Programmed death ligand 1 (PD-L1) plays a vital part in DC tolerogenicity induced by IFN-γ.

Interferon-γ (IFN-γ) is the sole representative of type II IFNs, with well recognized role in numerous inflammatory processes. Lately, its significant pleiotropic nature has been recognized in many scenarios, where IFN-γ contributes to maintenance or induction of tolerogenic responses in context of various immune cell types. In this manuscript we demonstrate, that IFN-γ-mediated induction of programmed death ligand 1 (PD-L1) on human monocyte-derived dendritic cells (DCs) represents an important tolerogenic aspect in immunological network of type II IFNs. When fully differentiated, immature DCs were treated with increasing concentrations of IFN-γ there was no sign of maturation, as revealed by CD80, CD83 and CD86 expression. In terms of co-stimulatory receptor response, we did observe a dose-dependent increase in CD40 expression. Phenotypic analysis of inhibitory molecules revealed that PD-L1 expression is particularly sensitive to IFN-γ, as its expression can be induced almost 10-fold in comparison to non-treated DCs. Functional analysis of such PD-L1high DCs revealed significant immunosuppressive properties in a mixed lymphocyte reaction with whole or memory CD4+ T cells. When IFN-γ treated DCs were co-cultured with naive CD4+CD45RA+ T cells, they induced an increased percentage of CD4+CD25+CD127-FoxP3+ Tregs. Inhibition of PD-1/PD-L1 axis using neutralizing anti-PD-L1 mAbs, reversed the immunosuppressive effect of IFN-γ-treated DCs to suppress CD4+ T cell proliferation and to induce Tregs. In summary, our findings demonstrate the importance of IFN-γ-mediated tolerogenic effects, exerted on DCs by inducing increased expression of PD-L1, which enhances their regulatory function.

Novel induction of CD40 expression by tumor cells with RAS/RAF/PI3K pathway inhibition augments response to checkpoint blockade.

BACKGROUND: While immune checkpoint blockade (ICB) is the current first-line treatment for metastatic melanoma, it is effective for ~ 52% of patients and has dangerous side effects. The objective here was to identify the feasibility and mechanism of RAS/RAF/PI3K pathway inhibition in melanoma to sensitize tumors to ICB therapy. METHODS: Rigosertib (RGS) is a non-ATP-competitive small molecule RAS mimetic. RGS monotherapy or in combination therapy with ICB were investigated using immunocompetent mouse models of BRAFwt and BRAFmut melanoma and analyzed in reference to patient data. RESULTS: RGS treatment (300 mg/kg) was well tolerated in mice and resulted in ~ 50% inhibition of tumor growth as monotherapy and ~ 70% inhibition in combination with αPD1 + αCTLA4. RGS-induced tumor growth inhibition depends on CD40 upregulation in melanoma cells followed by immunogenic cell death, leading to enriched dendritic cells and activated T cells in the tumor microenvironment. The RGS-initiated tumor suppression was partially reversed by either knockdown of CD40 expression in melanoma cells or depletion of CD8+ cytotoxic T cells. Treatment with either dabrafenib and trametinib or with RGS, increased CD40+SOX10+ melanoma cells in the tumors of melanoma patients and patient-derived xenografts. High CD40 expression level correlates with beneficial T-cell responses and better survival in a TCGA dataset from melanoma patients. Expression of CD40 by melanoma cells is associated with therapeutic response to RAF/MEK inhibition and ICB. CONCLUSIONS: Our data support the therapeutic use of RGS + αPD1 + αCTLA4 in RAS/RAF/PI3K pathway-activated melanomas and point to the need for clinical trials of RGS + ICB for melanoma patients who do not respond to ICB alone. TRIAL REGISTRATION: NCT01205815 (Sept 17, 2010).

Single-cell analysis can define distinct evolution of tumor sites in follicular lymphoma.

