B-cell Proliferative Responses Research Articles

Linking signaling and selection in the germinal center.

Germinal centers (GC) are sites of rapid B-cell proliferation in response to certain types of immunization. They arise in about 1week and can persist for several months. In GCs, B cells differentiate in a unique way and begin to undergo somatic mutation of the Ig V regions at a high rate. GC B cells (GCBC) thus undergo clonal diversification that can affect the affinity of the newly mutant B-cell receptor (BCR) for its driving antigen. Through processes that are still poorly understood, GCBC with higher affinity are selectively expanded while those with mutations that inactivate the BCR are lost. In addition, at various times during the extended GC reaction, some GCBC undergo differentiation into either long-lived memory B cells (MBC) or plasma cells. The cellular and molecular signals that govern these fate decisions are not well-understood, but are an active area of research in multiple laboratories. In this review, we cover both the history of this field and focus on recent work that has helped to elucidate the signals and molecules, such as key transcription factors, that coordinate both positive selection as well as differentiation of GCBC.

Functionalized porous silica&maghemite core-shell nanoparticles for applications in medicine: design, synthesis, and immunotoxicity.

AimTo determine cytotoxicity and effect of silica-coated magnetic nanoparticles (MNPs) on immune response, in particular lymphocyte proliferative activity, phagocytic activity, and leukocyte respiratory burst and in vitro production of interleukin-6 (IL-6) and 8 (IL-8), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF).MethodsMaghemite was prepared by coprecipitation of iron salts with ammonia, oxidation with NaOCl and modified by tetramethyl orthosilicate and aminosilanes. Particles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). Cytotoxicity and lymphocyte proliferative activity were assessed using [3H]-thymidine incorporation into DNA of proliferating human peripheral blood cells. Phagocytic activity and leukocyte respiratory burst were measured by flow cytometry; cytokine levels in cell supernatants were determined by ELISA.Resultsγ-Fe2O3&SiO2-NH2 MNPs were 13 nm in size. According to TEM, they were localized in the cell cytoplasm and extracellular space. Neither cytotoxic effect nor significant differences in T-lymphocyte and T-dependent B-cell proliferative response were found at particle concentrations 0.12-75 μg/cm2 after 24, 48, and 72 h incubation. Significantly increased production of IL-6 and 8, and GM-CSF cytokines was observed in the cells treated with 3, 15, and 75 µg of particles/cm2 for 48 h and stimulated with pokeweed mitogen (PHA). No significant changes in TNF-α and IFN-γ production were observed. MNPs did not affect phagocytic activity of monocytes and granulocytes when added to cells for 24 and 48 h. Phagocytic respiratory burst was significantly enhanced in the cultures exposed to 75 µg MNPs/cm2 for 48 h.ConclusionsThe cytotoxicity and in vitro immunotoxicity were found to be minimal in the newly developed porous core-shell γ-Fe2O3&SiO2-NH2 magnetic nanoparticles.

Local induction of IgT responses to pathogens and microbiota in the gill of rainbow trout

