Leopoldo Santos-Argumedo

Arrest of B Lymphocyte Terminal Differentiation by CD40 Signaling: Mechanism for Lack of Antibody-Secreting Cells in Germinal Centers

Despite extensive research, the role of CD40 signaling in B cell terminal differentiation remains controversial. Here we show that CD40 engagement arrests B cell differentiation prior to plasma cell formation. This arrest is manifested at a molecular level as a reduction in mRNA levels of secretory immunoglobulin gene products such as mu(s) and J chain as well as the loss of the transcriptional regulator BLIMP-1. Furthermore, the inhibition of B cell differentiation by CD40 engagement could not be overcome by either mitogens or cytokines, but could be reversed by antibodies that interfere with the CD40/gp39 interaction. These data suggest that secretory immunoglobulin is not produced by B cells that are actively engaged by gp39-expressing T cells.

CD38 is expressed selectively during the activation of a subset of mature T cells with reduced proliferation but improved potential to produce cytokines.

CD38 is an ∼45‐kDa type II transmembrane glycoprotein expressed by hematopoietic and nonhematopoietic cells. Its surface expression is under complex control and varies during lymphocyte development, activation, and differentiation, suggesting an important role in these processes. Murine CD38 has been mainly characterized on B lymphocytes, and in humans, the molecule has been studied in T cells. This paper provides evidences that murine CD38 is regulated tightly during T cell activation and differentiation. On the periphery, a subset of mature T lymphocytes was identified by the expression of CD38. These cells showed an activated phenotype; they were larger and more granular than their negative counterparts. In accord with this observation, in vitro‐activated T cells up‐regulated CD38. Memory T lymphocytes also were CD38‐positive. It is interesting that T cells expressing high levels of CD38 had a reduced, proliferative capacity but displayed an improved potential to produce interleukin‐2 and interferon‐γ, suggesting a role of this molecule during T cell activation and differentiation.

IL-12Rβ1 deficiency: mutation update and description of the IL12RB1 variation database

IL‐12Rβ1 deficiency is an autosomal recessive disorder characterized by predisposition to recurrent and/or severe infections caused by otherwise poorly pathogenic mycobacteria and salmonella. IL‐12Rβ1 is a receptor chain of both the IL‐12 and the IL‐23 receptor and deficiency of IL‐12Rβ1 thus abolishes both IL‐12 and IL‐23 signaling. IL‐12Rβ1 deficiency is caused by bi‐allelic mutations in the IL12RB1 gene. Mutations resulting in premature stop codons, such as nonsense, frame shift, and splice site mutations, represent the majority of IL‐12Rβ1 deficiency causing mutations (66%; 46/70). Also every other morbid mutation completely inactivates the IL‐12Rβ1 protein. In addition to disease‐causing mutations, rare and common variations with unknown functional effect have been reported in IL12RB1. All these variants have been deposited in the online IL12RB1 variation database (www.LOVD.nl/IL12RB1). In this article, we review the function of IL‐12Rβ1 and molecular genetics of human IL12RB1.

Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

Background When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times (“probing”) before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells. Methodology/Principal Findings Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 (IRF7), when compared with mock-infected fibroblasts. Conclusions/Significance In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and provide viral particles that may contribute to subsequent viral dissemination.

CD38 Signaling Regulates B Lymphocyte Activation via a Phospholipase C (PLC)-γ2-Independent, Protein Kinase C, Phosphatidylcholine-PLC, and Phospholipase D-Dependent Signaling Cascade

The CD38 cell surface receptor is a potent activator for splenic, B lymphocytes. The molecular mechanisms regulating this response, however, remain incompletely characterized. Activation of the nonreceptor tyrosine kinase, Btk, is essential for CD38 downstream signaling function. The major Btk-dependent substrate in B cells, phospholipase C-γ2 (PLC-γ2), functions to generate the key secondary messengers, inositol-1,4,5 trisphosphate and diacylglycerol. Surprisingly, CD38 ligation results in no detectable increase in phosphoinositide metabolism and only a minimal increase in cytosolic calcium. We hypothesized that Btk functioned independently of PLC-γ2 in the CD38 signaling pathway. Accordingly, we demonstrate that CD38 cross-linking does not result in the functional phosphorylation of PLC-γ2 nor an increase in inositol-1,4,5 trisphosphate production. Furthermore, splenic B cells exhibit a normal CD38-mediated, proliferative response in the presence of the phosphoinositide-PLC inhibitor, U73122. Conversely, protein kinase C (PKC) β-deficient mice, or PKC inhibitors, indicated the requirement for diacylglycerol-dependent PKC isoforms in this pathway. Loss of PKC activity blocked CD38-dependent, B cell proliferation, NF-κB activation, and subsequent expression of cyclin-D2. These results suggested that an alternate diacylglycerol-producing phospholipase must participate in CD38 signaling. Consistent with this idea, CD38 increased the enzymatic activity of the phosphatidylcholine (PC)-metabolizing enzymes, PC-PLC and phospholipase D. The PC-PLC inhibitor, D609, completely blocked CD38-dependent B cell proliferation, IκB-α degradation, and cyclin-D2 expression. Analysis of Btk mutant B cells demonstrated a partial requirement for Btk in the activation of both enzymes. Taken together, these data demonstrate that CD38 initiates a novel signaling cascade leading to Btk-, PC-PLC-, and phospholipase D-dependent, PLC-γ2-independent, B lymphocyte activation.

