Cell Ig Production Research Articles

T Follicular Regulatory (TFR) Cell Deficiency during Chronic Graft-Versus-Host Disease Is Improved By Low-Dose IL-2 Therapy

Background: Chronic Graft versus Host Disease (cGVHD) reflects the inability to achieve immune tolerance after allogeneic stem cell transplantation (allo-SCT) resulting in the persistence of allo and auto-reactive T cells and B cells. We previously showed that regulatory T cells (Treg) are decreased in patients with cGVHD and may be rescued by low dose IL-2 therapy resulting in improved Treg number and survival and clinical improvement of cGVHD symptoms. More recently, we showed that circulating T follicular helper cells (cTFH) have increased function during cGVHD, thus stimulating B cells to produce auto and allo-antibodies involved in cGVHD. T follicular regulatory cells (TFR) regulate the germinal center (GC) reaction and suppress B cell differentiation and immunoglobulin secretion. TFR reside primarily in lymphoid tissues but also circulate in small numbers. In the present study we characterized the numbers and function of blood CD3+CD4+CD45RA-CD25hiFoxP3+CXCR5+ TFR in healthy donors (HD; N=18) and allo-SCT patients (N=64).Results: Patients undergoing allo-SCT showed a significant reduction of circulating TFR (cTFR) compared to HD (median: 0.08% vs 0.34% of CD4+T respectively, p<10-4). Moreover, patients with active cGVHD (N=37) had significantly lower cTFR frequency compared to patients with no cGVHD (N=15) (median: 0.034 vs 0.18% of CD4+, p=0.0012) and resolved cGVHD (N=12) (median: 0.034 vs 0.14, p=0.02). Interestingly, cTFR values tended to be negatively associated with cGVHD grade (median cTFR %: 0.11 vs 0.029, p=0.004 for mild vs moderate; 0.11 vs 0.012, p=0.005 for mild vs severe; 0.029 vs 0.012, p=0.2 for moderate vs severe). The numeric reduction of cTFR in cGVHD patients appears to be a consequence of decreased survival as attested by lower BCL-2 expression (median: 1831 vs 1939, p=0.037 in active cGVHD and without cGVHD, respectively). This favors the survival of TFH effector cells (CD4+CD45RA-CXCR5+ excluding CD25hiFoxP3+) as measured by the ratio of expression of BCL-2 in effectors/regulators in active compared to no cGVHD (BCL-2 E/R ratio: 1.26 vs 1.14, p=0.0013 respectively). Similar to tonsil TFR, blood TFR exhibit suppressive functions in in vitro assays. Purified TFR were co-cultured with TFH and memory B cells at different ratios. The addition of TFR was associated with a significant reduction of plasmablast (PB) generation (CD27hiCD38hiCD19+ cells) (Figure 1A, t-test: p=0.0087) and IgG production (Figure 1B, t-test: p=0.0087). Human cTFR in vivo display similar suppressive function toward B cells, as analysis of active cGVHD patients revealed an inverse correlation between TFR and PB frequency in the peripheral blood (Spearman test: r = -0.49, p=0.003) (Figure 1C). As IL-2 has been shown to enhance regulatory T cells, we assessed its impact on the GC reaction in vitro . Purified TFH and memory B cells from HD were co-cultured at different doses of IL-2 from 0 to 100 IU/mL, simulating a germinal center reaction in vitro . Low dose IL-2 starting at 10 IU/mL was associated with a suppressive effect on PB generation and IgG production that peaked at 50 IU/mL reflecting the high expression level of IL-2 high affinity receptor (CD25). We also evaluated the capacity of IL-2 to restore in vivo cTFR:cTFH ratio using mass cytometry (CyTOF) in a cohort of patients receiving IL-2 (1x106 IU/M2/day) for 12 weeks as previously described (Koreth et al, Blood 2016). We observed an improvement of blood cTFR:cTFH ratio over the course of IL-2 therapy, with a maximum ratio observed at 2 weeks, and subsequently decreasing to reach baseline values after stopping IL-2 (Figure 2).Conclusion: Our results confirm that blood TFR exert a suppressive function as evidence by an inverse correlation with PB and in vitro suppression of memory B cell Ig production. This suppressive subset is deficient in patients with cGVHD due to altered TFR survival, thus favoring imbalance toward TFH effectors which may promote a GC reaction in vivo resulting in PB and memory B cell pathogenic Ig production. By enhancing TFR, low-dose IL-2 therapy promotes effective control of the GC reaction. [Display omitted] DisclosuresForcade:Neovii: Other: Travel grant. Koreth:Millennium Pharmaceuticals: Research Funding; Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Roche: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Affimed: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sigma Tau: Research Funding; Otsuka: Research Funding; Pfizer: Consultancy, Research Funding; Infinity: Consultancy; Tensha: Research Funding; Genmab: Consultancy. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Evidence for the Presence of Long-Lived Plasma Cells in Nasal Polyps.