Gas exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin (Ig) responses against pathogens in specialized respiratory organs have only been described in tetrapods. We have previously shown that IgT is an Ig specialized in gut and skin mucosal immunity. Thus, we tested the hypothesis that IgT might play a pivotal role in mucosal immunity of teleost gills. In this study, we provide the first structural and functional characterization of all teleost Igs, including secreted IgD, IgM and IgT at a mucosal surface of a teleost fish. Here we show that IgT B cells represent the major B cell subset in the gill filaments. In contrast to reported results by others, we found that all gill B cells expressing surface IgM, also expressed surface IgD and that the percentage of B cells solely expressing either IgD or IgM was negligible. We found that the majority of bacterial microbiota in the gill mucosa is coated with IgT and, to a much lesser degree with IgM and IgD. More crucially, significant specific-IgT immune responses against Ichthyophthirius multifiliis (Ich) and Flavobacterium columnare were measured in the gill mucus, while pathogen-specific IgM responses were almost exclusively detected in the serum. Pathogen-specific IgD titers were absent both in gill mucus and serum. Importantly, we found significant IgT B-cell proliferative responses in the gill but not in the spleen or head kidney of fish that survived Ich infection. Moreover we also found that Ichand F. columnare-specific IgT titers were locally produced by gill explants of survivor fish while they were absent in the spleen or head kidney explants of the same animals. In addition to showing that IgT is the main Ig player in gill mucosal immunity, the observed generation of local IgT B cell proliferative and pathogen-specific IgT responses in the gills provides the first demonstration of locally induced B cell and secretory Ig responses in the mucosa of a teleost. Moreover, this represents the first study in which a bacterial pathogen is shown to induce dominant IgT responses in a fish mucosal site, thus strongly suggesting that IgT is induced by a variety of pathogens in addition to parasites. Our findings also have special relevance from an applied perspective as they may lead to the development of fish vaccines and immunostimulants that have the capacity to induce gill IgT mucosal immune responses.

Association of B-cell activating factor receptor deficiency with the P21R polymorphism and common variable immunodeficiency

Association of B-cell activating factor receptor deficiency with the P21R polymorphism and common variable immunodeficiency

Duvelisib (IPI-145) Inhibits Malignant B-Cell Proliferation and Disrupts Signaling from the Tumor Microenvironment through Mechanisms That Are Dependent on PI3K-δ and PI3K-γ

Background: The neoplastic B cells of indolent non-Hodgkin lymphoma (iNHL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) rely upon the support of non-neoplastic cells within their microenvironment for proliferation and survival. Support cells include T cells, myeloid-derived cells, and mesenchymal stromal cells, which provide phosphoinositide-3 kinase (PI3K)-dependent survival and growth signals for the neoplastic cells, as well as signals that maintain the tumor microenvironment (TME). Duvelisib (IPI-145) is an oral inhibitor of PI3K-δ,γ in clinical development for iNHL and CLL/SLL. To better understand the roles of the PI3K-δ and PI3K-γ isoforms in mediating signaling between the tumor and TME cells in B-cell malignancies, Infinity’s highly potent (low nM) PI3K isoform-selective compounds that target either PI3K-δ or PI3K-γ with >100-fold selectivity over the other PI3K isoforms were utilized in in vitro experiments.Methods/Results: A mixture of cytokines (CD40L/IL-2/IL-10) was utilized in an assay that recapitulated TME-induced malignant B-cell proliferative responses. Duvelisib inhibited CD40L/IL-2/IL-10-induced proliferation of primary CLL cells with an average IC50 in the sub-nanomolar range. The use of PI3K isoform-selective inhibitory compounds revealed these proliferative signals are PI3K-δ dependent, as the PI3K-δ-selective inhibitor was more active than the PI3K-γ-selective inhibitor.While these experiments established the direct PI3K-δ dependence of TME-derived cytokines on CLL cell proliferation, the role of PI3K-γ in key functions such as the directed migration of normal immune cells of the TME was also tested. We hypothesized that chemokines that recruit immune cells to the TME would signal through G-protein coupled receptors linked to PI3K-γ. The stromally-derived chemokine CXCL12 resulted in upregulation of phospho (p)-AKT in both the CD3+ T-cell and CLL-cell populations in CLL patient total peripheral blood mononuclear cells (PBMCs). Using isoform-selective inhibitors, the increase in CXCL12-induced pAKT in CD3+ T cells was found to be mediated by PI3K-γ. Interestingly, within the malignant B-cell population, the increase in CXCL12-induced pAKT was PI3K-δ dependent, suggesting that CXCL12 signals through different PI3K isoforms in these varying cell types. Chemotactic assays demonstrated reduced migration of total CLL PBMCs towards CXCL12 in the presence of combined PI3K-δ and PI3K-γ inhibition by duvelisib. Flow cytometric analyses of the migrating populations revealed that the greatest effect of duvelisib on CXCL12-induced migration occurred primarily within the T-cell population. Utilizing PI3K isoform-selective compounds, the inhibition of T-cell migration toward CXCL12 was found to be a PI3K-γ mediated process, as the PI3K-γ-selective inhibitor was more potent than the PI3K-δ-selective inhibitor in blocking T-cell migration.Myeloid-derived cells and mesenchymal stromal cells can also support CLL cell survival as components of the TME. Recent reports suggest that CLL cytoprotective nurse-like cells may have an M2 polarization and be similar to the immunosuppressive myeloid-derived suppressor cells found in some solid tumors [Gianonni et al. Haematologica 2014, 99(6)]. To model these TME components, mouse bone marrow cells were differentiated into macrophages with murine MCSF and IL-4 (M2–polarized). CXCL12-induced pAKT in these M2 cells, which express CXCR4, was more potently inhibited by duvelisib and the PI3K-γ-selective inhibitor than the PI3K-δ-selective inhibitor. Finally, co-cultures of M2 macrophages with CLL cells led to extended CLL cell survival. These data show that CXCL12 mediated-M2 activation is dependent upon PI3K-γ and that M2-cells can act to support CLL cell survival.Conclusions: T cells and myeloid cells provide a survival and proliferative advantage to malignant CLL cells within the TME. The role of PI3K-γ in the migration and activation of these cells supports the potential for therapeutic benefit from inhibition of PI3K-γ. By inhibiting both the PI3K-δ and PI3K-γ isoforms, duvelisib is uniquely positioned to inhibit key signals important in the pathogenesis of B-cell malignancies. DisclosuresPeluso:Infinity Pharmaceuticals, Inc.: Employment. Faia:Infinity Pharmaceuticals, Inc.: Employment. Winkler:Infinity Pharmaceuticals, Inc.: Employment. Patel:Infinity Pharmaceuticals, Inc.: Employment. Brophy:Infinity Pharmaceuticals, Inc.: Employment. White:Infinity Pharmaceuticals, Inc.: Employment. Douglas:Infinity Pharmaceuticals, Inc.: Employment. Stern:Infinity Pharmaceuticals, Inc.: Employment. Palombella:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment.

