Expression Of Costimulatory Molecules Research Articles

IL-10 Dampens the Th1 and Tc Activation through Modulating DC Functions in BCG Vaccination.

BCG, the only registered vaccine against Mycobacterial Tuberculosis (TB) infection, has been questioned for its protective efficacy for decades. Although lots of efforts were made to improve the BCG antigenicity, few studies were devoted to understand the role of host factors in the variability of the BCG protection. Using the IL-10KO mice and pulmonary tuberculosis infection model, we have addressed the role of IL-10 in the BCG vaccination efficacy. The data showed that IL-10-deficient dendritic cells (DCs) could promote the immune responses through upregulation of the surface costimulatory molecule expression and play an orchestra role through activating CD4+T cell. IL-10-deficient mice had higher IFN γ, TNF α, and IL-6 production after BCG vaccination, which was consistent with the higher proportion of IFN γ+CD3+, IFN γ+CD4+, and IFN γ+CD8+ T cells in the spleen. Particularly, the BCG-vaccinated IL-10KO mice showed less inflammation after TB challenge compared to WT mice, which was supported by the promoted Th1 and Tc, as well as the downregulated Treg responses in IL-10 deficiency. In a conclusion, we demonstrated the negative relationship between Th1/Tc responses with IL-10 production. IL-10 deficiency restored the type 1 immune response through DC activation, which provided better protection against TB infection. Hence, our study offers the first experimental evidence that, contrary to the modulation of BCG, host immunity plays a critical role in the BCG protective efficacy against TB.

Reduction of immunosuppressive tumor microenvironment in cholangiocarcinoma by ex vivo targeting immune checkpoint molecules

Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs. We isolated TILs from resected tumors of patients with cholangiocarcinoma and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TILs, and determined whether targeting these molecules improved ex vivo functions of TILs. Proportions of cytotoxic T cells and natural killer cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margins, and natural killer cells were excluded from the tumors. The co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype. Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for patients with cholangiocarcinoma. The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking "immune checkpoint" molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in patients with cholangiocarcinoma.

Resiquimod-Mediated Activation of Plasmacytoid Dendritic Cells Is Amplified in Multiple Sclerosis.

Background: Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. The cause of multiple sclerosis is unknown but there are several evidences that associate the genetic basis of the disease with environmental causes. An important association between viral infection and development of MS is clearly demonstrated. Viruses have a strong impact on innate immune cells. In particular, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs), are able to respond to viruses and to activate the adaptive immune response. Methods: In this study we mimic viral infection using synthetic single-strand RNA, Resiquimod, and we compared the response of both DC subsets derived from healthy donors and MS patients by characterizing the expression of costimulatory molecules on the DC surface. Results: We found that pDCs from MS patients express higher levels of OX40-L, HLA-DR, and CD86 than healthy donors. Moreover, we found that blood cells from MS patients and healthy donors upon Resiquimod-stimulation are enriched in a subpopulation of pDCs, characterized by a high amount of costimulatory molecules. Conclusion: Overall, these results indicate that activation of pDCs is enhanced in MS, likely due to a latent viral infection, and that costimulatory molecules expressed on pDCs could mediate a protective response against the viral trigger of autoimmunity.

ERdj5 in Innate Immune Cells Is a Crucial Factor for the Mucosal Adjuvanticity of Cholera Toxin.

Cholera toxin (CT) is one of most strong mucosal adjuvants, but it cannot be clinically used owing to its toxicity. The cytosolic A1 subunit of CT (CTA1) is the molecule responsible for its immunostimulatory activity, which increases the concentration of cyclic AMP and causes the induction of pro-inflammatory cytokines in innate immune cells. However, the importance of endoplasmic reticulum (ER) molecules involved in CTA1 retro-translocation to induce immune responses remained to be investigated. ERdj5 is an ER protein which is expected to transfer CTA1 to the Hrd1 complex for the retro-translocation of CTA1. In this study, we investigated the physiological relevance of ERdj5 in immune stimulation by CT. ERdj5-knockout (ERdj5 KO) mice had decreased production of antigen-specific IgG in the serum and IgA in the mucosal secretion after intranasal immunization with Ag and CT. Especially, IgG2c isotypes were specifically reduced in the absence of ERdj5. ERdj5 KO dendritic cells (DCs) failed to full activation with decreased expression of costimulatory molecules, such as MHC class II, CD80, and CD 86. In ERdj5 KO DCs, secretion of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, was reduced. The cytokine signatures of several helper T cells were reduced in ERdj5 KO mice following intranasal CT immunization. The absence of ERdj5 affects the immunostimulatory properties of CT but does not affect the response to the CTB pentamer, the response to alum, total antibody production, or cytokine release from DCs exposed to CpG. Interestingly, CT enhanced the expression of ER stress proteins in ERdj5 KO innate immune cells. These results suggested that ERdj5 contributed as a decisive factor to the immunostimulatory capacity of CT via CTA1 retro-translocation.