PurposePlasma cells and immunoglobulins (Igs) play a pivotal role in the induction and maintenance of chronic inflammation in nasal polyps. During secondary immune responses, plasma cell survival and Ig production are regulated by the local environment. The purpose of the present study was to investigate the presence of long-lived plasma cells (LLPCs) and specific survival niches for LLPCs in human nasal polyps.MethodsNasal mucosal samples were cultured with an air-liquid interface system and the Ig levels in culture supernatants were analyzed by enzyme-linked immunosorbent assay. The characteristics of LLPCs in nasal polyps were determined by immunohistochemistry and immunofluorescence. The expression of neurotrophins as well as their receptors was detected by quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and Western blotting.ResultsThe numbers of CD138+ total plasma cells and BCL2+ plasma cells were increased in both eosinophilic and non-eosinophilic nasal polyps compared with those in normal tissues. The production of IgG, IgA, and IgE was detected in culture supernatants even after a 32-day culture of nasal polyps. Although the total numbers of plasma cells were decreased in nasal polyps after culture, the numbers of BCL2+ plasma cells remained stable. The expression of nerve growth factor (NGF) as well as tropomyosin receptor kinase (Trk) A, a high-affinity receptor for NGF, was upregulated in both eosinophilic and non-eosinophilic nasal polyps. In addition, BCL2+ plasma cell numbers were positively correlated with NGF and TrkA mRNA expression in nasal mucosal tissues. Polyp plasma cells had the expression of TrkA.ConclusionsHuman nasal polyps harbor a population of LLPCs and NGF may be involved in their prolonged survival. LLPCs may be a novel therapeutic target for suppressing the local Ig production in nasal polyps.

Toll-like Receptor 1/2 Agonist Pam3CSK4 Suppresses Lipopolysaccharide-driven IgG1 Production while Enhancing IgG2a Production by B Cells.

Interaction between pathogen-associated molecular patterns and pattern recognition receptors triggers innate and adaptive immune responses. Several studies have reported that toll-like receptors (TLRs) are involved in B cell proliferation, differentiation, and Ig class switch recombination (CSR). However, roles of TLRs in B cell activation and differentiation are not completely understood. In this study, we investigated the direct effect of stimulation of TLR1/2 agonist Pam3CSK4 on mouse B cell viability, proliferation, activation, Ig production, and Ig CSR in vitro. Treatment with 0.5 µg/ml of Pam3CSK4 only barely induced IgG1 production although it enhanced B cell viability. In addition, high-dosage Pam3CSK4 diminished IgG1 production in a dose-dependent manner, whereas the production of other Igs, cell viability, and proliferation increased. Pam3CSK4 additively increased TLR4 agonist lipopolysaccharide (LPS)-induced mouse B cell growth and activation. However, interestingly, Pam3CSK4 abrogated LPS-induced IgG1 production but enhanced LPS-induced IgG2a production. Further, Pam3CSK4 decreased LPS-induced germline γ1 transcripts (GLTγ1)/GLTε expression but increased GLTγ2a expression. On the other hand, Pam3CSK4 had no effect on LPS-induced plasma cell differentiation. Taken together, these results suggest that TLR1/2 agonist Pam3CSK4 acts as a potent mouse B cell mitogen in combination with TLR4 agonist LPS, but these 2 different TLR agonists play diverse roles in regulating the Ig CSR of each isotype, particularly IgG1/IgE and IgG2a.