Tumor heterogeneity complicates biomarker development and fosters drug resistance in solid malignancies. In lymphoma, our knowledge of site-to-site heterogeneity and its clinical implications is still limited. Here, we profiled 2 nodal, synchronously acquired tumor samples from 10 patients with follicular lymphoma (FL) using single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing, and flow cytometry. By following the rapidly mutating tumor immunoglobulin genes, we discovered that BCR subclones were shared between the 2 tumor sites in some patients, but in many patients, the disease had evolved separately with limited tumor cell migration between the sites. Patients exhibiting divergent BCR evolution also exhibited divergent tumor gene-expression and cell-surface protein profiles. While the overall composition of the tumor microenvironment did not differ significantly between sites, we did detect a specific correlation between site-to-site tumor heterogeneity and T follicular helper (Tfh) cell abundance. We further observed enrichment of particular ligand-receptor pairs between tumor and Tfh cells, including CD40 and CD40LG, and a significant correlation between tumor CD40 expression and Tfh proliferation. Our study may explain discordant responses to systemic therapies, underscores the difficulty of capturing a patient's disease with a single biopsy, and furthers our understanding of tumor-immune networks in FL.

Runx proteins mediate protective immunity against Leishmania donovani infection by promoting CD40 expression on dendritic cells.

The level of CD40 expression on dendritic cells (DCs) plays a decisive role in disease protection during Leishmania donovani (LD) infection. However, current understanding of the molecular regulation of CD40 expression remains elusive. Using molecular, cellular and functional approaches, we identified a role for Runx1 and Runx3 transcription factors in the regulation of CD40 expression in DCs. In response to lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα) or antileishmanial drug sodium antimony gluconate (SAG), both Runx1 and Runx3 translocated to the nucleus, bound to the CD40 promoter and upregulated CD40 expression on DCs. These activities of Runx proteins were mediated by the upstream phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Notably, LD infection attenuated LPS- or TNFα-induced CD40 expression in DCs by inhibiting PI3K-Akt-Runx axis via protein tyrosine phosphatase SHP-1. In contrast, CD40 expression induced by SAG was unaffected by LD infection, as SAG by blocking LD-induced SHP-1 activation potentiated PI3K-Akt signaling to drive Runx-mediated CD40 upregulation. Adoptive transfer experiments further showed that Runx1 and Runx3 play a pivotal role in eliciting antileishmanial immune response of SAG-treated DCs in vivo by promoting CD40-mediated type-1 T cell responses. Importantly, antimony-resistant LD suppressed SAG-induced CD40 upregulation on DCs by blocking the PI3K-Akt-Runx pathway through sustained SHP-1 activation. These findings unveil an immunoregulatory role for Runx proteins during LD infection.

Transcriptional Regulation of CD40 Expression by 4 Ribosomal Proteins via a Functional SNP on a Disease-Associated CD40 Locus.

Previously, using FREP-MS, we identified a protein complex including eight proteins that specifically bind to the functional SNP (fSNP) rs6032664 at a CD40 locus associated with autoimmune diseases. Among these eight proteins, four are ribosomal proteins RPL26, RPL4, RPL8, and RPS9 that normally make up the ribosomal subunits involved in the cellular process of protein translation. So far, no publication has shown these ribosomal proteins function as transcriptional regulators. In this work, we demonstrate that four ribosomal proteins: RPL26, RPL4, RPL8, and RPS9 are bona fide CD40 transcriptional regulators via binding to rs6032664. In addition, we show that suppression of CD40 expression by RPL26 RNAi knockdown inactivates NF-κB p65 by dephosphorylation via NF-κB signaling pathway in fibroblast-like synoviocytes (FLS), which further reduces the transcription of disease-associated risk genes such as STAT4, CD86, TRAF1 and ICAM1 as the direct targets of NF-κB p65. Based on these findings, a disease-associated risk gene transcriptional regulation network (TRN) is generated, in which decreased expression of, at least, RPL26 results in the downregulation of risk genes: STAT4, CD86, TRAF1 and ICAM1, as well as the two proinflammatory cytokines: IL1ß and IL6 via CD40-induced NF-κB signaling. We believe that further characterization of this disease-associated TRN in the CD40-induced NF-κB signaling by identifying both the upstream and downstream regulators will potentially enable us to identify the best targets for drug development.

Diabetes enhances translation of Cd40 mRNA in murine retinal Müller glia via a 4E-BP1/2-dependent mechanism.