Divergence of primary cognate B- and T-cell proliferative responses to subcutaneous and intravenous immunization with virus-like particles.

A major advantage of virus-like particle (VLP) vaccines against HIV is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter the envelope protein in its natural conformation. For the induction of affinity-matured antibodies, the B-cells must also obtain help from T-cells that are restricted by linear epitopes. Using B- and T-cell transgenic mouse models, we compared the efficacy of modified HIV-VLPs delivered by subcutaneous and intravenous immunization to stimulate primary B- and T-cell proliferative responses in different lymphoid organs. VLPs containing an influenza virus hemagglutinin epitope within the HIV-Gag protein induced comparable primary cognate T-cell proliferative responses in the draining lymph node and the spleen, irrespective of the delivery route. In contrast, after subcutaneous immunization with HIV-Gag VLPs containing hen egg lysozyme (HEL) on their surface, the proliferative response of transgenic HEL-specific B-cells was restricted to the draining lymph nodes, while intravenous VLP immunization primarily induced a B-cell proliferative response in the spleen. In vitro co-culture experiments further revealed that the presentation of VLP-associated surface antigens by dendritic cells to cognate B-cells is inefficient. This is consistent with a direct triggering of the B-cell proliferative response by the VLPs and suggests that HIV VLPs may indeed be suitable to directly promote the expansion of B-cells specific for conformational epitopes that are unique to functionally-active Env spikes on the virion. Further investigations are warranted to explore potential differences in the quality and protective potency of HIV-specific antibody responses induced by the two routes.

