Gabriela López-Herrera

Brutonˈs tyrosine kinase—an integral protein of B cell development that also has an essential role in the innate immune system

Btk is the protein affected in XLA, a disease identified as a B cell differentiation defect. Btk is crucial for B cell differentiation and activation, but its role in other cells is not fully understood. This review focuses on the function of Btk in monocytes, neutrophils, and platelets and the receptors and signaling cascades in such cells with which Btk is associated.

Increased Pro-inflammatory Cytokine Production After Lipopolysaccharide Stimulation in Patients with X-linked Agammaglobulinemia

PurposeTo evaluate the lipopolysaccharide (LPS)-induced pro-inflammatory cytokine response by peripheral blood mononuclear cells (PBMCs) from XLA patients.MethodsThirteen patients with XLA were included in the study. LPS-induced TNF-α, IL-1β, IL-6, and IL-10 production was determined in PBMCs from patients and matched healthy controls by ELISA. Cytokine production was correlated with the severity of mutation, affected domain and clinical characteristics.ResultsIn response to LPS, PBMCs from XLA patients produced significantly higher amounts of pro-inflammatory cytokines and IL-10 compared to controls, and this production was influenced neither by the severity of the mutation nor the affected domain. PBMCs from patients with a history of more hospital admissions before their diagnosis produced higher levels of TNF-α. PBMCs from patients with lower serum IgA levels showed a higher production of TNF-α and IL-1β. Less severe (punctual) mutations in the Btk gene were associated with higher serum IgG levels at diagnosis.ConclusionsOur results demonstrate a predominantly inflammatory response in XLA patients after LPS stimulation and suggest a deregulation of TLR signaling in the absence of Btk. This response may be influenced by environmental factors.

Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase

Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B‐cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X‐linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein–protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK‐deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR‐crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK.

Clinical and mutational features of X-linked agammaglobulinemia in Mexico.

X-linked agammaglobulinemia (XLA) is caused by BTK mutations, patients typically show <2% of peripheral B cells and reduced levels of all immunoglobulins; they suffer from recurrent infections of bacterial origin; however, viral infections, autoimmune-like diseases, and an increased risk of developing gastric cancer are also reported. In this work, we report the BTK mutations and clinical features of 12 patients diagnosed with XLA. Furthermore, a clinical revision is also presented for an additional cohort of previously reported patients with XLA. Four novel mutations were identified, one of these located in the previously reported mutation refractory SH3 domain. Clinical data support previous reports accounting for frequent respiratory, gastrointestinal tract infections and other symptoms such as the occurrence of reactive arthritis in 19.2% of the patients. An equal proportion of patients developed septic arthritis; missense mutations and mutations in SH1, SH2 and PH domains predominated in patients who developed arthritis.

Impaired selective cytokine production by CD4+ T cells in Common Variable Immunodeficiency associated with the absence of memory B cells

Common Variable Immunodeficiency (CVID) is a primary immunodeficiency characterized by B cell dysfunction and decreased serum immunoglobulin. CVID patients are classified by the absence or presence of memory B cells. In addition, T cell defects have been demonstrated in only a proportion of CVID patients. The aim of this study was to evaluate the function of CD4(+) T cells from CVID patients and its association with memory B cells. Patients were classified according to their Freiburg groups: group Ia and Ib, with decreased switched memory B cells (<0.4 of PBL), and group II, with normal B cell subsets. Their T cell function was evaluated after stimulation. We observed normal and even increased CD4(+) T cell proliferation in group Ia (p=0.0277). The proliferation positively correlated with the clinical severity score (r=0.4796). We observed lower levels of IL-17A and IL-10 in group Ia (p=0.0177, 0.0109) and Ib (p=0.0009, 0.0084) patients. Group Ib patients also had low levels of IL-13 and IL-9 (p=0.0169, 0.010). Group II patients had similar cytokine production to that of the controls. BAFFR expression was reduced in groups Ia (p=0.0001) and Ib (p=0.0002) and showed an inverse correlation with the severity score (p=0.0262; r=0.5371). ICOS expression was reduced in group Ia (p=0.0364), and PD-1 was increased in group Ib (p=0.0432) patients. This study shows a selective impairment in cytokine production in group Ia patients, which was more extensive than in group Ib patients. The impairment was associated with BAFFR expression in B cells, with ICOS and PD-1 in T cells and, remarkably, with the absence of memory B cells and with the disease severity. Our results suggest that the evaluation of cytokine expression by T cells in combination with the study of B cell memory could be important for understand the pathogenesis of CVID patients.

