The Effect of Akkermansia muciniphila and Its Outer Membrane Vesicles on MicroRNAs Expression of Inflammatory and Anti-inflammatory Pathways in Human Dendritic Cells.

Probiotics play a crucial role in immunomodulation by regulating dendritic cell (DC) maturation and inducing tolerogenic DCs. Akkermansia muciniphila affects inflammatory response by elevating inhibitory cytokines. We aimed to evaluate whether Akkermansia muciniphila and its outer membrane vesicles (OMVs) affect microRNA-155, microRNA-146a, microRNA-34a, and let-7i expression of inflammatory and anti-inflammatory pathways. Peripheral blood mononuclear cells (PBMCs) were isolated from the healthy volunteers. To produce DCs, monocytes were cultivated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were allocated into six subgroups: DC + Lipopolysaccharide (LPS), DC + dexamethasone, DC + A. muciniphila (MOI 100, 50), DC + OMVs (50 µg/ml), and DC + PBS. The surface expression of human leukocyte antigen-antigen D related (HLA-DR), CD86, CD80, CD83, CD11c, and CD14 was examined using flow cytometry, and the expression of microRNAs was assessed using qRT-PCR, and the levels of IL-12 and IL-10 were measured using ELISA. A. muciniphila (MOIs 50, 100) could significantly decrease IL-12 levels relative to the LPS group. The IL-10 levels were decreased in the DC + LPS group than the DC + dexamethasone group. Treatment with A. muciniphila (MOI 100) and OMVs could elevate the concentrations of IL-10. DC treatment with LPS led to a significant increment in the expression of microRNA-155, microRNA-34a, and microRNA-146a. The expression of these microRNAs was reversed by A. muciniphilia and its OMVs treatment. Let-7i increased in treatment groups compared to the DC + LPS group. A. muciniphilia (MOI 50) had a substantial effect on the expression of HLA-DR, CD80, and CD83 on DCs. Therefore, DCs treatment with A. muciniphila led to induce tolerogenic DCs and the production of anti-inflammatory IL-10.

[Effect of Xijiao Dihuang Combined Prescription on Human Dendritic Cell Function Induced by Lipopolysaccharide].

OBJECTIVE: To observe the effects of drug-containing serum of Xijiao Dihuang combined prescription(XJDH) on the related functions of dendritic cells(DCs) induced in vitro, and to explore the mechanisms underlying the effectiveness of XJDH treatment on primary immune thrombocytopenia(ITP). METHODS: Peripheral blood samples were colle-ted from 6 healthy volunteers. Mononuclear cells were isolated by density gradient centrifugation, and CD14+ mononuclear cells were collected by the magnetic separation technique. CD14+ mononuclear cells were induced into immature DCs by recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin 4 (IL-4). Immature DCs were divided into three groups: control group, model group and XJDH group. CCK-8 assay was used to determine the intervention concentration and time of drug-containing serum. Lipopolysaccharide(LPS) with the final concentration of 1 µg/ml was added to model group and XJDH group respectively for 24 h to induce DCs maturation. Normal rat serum was added to control group and model group, and XJDH was added to XJDH group for 24 h. Flow cytometry was used to detect the levels of CD80, CD83 and HLA-DR on the surface of DCs. Western blot was used to detect the expression of TLR4 and NF-κB, and levels of IL-6, IL-12 and TNF-α in cell supernatant was detected by ELISA. RESULTS: Compared with the control group, LPS stimulation increased the expression of CD80, CD83 and HLA-DR, with subsequent increasing expression of TLR4 and NF-κB, as well as IL-6, IL-12 and TNF-α increased(P<0.05). In comparison with model group, the expression of DCs surface molecules CD80, CD83 and HLA-DR, DCs' expression of TLR4 and NF-κB protein, and the levels of IL-6, IL-12 and TNF-α in the cell supernatant of XJDH group decreased after the intervention of XJDH (P<0.05). CONCLUSION: Drug containing serum of Xijiao Dihuang combined prescription can down-regulate TLR4/NF-κB signaling pathway related protein expression, inhibit DCs maturation, and reduce proinflammatory factor secretion, which may be one of the mechanisms of drug-containing serum of Xijiao Dihuang combined prescription in the treatment of immune thrombocytopenia.

Myd88 knockdown with RNA interference induces in vitro immune hyporesponsiveness in dendritic cells from rhesus monkeys.

Immature dendritic cells (imDCs) are activated and mature to initiate an adaptive immune response, resulting in allograft rejection and transplantation failure. Myeloid differentiation factor 88 (Myd88) is a key factor in the Toll-like receptor (TLR) signaling pathway. Here, we investigated the effect of Myd88 silencing on DC function and immune response. CD34 + cells were isolated from the bone marrow of rhesus monkeys by the immunomagnetic bead method and then infected with an adenovirus expressing Myd88-specific short hairpin RNA (sh-Myd88). sh-NC (nontargeting negative control)- or sh-Myd88-infected DCs were treated with lipopolysaccharide (LPS) for another 48 h to induce DCS maturation. The maturation of DCs was identified by immunofluorescence staining for MHCII, CD80, and CD86. DC apoptosis was examined using Annexin V/PI staining. DC-related cytokine levels (IFN-γ and IL-12) were assessed by ELISA. A mixed lymphocyte reaction (MLR) was performed to test the effect of Myd88-silenced DCs on T lymphocytes in vitro. The results showed that compared with control or sh-NC-infected DCs, Myd88-silenced DCs had lower MHCII, CD80, CD86, and DC-related cytokine (IFN-γ and IL-12) levels. Myd88 did not affect the apoptosis of DCs. MLR demonstrated that Myd88 silencing could effectively block LPS-activated T cell proliferation in vitro. These data were consistent with the characteristics of tolerogenic DCs. In conclusion, our data indicated that Myd88 silencing could inhibit the maturation of imDCs and alleviate immune rejection, which provides a reference for immune tolerance in clinical liver transplantation.