Phosphoproteomic analyses reveal that galectin-1 augments the dynamics of B-cell receptor signaling

B-cell activation is important for mounting humoral immune responses and antibody production. Galectin-1 has multiple regulatory functions in immune cells. However, the effects of galectin-1 modulation and the mechanisms underlying the coordination of B-cell activation are unclear. To address this issue, we applied label-free quantitative phosphoproteomic analysis to investigate the dynamics of galectin-1-induced signaling in comparison with that following anti-IgM treatment. A total of 3247 phosphorylation sites on 1245 proteins were quantified, and 70-80% of the 856 responsive phosphoproteins were commonly activated during various biological functions. The similarity between galectin-1- and anti-IgM-elicited B-cell receptor (BCR) signaling pathways was also revealed. Additionally, the mapping of the 149 BCR-responsive phosphorylation sites provided complementary knowledge of BCR signaling. Compared to anti-IgM induction, the phosphoproteomic profiling of BCR signaling, along with validation by western blot analysis and pharmacological inhibitors, revealed that the activation of Syk, Btk, and PI3K may be dominant in galectin-1-mediated activation. We further demonstrated that the proliferation of antigen-primed B cells was diminished in the absence of galectin-1 in an animal model. Together, these findings provided evidence for a new role and insight into the mechanism of how galectin-1 augments the strength of the immunological synapse by modulating BCR signaling. The current study revealed the first systematic phosphorylation-mediated signaling network and its dynamics in B cell activation. The comparative phosphoproteomic analysis on the dynamics of galectin-1 induced activation profiles not only showed that exogenously added galectin-1 augmented B-cell activation but also revealed its relatively enhanced activation in PI3K pathway. Together with proliferation assay, we further delineated that galectin-1 is important for B-cell proliferation in response to antigen challenge. Our phosphoproteomic study reveals a new role for galectin-1 in augmenting the strength of immunological synapse by modulating BCR signaling.

B-cell development and functions and therapeutic options in adenosine deaminase–deficient patients

Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor. We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function. Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation. Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT. ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.

Immunomodulatory Activity of Recombinant Ricin Toxin Binding Subunit B (RTB)

Ricin toxin binding subunit B (RTB) is one of the subunits of the ricin protein. RTB has been used as adjuvant, but little is known about its mechanism. In this study, we found that RTB increased not only nitric oxide (NO) release, but also tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in mouse macrophage cell line RAW264.7 cells. They subsequently exhibited enhanced ConA-induced T-cell and LPS-induced B-cell proliferative responses. We also examined the cytokines that were produced from splenocytes following in vitro RTB administration. Increased levels of IL-2, interferon (IFN)-γ and TNF-α were observed, while IL-4 and IL-5 were unaffected. These results demonstrate that recombinant RTB can act on the immune system and activate T-cells by introducing a Th1 immune response. Th1 cells might be the primary cellular target affected by RTB. Our results suggest that the recombinant RTB can promote the activation of macrophages and has a beneficial effect on immunomodulatory activity.

Toll-Like Receptor 2 and Toll-Like Receptor 4-Dependent Activation of B Cells by a Polysaccharide from Marine Fungus Phoma herbarum YS4108

Various natural polysaccharides are capable of activating the immune system and therefore can be employed as biological response modifiers in anti-tumor therapy. We previously found a homogenous polysaccharide from the mycelium of marine fungus Phoma herbarum YS4108, named YCP, exhibiting strong in vivo antitumor ability via enhancement of the host immune responses. To further elucidate the role of YCP as a biological response modifier, the immunomoduating activities of YCP in B cells was investigated in the current study. We demonstrated that stimulation of YCP with murine splenic B cells resulted in cell proliferation and generation of IgM antibody response. Binding of YCP to B cells was a direct, saturable and reversible event and required TLR2 and TLR4 involvement. TLR2 and TLR4 defunctionalization by either antibody blocking or allele-specific mutation significantly impaired the B-cell proliferative and IgM responses to YCP. YCP interaction with TLR2 and TLR4 led to the activation of intracellular p38, ERK and JNK, as well as the translocation of transcriptional factor NF-κB into nucleus. Furthermore, specific inhibitors of p38, ERK, JNK and NF-κB could attenuate the ability of YCP to induce B cell proliferation and IgM production. Taken together, this study has indicated for the first time the immunostimulating properties of YCP on B cells through a receptor-mediated mechanism, which involves TLR2 and TLR4 and resultant activation of MAPK and NF-κB signaling pathways, thereby highlighting the role of YCP as an efficacious biological response modifier in oncologic immunotherapy.