Activation of the immune costimulatory molecule cluster of differentiation 40 (CD40) in Müller glia has been implicated in the initiation of diabetes-induced retinal inflammation. Results from previous studies support that CD40 protein expression is elevated in Müller glia of diabetic mice; however, the mechanisms responsible for this increase have not been explored. Here, we evaluated the hypothesis that diabetes augments translation of the Cd40 mRNA. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation and increased Cd40 mRNA translation. TMG administration also promoted Cd40 mRNA association with Müller cell-specific ribosomes isolated from the retina of RiboTag mice. Similar effects on O-GlcNAcylation and Cd40 mRNA translation were also observed in the retina of a mouse model of type 1 diabetes. In cultured cells, TMG promoted sequestration of the cap-binding protein eIF4E (eukaryotic translation in initiation factor 4E) by 4E-BP1 (eIF4E-binding protein 1) and enhanced cap-independent Cd40 mRNA translation as assessed by a bicistronic reporter that contained the 5'-UTR of the Cd40 mRNA. Ablation of 4E-BP1/2 prevented the increase in Cd40 mRNA translation in TMG-exposed cells, and expression of a 4E-BP1 variant that constitutively sequesters eIF4E promoted reporter activity. Extending on the cell culture results, we found that in contrast to WT mice, diabetic 4E-BP1/2-deficient mice did not exhibit enhanced retinal Cd40 mRNA translation and failed to up-regulate expression of the inflammatory marker nitric-oxide synthase 2. These findings support a model wherein diabetes-induced O-GlcNAcylation of 4E-BP1 promotes Cd40 mRNA translation in Müller glia.

Particles from the Echinococcus granulosus Laminated Layer Inhibit CD40 Upregulation in Dendritic Cells by Interfering with Akt Activation.

The larval stage of the cestode Echinococcus granulosus causes cystic echinococcosis in humans and livestock. This larva is protected by the millimeter-thick, mucin-based laminated layer (LL), from which materials have to be shed to allow parasite growth. We previously reported that dendritic cells (DCs) respond to microscopic pieces of the mucin gel of the LL (pLL) with unconventional maturation phenotypes, in the absence or presence of Toll-like receptor (TLR) agonists, including lipopolysaccharide (LPS). We also reported that the presence of pLL inhibited the activating phosphorylation of the phosphatidylinositol 3-kinase (PI3K) effector Akt induced by granulocyte-macrophage colony-stimulating factor or interleukin-4. We now show that the inhibitory effect of pLL extends to LPS as a PI3K activator, and results in diminished phosphorylation of GSK3 downstream from Akt. Functionally, the inhibition of Akt and GSK3 phosphorylation are linked to the blunted upregulation of CD40, a major feature of the unconventional maturation phenotype. Paradoxically, all aspects of unconventional maturation induced by pLL depend on PI3K class I. Additional components of the phagocytic machinery are needed, but phagocytosis of pLL particles is not required. These observations hint at a DC response mechanism related to receptor-independent mechanisms proposed for certain crystalline and synthetic polymer-based particles; this would fit the previously reported lack of detection of molecular-level motifs necessary of the effects of pLL on DCs. Finally, we report that DCs exposed to pLL are able to condition DCs not exposed to the material so that these cannot upregulate CD40 in full in response to LPS.

CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation.

CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets.

B-cell subsets imbalance and reduced expression of CD40 in ataxia-telangiectasia patients

BACKGROUND: Ataxia-telangiectasia (AT) is a well-known primary immunodeficiency with recurrent sinopulmonary infections and variable abnormalities in both the humoral and cellular immune system. Dysfunctions in immunoglobulin production, reduced number of B cells, and B-cell receptor excision circles copies have been reported. We aimed to understand the immunological mechanisms involving the humoral compartment in AT patients by analysing peripheral blood B cells subsets, B-T lymphocyte cooperation through the expression of CD40 and CD40 ligand (CD40L), and cytokines involved in class-switch recombination production. METHODS: We compared the proportion of B-cell subsets, the expression of CD40/CD40L, and the plasma levels of IL-6 and IFN-gamma of 18 AT patients and 15 healthy age-sex-matched controls using flow cytometry. RESULTS: We found that some steps in peripheral B cell development were altered in AT with a pronounced reduction of cell-surface CD40 expression. The proportions of transitional and naïve-mature B cells were reduced, whereas CD21-low, natural effector memory, IgM-only memory, and IgG atypical memory B cells were present in a higher proportion. CONCLUSIONS: These findings revealed a disturbed B-cell homeostasis with unconventional maturation of B lymphocyte memory cells, which can explain the consequent impairment of humoral immunity

No disponible

B-cell subsets imbalance and reduced expression of CD40 in ataxia-telangiectasia patients.