Transcriptomic Analysis Reveals Significant B Lymphocyte Suppression in Corticosteroid-Treated Hosts with Pneumocystis Pneumonia.

Pneumocystis pneumonia (PCP) is an opportunistic, infectious disease that is prevalent in immunosuppressed hosts. Corticosteroid treatment is the most significant risk factor for patients with PCP who are human immunodeficiency virus negative, although little is known about how corticosteroids alter the host defense against Pneumocystis infection. In the present study, we used transcriptome analysis to examine the immune response in the lungs of corticosteroid-treated PCP mice. The results showed down-regulation in the genes related to both native immunity, such as antigen processing and presentation, inflammatory response, and phagocytosis, as well as B and T lymphocyte immunity. The repression of gene expression, corresponding to B cell immunity, including B cell signaling, homeostasis, and Ig production, was prominent. The finding was confirmed by quantitative PCR of mouse lungs and the peripheral blood of patients with PCP. Flow cytometry also revealed a significant depletion of B cells in corticosteroid-treated PCP mice. Our study has highlighted that corticosteroid treatment suppresses the B cell immunity in the PCP host, which is likely one of the main reasons that corticosteroid treatment may stimulate PCP development.

Hedgehog and retinoid signaling alters multiple myeloma microenvironment and generates bortezomib resistance.

Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in bortezomib (BTZ) resistance. However, the mechanisms involved in these interactions are not completely understood. We previously showed that expression of CYP26 in BM stromal cells maintains a retinoic acid-low (RA-low) microenvironment that prevents the differentiation of normal and malignant hematopoietic cells. Since a low secretory B cell phenotype is associated with BTZ resistance in MM and retinoid signaling promotes plasma cell differentiation and Ig production, we investigated whether stromal expression of the cytochrome P450 monooxygenase CYP26 modulates BTZ sensitivity in the BM niche. CYP26-mediated inactivation of RA within the BM microenvironment prevented plasma cell differentiation and promoted a B cell-like, BTZ-resistant phenotype in human MM cells that were cocultured on BM stroma. Moreover, paracrine Hedgehog secretion by MM cells upregulated stromal CYP26 and further reinforced a protective microenvironment. These results suggest that crosstalk between Hedgehog and retinoid signaling modulates BTZ sensitivity in the BM niche. Targeting these pathological interactions holds promise for eliminating minimal residual disease in MM.

Group 2 Innate Lymphoid Cells Promote an Early Antibody Response to a Respiratory Antigen in Mice.

Innate lymphoid cells (ILCs) are a new family of immune cells that play important roles in innate immunity in mucosal tissues, and in the maintenance of tissue and metabolic homeostasis. Recently, group 2 ILCs (ILC2s) were found to promote the development and effector functions of Th2-type CD4(+) T cells by interacting directly with T cells or by activating dendritic cells, suggesting a role for ILC2s in regulating adaptive immunity. However, our current knowledge on the role of ILCs in humoral immunity is limited. In this study, we found that ILC2s isolated from the lungs of naive BALB/c mice enhanced the proliferation of B1- as well as B2-type B cells and promoted the production of IgM, IgG1, IgA, and IgE by these cells in vitro. Soluble factors secreted by ILC2s were sufficient to enhance B cell Ig production. By using blocking Abs and ILC2s isolated from IL-5-deficient mice, we found that ILC2-derived IL-5 is critically involved in the enhanced production of IgM. Furthermore, when adoptively transferred to Il7r(-/-) mice, which lack ILC2s and mature T cells, lung ILC2s promoted the production of IgM Abs to a polysaccharide Ag, 4-hydroxy-3-nitrophenylacetyl Ficoll, within 7 d of airway exposure in vivo. These findings add to the growing body of literature regarding the regulatory functions of ILCs in adaptive immunity, and suggest that lung ILC2s promote B cell production of early Abs to a respiratory Ag even in the absence of T cells.