Atractylodin Suppresses Dendritic Cell Maturation and Ameliorates Collagen-Induced Arthritis in a Mouse Model.

The aim of this study was to evaluate the immunomodulatory effects of atractylodin, a polyethylene alkyne, on the maturation of bone marrow-derived dendritic cells (BM-DC) as well as its antirheumatic effect on collagen-induced arthritis (CIA) in DBA/1 mice. Our results indicate that atractylodin effectively suppressed the secretion of pro-inflammatory cytokines, expression of costimulatory molecules, and p38 MAPK, ERK, and NF-κBp65 signaling pathways in LPS-incubated dendritic cells (DCs). Additionally, the proliferation and cytokine secretion (IFN-γ and IL-17A) of CD8+ and CD4+ T cells were reduced. In a murine CIA model, intraperitoneal injection of atractylodin significantly alleviated the severity of the disease progression, as indicated by reduced paw swelling, clinical arthritis scores, and pathological changes of joint tissues. In addition, the overall proliferation of T cells stimulated by type II collagen and the abundance of Th1 and Th17 in the spleens were also significantly decreased with atractylodin treatments. Furthermore, atractylodin significantly downregulated the expression levels of CD40, CD80, and CD86 of DCs in the spleens. In conclusion, this study shows for the first time that atractylodin has potential to manipulate the maturation of BM-DCs and should be further explored as a therapeutic agent in the treatment of rheumatoid arthritis (RA).

Paraquat Preferentially Induces Apoptosis of Late Stage Effector Lymphocyte and Impairs Memory Immune Response in Mice.

Paraquat (PQ) is a toxic non-selective herbicide. To date, the effect of PQ on memory immune response is still unknown. We investigated the impact of PQ on memory immune response. Adult C57BL/6 mice were subcutaneously injected with 2 mg/kg PQ, 20 mg/kg PQ or vehicle control every three days for two weeks. A single injection of keyhole limpet hemocyanin (KLH) at day four after the initial PQ treatment was used to induce a primary immune response; a second KLH challenge was performed at three months post the first KLH immunization to induce a secondary immune response. In steady state, treatment with 20 mg/kg PQ reduced the level of serum total IgG, but not that of IgM; treatment with 20 mg/kg PQ decreased the number of effector and memory lymphocytes, but not naïve or inactivated lymphocytes. During the primary immune response to KLH, treatment with 20 mg/kg PQ did not influence the proliferation of lymphocytes or expression of co-stimulatory molecules. Instead, treatment with 20 mg/kg PQ increased the apoptosis of lymphocytes at late stage, but not early stage of the primary immune response. During the secondary immune response to KLH, treatment with 20 mg/kg PQ reduced the serum anti-KLH IgG and KLH-responsive CD4 T cells and B cells. Moreover, effector or activated lymphocytes were more sensitive to PQ-induced apoptosis in vitro. Treatment with 2 mg/kg PQ did not impact memory immune response to KLH. Thus, treatment with 20 mg/kg PQ increased apoptosis of late stage effector cells to yield less memory cells and thereafter impair memory immune response, providing a novel understanding of the immunotoxicity of PQ.

