Cell Immunoglobulin Production Research Articles

Why Proteasome Inhibitors Cannot ERADicate Multiple Myeloma

Proteasome inhibitors are key parts of our armamentarium against multiple myeloma, but the disease can become resistant through poorly defined mechanisms. In this issue of Cancer Cell, Leung-Hagesteijn and colleagues describe XBP1s(-) subpopulations of tumor cells that are resistant to bortezomib and may account for therapeutic failures in the clinic.

Xbp1s-Negative Tumor B Cells and Pre-Plasmablasts Mediate Therapeutic Proteasome Inhibitor Resistance in Multiple Myeloma

Proteasome inhibitor (PI) resistance mechanisms in multiple myeloma (MM) remain controversial. We report the existence of a progenitor organization in primary MM that recapitulates maturation stages between B cells and plasma cells and that contributes to clinical PI resistance. Xbp1s(-) tumor B cells and pre-plasmablasts survive therapeutic PI, preventing cure, while maturation arrest of MM before the plasmablast stage enables progressive disease on PI treatment. Mechanistically, suppression of Xbp1s in MM is shown to induce bortezomib resistance via de-commitment to plasma cell maturation and immunoglobulin production, diminishing endoplasmic reticulum (ER) front-loading and cytotoxic susceptibility to PI-induced inhibition of ER-associated degradation. These results reveal the tumor progenitor structure in MM and highlight its role in therapeutic failure.

FRI0008 IL-21 regulates B cell proliferation and differentiation in rheumatoid arthritis

BackgroundInterleukin (IL)-21 is a member of type I cytokine family. Recent studies have indicated that IL-21 is an important regulator for human B cell activation, proliferation, plasma cell (PC) differentiation,...

Avian Resistance to Campylobacter jejuni Colonization Is Associated with an Intestinal Immunogene Expression Signature Identified by mRNA Sequencing

Campylobacter jejuni is the most common cause of human bacterial gastroenteritis and is associated with several post-infectious manifestations, including onset of the autoimmune neuropathy Guillain-Barré syndrome, causing significant morbidity and mortality. Poorly-cooked chicken meat is the most frequent source of infection as C. jejuni colonizes the avian intestine in a commensal relationship. However, not all chickens are equally colonized and resistance seems to be genetically determined. We hypothesize that differences in immune response may contribute to variation in colonization levels between susceptible and resistant birds. Using high-throughput sequencing in an avian infection model, we investigate gene expression associated with resistance or susceptibility to colonization of the gastrointestinal tract with C. jejuni and find that gut related immune mechanisms are critical for regulating colonization. Amongst a single population of 300 4-week old chickens, there was clear segregation in levels of C. jejuni colonization 48 hours post-exposure. RNAseq analysis of caecal tissue from 14 C. jejuni-susceptible and 14 C. jejuni-resistant birds generated over 363 million short mRNA sequences which were investigated to identify 219 differentially expressed genes. Significantly higher expression of genes involved in the innate immune response, cytokine signaling, B cell and T cell activation and immunoglobulin production, as well as the renin-angiotensin system was observed in resistant birds, suggesting an early active immune response to C. jejuni. Lower expression of these genes in colonized birds suggests suppression or inhibition of a clearing immune response thus facilitating commensal colonization and generating vectors for zoonotic transmission. This study describes biological processes regulating C. jejuni colonization of the avian intestine and gives insight into the differential immune mechanisms incited in response to commensal bacteria in general within vertebrate populations. The results reported here illustrate how an exaggerated immune response may be elicited in a subset of the population, which alters host-microbe interactions and inhibits the commensal state, therefore having wider relevance with regard to inflammatory and autoimmune disease.

DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation

DOCK8 and MyD88 have been implicated in serologic memory. Here we report antibody responses were impaired and CD27+ memory B cells were severely reduced in DOCK8-deficient patients. Toll-like receptor 9 (TLR9)- but not CD40-driven B cell proliferation and immunoglobulin production were severely reduced in DOCK8-deficient B cells. In contrast, TLR9-driven expression of AICDA, CD23 and CD86, and activation of NF-κB, p38 and Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. Following TLR9 ligation, DOCK8 became tyrosine phosphorylated by Pyk2, bound the Src family kinase Lyn and linked TLR9 to a Src-Syk-STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

B cell development and immunoglobulin production are altered in the absence of IκBNS (109.4)

Abstract NF-κB transcription factors are key mediators of immune function as well as inflammatory responses. The activation of NF-κB is regulated by several IκB proteins, a family comprising classical cytoplasmic as well as nuclear IκBs. In this study, we focused on the role of IκBNS, a nuclear IκB protein, in B lymphocyte development and function. Analysis of lymphoid tissues from IκBNS knockout (KO) mice revealed that a lack of IκBNS affects the development of peritoneal B-1 cells and splenic marginal zone B cells. Development of B-2 cells was essentially normal. We also found that B cell proliferative activity and immunoglobulin production were altered in the absence of IκBNS. These defects were confirmed by the analysis of B cells in bone marrow chimeric mice constructed by the transfer of wild type or IκBNS KO bone marrow cells into B cell-deficient mice. Furthermore, histological analysis of spleens from chimeric mice revealed that re-constitution of B cell areas, marginal zones and germinal centers were markedly reduced in IκBNS KO cell recipient mice. Examination of the functions of IκBNS KO B cell subsets suggested that IκBNS is important for antibody production by both marginal zone and follicular B cells. These results indicate that IκBNS plays a key role in peripheral B cell development and function.

Immune Modulation Effect of Pig Placenta Extracts in a Mouse Model: Putative Use as a Functional Food Supplement

This study was performed to establish an effective extraction method of pig placenta extract that could be used for a putative functional food supplement with immunomodulatory effects. In the present study, we used different temperatures (4, 37, 60, 80, and <TEX>$100^{\circ}C$</TEX>) and different solvents (chloroform, NaOH, and phosphate buffered saline [PBS]) to extract the pig placenta. Among the different placenta extracts yielded by the different extraction methods, placenta extract (PE) in PBS at <TEX>$80^{\circ}C$</TEX> for 30 min (referred to as PE-PBS80) showed a significant increase of nitric oxide production of up to 22.97 <TEX>${\mu}M/10^5$</TEX> cells at a 1 mg/mL dose (p<0.05 ) in J774A.1 cells than other extracts and control tested. Using PE-PBS80, further animal challenges were performed to identify the immune-enhanced effects. As a result, orally administered PE-PBS80 showed a significant increase in blood T and B cell activities and immunoglobulin (IgG and IgM) production. IgG and IgM levels increased to 41.53 mg/mL at a 20 mg dose on day 7 and to 27.38 mg/mL at a 10 mg dose on day 14, respectively (p<0.05). Furthermore, PE-PBS80 was also able to significantly enhance the immune modulator cytokine levels (p<0.05) compared to the control and vehicle treatments. Among the evaluated cytokines, the tumor necrosis factor-<TEX>${\alpha}$</TEX> (TNF-<TEX>${\alpha}$</TEX>) level increased to 28.89 pg/mL at extract doses of 20 and 50 mg, the interleukin-<TEX>$1{\beta}$</TEX> (IL-<TEX>$1{\beta}$</TEX>) level increased to 21.52 pg/mL at extract doses of 10, 20, 50 and 75 mg and the interferon (IFN)-<TEX>${\gamma}$</TEX> level increased to 18.24 pg/mL at extract doses of 10, 20, and 50 mg. Therefore, this study presents an effective method for extracting pig placenta extracts and also demonstrates that pig placenta extracts had significant immunomodulatory effects not only at the cellular level but also in a mouse model, suggesting that this material could be used as an excellent candidate functional food supplement.