Deleted in Breast Cancer 1 Suppresses B Cell Activation through RelB and Is Regulated by IKKα Phosphorylation.

Alternative NF-κB signaling is crucial for B cell activation and Ig production, and it is mainly regulated by the inhibitor of κ B kinase (IKK) regulatory complex. Dysregulation of alternative NF-κB signaling in B cells could therefore lead to hyperactive B cells and Ig overproduction. In our previous, study we found that deleted in breast cancer 1 (DBC1) is a suppressor of the alternative NF-κB pathway to attenuate B cell activation. In this study, we report that loss of DBC1 results in spontaneous overproduction of Ig in mice after 10 mo of age. Using a double mutant genetic model, we confirm that DBC1 suppresses B cell activation through RelB inhibition. At the molecular level, we show that DBC1 interacts with alternative NF-κB members RelB and p52 through its leucine zipper domain. In addition, phosphorylation of DBC1 at its C terminus by IKKα facilitates its interaction with RelB and IKKα, indicating that DBC1-mediated suppression of alternative NF-κB is regulated by IKKα. Our results define the molecular mechanism of DBC1 inhibition of alternative NF-κB activation in suppressing B cell activation.

Increased IL-21 expression in chronic rhinosinusitis with nasalpolyps.

IL-21 is a key cytokine for regulating B cell immunity, which is involved in several inflammatory conditions. This study sought to define a role for IL-21 in activated B lymphocytes and enhanced tissue eosinophilia in NP tissues during the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). NP and uncinate process tissues were collected from 64 CRSwNP patients, 25 CRSsNP patients, and 29 control subjects. IL-21 expression was examined using IHC staining, qRT-PCR, flow cytometry, and ELISA, and its clinical implication was evaluated. Moreover, the effects of IL-21 on B cell differentiation and Ig production in cultured NP cells were examined in vitro. The mRNA and protein levels of IL-21 were significantly increased in polyp tissues compared with control tissues (P < 0.05). Polyp IL-21 level was significantly associated with polyp size, tissue eosinophilia and asthma comorbidity, and recurrence after surgery (P < 0.05). Both Th1 and Th17 cells were the main cellular sources of IL-21 in polyp tissues. The percentage of IL-21(+) CD4(+) cells was significantly higher in polyp tissues compared with control tissues and matched PBMCs (P < 0.01). Accordingly, the percentage of CD19(+) CD20(+/-) CD38(high) cells was significantly higher in polyp tissues compared with control tissues (P < 0.01). Moreover, recombinant IL-21 significantly increased the percentage of CD19(+) CD20(+/-) CD38(high) cells (plasmablasts) and IgG and IgA production in cultured NP cells in vitro (P < 0.05). Increased IL-21 level in polyp tissues was associated with disease severity, local B cell activation, and immunoglobulin production, suggesting that IL-21 might play an important role in promoting persistent mucosal inflammation in CRSwNP patients.

DBC1 is a suppressor of B cell activation by negatively regulating alternative NF-κB transcriptional activity.