Adult-onset manifestation of idiopathic T-cell lymphopenia due to a heterozygous RAG1 mutation

Adult-onset manifestation of idiopathic T-cell lymphopenia due to a heterozygous RAG1 mutation

Targeting antiapoptotic A1/Bfl-1 by in vivo RNAi reveals multiple roles in leukocyte development in mice

AbstractGene-targeting studies in mice have identified the essential roles of most prosurvival Bcl-2 family members in normal physiology and under conditions of stress. The function of one member, Bcl2a1/Bfl-1/A1, is only poorly understood because of quadruplication of its gene locus in mice, hindering conventional knockout studies. To overcome this problem, we generated mouse models allowing traceable constitutive or reversible ablation of A1 in the hematopoietic system by RNA interference. Knockdown of A1 impaired early stages of T-cell differentiation, B-cell homeostasis, and sensitized transitional as well as follicular B cells to apoptosis induced by ligation of the B-cell receptor. As a consequence, B-cell proliferation in response to mitogens was severely impaired, whereas that of T cells appeared unaffected. Furthermore, depending on the extent of A1 knockdown, granulocytes showed increased spontaneous death in culture or failed to accumulate in significant numbers in vivo. These models highlight the critical role of A1 in leukocyte development and homeostasis, constituting valuable tools for investigating presumed roles of this Bcl-2 family member in immunity, tumorigenesis, and drug resistance.

Survey: Immune function and immunotoxicity assessment in dogs

While immunotoxicology evaluations are often conducted in either rodents or non-human primates, findings in standard toxicology studies may trigger additional investigations in dogs. A survey sponsored by the HESI Immunotoxicology Technical Committee, and described herein, was conducted to gather information regarding the extent and nature of immunology and immunotoxicity assessments available in the dog, and the need thereof. The survey was issued via e-mail to scientists affiliated with 39 organizations in industry and academia, including contract research organizations, academic research organizations, pharmaceutical companies, and veterinary practices. Fifteen institutions responded, including 10 biotechnology or pharmaceutical industry organizations, 4 contract research organizations, and 1 academic institution. Responses indicated that indeed, immunological assessments in dogs are necessary for research and/or toxicology purposes. The survey demonstrated that multiple types of assays are used in the dog model, including assessment of T-cell-dependent antibody responses, immunoglobulins, complement CH50, cytokines and cytokine mRNAs, lymphocyte proliferation in response to T-cell mitogens, neutrophil activation, phagocytosis, and immunophenotyping of several cell types. The survey also revealed that certain assays/endpoints are not available in the dog (complement components, NK immunophenotyping, T-cell activation and memory immunophenotyping) or require further optimization (ex vivo cytolysis assays such as CTL and NK function, B-cell proliferation in response to LPS). In addition, the survey indicated that a greater understanding of the specificity of the available immunophenotyping reagents is needed.

