Structural Activation of Pro-inflammatory Human Cytokine IL-23 by Cognate IL-23 Receptor Enables Recruitment of the Shared Receptor IL-12Rß1.

Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rß1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.

C-type lectin-like receptor LOX-1 promotes dendritic cell-mediated class-switched B cell responses.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a pattern-recognition receptor for a variety of endogenous and exogenous ligands. However, LOX-1 function in the host immune response is not fully understood. Here, we report that LOX-1 expressed on dendritic cells (DCs) and B cells promotes humoral responses. On B cells LOX-1 signaling upregulated CCR7, promoting cellular migration toward lymphoid tissues. LOX-1 signaling on DCs licensed the cells to promote B cell differentiation into class-switched plasmablasts and led to downregulation of chemokine receptor CXCR5 and upregulation of chemokine receptor CCR10 on plasmablasts, enabling their exit from germinal centers and migration toward local mucosa and skin. Finally, we found that targeting influenza hemagglutinin 1 (HA1) subunit to LOX-1 elicited HA1-specific protective antibody responses in rhesus macaques. Thus, LOX-1 expressed on B cells and DC cells has complementary functions to promote humoral immune responses.

Blood-based test for diagnosis and functional subtyping of familial Mediterranean fever.

BACKGROUND AND OBJECTIVE: Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease (AID) worldwide. The disease is caused by mutations in the MEFV gene encoding the inflammasome sensor Pyrin. Clinical diagnosis of FMF is complicated by overlap in symptoms with other diseases, and interpretation of genetic testing is confounded by the lack of a clear genotype-phenotype association for most of the 340 reported MEFV variants. In this study, the authors designed a functional assay and evaluated its potential in supporting FMF diagnosis. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with Pyrin-associated autoinflammation with an FMF phenotype (n=43) or with autoinflammatory features not compatible with FMF (n=8), 10 asymptomatic carriers and 48 healthy donors. Sera were obtained from patients with distinct AIDs (n=10), and whole blood from a subset of patients and controls. The clinical, demographic, molecular genetic factors and other characteristics of the patient population were assessed for their impact on the diagnostic test read-out. Interleukin (IL)-1ß and IL-18 levels were measured by Luminex assay. RESULTS: The ex vivo colchicine assay may be performed on whole blood or PBMC. The functional assay robustly segregated patients with FMF from healthy controls and patients with related clinical disorders. The diagnostic test distinguished patients with classical FMF mutations (M694V, M694I, M680I, R761H) from patients with other MEFV mutations and variants (K695R, P369S, R202Q, E148Q) that are considered benign or of uncertain clinical significance. CONCLUSION: The ex vivo colchicine assay may support diagnosis of FMF and functional subtyping of Pyrin-associated autoinflammation.

Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion.

Although a fraction of human blood memory CD4(+) T cells expresses chemokine (C-X-C motif) receptor 5 (CXCR5), their relationship to T follicular helper (Tfh) cells is not well established. Here we show that human blood CXCR5(+)CD4(+) T cells share functional properties with Tfh cells and appear to represent their circulating memory compartment. Blood CXCR5(+)CD4(+) T cells comprised three subsets: T helper 1 (Th1), Th2, and Th17 cells. Th2 and Th17 cells within CXCR5(+), but not within CXCR5(-), compartment efficiently induced naive B cells to produce immunoglobulins via interleukin-21 (IL-21). In contrast, Th1 cells from both CXCR5(+) and CXCR5(-) compartments lacked the capacity to help B cells. Patients with juvenile dermatomyositis, a systemic autoimmune disease, displayed a profound skewing of blood CXCR5(+) Th cell subsets toward Th2 and Th17 cells. Importantly, the skewing of subsets correlated with disease activity and frequency of blood plasmablasts. Collectively, our study suggests that an altered balance of Tfh cell subsets contributes to human autoimmunity.

Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.

Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1ß and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.

Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor.

BACKGROUND: Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFß-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS: Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS: ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFß1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS: Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

A T cell-dependent mechanism for the induction of human mucosal homing immunoglobulin A-secreting plasmablasts.