CD40 and BAFFR signaling play important roles in B cell proliferation and Ig production. In this study, we found that B cells from mice with deletion of Dbc1 gene (Dbc1(-/-)) show elevated proliferation, and IgG1 and IgA production upon in vitro CD40 and BAFF, but not BCR and LPS stimulation, indicating that DBC1 inhibits CD40/BAFF-mediated B cell activation in a cell-intrinsic manner. Microarray analysis and chromatin immunoprecipitation experiments reveal that DBC1 inhibits B cell function by selectively suppressing the transcriptional activity of alternative NF-κB members RelB and p52 upon CD40 stimulation. As a result, when immunized with nitrophenylated-keyhole limpet hemocyanin, Dbc1(-/-) mice produce significantly increased levels of germinal center B cells, plasma cells, and Ag-specific Ig. Finally, loss of DBC1 in mice leads to higher susceptibility to experimental autoimmune myasthenia gravis. Our study identifies DBC1 as a novel regulator of B cell activation by suppressing the alternative NF-κB pathway.

Costimulatory Pathways in Kidney Transplantation: Pathogenetic Role, Clinical Significance and New Therapeutic Opportunities

Costimulatory pathways play a key role in immunity, providing the second signal required for a full activation of adaptive immune response. Different costimulatory families (CD28, TNF-related, adhesion and TIM molecules), characterized by structural and functional analogies, have been described. Costimulatory molecules modulate T cell activation, B cell function, Ig production, cytokine release and many other processes, including atherosclerosis. Patients suffering from renal diseases present significant alterations of the costimulatory pathways, which might make them particularly liable to infections. These alterations are further pronounced in patients undergoing kidney transplantation. In these patients, different costimulatory patterns have been related to distinct clinical features. The importance that costimulation has gained during the last years has led to development of several pharmacological approaches to modulate this critical step in the immune activation. Different drugs, mainly monoclonal antibodies targeting various costimulatory molecules (i.e. anti-CD80, CTLA-4 fusion proteins, anti-CD154, anti-CD40, etc.) were designed and tested in both experimental and clinical studies. The results of these studies highlighted some criticisms, but also some promising findings and now costimulatory blockade is considered a suitable strategy, with belatacept (a CTLA-4 fusion protein) being approved as the first costimulatory blocker for use in renal transplantation. In this review, we summarize the current knowledge on costimulatory pathways in the setting of kidney transplantation. We describe the principal costimulatory molecule families, their role and clinical significance in patients undergoing renal transplantation and the new therapeutic approaches that have been developed to modulate the costimulatory pathways.

FRI0014 Il-21 is upregulated in sle and promotes tfh-mediated b cell maturation and ig production

BackgroundThe dysregulation of germinal centers and ectopic follicles, resulting in hyperactive B cells, is thought to be a key mechanism leading to systemic lupus erythematosus (SLE). Interleukin-21 (IL-21) is a...

Identification of B Cell Defects Using Age-Defined Reference Ranges for In Vivo and In Vitro B Cell Differentiation

Primary immunodeficiencies consist to a large extent of B cell defects, as indicated by inadequate Ab levels or response upon immunization. Many B cell defects have not yet been well characterized. Our objective was to create reliable in vivo and in vitro assays to routinely analyze human B cell differentiation, proliferation, and Ig production and to define reference ranges for different age categories. The in vitro assays were applied to classify the developmental and/or functional B cell defects in patients previously diagnosed with common variable immunodeficiency. Apart from standard immunophenotyping of circulating human B cell subsets, an in vitro CFSE dilution assay was used for the assessment of proliferative capacity comparing T cell-dependent and T cell-independent B cell activation. Plasmablast/plasma cell differentiation was assessed by staining for CD20, CD38, and CD138, and measurement of in vitro Ig secretion. At young age, B cells proliferate upon in vitro activation, but neither differentiate nor produce IgG. These latter functions reached adult levels at 5 and 10 y of age for T cell-dependent versus T cell-independent stimulations, respectively. The capacity of B cells to differentiate into plasmablasts and to produce IgG appeared to be contained within the switched memory B cell pool. Using these assays, we could categorize common variable immunodeficiency patients into subgroups and identified a class-switch recombination defect caused by an UNG mutation in one of the patients. We defined age-related reference ranges for human B cell differentiation. Our findings indicate that in vivo B cell functionality can be tested in vitro and helps to diagnose suspected B cell defects.