Activated human renal tubular cells inhibit autologous immune responses

Renal proximal tubule epithelial cells (PTEC) respond and contribute to the pathological process in a range of kidney diseases. Within this disease setting, PTEC up-regulate surface antigens which may enable them to act as non-professional antigen-presenting cells and become targets for infiltrating T cells in the context of disease and allograft rejection. In order to define, for the first time, whether PTEC modulate immune responses within the autologous human system, we monitored their interaction with T and B cells in the presence of stimuli which mimic immunological signalling. The expression of PTEC surface antigen in response to inflammatory mediators was monitored by flow cytometry. Purified T and B lymphocyte subsets and peripheral blood mononuclear cells were cultured in the presence or absence of autologous activated PTEC, and their responses to specific activators were monitored by proliferation, cytokine secretion and surface antigen expression. Some experiments were performed in the presence of blocking antibodies to PD-L1. The presence of activated primary autologous PTEC resulted in significantly decreased T- and B-cell proliferative responses, which were only partly mediated by programmed death ligand 1. This modulation was not induced by a decrease in activation markers or an increase in T regulatory cells but was accompanied by strong significant skewing of cytokine profiles. Significant decreases in gamma-interferon, interleukin-2 and tumour necrosis factor and increases in interleukin-4 were detected in the presence of PTEC, indicating that these cells induce a shift away from an inflammatory Th1 effector profile to a Th2 type profile. Human PTEC do modulate autologous immune responses. We hypothesize that such mechanisms may have developed to help dampen inflammatory responses and macrophage activation seen within kidney interstitium in many immune-mediated kidney diseases.

The C104R mutant impairs the function of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) through haploinsufficiency

TNFRSF13B, which encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), is mutated in 10% of patients with common variable immunodeficiency. One of the 2 most common TACI mutations in common variable immunodeficiency, C104R, abolishes ligand binding and is found predominantly in the heterozygous state. The murine TACI mutant C76R is the equivalent of the human TACI mutant C104R. We sought to define the consequence of the C76R mutation on TACI function in mice that express both wild-type TACI and the murine C76R mutant. Transgenic mice that express murine TACI C76R, the counterpart of human TACI C104R, on the TACI(+/-) B6/129 background (C76R/TACI(+/-) mice) were constructed. Serum immunoglobulins and antibody responses to the type II T-independent antigen trinitrophenylated (TNP)-Ficoll were determined by means of ELISA. B-cell proliferation in response to a proliferation-inducing ligand was determined based on tritiated thymidine incorporation into DNA. IgG1 secretion by B cells in response to a proliferation-inducing ligand plus IL-4 was determined by means of ELISA. C76R/TACI(+/-) mice had significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll compared with TACI(+/+) B6/129 control animals, and their B cells were impaired in their capacity to proliferate and secrete IgG1 in response to TACI ligation. Unexpectedly, TACI(+/-) mice had similarly impaired B-cell function as C76R/TACI(+/-) littermates. Impaired TACI function caused by haploinsufficiency was confirmed in TACI(+/-) mice on the C57BL/6 background. These results suggest that the human TACI mutant C104R might impair TACI function in heterozygotes through haploinsufficiency.

Inhibition of B-Cell Activation by Retinal Pigment Epithelium

To determine whether retinal pigment epithelial (RPE) cells can inhibit B-cell activation in vitro. Primary cultured RPE cells were established from normal C57BL/6 mice. Activated target B cells were established from splenic B cells stimulated with anti-mouse CD40 antibody and lipopolysaccharide (LPS) in the presence of recombinant interleukin 4 (rIL-4). B-cell activation was assessed by examining proliferation through [(3)H]-thymidine incorporation or carboxyfluorescein succinimidyl ester dilution, and antibody production was determined by ELISA. Expression of costimulatory molecules and the receptors on B cells was evaluated by flow cytometry. Neutralizing anti-TGFβ antibodies were used in the assay. Addition of primary cultured RPE cells suppressed B-cell proliferation in response to anti-CD40, LPS, and rIL-4 stimulation. Similarly, antibody production by these activated B cells was suppressed. Suppression of B-cell activation was mediated by a soluble factor because supernatants from cultured RPE cells were sufficient to inhibit B-cell responses. Moreover, TGFβ was identified as the soluble mediator given that RPE-supernatants failed to suppress B-cell activation if pretreated with neutralizing anti-TGFβ antibodies. Cultured RPE cells suppress the activation of B cells in vitro. These data support the hypothesis that retinal pigment epithelium has immunosuppressive properties that are capable of suppressing B-cell activation.

