 21low), whereas others do not (CVID 21norm). Altered B-cell receptor (BCR) signaling might contribute to the defective memory formation observed in patients with CVID. OBJECTIVE: We sought to investigate canonical nuclear factor of κ light chain (NF-κB) signaling in B cells from patients with CVID as a central pathway in B-cell differentiation. METHODS: Degradation of inhibitor of κBα (IκBα) and p65 phosphorylation, nuclear translocation of p65, and regulation of target genes and cell function were investigated after different modes of B-cell stimulation. RESULTS: BCR-mediated canonical NF-κB signaling was impaired in all mature naive CVID-derived B cells. This impairment was more profound in naive B cells from CVID 21low patients than CVID 21norm patients and most pronounced in CD21low B cells. The signaling defect translated into reduced induction of Bcl-xL and IκBα, 2 bona fide target genes of the canonical NF-κB pathway. CD40 ligand- and Toll-like receptor 9-mediated signaling were less strongly altered. Signaling in CD21low B cells but not CD21+ B cells of patients with HIV was similarly affected. CONCLUSION: Combined with the previous description of disturbed Ca2+ signaling, the discovery of NF-κB signaling defects, especially in CVID 21low patients, suggests a broad underlying signaling defect affecting especially BCR-derived signals. Given the immune phenotype of monogenic defects affecting Ca2+ and NF-κB signaling, the latter is more likely to contribute to the humoral deficiency. The strongly disturbed BCR signaling of CD21low B cells is characteristic for this cell type and independent of the underlying disease.

Increase of circulating α4ß7+ conventional memory CD4 and regulatory T cells in patients with common variable immunodeficiency (CVID).

This study investigated whether circulating α4ß7+ expressing T cells could serve as a potential marker for gastrointestinal (GI) disease activity in patients with CVID. The analysis of α4ß7+ T cells in the peripheral blood of 36 patients and 22 healthy donors (HD) revealed increased percentages of α4ß7+ conventional memory CD4 T cells and Tregs, but not among CD8 T-cell populations in patients with CVID compared to HD. No differences between patients with and without chronic or acute GI symptoms were observed. EUROClass smB- and 21lo patients, had higher percentages of α4ß7+ memory CD4 T cells compared to HD and smB+ or 21norm patients, respectively. In summary, the detection of α4ß7+ T cells in the peripheral blood did not correlate with active or chronic gastrointestinal disease. The increase of these cells in smB- and 21lo patients adds another piece to the immune dysregulation observed in these patients.

STAT3 gain-of-function mutations associated with autoimmune lymphoproliferative syndrome like disease deregulate lymphocyte apoptosis and can be targeted by BH3 mimetic compounds.

Autoimmune lymphoproliferative syndrome (ALPS) is typically caused by mutations in genes of the extrinsic FAS mediated apoptotic pathway, but for about 30% of ALPS-like patients the genetic diagnosis is lacking. We analyzed 30 children with ALPS-like disease of unknown cause and identified two dominant gain-of-function mutations of the Signal Transducer And Activator Of Transcription 3 (STAT3, p.R278H, p.M394T) leading to increased transcriptional activity. Hyperactivity of STAT3, a known repressor of FAS, was associated with decreased FAS-mediated apoptosis, mimicking ALPS caused by FAS mutations. Expression of BCL2 family proteins, further targets of STAT3 and regulators of the intrinsic apoptotic pathway, was disturbed. Cells with hyperactive STAT3 were consequently more resistant to intrinsic apoptotic stimuli and STAT3 inhibition alleviated this effect. Importantly, STAT3-mutant cells were more sensitive to death induced by the BCL2-inhibitor ABT-737 indicating a dependence on anti-apoptotic BCL2 proteins and potential novel therapeutic options.

Preferential Reduction of Circulating Innate Lymphoid Cells Type 2 in Patients with Common Variable Immunodeficiency with Secondary Complications Is Part of a Broader Immune Dysregulation.