BACKGROUND: Ataxia-telangiectasia (AT) is a well-known primary immunodeficiency with recurrent sinopulmonary infections and variable abnormalities in both the humoral and cellular immune system. Dysfunctions in immunoglobulin production, reduced number of B cells, and B-cell receptor excision circles copies have been reported. We aimed to understand the immunological mechanisms involving the humoral compartment in AT patients by analysing peripheral blood B cells subsets, B-T lymphocyte cooperation through the expression of CD40 and CD40 ligand (CD40L), and cytokines involved in class-switch recombination production. METHODS: We compared the proportion of B-cell subsets, the expression of CD40/CD40L, and the plasma levels of IL-6 and IFN-γ of 18 AT patients and 15 healthy age-sex-matched controls using flow cytometry. RESULTS: We found that some steps in peripheral B cell development were altered in AT with a pronounced reduction of cell-surface CD40 expression. The proportions of transitional and naïve-mature B cells were reduced, whereas CD21-low, natural effector memory, IgM-only memory, and IgG atypical memory B cells were present in a higher proportion. CONCLUSIONS: These findings revealed a disturbed B-cell homeostasis with unconventional maturation of B lymphocyte memory cells, which can explain the consequent impairment of humoral immunity.

Clinical significance of costimulatory molecules CD40/CD40L and CD134/CD134L in coronary heart disease: A case-control study.

The aim of the study was to evaluate the potential role of CD40/CD40 ligand (CD40L) and CD134/CD134 ligand (CD134L) in the development of coronary heart disease (CHD) via the performance of a case-control study.The research objects were 234 cases of CHD patients and 120 cases of well-matched normal controls. Following the separation of peripheral blood mononuclear cells (PBMCs), real-time quantitative PCR (qRT-PCR), Western blot, immunohistochemistry, and flow cytometry were applied for the detection of mRNA levels and expression levels of CD40/CD40L and CD134/CD134L; meanwhile, intercellular adhesion molecule-1 (ICAM-1) and Fas protein mRNA levels were detected using qRT-PCR.There was no statistical difference in the comparison of baseline characteristics between groups, indicating comparability between groups. qRT-PCR and Western blot analysis indicated that CD40/CD40L and CD134/CD134L mRNA and protein expression levels were all increased in the CHD group than those in the control group. Flow cytometry further confirmed the similar tendency. Meanwhile, ICAM-1 and Fas protein mRNA levels were elevated in the CHD group and positively correlated with the above parameters. Furthermore, CD40/CD40L expression rates were negatively correlated with gender and different types of CHD. Meanwhile, CD134/CD134L expressions were also higher in male patients, in patients with family history, previous history of hypertension, diabetes, and cerebrovascular diseases.CD40/CD40L and CD134/CD134L are increased and may have potential correlation with clinical pathological features of patients with CHD. Further in-depth exploration of costimulatory molecules for CHD guidance as well as intrinsic mechanisms are needed combined with in vivo and in vitro experiments.

Regulatory role of cytosolic phospholipase A2 alpha in the induction of CD40 in microglia.

BACKGROUND: The aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A2 alpha (cPLA2α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA2α has a direct, participatory role in the molecular events leading to CD40 induction. METHODS: Cultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA2α in the events leading to CD40 protein induction. RESULTS: Addition of LPS or IFNγ caused a significant upregulation of cPLA2α and of CD40, while prevention of cPLA2α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA2α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA2α detected by its phosphorylated form, while addition of IFNγ induced cPLA2α activation at a later time scale (4 h). The activation of cPLA2α is mediated by ERK activity. Suppression of cPLA2α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-κB detected by the phosphorylation of p65 on serine 536 at 15 min by LPS and at 4 h by IFNγ. Inhibition of NOX2 prevented NF-κB activation and CD40 induction but did not affect cPLA2α activation, suggesting cPLA2α is located upstream to NOX2 and NF-κB. The activation of cPLA2 by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA2α by IFNγ was mediated by the secreted TNF-α at 4 h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA2α inhibitors, indicating that STAT1α is not under cPLA2α regulation. CONCLUSIONS: Our results show for the first time that cPLA2 upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-κB. Cumulatively, our results indicate that cPLA2α may serve as a pivotal amplifier of the inflammatory response in the CNS.

Capparis spinosa Fruit Ethanol Extracts Exert Different Effects on the Maturation of Dendritic Cells.