Evidence of a Functional B-Cell Immunodeficiency in Adults Who Experience Serogroup C Meningococcal Disease

After adolescence, the incidence of meningococcal disease decreases with age as a result of the cumulative immunizing effect of repeated nasopharyngeal colonization. Nevertheless, some adults succumb to meningococcal disease, so we hypothesized that this is due to a subtle functional immunological defect. Peripheral blood lymphocytes derived from survivors of serogroup C meningococcal disease and from age- and sex-matched controls were incubated with a polyclonal B-cell activator containing anti-immunoglobulin D (alpha-delta-dex) employed to mimic antigen-specific stimuli encountered during immune responses to bacterial polysaccharides, with and without T-cell activation (using anti-CD3/anti-CD28). Subsequent proliferation and activation of T and B lymphocytes were measured. In patients, T-cell responses to polyclonal stimuli and the delivery of T-cell help to B cells were unimpaired. Levels of B-cell proliferation in response to alpha-delta-dex stimulation alone were low in all samples but were significantly lower in patients than in controls, and these differences were more pronounced with the addition of T-cell help. The data are consistent with the presence of a subtle immunodeficiency in adults who have exhibited susceptibility to meningococcal disease. This defect is manifested as an impaired B-cell response to T-cell-independent type 2 antigens analogous to bacterial capsular polysaccharide.

Immunomodulatory Effects of Dietary Potassium Perfluorooctane Sulfonate (PFOS) Exposure in Adult Sprague-Dawley Rats

Perfluorooctanesulfonate (PFOS) is a stable and environmentally persistent metabolic or degradation product of perfluorooctanyl compounds that were manufactured for a variety of industrial and consumer applications. PFOS itself was sold for use as a surfactant. The structurally related contaminants perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), and N-ethyl perfluorooctane sulfonamide (N-EtPFOSA) were shown to suppress immune responses in laboratory rodents. Relatively low doses of PFOS were found to be immunosuppressive in mice. To assess effects of PFOS on the rat immune system at doses known to alter hepatic function, changes in the morphology and function of immune tissues and cells were measured in adult rats exposed to PFOS in their diet for 28 d at levels ranging from 2 to 100 mg PFOS/kg diet (corresponding to approximately 0.14 to 7.58 mg/kg body weight [bw]/d) and compared to those receiving control diet. Body weight reductions were significant in male and female rats exposed to 50 and 100 mg PFOS/kg diet. Liver/body weight was significantly increased in females exposed to 2 mg PFOS/kg diet and in males exposed to 20 mg PFOS/kg diet. Female rats exposed to 100 mg PFOS/kg diet exhibited a significant increase in spleen weight relative to body weight; these changes lacked a histologic correlate and were not observed in males. While thymus weights relative to body weights were not affected, numbers of apoptotic lymphocytes rose in thymus with increasing dietary PFOS. There was a significant dose-related increase in total peripheral blood lymphocyte numbers in female but not male rats. In both genders the percentages of cells within lymphocyte subclasses were altered. There was a significant trend toward increasing T and T‐helper (Th) cells and decreasing B cells with higher PFOS dose. Serum total immunoglobulin (Ig) G1 levels were significantly reduced in males exposed to 2 and 20 mg PFOS/kg diet. The ability of male and female rats to mount delayed-type hypersensitivity (DTH) responses to the T-cell-dependent antigen keyhole limpet hemocyanin (KLH) was not altered by PFOS. There was a significant trend toward elevated KLH-specific IgG in serum from male rats exposed to increasing levels of PFOS in diet. Splenic T- and B-cell proliferation in response to ex vivo mitogen exposure was unaffected by exposure to dietary PFOS. In conclusion, changes in immune parameters in rat did not manifest as functional alterations in response to immune challenge with KLH and may be secondary to hepatic-mediated effects of PFOS in this model.