PF231 REDIRECTED CYTOTOXICITY AND PROLIFERATION OF T CELLS INDUCED BY BITE® ANTIBODY CONSTRUCTS ARE MODULATED BY THE EXPRESSION PROFILE OF COSTIMULATORY AND COINHIBITORY MOLECULES ON TARGET CELLS IN VITRO

Background:BiTE® antibody constructs represent a novel immunotherapeutic strategy relying on the recruitment of T cells against tumor cells independent of TCR specificity. In Acute Myeloid Leukemia (AML), CD33 represents a validated target antigen with high prevalence in expression in >90 % of primary AML samples (Krupka et al. 2014). A CD33 specific BiTE® antibody construct was developed (AMG 330) and showed cytotoxicity against primary AML in vitro (Krupka et al. 2016). AMG 330 is being evaluated in a ongoing dose escalation of a first in human phase 1 study in relapsed/refractory AML with encouraging early evidence of tolerability and anti‐leukemic activity in heavily pretreated patients (Ravandi et al. 2018). To better understand molecular mechanisms mediating AMG 330 effects, we created a novel in vitro model system to characterize the effects of costimulatory and coinhibitory molecules on redirected cytotoxicity and induced proliferation of T cells.Aims:We hypothesized that BiTE®‐mediated cytotoxicity and induction of T‐cell proliferation is influenced by the checkpoint expression profile on target cells. We assume that the ratio of positive and negative checkpoint molecules on target cells is an important modulator of AMG 330‐mediated cytotoxicity against AML cells.Methods:To dissect mechanistic aspects of T cell recruitment a stable expression system was established utilizing murine Ba/F3 cells expressing human CD33 ± CD80 ± CD86 ± PD‐L1. Co‐cultures of murine Ba/F3 transfectants and human healthy donor (huHD) T cells were performed in presence of AMG 330 or a control BiTE® (cBiTE®) (5 ng/ml). For some experiments, huHD T cells were separated into naive (CD45RA+/CCR7+) vs memory (CD45RADIM) cells using fluorescence‐activated cell sorting. Further co‐cultures were performed by mixing varying proportions of Ba/F3 CD33+ CD80+ PD‐L1 ± and Ba/F3 CD33+ PD‐L1+ cells with huHD T cells (AMG 330/cBite c = 0.5 ng/ml). Proportion of Ba/F3 CD33+ PD‐L1+ cells was varied between 0–100 %. After 3 days, specific lysis was determined by flow cytometry and calculated as % specific lysis = 100 × (1–live CD33+ cellsAMG 330/live CD33+ cellscBiTE). T‐cell proliferation was defined as number of CD2+ cells on day 3 compared to day 0.Results:Murine Ba/F3 cells expressing only human CD33 were not lysed upon addition of AMG 330 and huHD T cells. Cytotoxicity could be observed with cells additionally expressing human CD80+CD86 >> CD80 > CD86 (Table 1, A). There was a direct correlation of AMG 330 mediated T‐cell proliferation and cytotoxicity. Memory T cells showed increased cytotoxicity compared to naive T cells against Ba/F3 cell lines expressing the different sets of human molecules. The influence of co‐inhibition was investigated by additionally transducing PD‐L1 into the different Ba/F3 cells. This led to a reduced AMG 330‐mediated cytotoxicity in all PD‐L1 expressing Ba/F3 cells and was accompanied by a reduction in T‐cell proliferation (Table 1, A). When mixing Ba/F3 CD33+ CD80+ PD‐L1 ± with Ba/F3 CD33+ PD‐L1+ cells we observed a decrease in T‐cell proliferation and mediated cytotoxicity with increased percentage of Ba/F3 CD33+ PD‐L1+ cells (Table 1, B).Summary/Conclusion:In summary, this data supports the hypothesis T‐cell proliferation and thereto related cytotoxicity induced by redirection through BiTE® antibody constructs are influenced by the expression of costimulatory and coinhibitory molecules. Our data support the notion that the checkpoint profile on AML and its distribution among target cells, rather than one molecule by itself on a single cell level, may modulate T‐cell response to AMG 330.image

Expression of Toll-like receptors and costimulatory molecules in splenic B cells in a normal and abortion-prone murine pregnancy model.