Capparis spinosa L. (C. spinosa) has been used as food and traditional medicine and shows anti-inflammatory and anti-oxidant activities. Here, we prepared the C. spinosa fruit ethanol extracts (CSEs) using different procedures and investigated the effects of CSE on the maturation of mouse bone marrow-derived dendritic cells (DCs) in the absence or presence of lipopolysaccharide (LPS). DC maturation and cytokine production were detected by flow cytometry and ELISA, respectively. We obtained three different CSEs and dissolved in water or DMSO, named CSE2W, CSEMW, CSE3W, CSE2D, CSEMD, and CSE3D, respectively. These CSEs showed different effects on DC maturation. CSEMW and CSEMD significantly increased the expressions of CD40, CD80, and CD86, in a dose-dependent manner. CSE2W and CSE2D also showed a modest effect on DC maturation, which enhanced the expression of CD40. CSE3W and CSE3D did not change DC maturation but suppressed LPS-induced DC maturation characterized by the decreased levels of CD40 and CD80. CSE3W and CSE3D also significantly inhibited the secretions of IL-12p40, IL-6, IL-1ß, and TNF-α induced by LPS. CSE3W further increased the level of IL-10 induced by LPS. Moreover, CSE3D suppressed LPS-induced DC maturation in vivo, which decreased the expressions of CD40 and CD80. These results suggested that CSE3W and CSE3D might be used to treat inflammatory diseases.

Curcumin reduces lung inflammation via Wnt/ß-catenin signaling in mouse model of asthma.

OBJECTIVES: Asthma is a chronic inflammatory, heterogeneous airway disease affecting millions of people around the world. Curcumin has been found to have anti-inflammatory and antifibrosis effects. Researchers reported that curcumin regulated Wnt/ß-catenin signaling in lots of cells. However, whether curcumin regulates the levels of Wnt/ß-Catenin signaling in lung tissues and DCs (dendritic cells) remains unclear. In this study, we assessed the effects of curcumin on DCs and asthma. METHODS: C57BL/6 mice immunized with OVA (ovalbumin) were challenged thrice with an aerosol of OVA every second day for 8 days. Dexamethasone or curcumin was administered intraperitoneally to OVA-immunized C57BL/6 mice on day 24 once a day for 9 days. Mice were analyzed for effects of curcumin on asthma, inflammatory cell infiltration and cytokine levels in lung tissue. DCs were isolated from mouse bone morrow. The surface markers CD40, CD86 and CD11c of DCs was detected by FACS (fluorescence activated cell sorting) and the function of DCs was detected by mixed lymphocyte reaction. The expression of GSK-3ß and ß-catenin was detected by Western Blot. RESULTS: Results showed that OVA increased the number of inflammatory factors in BALF (bronchoalveolar lavage fluid), elevated lung inflammation scores in mice. Curcumin dose-dependently reversed the alterations induced by OVA in the asthmatic mice. Curcumin activated Wnt/ß-catenin signaling pathway in DCs and asthmatic mouse lungs. CONCLUSIONS: Curcumin could influence the morphology and function of DCs, ease asthma symptom and inflammatory reaction through the activation of Wnt/ß-catenin signaling. These results provide new evidence new evidence for application of curcumin on asthma.

Human Basophils Modulate Plasma Cell Differentiation and Maturation.

Basophils represent <1% of circulating leukocytes. They play a crucial role during allergy and helminth-induced Th2 responses. However, recent data also suggest a contribution to the pathogenesis of autoimmune diseases. Basophils from patients with systemic lupus erythematosus show an activated phenotype, correlating to disease activity. Furthermore, murine basophils or their mediators enhance memory responses and plasma cell (PC) survival, suggesting that they directly modulate the function of B cells. This is highly relevant with respect to human allergy and autoimmunity because a possible modulation of B cell differentiation by basophils could point to new therapeutic targets. Therefore, the interaction between human B cells and basophils and the mechanism underlying this interaction were investigated in detail. Using two different methods to induce PC differentiation, we found that human basophils enhance B cell proliferation, class switching, differentiation into PC, maturation of PC, and production of Igs, especially IgG. Basophil supernatants enhanced the expression of the B cell markers CD23 and CD40, which are important for B cell differentiation into IgG-producing PC. This was mainly IL-4 dependent. IL-3 amplified the number of PC in vitro, and acted synergistically with basophils in enhancing Ab production. Thus, human basophils modulate B cell differentiation into Ab-producing PC. Their contribution as modulators and effectors during allergy and autoimmunity should be considered when designing new therapeutic options.