First Report of the Hyper-IgM Syndrome Registry of the Latin American Society for Immunodeficiencies: Novel Mutations, Unique Infections, and Outcomes

Hyper-IgM (HIGM) syndrome is a heterogeneous group of disorders characterized by normal or elevated serum IgM levels associated with absent or decreased IgG, IgA and IgE. Here we summarize data from the HIGM syndrome Registry of the Latin American Society for Immunodeficiencies (LASID). Of the 58 patients from 51 families reported to the registry with the clinical phenotype of HIGM syndrome, molecular defects were identified in 37 patients thus far. We retrospectively analyzed the clinical, immunological and molecular data from these 37 patients. CD40 ligand (CD40L) deficiency was found in 35 patients from 25 families and activation-induced cytidine deaminase (AID) deficiency in 2 unrelated patients. Five previously unreported mutations were identified in the CD40L gene (CD40LG). Respiratory tract infections, mainly pneumonia, were the most frequent clinical manifestation. Previously undescribed fungal and opportunistic infections were observed in CD40L-deficient patients but not in the two patients with AID deficiency. These include the first cases of pneumonia caused by Mycoplasma pneumoniae, Serratia marcescens or Aspergillus sp. and diarrhea caused by Microsporidium sp. or Isospora belli. Except for four CD40L-deficient patients who died from complications of presumptive central nervous system infections or sepsis, all patients reported in this study are alive. Four CD40L-deficient patients underwent successful bone marrow transplantation. This report characterizes the clinical and genetic spectrum of HIGM syndrome in Latin America and expands the understanding of the genotype and phenotype of this syndrome in tropical areas.

CD44-stimulated dendrite formation (‘spreading’) in activated B cells

A rat monoclonal antibody (mAb) (NIM‐R8), insolubilized by binding to plastic plates, induced a rapid and extensive formation of dendrite processes (‘spreading’) in B lymphocytes activated by anti‐IgM and interleukin‐4 (IL‐4) or anti‐CD38 and IL‐4. In contrast, resting B cells were unable to spread similarly on the NIM‐R8‐coated plates. The NIM‐R8 antibody recognized a 90 000 MW surface glycoprotein (gp90) present on both B and T lymphocytes. The expression of this molecule was greatly increased after polyclonal (lipopolysaccarhide, anti‐IgM plus IL‐4 or concanavalin A) activation. The NIM‐R8 mAb with or without IL‐2 or IL‐4 was unable to induce proliferation of splenic lymphocytes. Following the demonstration that the NIM‐R8 mAb recognizes the murine equivalent of human CD44, the induction of spreading of activated B lymphocytes was studied using a panel of mAb recognizing different epitopes of murine CD44. All of these different mAb induced similar spreading of activated B cells. The ligand‐inducible spreading of activated B lymphocytes may be an important mechanism for providing an increased cell‐surface area for cell–cell or cell–matrix interactions, and thus may be an important factor controlling the response of activated lymphocytes.

Translating innate response into long-lasting antibody response by the intrinsic antigen-adjuvant properties of papaya mosaic virus

Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long‐lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen‐associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long‐lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro‐inflammatory cytokines, up‐regulation of co‐stimulatory molecules on dendritic cells and macrophages, and long‐lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen‐presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long‐lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.

Myosin 1c Participates in B Cell Cytoskeleton Rearrangements, Is Recruited to the Immunologic Synapse, and Contributes to Antigen Presentation

Myosin 1c (Myo1c) is a member of the unconventional class I myosins of vertebrates, which directly link the plasma membrane with the microfilament cortical web. Although this molecular motor has been implicated in cell functions such as cytoskeleton organization, cell motility, nuclear transcription, and endocytosis, its role in hematopoietic cells is largely unknown. In this study, we show that Myo1c is abundantly expressed in murine B lymphocytes and is preferentially located at the plasma membrane, especially in peripheral processes such as microvilli. We observed that this motor concentrates at the growing membrane protrusions generated during B cell spreading and that it is actively recruited to the immune synapse. Interestingly, Myo1c was detected in lipid rafts of B cells and showed strong colocalization with MHC-II, particularly after cross-linking of these molecules. By transfection of a dominant negative form of Myo1c or specific siRNA, we also detected alterations in the spreading and Ag-presenting ability of these cells. The data suggest that Myo1c is involved in the cytoskeleton dynamics and membrane protein anchoring or sorting in B lymphocytes.

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.