Relationship of Blood Mercury Levels to Health Parameters in the Loggerhead Sea Turtle ( Caretta caretta )

BackgroundMercury is a pervasive environmental pollutant whose toxic effects have not been studied in sea turtles in spite of their threatened status and evidence of immunosuppression in diseased populations.ObjectivesIn the present study we investigate mercury toxicity in loggerhead sea turtles (Caretta caretta) by examining trends between blood mercury concentrations and various health parameters.MethodsBlood was collected from free-ranging turtles, and correlations between blood mercury concentrations and plasma chemistries, complete blood counts, lysozyme, and lymphocyte proliferation were examined. Lymphocytes were also harvested from free-ranging turtles and exposed in vitro to methylmercury to assess proliferative responses.ResultsBlood mercury concentrations were positively correlated with hematocrit and creatine phosphokinase activity, and negatively correlated with lymphocyte cell counts and aspartate amino-transferase. Ex vivo negative correlations between blood mercury concentrations and B-cell proliferation were observed in 2001 and 2003 under optimal assay conditions. In vitro exposure of peripheral blood leukocytes to methylmercury resulted in suppression of proliferative responses for B cells (0.1 μg/g and 0.35 μg/g) and T cells (0.7 μg/g).ConclusionsThe positive correlation between blood mercury concentration and hematocrit reflects the higher affinity of mercury species for erythrocytes than plasma, and demonstrates the importance of measuring hematocrit when analyzing whole blood for mercury. In vitro immunosuppression occurred at methylmercury concentrations that correspond to approximately 5% of the individuals captured in the wild. This observation and the negative correlation found ex vivo between mercury and lymphocyte numbers and mercury and B-cell proliferative responses suggests that subtle negative impacts of mercury on sea turtle immune function are possible at concentrations observed in the wild.

Preferential depletion of a splenic marginal zone-like peripheral blood CD27+B220- memory B cell population in HIV-1 infected individuals

In addition to CD4+ T cell depletion, HIV-1 infection is characterized by changes in humoral immunity including hypergammaglobulinemia, polyclonal B cell activation and loss of circulating memory B lymphocytes. Expression of the CD45 isoform B220 has recently been described on CD27- and a subset of CD27+ human B lymphocytes. The aim of this study was to evaluate B cell defects in HIV infection in more detail. We studied the frequency of memory B cells, based on the expression of B220, in HIV infected individuals (n = 27) and healthy controls (n = 22) and characterized their functional properties. RNA expression of TLR9 and activation-induced cytidine deaminase (AICD) was analyzed by Real-time PCR from B cells sorted according to their memory phenotype. In addition, B-cell proliferative responses and immunoglobulin secretion in response to SAC and ODN treatment were monitored. We found that the previously described reduction in the frequency of CD27+ B cells in HIV-infected individuals affects preferentially the subset characterized by the lack of B220 expression. The proportion of CD27+B220- B cells expressing surface IgD (28.8%) and IgM (29.5%) was reduced in the HIV+ group compared to healthy controls (53.9% and 59.8%, respectively). This depletion did not correlate with either CD4 counts or viral load, and was not reversed by antiretroviral therapy. We found that CD27+B220- B cells have a splenic marginal zone like immunophenotype (IgMhiIgDloCD21+CD23-), express TLR9, and proliferate and secrete IgG and IgM in response to B cell specific ODN. In contrast, CD27+B220+ B cells are IgMloIgDhiCD21+CD23+, express AICD and proliferate in response to SAC but do not secrete immunoglobulins. The lack of B220 expression on CD27+ B cells defines a distinct memory B cell compartment that is preferentially depleted in HIV-infected individuals. The phenotype, pattern of gene expression and functional properties of the preferentially depleted CD27+B220- B cells suggest that these cells are splenic marginal zone-like circulating memory B cells. The lack of these cells in HIV patients may play an important role in the defective immunity against T-independent pathogens and may impair the humoral control of HIV-1 propagation.