IL-21 neutralizing antibody inhibits human TFH proliferation and TFH-mediated maturation of naïve B cells (P5220)

Abstract The hallmark of T follicular helper (TFH) cells is the ability to help B cells generate humoral immune responses. Recent evidence suggests that aberrant function of TFH cells plays a key role in the generation of autoantibodies, which can inflict autoimmune pathologies. TFH cells are characterized by their unique master regulator (Bcl6), surface marker expression (CXCR5 and ICOS), and production of the pro-inflammatory cytokine interleukin-21 (IL-21). In mice, IL-21 is described as a regulator of TFH cell differentiation and B cell maturation, but its role in human TFH cells remains incompletely understood. In the present study, we sorted TFH subsets from human tonsils or TFH-like cells from human blood to characterize IL-21 production and the functional consequences of blocking IL-21 with a neutralizing antibody. IL-21 was highest in TFHHI cells (CXCR5++ICOS++), detectable in TFHINT cells (CXCR5+ICOS+), and minimal in non-TFH cells. Anti-IL-21 caused a moderate decrease in anti-CD3 stimulated TFH proliferation. Peripheral CXCR5+ TFH-like cells also had elevated IL-21 production, but anti-IL-21 had little effect on TFH-like proliferation. When TFHINT, TFHHI, or TFH-like cells were co-incubated with naïve B cells, anti-IL-21 decreased B cell maturation and Ig production in a dose-dependent manner. Together, these findings indicate that anti-IL-21 inhibits TFH generation and B cell help, and is a promising therapeutic for autoimmune disorders involving autoantibody production.

Pathogen-Triggered Activation of Plasmacytoid Dendritic Cells Induces IL-10–Producing B Cells in Response to Staphylococcus aureus

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.

Kinetic Analysis of CpG-Induced Mouse B Cell Growth and Ig Production

Immune cells express toll-like receptors (TLRs) and respond to molecular patterns of various pathogens. CpG motif in bacterial DNA activates innate and acquired immune systems through binding to TLR9 of immune cells. Several studies reported that CpG can directly regulate B cell activation, differentiation, and Ig production. However, the role of CpG in B cell growth and Ig production is not fully understood. In this study, we analyzed the effect of CpG on the kinetics of mouse B cell viability, proliferation, and Igs production. Overall, CpG enhanced mouse B cell growth and production of Igs in a dose-dependent manner. Unlike LPS, 100 nM CpG (high dose) did not support TGF-β1-induced IgA and IgG2b production. Moreover, 100 nM CpG treatment abrogated either LPS-induced IgM or LPS/TGF-β1-induced IgA and IgG2b production, although B cell growth was enhanced by CpG under the same culture conditions. We subsequently found that 10 nM CpG (low dose) is sufficient for B cell growth. Again, 10 nM CpG did not support TGF-β1-induced IgA production but, interestingly enough, supported RA-induced IgA production. Further, 10 nM CpG, unlike 100 nM, neither abrogated the LPS/TGF-β1-nor the LPS/RA-induced IgA production. Taken together, these results suggest that dose of CpG is critical in B cell growth and Igs production and the optimal dose of CpG cooperates with LPS in B cell activation and differentiation toward Igs production.

Lactoferrin Stimulates Mouse Macrophage to Express BAFF via Smad3 Pathway

B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and stimulates B cell proliferation, differentiation, survival, and Ig production. In this study, we explored the effect of lactoferrin (LF) on BAFF expression by murine macrophages. We determined the level of BAFF expression at the transcriptional and protein levels using RT-PCR and ELISA, respectively. LF markedly enhanced BAFF expression in mouse macrophages at both the transcriptional and protein levels. Overexpression of Smad3/4 further increased LF-induced BAFF transcription while DN-Smad3 abolished the LF-induced BAFF expression. These results demonstrate that LF can enhance BAFF expression through Smad3/4 pathway.