Hemophagocytic Lymphohistiocytosis as a Complication in Patients with MSMD

Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder characterized by deregulated immune activation of macrophages and overproduction of pro-inflammatory cytokines such as IFN-γ and IL-6 [1]. These cytokines contribute to clinical features frequently observed in HLH patients, which include prolonged fever, hematological cytopenias, liver failure, seizures, and abnormal natural killer (NK) cell function; the last criterion is one of the most important ones for the categorization of HLH into familial and acquired forms [2]. Patients with the familial form have an identifiable genetic defect and often present the disease at a young age or have additional affected family members. Primary immunodeficiencies are associated with HLH; nevertheless, MSMD has not been related with HLH before despite having two components that are important for HLH pathogenesis, such as mycobacterial infection and altered T cell function. Here, we report three patients with MSMD and a diagnosis of HLH, two of them previously reported by our group with IL12Rβ1 deficiency and impaired responses to IL-12 [3]. The third patient harbors an unreported mutation in STAT1 (c.208 C>T; p.R70C) (Fig. 1a) and when we evaluated the response of patient’s cells to IFN-γ in terms of the production of IL12p40, the levels of IL12p40 from patient cells were absent (Fig. 1b). However, we analyzed the recruitment of STAT-1 into the nucleus by immunofluorescence in primary fibroblasts from the patient and a control, observing that this process was not affected (Fig. 1c). These data suggest that the allele c.208C>T impairs the response to IFN type II but does not affect the STAT-1 recruitment into the nucleus. All patients presented with fever, splenomegaly, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia all of which are compatible with HLH. We analyzed the subsets of leukocytes in patients 1 and 3 and found that the numbers of granulocytes, monocytes, and T, B, NK lymphocytes were normal (Fig. 1d). Since the function of NK cells is implicated in the etiology of HLH, we evaluated the cytotoxic activity of NK cells from patients 1 and 3 by co-culture with K562 cells and evaluated their propidium iodide (PI) incorporation. The percentage of K562 cells positive for PI after contact with the PBMCs from patients was comparable to the observed with the control. The perforin content in the NK cells, measured by flow cytometry, was in the normal range (Fig. 1e, f). Taken together, these data do not suggest any defect in NK cells function on these patients. On the other hand, Sepulveda et al. recently showed that NK cells are necessary to reduce the infiltration of CD8 T cells and macrophages activated in the livers and spleens of mice with HLH-like syndrome [4]. This regulatory mechanism of NK cells is perforin-dependent and is directed against activated CD8 Tcells. The authors suggest that this regulatory activity could be directed by limiting the help of CD4 T cells during early stages of CD8 T cell activation and differentiation, and/or by direct killing of early activated CD8 Tcells and activated macrophages, thus limiting the destruction of tissue. Strikingly, the beneficial effect of NK cell cytotoxicity in reducing HLH-like pathology was not related to a decrease in serum IFN-γ levels. This emphasizes that an infection is required to trigger acquired HLH syndrome but the course of the disease is a result from failure of the correct immunoregulatory mechanisms. In this context, participation of cytokines such * Noé Ramírez-Alejo noeramirez@cinvestav.mx

CD38 protein deficiency induces autoimmune characteristics and its activation enhances IL-10 production by regulatory B cells