CD40 functional gene polymorphisms and mRNA expression in rheumatoid arthritis patients from western Mexico.

The CD40 pathway is involved in the development and pathogenesis of autoimmune diseases, including rheumatoid arthritis (RA). Two single nucleotide polymorphisms (SNPs) in the CD40 gene, rs1883832 and rs4810485, are associated with susceptibility to inflammatory and autoimmune diseases and are thought to alter CD40 expression at the mRNA and protein level. This study assessed for the first time the association of these SNPs with RA and CD40 mRNA levels in a western Mexican population. A total of 278 RA patients and 318 control subjects were included. Genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, and CD40 mRNA expression was determined by real-time quantitative PCR. No significant differences in genotype and allele frequencies were identified between the RA patients and controls. When stratified by genotype, these SNPs were not found to be associated with the presence of autoantibodies or the clinical activity of the disease. CD40 mRNA levels were elevated 1.5-fold in RA patients compared to control subjects; however, no clear tendencies were observed following stratification by genotype. These results suggest that the CD40 SNPs rs1883832 and rs4810485 are not RA susceptibility markers in the western Mexican population. Further studies are needed to clarify their roles in CD40 mRNA expression.

Increased CD40 Expression Enhances Early STING-Mediated Type I Interferon Response and Host Survival in a Rodent Malaria Model.

Both type I interferon (IFN-I) and CD40 play a significant role in various infectious diseases, including malaria and autoimmune disorders. CD40 is mostly known to function in adaptive immunity, but previous observations of elevated CD40 levels early after malaria infection of mice led us to investigate its roles in innate IFN-I responses and disease control. Using a Plasmodium yoelii nigeriensis N67 and C57BL/6 mouse model, we showed that infected CD40-/- mice had reduced STING and serum IFN-ß levels day-2 post infection, higher day-4 parasitemia, and earlier deaths. CD40 could greatly enhance STING-stimulated luciferase signals driven by the IFN-ß promoter in vitro, which was mediated by increased STING protein levels. The ability of CD40 to influence STING expression was confirmed in CD40-/- mice after malaria infection. Substitutions at CD40 TRAF binding domains significantly decreased the IFN-ß signals and STING protein level, which was likely mediated by changes in STING ubiquitination and degradation. Increased levels of CD40, STING, and ISRE driven luciferase signal in RAW Lucia were observed after phagocytosis of N67-infected red blood cells (iRBCs), stimulation with parasite DNA/RNA, or with selected TLR ligands [LPS, poly(I:C), and Pam3CSK4]. The results suggest stimulation of CD40 expression by parasite materials through TLR signaling pathways, which was further confirmed in bone marrow derived dendritic cells/macrophages (BMDCs/BMDMs) and splenic DCs from CD40-/-, TLR3-/- TLR4-/-, TRIF-/-, and MyD88-/- mice after iRBC stimulation or parasite infection. Our data connect several signaling pathways consisting of phagocytosis of iRBCs, recognition of parasite DNA/RNA (possibly GPI) by TLRs, elevated levels of CD40 and STING proteins, increased IFN-I production, and longer host survival time. This study reveals previously unrecognized CD40 function in innate IFN-I responses and protective pathways in infections with malaria strains that induce a strong IFN-I response, which may provide important information for better understanding and management of malaria.

BAFF upregulates CD28/B7 and CD40/CD154 expression and promotes mouse T and B cell interaction in vitro via BAFF receptor.