Cannabinoid Receptor 2 Is Critical for the Homing and Retention of Marginal Zone B Lineage Cells and for Efficient T-Independent Immune Responses

The endocannabinoid system has emerged as an important regulator of immune responses, with the cannabinoid receptor 2 (CB2) and its principle ligand 2-archidonoylglycerol playing a major role. How CB2 regulates B cell functions is not clear, even though they express the highest levels of CB2 among immune cell subsets. In this study, we show that CB2-deficient mice have a significant reduction in the absolute number of marginal zone (MZ) B cells and their immediate precursor, transitional-2 MZ precursor. The loss of MZ lineage cells in CB2(-/-) mice was shown to be B cell intrinsic using bone marrow chimeras and was not due to a developmental or functional defect as determined by B cell phenotype, proliferation, and Ig production. Furthermore, CB2(-/-) B cells were similar to wild type in their apoptosis, cell turnover, and BCR and Notch-2 signaling. We then demonstrated that CB2(-/-) MZ lineage B cells were less efficient at homing to the MZ and that their subsequent retention was also regulated by CB2. CB2(-/-) mice immunized with T-independent Ags produced significantly less Ag-specific IgM. This study demonstrates that CB2 positively regulates T-independent immune responses by controlling the localization and positioning of MZ lineage cells to the MZ.

Immunologic Studies in Humans.

Immunologic Studies in Humans.

Activin A stimulates mouse macrophages to express APRIL via the Smad3 and ERK/CREB pathways

A proliferation-inducing ligand (APRIL) is primarily expressed by macrophages and dendritic cells, and stimulates B cell proliferation, differentiation, survival, and Ig production. In the present study, we investigated the role and signaling mechanisms of activin A in APRIL expression by mouse macrophages. Activin A markedly enhanced APRIL expression in mouse macrophages at both the transcriptional and protein levels. Overexpression of dominant-negative (DN)-Smad3 and SB431542 abrogated activin-induced APRIL transcription. Furthermore, activin A induced Smad3 phosphorylation. These results indicate that activin A enhances APRIL expression through both activin receptor-like kinase 4 (ALK4) and Smad3. In a subsequent analysis of activin A signaling, it was found that PD98059, an extracellular signal-related kinase (ERK) inhibitor, eliminated activin A-induced APRIL expression. On the other hand, overexpression of cAMP responsive element-binding protein (CREB), a molecule downstream of ERK, augmented activin A-induced APRIL expression, and this effect could be abolished by PD98059. This finding that activin A induces ERK and CREB phosphorylation suggests that ERK and CREB act as intermediates in APRIL expression. Taken together, these results demonstrate that activin A can enhance APRIL expression through two different pathways, Smad3 and ERK/CREB.

Activin A Stimulates Mouse APCs to Express BAFF via ALK4-Smad3 Pathway

BackgroundB cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells, and stimulates B cell proliferation, differentiation, survival, and Ig production. In the present study, we explored the effect of activin A on BAFF expression by APCs.MethodsTo investigate the effect of activin A on BAFF expression by mouse APCs, we measured the level of BAFF expression at the transcriptional and protein levels using RT-PCR and ELISA.ResultsActivin A markedly enhanced BAFF expression in mouse macrophages and dendritic cells at both the transcriptional and protein levels. SB431542, an activin receptor-like kinase 4 (ALK4) inhibitor, completely abrogated activin A-induced BAFF transcription. Furthermore, overexpression of DN-Smad3 abolished activin-induced BAFF expression at the transcriptional and protein levels.ConclusionThese results demonstrate that activin A can enhance BAFF expression through ALK4-Smad3 pathway.