Mucosal immunoglobulin A (IgA) secreted by local plasma cells (PCs) is a critical component of mucosal immunity. Although IgA class switching can occur at mucosal sites, high-affinity PCs are optimally generated in germinal centers (GCs) in a T cell-dependent fashion. However, how CD4(+) helper T cells induce mucosal-homing IgA-PCs remains unclear. Here, we show that transforming growth factor beta1 (TGFbeta1) and interleukin 21 (IL-21), produced by follicular helper T cells (Tfh), synergized to generate abundant IgA-plasmablasts (PBs). In the presence of IL-21, TGFbeta1 promoted naive B cell proliferation and differentiation and overrode IL-21-induced IgG class switching in favor of IgA. Furthermore, TGFbeta1 and IL-21 downregulated CXCR5 while upregulating CCR10 on plasmablasts, enabling their exit from GCs and migration toward local mucosa. This was supported by the presence of CCR10(+)IgA(+)PBs in tonsil GCs. These findings show that Tfh contribute to mucosal IgA. Thus, mucosal vaccines should aim to induce robust Tfh responses.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1ß production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

House dust mite-driven asthma and allergen-specific T cells depend on B cells when the amount of inhaled allergen is limiting.

BACKGROUND: Allergic asthma is a CD4 TH2-lymphocyte driven disease characterized by airway hyperresponsiveness and eosinophilia. B cells can present antigens to CD4 T cells and produce IgE immunoglobulins that arm effector cells; however, mouse models are inconclusive on whether B cells are necessary for asthma development. OBJECTIVES: We sought to address the role of B cells in a house dust mite (HDM)-driven TH2-high asthma mouse model. METHODS: Wild-type and B cell-deficient muMT mice were sensitized and challenged through the airways with HDM extracts. The antigen-presenting capacities of B cells were studied by using new T-cell receptor transgenic 1-DER mice specific for the Der p 1 allergen. RESULTS: In vitro-activated B cells from HDM-exposed mice presented antigen to 1-DER T cells and induced a TH2 phenotype. In vivo B cells were dispensable for activation of naive 1-DER T cells but necessary for full expansion of primed 1-DER T cells. At high HDM challenge doses, B cells were not required for development of pulmonary asthmatic features yet contributed to TH2 expansion in the mediastinal lymph nodes but not in the lungs. When the amount of challenge allergen was decreased, muMT mice had reduced asthma features. Under these limiting conditions, B cells contributed also to expansion of TH2 effector cells in the lungs and central memory T cells in the mediastinal lymph nodes. CONCLUSION: B cells are a major part of the adaptive immune response to inhaled HDM allergen, particularly when the amount of inhaled allergen is low, by expanding allergen-specific T cells.

The immunophenotypic fingerprint of patients with primary antibody deficiencies is partially present in their asymptomatic first-degree relatives.

The etiology of primary antibody deficiencies is largely unknown. Beside rare monogenic forms, the majority of cases seem to have a more complex genetic basis. Whereas common variable immunodeficiency has been investigated in depth, there are only a few reports on milder primary antibody deficiencies such as idiopathic primary hypogammaglobulinemia and IgG subclass deficiency. We performed flow cytometric immunophenotyping in 33 patients with common variable immunodeficiency, 23 with idiopathic primary hypogammaglobulinemia and 21 with IgG subclass deficiency, as well as in 47 asymptomatic first-degree family members of patients and 101 unrelated healthy controls. All three groups of patients showed decreased memory B- and naïve T-cell subsets and decreased B-cell activating factor receptor expression. In contrast, circulating follicular helper T-cell frequency and expression of inducible T-cell co-stimulator and chemokine receptors were only significantly altered in patients with common variable immunodeficiency. Asymptomatic first-degree family members of patients demonstrated similar, albeit intermediate, alterations in naïve and memory B- and T-cell subsets. About 13% of asymptomatic relatives had an abnormal peripheral B-cell composition. Furthermore, asymptomatic relatives showed decreased levels of CD4+ recent thymic emigrants and increased central memory T cells. Serum IgG and IgM levels were also significantly lower in asymptomatic relatives than in healthy controls. We conclude that, in our cohort, the immunophenotypic landscape of primary antibody deficiencies comprises a spectrum, in which some alterations are shared between all primary antibody deficiencies whereas others are only associated with common variable immunodeficiency. Importantly, asymptomatic first-degree family members of patients were found to have an intermediate phenotype for peripheral B- and T-cell subsets.