The regulatory role of B lymphocytes in the pregnancy-induced maternal immune response is not well recognized. B lymphocytes function as antigen-presenting cells (APCs) and regulate Toll-like receptors and costimulatory molecule expression in response to intrinsic and extrinsic signals. Therefore, the aim of this study was to determine the expression of TLR2, TLR4, TLR9, and MHC class II and the costimulatory molecules CD80, CD86, and CD40 in splenic B cells in a normal and abortion-prone murine pregnancy model. The expression level of these molecules on female splenic B cells was investigated using real-time PCR and flow cytometry. The analysis was performed on the 3rd and 14th day of normal (CBA/JxBALB/c) and abortion-prone (CBA/JxDBA/2J) murine pregnancy. The expression of Tlr9, Cd86, and H2-Ab1 in splenic B cells on the 3rd day after mating was upregulated, whereas Tlr2 was downregulated in abortion-prone females. On day 14, we observed lower expression levels of Tlr4 and Cd80 and higher expression levels of Cd86 in CBA/J females mated with DBA/2J males. At the protein level, the differences were observed only on day 3 of pregnancy. TLR4 and CD40 molecules were upregulated in splenic B cells, while TLR9 and CD86 were downregulated in abortion-prone mice. Differential expression of TLRs and costimulatory molecules in splenic B cells in abortion-prone and normal pregnancies suggests the involvement of these cells in the regulation of the immune response at the periphery in pregnant females.

Anti-inflammatory modulation of human myeloid-derived dendritic cell subsets by lenalidomide

Although immunomodulatory drugs (IMiDs) were originally developed as anti-inflammatory drugs, they are effective for multiple myeloma. In order to gain further insights into the immunomodulatory mechanisms of IMiDs for the treatment of inflammatory disorders and myeloma, we investigated the influence of a representative IMiD, lenalidomide, on human primary dendritic cell (DC) subsets: myeloid-derived CD1c+ DCs, CD141+ DCs, and plasmacytoid DCs. Lenalidomide did not affect the viability or expression of costimulatory molecules, but it potently suppressed the production of the key inflammatory cytokines IL-12 and IL-23, and enhanced the production of the anti-inflammatory cytokine IL-10 by CD1c+ DCs. Lenalidomide also suppressed the production of IFN-α by CD141+ DCs but not that by plasmacytoid DCs. Lenalidomide likely targets pathways downstream of the nuclear translocation of the transcription factors nuclear factor κB (NF-κB) and IFN regulatory 5 (IRF5) in CD1c+ DCs. Consistent with the direct immunomodulatory effects on DCs, lenalidomide decreased the capacity of CD1c+ DCs to induce differentiation of naïve CD4+ T cells into effector cells producing immune activating and myeloma-promoting cytokines. This study demonstrated that lenalidomide has anti-inflammatory effects via the modulation of cytokine production by human myeloid-derived DCs. Such effects on DCs may allow for beneficial immunomodulation aiding in the treatment of inflammatory disorders and multiple myeloma.

Monoclonal microbial EDP1503 to induce antitumor responses via gut-mediated activation of both innate and adaptive immunity.

e14241 Background: Systemic immunity is regulated by interactions of commensal bacteria with immune cells in the gut. Enrichment of specific intestinal microbes has been shown to enhance the anti-tumor response to PD-1 blockade in both murine models and cancer patients. Here we report that oral administration of a monoclonal microbial, EDP1503, induces systemic anti-tumor immunity. Methods: The mechanism of action and efficacy of EDP1503 was investigated in isograft tumor models and a variety of ex vivo and in vitro studies in murine and human cells. Results: EDP1503 increases expression of costimulatory molecules on CD11c+ dendritic cells (DCs) within the mesenteric LNs with an accompanying increase in proinflammatory CD103+ DCs within tumor draining lymph nodes. In addition, EDP1503 amplifies both myeloid and lymphocyte responses via production of DC-derived growth factors, M1 polarization of macrophages, and production of the lymphocyte-recruiting chemokines, CXCL9 and CXCL10. Mechanistically, EDP1503 triggers specific pattern recognition receptors and induces proinflammatory responses in antigen presenting cells. Moreover, in vivo, treatment of mice with EDP1503 results in decreased tumor volume and delayed tumor growth. The prominent anti-tumor effects of EDP1503 are further augmented by combination with anti-PD-1 neutralizing Abs. Dissection of the tumor microenvironment reveals increased activated Ki67+ NK cells and CD8+ T cells producing IFNg. Conclusions: Together, these data clearly demonstrate the ability of an orally delivered non-colonizing monoclonal microbe to enhance innate and adaptive anti-tumor immunity and substantiates the rationale for ongoing clinical trials. EDP1503 is currently in Phase 1b/2 studies (NCT03775850; NCT03595683) with enrollment open at multiple sites.