Effect of vitamin D3 supplementation on peripheral B cell differentiation and isotype switching in patients with multiple sclerosis

Background: Vitamin D has been proposed as a promoter of immune homeostasis in multiple sclerosis (MS). During the past decade, the focus of the effects of vitamin D has been on dendritic cells and on T cells. Since there is an increasing interest in the role of B cells in the pathophysiology of MS, we studied the role of vitamin D on B cells in vivo in patients with MS. Objective: We explored the effects of 12 weeks high-dose vitamin D3 supplementation on peripheral B cell differentiation, immunoglobulin production and levels of B cell activating factor (BAFF) in 15 patients with MS. Methods: Circulating B cell subsets were characterized by flow cytometry. Plasma immunoglobulin levels were assessed by nephelometry. Plasma BAFF levels were assessed by enzyme-linked immunosorbent assay (ELISA). Results: Although a significant increase serum 25-hydroxyvitamin D was induced, we found no significant shift in B cell differentiation, isotype switching, or plasma BAFF levels. Conclusion: In patients with MS, supplementation of high doses vitamin D3 does not have substantial effects on phenotypic markers of B cell differentiation in circulating B cells. Future studies may unravel more subtle changes in the B cell compartment, either in the circulation or in the central nervous system.

Glutamate signaling through the kainate receptor enhances human immunoglobulin production

CD23 is implicated as a regulator of IgE synthesis. A soluble form of CD23 (sCD23) is released following cleavage by ADAM10 and enhanced sCD23 is correlated with increased IgE. In the CNS, signaling through the kainate receptor (KAR) increases ADAM10. In B cells, activation of KARs produced a significant increase in ADAM10 and sCD23 release as well as an increase in B cell proliferation and immunoglobulin production. In addition, ADAM10 inhibitors reduce IgE synthesis from in vitro cultures of human B cells. Thus, we report for the first time the unique presence of the kainate receptor in B cells and that activation of KARs could serve as a novel mechanism for enhancing B cell activation.

Liver X receptors and immune regulation.

Recent studies suggest that homeostasis of lipid metabolism is crucial for the function of various immune cells. Oxygenated derivatives of cholesterol (oxysterols) are well-known regulators of lipid metabolism and have diverse functions, such as inhibition of cholesterol synthesis, efflux of intracellular cholesterol, synthesis of cholesterol esters, and activation of liver X receptors (LXRs). In this review, we introduce novel roles of the oxysterol receptors LXRs in the immune system, including regulation of inflammatory responses, T cell expansion, immunoglobulin production, and antitumor responses. We also discuss lipid-mediated signaling as a potential target for treatment of immune diseases.

Therapeutic Implications of the IL-21: IL-4 Receptor System in Children with Common Variable Immunodeficiency Syndrome

Interleukin-21 (IL-21) has been shown to restore immunoglobulin production together with Interleukin-4 (IL-4) in common variable immunodeficiency syndrome (CVID). Here, we elucidate the functional and structural properties of the corresponding IL-21R : IL-4R system. An in vitro cell culture system was established to study the molecular effects of IL-21 and IL-4 on B cell differentiation, class-switch recombination (CSR) and immunoglobulin (Ig) production in 32 paediatric patients with CVID. MHC haplotypes and the IL21 and IL21R gene were analysed by genotyping. Ternary complexes of the IL-21 respectively IL-4 receptor were set-up by homology modeling and ligand-interaction was examined by molecular dynamics (MD) simulation. Stimulation with IL-21, IL-4 and CD40L uniformly induced IgG and IgA production in B cells from all tested patients by initiation of both CSR and AID-independent Ig production. No mutations were found in the coding regions of the IL21 or IL21R genes and no distinct HLA allele or extended haplotype could be correlated with the amount of Ig production or the gene expression pattern induced by IL-21. MD simulations of the modelled receptor complexes showed that IL-4 and IL-21 are both able to bind to IL-4R and IL-21R complexes in an interchangeable manner. The function of the IL-21R : IL-4R system seems not to be related to the aetiology of CVID in paediatric patients and might be suitable for a regenerative therapy.

Follicular Dendritic Cell Makes Environmental Sense

Follicular dendritic cells (FDCs) are key organizers of B cell follicles and germinal centers. In this issue of Immunity, Suzuki et al. (2010) and Garin et al. (2010) identify the roles of Toll-like receptors in the responses of FDCs, providing a unique link between innate and adaptive immunity.

Immune-Modulatory Effects of Mesenchymal Stromal Cell Infusions for the Treatment of Factor VIII Inhibitor in Hemophilia A.

