Jordi Yagüe

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

B cell–helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen

Neutrophils utilize immunoglobulins (Igs) to clear antigen, but their role in Ig production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T-independent Ig responses to circulating antigen. Neutrophils colonized peri-MZ areas after post-natal mucosal colonization by microbes and enhanced their B-helper function upon receiving reprogramming signals from splenic sinusoidal endothelial cells, including interleukin 10 (IL-10). Splenic neutrophils induced Ig class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism involving the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and less preimmune Igs to T-independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial Ig defense by interacting with MZ B cells.Neutrophils use immunoglobulins to clear antigen, but their role in immunoglobulin production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T cell–independent immunoglobulin responses to circulating antigen. Neutrophils colonized peri-MZ areas after postnatal mucosal colonization by microbes and enhanced their B cell–helper function after receiving reprogramming signals, including interleukin 10 (IL-10), from splenic sinusoidal endothelial cells. Splenic neutrophils induced immunoglobulin class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism that involved the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and a lower abundance of preimmune immunoglobulins to T cell–independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial immunoglobulin defense by interacting with MZ B cells.

The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response

Assembly of the NLRP3 inflammasome activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1β and thereby serves a central role in the inflammatory response and in diverse human diseases. Here we found that upon activation of caspase-1, oligomeric NLRP3 inflammasome particles were released from macrophages. Recombinant oligomeric protein particles composed of the adaptor ASC or the p.D303N mutant form of NLRP3 associated with cryopyrin-associated periodic syndromes (CAPS) stimulated further activation of caspase-1 extracellularly, as well as intracellularly after phagocytosis by surrounding macrophages. We found oligomeric ASC particles in the serum of patients with active CAPS but not in that of patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3 inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.

Cryoglobulinemia: study of etiologic factors and clinical and immunologic features in 443 patients from a single center.

Cryoglobulins are immunoglobulins (Ig) that precipitate when serum is incubated at temperatures lower than 37 C. The existence of circulating cryoglobulins (cryoglobulinemia) is not always related to the presence of symptomatology, and the term “cryoglobulinemic syndrome” is used when patients present clinical manifestations (44). In 1933, Wintrobe and Buell (55) described cryoprecipitation as a laboratory phenomenon. In 1966, Meltzer and Franklin (27) described the typical clinical symptoms associated with cryoglobulinemia, particularly the triad of purpura, arthralgia, and weakness. In 1974, Brouet et al (7) classified cryoglobulins into 3 types: Type I cryoglobulins were those composed of single monoclonal immunoglobulins, and types II and III were those formed by monoclonal (type II) or polyclonal (type III) IgM with rheumatoid factor activity plus the corresponding antigen (usually polyclonal IgG). For this reason, types II and III are classically referred to as “mixed cryoglobulinemia.” Recently, new types of cryoglobulins have been described (41). Cryoglobulins have been observed in a wide variety of diseases, including malignancies, infections, and systemic autoimmune diseases (17, 40). When there is no demonstrable underlying disease, the condition is called “essential cryoglobulinemia.” Since the initial report (36) in 1990 of the association between cryoglobulinemia and hepatitis C virus (HCV) infection, it has become clear that most cases of socalled essential cryoglobulinemia are in fact associated with HCV infection. This has led to important changes in the etiology, classification, and treatment of cryoglobulinemia in the last 10 years. Several studies have analyzed cryoglobulinemia in patients with HCV infection, but studies performed in groups of patients covering all etiologies of cryoglobulinemia are scarce (7, 14, 17, 19, 30). In this study, we analyzed the etiology, clinical manifestations, and immunologic features of a large series of consecutive patients with cryoglobulinemia from a single center (Hospital Clinic, Barcelona, Spain).

High Incidence of NLRP3 Somatic Mosaicism in Patients With Chronic Infantile Neurologic, Cutaneous, Articular Syndrome: Results of an International Multicenter Collaborative Study

OBJECTIVE Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease. Although heterozygous germline gain-of-function NLRP3 mutations are a known cause of this disease, conventional genetic analyses fail to detect disease-causing mutations in ∼40% of patients. Since somatic NLRP3 mosaicism has been detected in several mutation-negative NOMID/CINCA syndrome patients, we undertook this study to determine the precise contribution of somatic NLRP3 mosaicism to the etiology of NOMID/CINCA syndrome. METHODS An international case-control study was performed to detect somatic NLRP3 mosaicism in NOMID/CINCA syndrome patients who had shown no mutation during conventional sequencing. Subcloning and sequencing of NLRP3 was performed in these mutation-negative NOMID/CINCA syndrome patients and their healthy relatives. Clinical features were analyzed to identify potential genotype-phenotype associations. RESULTS Somatic NLRP3 mosaicism was identified in 18 of the 26 patients (69.2%). Estimates of the level of mosaicism ranged from 4.2% to 35.8% (mean ± SD 12.1 ± 7.9%). Mosaicism was not detected in any of the 19 healthy relatives (18 of 26 patients versus 0 of 19 relatives; P < 0.0001). In vitro functional assays indicated that the detected somatic NLRP3 mutations had disease-causing functional effects. No differences in NLRP3 mosaicism were detected between different cell lineages. Among nondescript clinical features, a lower incidence of mental retardation was noted in patients with somatic mosaicism. Genotype-matched comparison confirmed that patients with somatic NLRP3 mosaicism presented with milder neurologic symptoms. CONCLUSION Somatic NLRP3 mutations were identified in 69.2% of patients with mutation-negative NOMID/CINCA syndrome. This indicates that somatic NLRP3 mosaicism is a major cause of NOMID/CINCA syndrome.

Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells

Innate lymphoid cells (ILCs) regulate stromal cells, epithelial cells and cells of the immune system, but their effect on B cells remains unclear. Here we identified RORγt+ ILCs near the marginal zone (MZ), a splenic compartment that contains innate-like B cells highly responsive to circulating T cell–independent (TI) antigens. Splenic ILCs established bidirectional crosstalk with MAdCAM-1+ marginal reticular cells by providing tumor-necrosis factor (TNF) and lymphotoxin, and they stimulated MZ B cells via B cell–activation factor (BAFF), the ligand of the costimulatory receptor CD40 (CD40L) and the Notch ligand Delta-like 1 (DLL1). Splenic ILCs further helped MZ B cells and their plasma-cell progeny by coopting neutrophils through release of the cytokine GM-CSF. Consequently, depletion of ILCs impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune system and circulatory system.

Crohn's Disease Patients Carrying Nod2/CARD15 Gene Variants Have an Increased and Early Need for First Surgery due to Stricturing Disease and Higher Rate of Surgical Recurrence

Objective:To study the predictive value of Nod2/CARD15 gene variants along with disease phenotypic characteristics for requirement of initial surgery and for surgical recurrence in Crohns disease (CD). Summary Background Data:Nod2/CARD15 gene variants play an important role in the susceptibility to CD. Studies of genotype-phenotype relationship suggest that these variants are associated with development of intestinal strictures. Preliminary reports analyzing the association between these variants and need for surgery have produced inconsistent results. Methods:A total of 170 CD patients were included prospectively in the study and followed up regularly for a mean of 7.4 ± 6.1 years. Clinical characteristics of CD, time and indication for surgery, and recurrence were registered. Nod2/CARD15 gene variants were determined by DNA sequencing analysis. Results:Surgery for stricturing disease was significantly more frequent in patients with Nod2/CARD15 variants in the univariate analysis (odds ratio [OR], 3.63; 95% confidence interval [CI], 1.42–9.27), and it was required at an earlier time (P = 0.004). Only Nod2/CARD15 variants (OR, 3.58; 95% CI, 1.21–10.5) and stricturing phenotype at diagnosis of CD (OR, 9.34; 95% CI, 2.56–33.3) were independent predictive factors of initial surgery for stricturing lesions in the multivariate analysis. Among 70 patients that required surgery, postoperative recurrence was also more frequent in patients with Nod2/CARD15 variants in the univariate and multivariate analysis (OR, 3.29; 95% CI, 1.13–9.56), and reoperation was needed at an earlier time (P = 0.03). Conclusion:Nod2/CARD15 variants are associated with early initial surgery due to stenosis and with surgical recurrence in Crohns disease. Patients with these variants could benefit from preventive and/or early therapeutic strategies.

The T cell repertoire may be biased in favor of MHC recognition

The receptors of two T cell hybridomas that recognize class I and class II major histocompatibility complex (MHC) molecules, respectively, have been compared. In both cases these receptors are hybrid molecules formed as a result of cellular fusion. The receptors contain the same alpha chain, contributed by the tumor cell fusion partner, and related beta chains, contributed by the normal T cell component. Thus, surprisingly, the same alpha chain can contribute to recognition of class I and class II MHC molecules. Moreover, the finding that in two independent examples hybrid receptor molecules created randomly by in vitro cell fusion recognize MHC supports the theory that the T cell repertoire has an intrinsic affinity for MHC.

Cryoglobulinemia in primary Sjögren's syndrome: Prevalence and clinical characteristics in a series of 115 patients

OBJECTIVES To determine the prevalence and nature of cryoglobulins in a large series of patients with primary Sjögrens syndrome (SS) and identify the clinical and immunologic features related to their presence. METHODS In a cross-sectional study, we investigated 115 consecutive patients (107 women and eight men) with primary SS. All patients fulfilled four or more of the preliminary diagnostic criteria for SS proposed by the European Community Study Group in 1993. Serum cryoglobulinemia was measured in all patients. Serum samples were obtained at 37 degrees C, and cryoglobulinemia was estimated by centrifugation after incubation at 4 degrees C for 7 days. The type of cryoglobulinemia was identified by agarose gel electrophoresis and immunofixation. RESULTS Cryoglobulins were detected in the sera of 18 (16%) of our patients with primary SS; most were IgMkappa monoclonal/IgG polyclonal. When compared with patients without cryoglobulins, those with cryoglobulins presented a higher prevalence of leukocytoclastic cutaneous vasculitis (56% v8%, P < .001), hypocomplementemia (75% v 2%; P < 0.001) and antibodies to hepatitis C virus (HCV) (47% v8%, P < .001). Liver involvement (clinical signs, biochemical features, or ultrasound/histological data of liver disease) was present in all patients (100%) with cryoglobulins and HCV infection but in only 11% of patients with cryoglobulins without HCV infection (P < .001). CONCLUSIONS Leukocytoclastic cutaneous vasculitis, hypocomplementemia, and HCV infection are associated with the presence of cryoglobulins in the sera of patients with primary SS. Testing for HCV infection is recommended for patients with SS and cryoglobulinemia because of its high prevalence and its strong association with liver disease.

Lipid transfer protein syndrome: clinical pattern, cofactor effect and profile of molecular sensitization to plant‐foods and pollens

Multiple plant‐food sensitizations with a complex pattern of clinical manifestations are a common feature of lipid transfer protein (LTP)‐allergic patients. Component‐resolved diagnosis permits the diagnosis of the allergen sensitization profile.