Macrophage immunomodulatory activity of the cationic polymer modified PLGA nanoparticles encapsulating Alhagi honey polysaccharide

In previous researches, the results showed that Alhagi honey polysaccharide-loaded poly(D,L-lactic-co-glycolic) acid nanoparticles (AHPP) as immune adjuvant enhanced Th1 immune responses. In order to further enhance the immune adjuvant activity and phagocytosis of the nanoparticles, three kinds of Alhagi honey polysaccharide-loaded cationic polymer modified PLGA nanoparticles were prepared to investigate the effects on macrophages in vitro. After treatment with the nanoparticles, the effects of phagocytosis, co-stimulatory molecules expression, nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines secretion were evaluated. The results showed that the surface structure of cationic polymer modified AHPP nanoparticles were not obviously changed, and the stability was greatly improved. Cationic polymer modified AHPP nanoparticles significantly stimulated phagocytic activity, MHCII+, CD86+, and CD80+ expression of macrophages. In addition, the levels of NO, iNOS, TNF-α, IFN-γ, IL-1β and IL-12 were enhanced in the peritoneal macrophages by stimulation with cationic polymer modified AHPP nanoparticles. Among them, polyethyleneimine modified PLGA nanoparticles (PEI-AHPP) showed the best effects on the expression of co-stimulatory molecules, and secretions of NO, iNOS, and cytokines. These results indicated that PEI-AHPP could enhance the activation of macrophages, and it could be potentially used as an AHP delivery system for the induction of strong immune responses.

Coronary artery bypass grafting surgery is associated with immunoparalysis of monocytes and dendritic cells

Abstract Background Exposure of patient blood to the bypass circuitry and re-perfusion of the heart at the end of coronary artery bypass grafting (CABG) is associated with activation of leucocytes, the complement cascade and cytokine secretion. Dendritic cells (DC) and monocytes are immunoregulatory cells that may be impacted by CABG contributing to an altered immune status. This study investigated changes in the DC and monocyte immune profile in CABG patients at 5 time-points: admission, peri-operative, ICU, day 3 (D3) and day 5 (D5). Methods At each time point, freshly collected whole blood from 50 CABG patients was used in an ex-vivo whole blood culture model. Lipopolysaccharide (LPS) was added in parallel to model an infectious complication. Expression of DC and monocyte co-stimulatory and adhesion molecules and production of intracellular inflammatory mediators was measured via flow cytometry. Results CABG resulted in significant perturbation of monocyte and DC responses. DC and monocytes were immunoparalysed as evidenced by failure to respond to LPS stimulation. Modulation of DC and monocyte costimulatory and adhesion molecule expression and inflammatory mediator production was associated with prolonged length of stay in intensive care and post-operative atrial fibrillation. While DC and monocyte expression of co-stimulatory and adhesion molecules recovered towards baseline by D5 post-surgery, their capacity to produce inflammatory mediators did not. Conclusions DC and monocyte responses were significantly impaired following CABG. Use of an ex-vivo model to assess immune competency of cardiac surgery patients may provide a tool for the early prediction of adverse outcomes.