Abstract 1299Poster Board I-321The current most serious treatment-related complication in hemophilia A is the development of antibodies to FVIII following exposure to FVIII replacement therapy. We have previously demonstrated that the injection of syngeneic mesenchymal stromal cells (MSCs) into hemophilia A mice with FVIII antibodies lead to a robust decrease in FVIII specific IgG levels because the secretome of MSCs suppresses plasma cell immunoglobulin production, induces plasmablast proliferation, and leads to IL-10-mediated blockade both in vitro and in vivo. Therefore, we wanted to pursue this study in a large animal model. In the current study, we harvested MSCs from bone marrow aspirates obtained from 2 hemophilia A dogs with both human and canine FVIII inhibitors. The isolated MSCs were expanded ex vivo for the infusions. Prior to the infusions, we performed in vitro characterization of the canine MSCs and found that the cells secrete a wide range of cytokines and chemokines including CC chemokine ligand 2 (CCL2) and CCL2 that has been cleaved to its truncated inhibitory form by proteolysis with matrix metalloproteinases (MMPs). These results are identical to those seen in the previous mouse experiments. Before the MSC infusions, the hemophilia A dogs with inhibitors were immunized by multiple intravenous infusions of both human FVIII and canine cryoprecipitate to further boost the development of FVIII antibodies. Expanded autologus MSCs (4.5-6.0×106 cells/Kg) were infused intravenously into the first dog twice, with a month in between each injection. After the second infusion, the titer of anti-human FVIII inhibitory activity was significantly decreased from 6.2 to 1.9 BUs and a transient but significant decrease from 3.5 to 2.2 BUs against canine FVIII. Second MSC infusion trial is currently ongoing in a second dog. In parallel with in vivo study, the inhibitory response mediated by MSCs on the FVIII antibody producing B cells was assessed by ELISPOT assay. In conclusion, MSCs may play an immunosuppressive role in modulating Immunoglobulin production by plasma cells via MMP processing of paracrine CCL2 secretion. This may represent a novel cellular therapy for attenuation of pathological humoral responses such as anti-FVIII antibody development. DisclosuresNo relevant conflicts of interest to declare.

Multiple bovine FcγRIIb sub-isoforms generated by alternative splicing

Receptors for the Fc portion of immunoglobulin molecules (FcR) provide an important and vital link between circulating antibody and cellular effector functions. These receptors have been well characterized in human and murine species, however few of these receptors have been investigated in livestock. FcγRII (CD32) is an FcR previously shown in mice and humans to exist in multiple isoforms, both activating (FcγRIIa, FcγRIIc) and inhibitory (FcγRIIb), on a wide variety of cells including B cells, T cells, dendritic cells, monocytes, macrophages and platelets. On B cells, FcγRIIb acts to suppress cell activation and immunoglobulin production by means of an intracellular immunoreceptor tyrosine-based inhibitory motif signaling domain. Two sub-isoforms of FcγRIIb, designated b1 and b2, distinguished by the inclusion of an additional cytoplasmic exon in the b1 form, have been demonstrated in humans and mice, whereas only one sequence corresponding to the human and mouse b2 isoform has been identified in cattle. In this study, the expression profile of FcγRIIb in bovine blood mononuclear cells was characterized by collecting blood samples from mature cattle of dairy and beef breeds, and determining their FcγRIIb mRNA expression profile by RT-PCR. Analysis revealed the presence of two uncharacterized bovine FcγRIIb transcripts in addition to the single previously published transcript. Analysis of the first unknown transcript revealed high homology with published human and murine FcγRIIb1 sequences. This transcript was present in all cell types examined, with little variation in primary sequence between individuals or among breeds. The second unknown sequence was found to be homologous to the murine FcγRIIb3 (IgG-binding protein or soluble FcγR in humans) sequence. This transcript appears to have a much more limited expression profile, which may indicate that expression varies with the cellular activation-state of the cell. These results indicate that cattle, like humans and mice, express multiple sub-isoforms of FcγRIIb. These findings add further complexity to the regulation of IgG-mediated immunity and provide new insight into the role Fc receptors play in antigen acquisition and presentation in cattle.

Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help

In general, humoral immune responses depend critically upon T cell help. In transplantation, prevention or treatment of humoral rejection therefore require drugs that ideally inhibit both B cell and T helper cell activity. Here, we studied the effects of commonly used immunosuppressive drugs [tacrolimus, cyclosporin, mycophenolic acid (MPA) and rapamycin] on T cell helper activity and on T cell-dependent B cell responses. T cells were activated polyclonally in the presence of immunosuppressive drugs in order to analyse the effect of these drugs on T cell proliferation, co-stimulatory ligand expression and cytokines. The impact of immunosuppressive drugs on T cell-dependent immunoglobulin production by B cells was addressed in T-B cell co-cultures. All drugs affected T cell proliferation and attenuated T cell co-stimulatory ligand (CD154 and CD278) expression when T cells were activated polyclonally. Tacrolimus, cyclosporin and rapamycin also attenuated B cell stimulatory cytokine mRNA levels in T cells. As a consequence, a decrease in immunoglobulin levels was observed in autologous T-B cell co-cultures, where T cell help is essential for immunoglobulin production. In contrast, when pre-activated T cells were used to stimulate autologous B cells, calcineurin inhibitors failed to inhibit B cell immunoglobulin production, whereas MPA and rapamycin did show inhibition. From these studies, it is evident that calcineurin inhibitors affect the humoral immune response by interfering with T helper signals, but not by targeting B cells directly. Furthermore, our studies support the necessity of intervening in T cell helper function to attenuate humoral responses.

IL‐21: roles in immunopathology and cancer therapy

Cytokines are secreted signalling molecules with decisive effects on haematopoiesis, innate and adaptive immunity, and immunopathology. Interleukin (IL)-21 is a novel cytokine produced by activated CD4(+) T cells and natural killer T (NKT) cells. IL-21 is part of a family of cytokines which include IL-2, -4, -7, -9 and -15 that all share the common IL-2 receptor gamma chain (gamma(c)) in their individual receptor complexes. IL-21 receptor (IL-21R) is widely expressed on both myeloid and lymphoid cell lineages and IL-21 actions include co-stimulation of B cell differentiation and immunoglobulin (Ig) production, co-mitogen of T cells, and stimulation of NK and CD8(+) T cell cytotoxic function. Initially, IL-21 was recognized for its anti-tumour effects in several preclinical tumour models, warranting its currently ongoing clinical development as a cancer immunotherapeutic. More recently, IL-21 has been associated with the development of a panel of autoimmune and inflammatory diseases, where neutralization of IL-21 has been suggested as a potential new therapy. In this review, we will cover the latest discoveries of IL-21 as a cancer therapy and its implications in immunopathologies.

Allogeneic Mesenchymal Stem Cells for Treatment of Experimental Autoimmune Encephalomyelitis

The use of allogeneic "universal donor" mesenchymal stromal cells (MSCs) may be a substantial clinical convenience for treatment of autoimmune ailments such as multiple sclerosis. We therefore tested whether allogeneic MSCs can be exploited for treatment of experimental autoimmune encephalomyelitis (EAE) in mice with otherwise intact immune system. Administration of allogeneic Balb/c-derived MSCs to C57Bl/6 mice with pre-established EAE led to a significant decrease in disease score over time comparable to that achieved with syngeneic MSCs, and was correlated with a significant blunting of immune cell infiltration to the spinal cord and reduced circulating levels of interferon-gamma (IFN-gamma) and interleukin-17. Pretreatment of allogeneic MSCs with IFN-gamma increased the expression levels of CCL2 as well as major histocompatibility complex I (MHCI) and MHCII, but also led to complete loss of suppressive activity in vivo that correlated with immune rejection. In conclusion, allogeneic MSCs can suppress the manifestations of EAE, yet retain the potential for alloimmunization.