IL-21 Is Increased in Nasal Polyposis and after Stimulation with Staphylococcus aureus Enterotoxin B.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is an inflammatory disease associated with lymphoid aggregates and local IgE production related to Staphylococcus aureus enterotoxins. T-follicular helper cells and their effector cytokine interleukin (IL)-21 play an important role in germinal center proliferation. METHODS: IL-21 was determined on the mRNA level by qPCR in nasal tissue of 3 groups of patients: control (n = 17), chronic rhinosinusitis without nasal polyposis (CRSsNP; n = 23), and CRSwNP (n = 35). The expression of IL-21 by CD4+ T cells was analyzed in tissue at baseline and after 24-h stimulation of tissue fragments with S. aureus enterotoxin B (SEB) using flow cytometry. Finally, human nasal IL-21+CXCR5+CD4+ T cells were isolated and coincubated with human blood naive B cells to investigate their functionality. RESULTS: IL-21 mRNA expression was increased in the CRSwNP group (p < 0.05) compared to the control group, and B-cell lymphoma-6 and B-lymphocyte-induced maturation protein-1 were upregulated in CRSwNP versus CRSsNP. Furthermore, SEB was able to increase IL-21 mRNA expression significantly (p < 0.01) in nasal polyps. Flow cytometry revealed that the source of IL-21 was predominantly CD4+ T cells and that IL-21+CD4+ T cells were significantly increased in polyp tissue and further increased after SEB stimulation. Finally, tissue CXCR5+CD4+ T cells derived from nasal polyp tissue were able to induce maturation of human naive B cells. CONCLUSIONS: IL-21- and IL-21-producing CD4+ T cells were increased in CRSwNP. In addition, SEB induced an increase in IL-21 and IL-21+CD4+ T cells, suggesting that S. aureus can modulate the function of Tfh cells in nasal polyps. We speculate that T-follicular helper cells and IL-21 are important in the pathophysiology of CRSwNP.

Genes associated with common variable immunodeficiency: one diagnosis to rule them all?

Common variable immunodeficiency (CVID) is a primary antibody deficiency characterised by hypogammaglobulinaemia, impaired production of specific antibodies after immunisation and increased susceptibility to infections. CVID shows a considerable phenotypical and genetic heterogeneity. In contrast to many other primary immunodeficiencies, monogenic forms count for only 2-10% of patients with CVID. Genes that have been implicated in monogenic CVID include ICOS, TNFRSF13B (TACI), TNFRSF13C (BAFF-R), TNFSF12 (TWEAK), CD19, CD81, CR2 (CD21), MS4A1 (CD20), TNFRSF7 (CD27), IL21, IL21R, LRBA, CTLA4, PRKCD, PLCG2, NFKB1, NFKB2, PIK3CD, PIK3R1, VAV1, RAC2, BLK, IKZF1 (IKAROS) and IRF2BP2 With the increasing number of disease genes identified in CVID, it has become clear that CVID is an umbrella diagnosis and that many of these genetic defects cause distinct disease entities. Moreover, there is accumulating evidence that at least a subgroup of patients with CVID has a complex rather than a monogenic inheritance. This review aims to discuss current knowledge regarding the molecular genetic basis of CVID with an emphasis on the relationship with the clinical and immunological phenotype.

Chronic and Invasive Fungal Infections in a Family with CARD9 Deficiency.

Chronic mucocutaneous or invasive fungal infections are generally the result of primary or secondary immune dysfunction. Patients with autosomal recessive CARD9 mutations are also predisposed to recurrent mucocutaneous and invasive fungal infections with Candida spp., dermatophytes (e.g., Trichophyton spp.) and phaeohyphomycetes (Exophiala spp., Phialophora verrucosa). We study a consanguineous family of Turkish origin in which three members present with distinct clinical phenotypes of chronic mucocutaneous and invasive fungal infections, ranging from chronic mucocutaneous candidiasis (CMC) in one patient, treatment-resistant cutaneous dermatophytosis and deep dermatophytosis in a second patient, to CMC with Candida encephalitis and endocrinopathy in a third patient. Two patients consented to genetic testing and were found to have a previously reported homozygous R70W CARD9 mutation. Circulating IL-17 and IL-22 producing T cells were decreased as was IL-6 and granulocyte/macrophage colony-stimulating factor (GM-CSF) secretion upon stimulation with Candida albicans. Patients with recurrent fungal infections in the absence of known immunodeficiencies should be analyzed for CARD9 gene mutations as the cause of fungal infection predisposition.