NLRC4 deficiency enhances macrophage and T cell responses in polymicrobial sepsis

Abstract Rationale Sepsis, a dysregulated systemic host inflammatory response to infection with organ failure, is leading cause of death in non-coronary intensive care unit worldwide. Recently, the NLRC4 inflammasome has been implicated in sensing pathogens and regulating inflammation during microbial infections. However, the role of NLRC4 in sepsis-induced immunosuppression, specifically macrophage dysfunction and T cell loss, is poorly understood. Methods We performed cecal ligation and puncture (CLP) to induce polymicrobial sepsis in wild type (C57BL6) and NLRC4-gene deficient (KO) mice. Cellular influx, bacterial burden, and host survival were measured. Macrophages were depleted using Clodronate liposomes. Depletion of lymphocytes, frequency of IFN-γ producing T cells, expression of costimulatory molecules in peritoneal macrophages were examined using flow cytometry. Results NLRC4 KO mice were protected from CLP-induced lethality. Furthermore, NLRC4 KO mice had reduced cellular influx, cytokine storm (IL-1b, TNF-a, MCP-1, CXCL-1, CXCL2), and bacterial burdens following CLP. NLRC4 KO mice were protected from sepsis-induced impairment of macrophage function and loss of CD4 and CD8 T cells. Peritoneal macrophages (CD11b+F4/80+) were protected from sepsis-induced loss of MHCII and CD86. Intriguingly, macrophage depletion prior to polymicrobial sepsis eliminated the NLRC4-mediated survival advantage. NLRC4 KO mice had increased frequency of IFN-γ producing T cells post-CLP. Conclusions These findings identify NLRC4 as a negative regulator of macrophage and T cell responses in sepsis and its selective inhibition as a potential therapeutic strategy to augment host immunity in sepsis.

Biodegradable Nanoparticles Containing Antigen and Vitamin D3 Efficiently Induce Antigen-specific Immune Suppression

Abstract Biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) encapsulated with antigens can efficiently deliver the antigens to antigen-presenting cells such as dendritic cells (DCs). Active form of vitamin D, 1α,25-dihydroxyvitamin D3 (VD3), is known to have various immunosuppressive effects. Encapsulation of VD3 within NPs would enhance the delivery of VD3 to APCs, while preventing the potential systemic side effects of VD3. In this study, we examined the effects of PLGA NPs encapsulated with ovalbumin (OVA) and VD3. PLGA NPs containing OVA and VD3 (NP[OVA+VD3]) were prepared by W1/O/W2 double emulsion solvent evaporation method. Bone marrow-derived DCs treated with NP[OVA+VD3] were decreased in the expression of co-stimulatory molecules, and produced higher levels of immunosuppressive cytokines, while producing lower levels of pro-inflammatory cytokines, compared with DCs treated with NP[OVA]. Addition of NP[OVA+VD3] to allogeneic MLR reduced the proliferation of allogenic T cells. In addition, NP[OVA+VD3]-treated DCs efficiently generated CD4+CD25+Foxp3+Treg cells from CD4+CD25− T cells. Furthermore, mice injected i.v. with NP[OVA+VD3] were suppressed in inducing OVA-specific cytotoxic T cells. These results show that phagocytic delivery of antigen and VD3 to APCs using biodegradable NPs efficiently suppresses antigen-specific immune responses.

Differential gene expression of the B Cell Receptor (BCR) signaling pathway during pregnancy in mice

Abstract The aim of this work was to characterize BCR signaling pathway gene expression in B cells during pregnancy. B-cell receptor (BCR) signaling regulates critical cell-fate decisions in B cells like activation, proliferation, expression of costimulatory molecules and cytokine receptors. Changes on the dynamic of BCR signaling pathway during pregnancy might have an impact on the way how the immune system fight against infections or regulate autoimmunity during this critical period of time. We performed a genome-wide transcriptome profile (GeneChip™ Mouse Gene 1.0 ST Array, Affymetrix) on pure splenic B cells isolated from pregnant and non-pregnant mice. Results were analyzed and significant gene expression regulation was confirmed by qPCR. In silico analysis showed that genes associated with B cell receptor signaling pathway were significantly down-regulated in B cells from pregnant mice compared to non-pregnant animals (≈1.5–2 fold changes, Limma eBayes test, p<0.0001, n=4). Further qPCR analysis showed that genes of the BCR pathway involved in B cell activation, proliferation and antibodies production were down-regulated (Unpaired t test, one-tailed, *p<0.05, n=8) in B cell from pregnant mice as compared to non-pregnant females. In contrast, genes of the BCR pathway that inhibits B cells activation presented no significant differences. We showed here that genes associated with BCR signaling leading to B cell activation and proliferation were significantly down-regulated during pregnancy. These results might help to understand why pregnant women are more susceptible to infections as well as some B cell associated autoimmune diseases experience a significant improvement in the symptoms during pregnancy.