Intravenous immunoglobulin preparations have no direct effect on B cell proliferation and immunoglobulin production

Intravenous immunoglobulin (IVIg) is used for treatment of a variety of immunological disorders and in transplantation. As one of its applications in transplantation is the reduction of donor specific antibodies in the circulation, we examined the direct effect of IVIg on essential parameters of human B cell responses in vitro. Purified human B cells, human B cell hybridomas and T cells were cultured in the presence of graded concentrations of IVIg to test its effect on their proliferative capacity. To address the effect of IVIg on immunoglobulin production, we designed a novel technique making use of quantitative polymerase chain reaction to assess IgM and IgG levels. IVIg failed to inhibit proliferation of human B cells and human B cell hybridomas. In contrast, when IVIg was added to T cell cultures, a dose-dependent reduction of the proliferative capacity was observed. IVIg did not affect the levels of IgM and IgG mRNA of activated B cells. Our data show that IVIg is not capable of directly inhibiting key B cell responses. Direct B cell inhibition by IVIg seems therefore unlikely, implying that alteration in humoral immunity by IVIg is due to indirect effects on T cells and/or interactions with circulating antibodies and complement factors.

Toll-like receptor 7 and 9 defects in common variable immunodeficiency

Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia, reduced numbers of peripheral blood isotype-switched memory B cells, and loss of plasma cells. Because Toll-like receptor (TLR) activation of B cells can initiate and potentially sustain normal B cell functions, we examined functional outcomes of TLR7 and TLR9 signaling in CVID B cells. TLR7-mediated, TLR7/8-mediated, and TLR9-mediated cell proliferation, isotype switch, and immunoglobulin production by control and CVID B cells or isolated naive and memory B cell subsets were examined. We quantitated TNF-alpha, IL-6, and IL-12 production in response to TLR1-9 ligands and measured IFN-alpha production by TLR7-stimulated PBMCs and isolated plasmacytoid dendritic cells (pDCs). IFN-beta mRNA expression by TLR3-stimulated fibroblasts was assessed. Unlike CD27(+) B cells of controls, TLR7-activated, TLR7/8-activated, or TLR9-activated CVID B cells or isolated CD27(+) B cells did not proliferate, upregulate CD27, or shed surface IgD. TLR-stimulated CVID B cells failed to upregulate activation-induced cytosine deaminase mRNA or produce IgG and IgA. TLR7-stimulated PBMCs and pDCs produced little or no IFN-alpha. Reconstituting IFN-alpha in TLR7-stimulated CVID B-cell cultures facilitated proliferation, CD27 upregulation, and isotype switch. These TLR defects are restricted because CVID PBMCs stimulated with TLR ligands produced normal amounts of TNF-alpha, IL-6, and IL-12; TLR3-mediated expression of IFN-beta by CVID fibroblasts was normal. Defective TLR7 and TLR9 signaling in CVID B cells and pDCs, coupled with deficient IFN-alpha, impairs CVID B cell functions and prevents TLR-mediated augmentation of humoral immunity in vivo.

ADAM10 regulates B cell development and immunoglobulin production (138.6)

Abstract Members of the disintegrin and metalloprotease (ADAM) family can regulate lymphocyte development and function by cleaving membrane receptors. Previous studies showed that ADAM10 cleaves the B cell receptors, CD23 and Notch2, in vitro. However, in the absence of in vivo models, the physiologic impact of these events has not been examined. Thus, we generated B cell-specific ADAM10 KO and transgenic (Tg) mice to determine the role of ADAM10 in B cell development and function. Notch signaling regulates the differentiation of common lymphoid progenitors (CLPs) by promoting T cell fate. Accordingly, overexpression of ADAM10 in CLPs completely blocked B cell development. Deletion of ADAM10 from B cells in ADAM10fl/fl, CD19-cre+ mice prevented differentiation of marginal zone B cells, which is dependent on Notch2 signaling. In addition, expression of CD23 on ADAM10null B cells was dramatically elevated while levels of soluble CD23 were reduced in serum. As a result, IgE production by ADAM10fl/fl, CD19-cre+ mice was remarkably suppressed upon immunization, mimicking the response of CD23Tg mice. Finally, production of IgG1 and IgG2b was profoundly inhibited, suggesting a novel role for ADAM10 in class-switching to IgG. These results illustrate the physiologic role of ADAM10 in Notch signaling and CD23 cleavage. Moreover, they identify ADAM10 as a critical regulator of B cell development and immunoglobulin production. AHA 0815066E, NIAID R01AI18697, NIAID U19AI077435