PURPOSE: Over a third of patients with common variable immunodeficiency (CVID) suffer from secondary complications like inflammatory organ disease, autoimmune manifestations, or lymphoproliferation contributing to increased morbidity and mortality in affected patients. Innate lymphoid cells (ILCs) have emerging roles in setting the milieu for physiological, but also pathological, immune responses and inflammation. We therefore sought to correlate the recently identified disturbed homeostasis of ILCs with alterations of the adaptive immune system in complex CVID patients (CVIDc). METHODS: We quantified peripheral blood ILC and T helper cell subsets of 58 CVID patients by flow cytometry and compared the results to the clinical and immunological phenotype. RESULTS: Total ILCs were significantly reduced in peripheral blood of CVIDc patients compared to healthy individuals, but not to CVID patients who suffered only from infections (CVIDio). This reduction was mainly due to a decrease in ILC2s, while ILC3s were relatively increased in CVIDc compared to CVIDio patients. This alteration in ILC phenotype was more prominent in patients with an expansion of CD21low B cells, but we could not detect an association of the altered ILC phenotype with a TH1-shift among circulating CD4 T cells, which was also prominent in CVIDc patients. CONCLUSION: We confirm a relative shift in ILCs of CVIDc patients towards ILC3s which was associated with the expansion of CD21low B cells, but not overtly with the relative expansion of TH1-like T cells. Given the relative abundance of TH1-like T cells compared to ILCs, these probably represent a more prominent source of the observed IFNγ-signature in CVIDc patients.

Deep intronic mis-splicing mutation in JAK3 gene underlies T-B+NK- severe combined immunodeficiency phenotype.

Severe combined immune deficiency (SCID) is a group of genetically heterogeneous diseases caused by an early block in T cell differentiation and present with life threatening infections, often within the first year of life. Janus kinase (JAK)3 gene mutations have been found to cause autosomal recessive T-B+ SCID phenotype. In this study we describe three patients with a novel deep intronic mis-splicing mutation in JAK3 as a cause of T-B+NK- SCID highlighting the need for careful evaluation of intronic regulatory elements of known genes associated with clearly defined clinical phenotypes. We present the cases and discuss the current literature.

Secondary Antibody Deficiency in Glucocorticoid Therapy Clearly Differs from Primary Antibody Deficiency.

PURPOSE: The aim of this study was to identify characteristics of hypogammaglobulinemia secondary to glucocorticoid therapy and their value in the differential diagnosis to primary forms of antibody deficiency. METHODS: We investigated prevalence and character of hypogammaglobulinemia in a cohort of 36 patients with giant cell arteritis (GCA) and polymyalgia rheumatica (PMR) on glucocorticoid therapy in comparison to a gender- and age-matched cohort of hospital controls. We therefore determined serum immunoglobulin levels as well as B- and T cell-subsets in the peripheral blood of all participants. In addition, prior serum immunoglobulin levels and clinical data of the GCA and PMR patients were extracted from the electronic patient data-base. RESULTS: 21/36 GCA/PMR patients on glucocorticoid treatment developed antibody deficiency. In 19 patients this included IgG and in 13 patients IgG was the only affected isotype. The reduction of IgG was persistent in nearly 50 % of these patients during the observed period. GCA/PMR patients had reduced circulating naive and transitional B cells (p = 0.0043 and p = 0.0002 respectively) while IgM, IgG and IgA memory B cells were preserved. Amongst T-cell subsets, we found a reduction of CD4 memory T cells (p < 0.0001), CD4 regulatory T cells (p = 0.0002) and few CD8 memory T-cell subtypes. CONCLUSION: Persistent humoral immunodeficiency occurs in about a quarter of GCA/PMR patients under glucocorticoid therapy. Because most patients have isolated IgG deficiency, preserved IgA production and class-switched memory B cells, by these markers this form of secondary hypogammaglobulinemia can be clearly distinguished from common variable immunodeficiency (CVID).

B-cell signaling in persistent polyclonal B lymphocytosis (PPBL).

Persistent polyclonal B lymphocytosis (PPBL) is a benign hematological disorder characterized by a selective expansion of circulating polyclonal marginal zone (MZ)-like B cells. Previous reports demonstrated that cases of PPBL showed poor activation, proliferation and survival of B cells in vitro, yet the underlying defect remains unknown. Here we report for the first time an attenuated activation of the canonical NF-κB (nuclear factor of kappa light polypeptide gene enhancer in B cells) and mitogen-activated protein kinase/extracellular signal-regulated kinase pathway after CD40 stimulation. This defect was selective, as alternative NF-κB signaling after CD40 stimulation and both B-cell receptor- and Toll-like receptor 9-mediated activation remained unaffected. Reduced canonical NF-κB activation resulted in decreased IκBα and CD40 expression in resting cells. In PPBL patients, expression of Bcl-xL in MZ-like B cells did not increase upon activation, consistent with the high apoptosis rates of PPBL-derived B cells that were observed in vitro. The B-cell phenotype of mice with selective knockouts of early components of the CD40 signaling pathway resembles PPBL, but sequencing corresponding genes in sorted MZ-like B cells of PPBL patients did not reveal relevant genetic alterations. Nevertheless, the frequently observed mutations in early signaling components of the NF-κB pathway in MZ lymphomas underline the relevance of our findings for the pathogenesis of PPBL.