Epigenetic priming underpins enhanced memory B cell differentiation

Abstract Immunological memory is a hallmark of the adaptive immune response. Memory B cells (MBC) have increased affinity, expression of costimulatory molecules, and capacity to proliferate and differentiate than their naïve counterparts; however, the epigenetic mechanisms that control enhanced MBC functions are currently unknown. Using a murine model of influenza infection, nucleoprotein (NP)-specific MBC and follicular naïve B cells (nB) were isolated and the chromatin accessibility and transcriptional landscape determined by ATAC and RNA-seq, respectively. MBC displayed distinct gene expression profiles from nB, including an upregulation of plasma cell (PC) signature genes and significant overall increases in total mRNA content. Furthermore, MBC display an open and primed chromatin conformation in gene regulatory regions that map to transcription factors controlling either PC (Prdm1 and Irf4), germinal center (Aicda), or MBC (Zbtb32) fates. These data reveal additional novel MBC-specific transcription factor networks and describe an epigenetic “antigen experience” signature that correlates with enhanced MBC function. To confirm the importance of these molecular changes, naïve and memory mice were challenged with a heterosubtypic influenza infection or isolated and cultured with CD40L, IL-4, and IL-5. In vivo, MBC form germinal centers earlier and to a higher frequency than nB. MBC also form significantly more NP+IgG+ PCs by flow cytometry and ELISPOT, corroborating the open chromatin signature. Ex vivo, MBCs upregulated primed transcription factors earlier than nB, validating the enhanced formation of CD138+ PCs. These data describe an epigenetic basis for enhanced the differentiation capacity and function of MBC.

The complex of poly-γ-glutamic acid and aluminum salts ameliorates the reduced efficacy of the influenza H1N1 vaccine in aged mice

Abstract Influenza is a highly contagious respiratory disease caused by the influenza A viruses and results in public health threat like seasonal epidemics and sporadic pandemics. Although vaccination is considered to be one of the most effective strategy for preventing the disease, the low efficacy of the influenza vaccine has been reported in the elderly. Here, we examined that the aging-related reduced efficacy of the influenza vaccine can be ameliorated by the complex of poly-γ-glutamic acid (γ-PGA) and alum (PGA/Alum), a novel adjuvant capable of inducing both humoral and cellular immune responses, using an aged mouse model. PGA/Alum induced activation of dendritic cells from aged mice, accompanied with increase of both expression of co-stimulatory molecules and production of pro-inflammatory cytokines. In addition, PGA/Alum enhanced the protective efficacy of pandemic influenza H1N1 (pH1N1) vaccine in aged mice, with earlier virus clearance after A/California/4/2009 (pH1N1) viral challenge, increased hemagglutination-inhibition titers, activity of cytotoxic T lymphocytes, and generation of multifunctional T lymphocytes. Taken together, these results suggest that PGA/Alum is a potent vaccine adjuvant that could improve the reduced vaccine efficacy of aging, thereby leading to prevention of influenza virus infection in the immune compromised populations.

Neutrophil Cytosolic Factor 1 in Dendritic Cells Promotes Autoreactive CD8+ T Cell Activation via Cross-Presentation in Type 1 Diabetes.

Aims: Reactive oxygen species (ROS) are critical in driving the onset of type 1 diabetes (T1D). Ablation of ROS derived from phagocytic NADPH oxidase 2 is protective against autoimmune diabetes in non-obese diabetic (NOD) mice. However, the mechanisms of NADPH oxidase 2-derived ROS in T1D pathogenesis need to be elucidated. Here, we have examined the role of Ncf1 (the regulatory subunit of NADPH oxidase 2) in dendritic cells (DC).Results: Ncf1-mutant DCs exhibit reduced ability to activate autoreactive CD8+ T cells despite no difference in co-stimulatory molecule expression or pro-inflammatory cytokine production. When provided with exogenous whole-protein antigen, Ncf1-mutant NOD DCs showed strong phagosome acidification and rapid antigen degradation, which lead to an absence of protein translocation into the cytoplasm and deficient antigenic peptide loading on MHC Class I molecules.Innovation: This study demonstrates that Ncf1 (p47phox) is required for activation and effector function of CD8+ T cells by acting both intrinsically within the T cell as well as within professional antigen presenting cells.Conclusion: ROS promote CD8+ T cell activation by facilitating autoantigen cross-presentation by DCs. ROS scavengers could potentially represent an important component of therapies aiming to disrupt autoantigen presentation and activation of CD8+ T cells in individuals at-risk for developing T1D.