Brucella ß 1,2 cyclic glucan is an activator of human and mouse dendritic cells.

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella ß 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella ß 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella ß 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.

Dual-targeting CD33/CD123 NANOBODY T-cell engager with potent anti-AML activity and good safety profile.

ABSTRACT: Novel therapies are needed for effective treatment of acute myeloid leukemia (AML). Relapse is common and salvage treatment with cytotoxic chemotherapy is rarely curative. CD123 and CD33, 2 clinically validated targets in AML, are jointly expressed on blasts and leukemic stem cells in >95% of patients with AML. However, their expression is heterogenous between subclones and between patients, which may affect the efficacy of single-targeting agents in certain patient populations. We present here a dual-targeting CD33/CD123 NANOBODY T-cell engager (CD33/CD123-TCE) that was designed to decrease the risk of relapse from possible single antigen-negative clones and to increase coverage within and across patients. CD33/CD123-TCE killed AML tumor cells expressing 1 or both antigens in vitro. Compared with single-targeting control compounds, CD33/CD123-TCE conferred equal or better ex vivo killing of AML blasts in most primary AML samples tested, suggesting a broader effectiveness across patients. In a disseminated cell-line-derived xenograft mouse model of AML, CD33/CD123-TCE cleared cancer cells in long bones and in soft tissues. As cytokine release syndrome is a well-documented adverse effect of TCE, the compound was tested in a cytokine release assay and shown to induce less cytokines compared to a CD123 single-targeting control. In an exploratory single-dose nonhuman primate study, CD33/CD123-TCE revealed a favorable PK profile. Depletion of CD123 and CD33 expressing cells was observed, but there were neither signs of cytokine release syndrome nor clinical signs of toxicity. Taken together, the CD33/CD123 dual-targeting NANOBODY TCE exhibits potent and safe anti-AML activity and promises a broad patient coverage.

Post-transplant food allergy in children is associated with liver and not with renal transplantation: a monocentric comparative study.

UNLABELLED: Food allergy is increasingly reported after paediatric liver transplantation. The underlying physiopathological mechanism remains incompletely understood. Therefore, we aimed to determine the incidence, clinical presentation, possible risk factors, and prognosis of post-transplant food allergy in children currently followed after liver and renal transplantation. The study population consists of 49 liver and 21 renal transplant patients transplanted between the age of 22 months and 15 years. Data were collected retrospectively from medical records and via a doctor's questionnaire taken from the parents in a monocentric setting. Post-transplant food allergy has developed in 13 liver transplant patients and in none of the renal transplant recipients. Within the liver transplant group, median age at liver transplantation is significantly lower in the food-allergic (10 months) versus non-food-allergic group (3.3 years; p = 0.002). The use of tacrolimus as primary maintenance immunosuppression is associated with food allergy (p = 0.032) and mean donor age is significantly lower in the food-allergic group (p = 0.009). Compared to the renal transplant group, median age at transplantation is significantly lower in the liver patients (p < 0.001). No significant differences are found in primary immunosuppressive regimens between renal and liver transplant patients. CONCLUSION: Post-transplant food allergy is an important clinical problem in children after liver transplantation which does not affect renal transplant patients despite similar immunosuppressive regimens. Within the group of liver transplant recipients, tacrolimus use, young age at time of transplant and younger donor age were associated with the development of food allergy.

The who, where, and when of IgE in allergic airway disease.

Allergic asthma and allergic rhinitis/conjunctivitis are characterized by a T(H)2-dominated immune response associated with increased serum IgE levels in response to inhaled allergens. Because IgE is a key player in the induction and maintenance of allergic inflammation, it represents a prime target for therapeutic intervention. However, our understanding of IgE biology remains fragmentary. This article puts together our current knowledge on IgE in allergic airway diseases with a special focus on the identity of IgE-secreting cells ("who"), their location ("where"), and the circumstances in which they are induced ("when"). We further consider the therapeutic implications of the insights gained.