AIM: B cell-activating factor belonging to the TNF family (BAFF) is a member of TNF family and required for peripheral B cell survival and homeostasis. BAFF has been shown to promote the proliferation of T and B cells. In this study we examined whether and how BAFF mediated the interaction between mouse T and B cells in vitro. METHODS: BAFF-stimulated B or T cells were co-cultured with T or B cells. The interactions between T and B cells were analyzed by measuring the expression of co-stimulatory molecules (CD28/CD80 or CD40/CD154), the proliferation and secretion of T and B cells and other factors. Two siRNAs against the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) were used to identify the receptors responsible for the actions of BAFF. RESULTS: BAFF-stimulated B cells significantly promoted the proliferation and activity of co-cultured T cells, and increased the percentages of CD4(+)CD28(+) and CD4(+)CD154(+) T cells. Similarly, BAFF-stimulated T cells significantly promoted the proliferation and activity of co-cultured B cells, and increased CD19(+)CD80(+) and CD19(+)CD40(+)B cell subpopulations. BAFF-R siRNA-silenced B cells showed significantly lower expression of CD40 and CD80 than the control B cells. When the BAFF-R siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was not increased. TACI siRNA-silenced B cells exhibited higher expression of CD40 and CD80 than the control B cells. When the TACI siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was significantly increased. CONCLUSION: BAFF upregulates CD28/B7 and CD40/CD154 expression, and promotes the interactions between T and B cells in a BAFF-R-dependent manner.

Encephalitozoon intestinalis Inhibits Dendritic Cell Differentiation through an IL-6-Dependent Mechanism.

Microsporidia are a group of intracellular pathogens causing self-limited and severe diseases in immunocompetent and immunocompromised individuals, respectively. A cellular type 1 adaptive response, mediated by IL-12, IFNγ, CD4+, and CD8+ T cells has been shown to be essential for host resistance, and dendritic cells (DC) play a key role at eliciting anti-microsporidial immunity. We investigated the in vitro response of DC and DC precursors/progenitors to infection with Encephalitozoon intestinalis (Ei), a common agent of human microsporidosis. Ei-exposed DC cultures up-regulated the surface expression of MHC class II and the costimulatory molecules CD86 and CD40, only when high loads of spores were used. A vigorous secretion of IL-6 but not of IL-1ß or IL-12p70 was also observed in these cultures. Ei-exposed DC cultures consisted of immature infected and mature bystander DC, as assessed by MHC class II and costimulatory molecules expression, suggesting that intracellular Ei spores deliver inhibitory signals in DC. Moreover, Ei selectively inhibited the secretion of IL-12p70 in LPS-stimulated DC. Whereas Ei-exposed DC promoted allogeneic naïve T cell proliferation and IL-2 and IFNγ secretion in DC-CD4+ T cell co-cultures, separated co-cultures with bystander or infected DCs showed stimulation or inhibition of IFNγ secretion, respectively. When DC precursors/progenitors were exposed to Ei spores, a significant inhibition of DC differentiation was observed without shifting the development toward cells phenotypically or functionally compatible with myeloid-derived suppressor cells. Neutralization experiments demonstrated that this inhibitory effect is IL-6-dependent. Altogether this investigation reveals a novel potential mechanism of immune escape of microsporidian parasites through the modulation of DC differentiation and maturation.

Cloning and high level expression of the biologically active extracellular domain of Macaca mulatta CD40 in Pichia pastoris.

The CD40-mediated immune response contributes to a wide variety of chronic inflammatory diseases. CD40 antagonists have potential as novel therapies for immune disorders. However, the CD40 pathway has not been well characterized in the rhesus monkey Macaca mulatta, which is a valuable animal model for human immune disease. An 834 bp transcript was cloned from peripheral blood mononuclear cells (PBMCs) of rhesus monkey using specific primers designed according to the predicted sequence of M. mulatta CD40 (mmCD40) in GenBank. Sequence analysis demonstrated that mmCD40 is highly homologous to human CD40 (hCD40), with an amino acid sequence identity of 94%. Genes encoding the extracellular domain of mmCD40 and the Fc fragment of the hIgG1 were inserted into a pPIC9K plasmid to produce mmCD40Ig by Pichia pastoris. Approximately 15-20 mg of the mmCD40Ig protein with ∼90% purity could be recovered from 1 L of culture. The purified mmCD40Ig protein can form dimers and can specifically bind CD40L-positive cells. Additionally, the mmCD40Ig protein can bind hCD40L protein in phosphate buffered saline and form a stable combination in a size-exclusion chromatography assay using a Superdex 200 column. Moreover, mmCD40Ig is as efficient as M. mulatta CTLA4Ig (mmCTLA4Ig) to suppress Con A-stimulated lymphocyte proliferation. Additionally, mmCD40Ig only showed mild immunosuppressive activity in a one-way mixed lymphocyte reaction (MLR) system. These results suggest that mmCD40Ig secreted by P. pastoris was productive and functional, and it could be used as a tool for pathogenesis and therapies for chronic inflammatory diseases in a M. mulatta model.