NLRP3 inflammasome-activating arginine-based liposomes promote antigen presentations in dendritic cells.

Purpose: The NLRP3 inflammasome activation has been proposed as a common mechanism for some adjuvants to boost the immune system, and cationic liposomes were reported to potentially activate the NLRP3 inflammasome. Herein, we questioned whether the NLRP3 inflammasome-activating cationic liposomes could promote antigen presentation and be applied as an immune adjuvant. In addition, we aimed to investigate the structure effect of lipid on triggering these immune responses.Materials and methods: A series of structurally similar lipids, consisting of arginine (Arg) head group and varied lengths of alkyl chains or spacers in between were used to prepare cationic liposomes. Lipopolysaccharide-primed human or murine macrophages or phorbol 12-myristate 13-acetate-primed THP-1 cells were treated with these liposomes, and interleukin (IL)-1β secretion was measured to quantify the NLRP3 inflammasome activation. Lysosome rupture was examined in THP-1 cells by the fluorescence loss of acridine orange, a lysosome dye. Further, chicken ovalbumin (OVA) was loaded on the liposome surface and applied to murine bone marrow-derived dendritic cells (BMDCs), which activate OT-I and OT-II lymphocytes upon major histocompatibility complex (MHC) class I- and class II-mediated antigen presentation, respectively. OT-I and OT-II cell division and IL-2 secretion were measured to evaluate the antigen presentation efficiency. The expressions of MHC molecules and co-stimulatory molecules ie, CD80, CD86, and CD40 on BMDCs were investigated by flow cytometry.Results: All the liposomes showed size distributions of 80–200 nm and zeta potentials of around 50 mV. A3C14 liposomes, consisting of Arg-C3-Glu2C14 lipids induced the most potent lysosome rupture and NLRP3 inflammasome activation. OVA-A3C14 also exhibited the most potent MHC class I- and class II-mediated antigen presentation in BMDCs without interfering MHC and co-stimulatory molecules.Conclusion: The hydrophobic moieties of arginine-based liposomes are crucial in stimulating innate immune cells. A3C14 liposomes were non-immunogenic but strongly activated innate immune cells and promoted antigen presentation, and therefore can be applied as immune adjuvants.

Immunomodulatory capability of VCG in Chlamydia immunity

Abstract Immunization with UV-inactivated Chlamydia trachomatis elementary bodies (UV-EB), the infectious form of Chlamydia, does not protect against infection with live Chlamydia. Vibrio cholerae ghosts (VCG), which are empty V. cholerae cell envelopes derived by genetic inactivation, constitute an effective delivery vehicle for cloned vaccine antigens and promotes the induction of protective immunity in the absence of added adjuvants. In this study, we sought to investigate the ability of VCG to enhance the innate and adaptive immune responsesinduced by UV-EB. Thus, bone marrow-derived dendritic cells (BMDC) were pulsed with UV-EB in the presence or absence of VCG and the induction of innate immune responses, T cell proliferation and ability to afford protection against Chlamydia following adoptive transfer into susceptible mice was evaluated. The results show that VCG efficiently modulated the immunostimulatory properties of DCs as indicated by increased secretion of proinflammatory cytokines and expression of toll-like receptors and co-stimulatory molecules associated with DC maturation. Also, UV-EB-pulsed DCs that were exposed to VCG and adoptively transferred into mice resulted in effective chlamydial antigen presentation and protective immunity. The results demonstrate that VCG activate the maturation of DCs leading to enhancement of innate and adaptive immunity to an otherwise non-protective antigen, suggesting their utility as adjuvants for enhancement of protective immunity to inactivated vaccine antigens.