CD38 is a transmembrane protein expressed in B lymphocytes, and is able to induce responses as proliferation, differentiation or apoptosis. Several reports propose that CD38 deficiency accelerates autoimmune processes in murine models of autoimmune diabetes, lymphoproliferation and rheumatoid arthritis. Other reports have shown elevated CD38 expression in B and T cells from patients with autoimmunity; however, the role of CD38 is still unclear in the development of autoimmunity. Recently, it has been characterized as CD1dhi CD5+ regulatory B cell subpopulation able to produce IL‐10, and the loss of these cells exacerbates the autoimmunity in murine models. Here, we report that CD38−/− mice exhibited elevated titres of ANAS, anti‐dsDNA autoantibodies from 12 months of age and were higher by 16 months of age and mice presented kidney damage. Interestingly, there is a reduction in the survival of CD38−/− mice compared to the WT. Furthermore, CD38 is highly expressed by CD1dhigh CD5+ regulatory B cells, and the agonistic anti‐CD38 stimulus plus LPS was able to increase the percentage of this cell subset and its ability to induce IL‐10 production. Together, these results suggest that CD38 could play a role in the control of autoimmune diseases through their expression on regulatory B cells.

Somatic mosaicism in B cells of a patient with autosomal dominant hyper IgE syndrome

Hyper IgE syndrome (HIES) is characterized by recurrent skin abscesses, eczema, pneumonia, and high levels of serum IgE. Nonimmunologic manifestations of HIES include a characteristic face, pathologic dentition, scoliosis, bone alterations, hyperextensible joints, and vascular abnormalities. Somatic mosaicism is defined by the presence of two or more populations of cells with different genotypes in one individual. In this report, we describe one patient with classical HIES and another patient with a mild phenotype, both harboring the same genetic mutation. The patient with a mild phenotype did not present the characteristic face, had normal production of IL‐17A by T CD4+ cells, but had low phosphorylation of STAT‐3 in B cells. Interestingly, the mutation found in B cells was absent in other cell types analyzed, in agreement with the presence of a somatic mosaic genotype. The clinical and functional differences observed between these patients justify the use of complementary tools for a better definition of the cases. These approaches allow for a better understanding of complex phenotypes associated with somatic mosaicisms, and present the possibility to analyze the role of B lymphocytes in the pathophysiology of this disease. This knowledge has an impact on not only the treatment but also the provision of appropriate genetic counseling.

Inmunodeficiencia común variable y su asociación con defectos en células B de memoria

Common variable immunodeficiency (CVID) is the most common symptomatic immunodeficiency in adulthood. CVID diagnosis is by exclusion and should be considered in patients of any age who have hypogammaglobulinemia of unknown origin. Numerous patients with CVID show alterations in the development of B lymphocytes, both in plasma cells and memory cells. The absence of memory B cells suggests an insufficient germinal reaction, which can be associated with a blockade of the transition of T1 cells into T2 in patients with IDCV, owing to B-cell activating factor (BAFF) receptor deficiency. In patients with IDCV, memory B cell alterations with isotype change favor the development of concomitant comorbidities such as lymphadenopathy, splenomegaly, autoimmunity and granulomatous disease, and multiple classifications that use memory B cells in common have therefore been made trying to generate a classification of patients with IDCV, as well as to establish prognostic factors.

Delayed diagnosis in X-linked agammaglobulinemia and its relationship to the occurrence of mutations in BTK non-kinase domains

ABSTRACT Background: X-linked agammaglobulinemia (XLA) is characterized by the absence of immunoglobulin and B cells. Patients suffer from recurrent bacterial infections from early childhood, and require lifelong immunoglobulin replacement therapy. Mutations in BTK (Bruton’s Tyrosine Kinase) are associated with this phenotype. Some patients that present XLA do not show typical clinical symptoms, resulting in delayed diagnosis due to the lack of a severe phenotype. This study presents a report of five XLA patients from four different families and attempts to determine a relationship between delayed diagnosis and the occurrence of BTK mutations. Methods: Samples from patients with antibody deficiency were analyzed to determine BTK expression, immunophenotyping and mutation analysis. Clinical and laboratory data was analyzed and presented for each patient. Results: Most patients presented here showed atypical clinical and laboratory data for XLA, including normal IgM, IgG, or IgA levels. Most patients expressed detectable BTK protein. Sequencing of BTK showed that these patients harbored missense mutations in the pleckstrin homology and Src-homology-2 domains. When it was compared to public databases, BTK sequencing exhibited a new change, along with three other previously reported changes. Conclusions: Delayed diagnosis and atypical manifestations in XLA might be related to mutation type and BTK expression.