Soluble CD80 Protein Delays Tumor Growth and Promotes Tumor-Infiltrating Lymphocytes.

Tumor cells use various immune-suppressive strategies to overcome antitumor immunity. One such method is tumor expression of programmed death ligand-1 (PD-L1), which triggers apoptotic death or anergy upon binding programmed death-1 (PD-1) on T cells. Our previous in vitro cellular studies with human and mouse PD-L1+ tumor cells demonstrated that a soluble form of the costimulatory molecule CD80 prevented PD-L1-mediated immune suppression and restored T-cell activation by binding PD-L1 and blocking interaction with PD-1. We now report that in vivo treatment of established syngeneic PD-L1+ CT26 colon carcinoma and B16F10 melanoma tumors with CD80-Fc delays tumor growth and promotes tumor-infiltrating T cells. Studies with PD-1-/- and CD28-/- mice demonstrate that soluble CD80 acts in vivo by simultaneously neutralizing PD-1 suppression and activating through CD28. We also report that soluble CD80 mediates its effects by activating transcription factors EGR1-4, NF-κB, and MAPK, downstream signaling components of the CD28 and T-cell receptor pathways. Soluble CD80 binds to CTLA-4 on activated human peripheral blood mononuclear cells. However, increasing quantities of CTLA-4 antagonist antibodies do not increase T-cell activation. These results indicate that soluble CD80 does not suppress T-cell function through CTLA-4 and suggest that CTLA-4 acts as a decoy receptor for CD80, rather than functioning as a suppressive signaling receptor. Collectively, these studies demonstrate that soluble CD80 has therapeutic efficacy in vivo in mouse tumor systems and that its effects are due to its ability to inhibit PD-1-mediated suppression while concurrently activating T cells through CD28. Cancer Immunol Res; 6(1); 59-68. ©2017 AACR.

Characterization of polysaccharides extracted from Platycodon grandiflorus (Jacq.) A.DC. affecting activation of chicken peritoneal macrophages.

Polysaccharides were isolated from Platycodon grandiflorus (Jacq.) A.DC. (PG) and the effects of three polysaccharides (PGPS80, PGPS60, PGPSt) on their immunological activities were studied. The structure identification of PGPSs was assessed using physicochemical and spectral methods. Results showed that PGPSt(2.67×105Da) compared to PGPS80(1.01×105Da) and PGPS60(1.12×105Da) has relatively higher average molecular weight(Mw) at the first peak with a narrower molecular weight distribution and all consisted of glucose, mannose, arabinose, galactose, xylose and rhamnose in different mass percentages. PGPS80 and PGPSt linked mainly by 1,3-and 1,6-ß-d-Galp residues. The immunological efficacy of PGPSs was performed on chicken peritoneal macrophages. Results showed that PGPSt significantly increased phagocytic rates, proliferation and NO production, stimulated macrophages to produce cytokines, including TNF-α, IL-1ß and IL-6 as well as stimulated macrophages to express the maturation markers CD80 and CD86. These findings suggest that PGPSt exerted significant immunological activity and might be associated with special characters.

Tumor suppressive effects of WEE1 gene silencing could not enhance immunopotentiation effects of CD80 and 4-1BBL co-stimulation in human T cells.

BACKGROUND: Activation of T cells against tumors by recruiting co-stimulatory molecules has been an attractive approach for cancer immunotherapy. Reports suggested that targeting different genes in tumors might also boost T cell-mediated tumor destruction. AIMS: We investigated whether in vitro WEE1 gene silencing in MDA-MB-468 and MCF7 breast cancer cell lines could enhance immunopotentiating effects of CD80 and 4-1BBL co-stimulation in human T cells. MATERIALS AND METHODS: WEE1 gene was specifically silenced in the cancer cells using shRNA technology. The co-stimulatory molecules were over-expressed on the surface of the cancer cells by recombinant non-replicative adenoviruses. The immune reaction of T cells in the co-culture with tumor cells was studied. IFN-g production was assessed by intracellular staining of T cells. To assess cytotoxic activity of CD8+ T cells, the CD107a mobilization-degranulation assay was performed. Expression of granzyme B, perforin and fasl were examined by real time PCR. RESULTS: T cell dual co-stimulation led to a significant increase in the frequency of IFN-g producing cells and higher percentages of degranulation in CD8+ T cells. It also resulted in higher expression levels of the cytotoxicity-related genes. WEE1 gene silencing in the target cells alone however, could not produce significant immune reactivation in the cultured T cells. Likewise, the immune responses of T cells neither improved nor suppressed when dually co-stimulated PBMCs were exposed to the cancer cells with silenced WEE1. CONCLUSIONS: In spite of antitumor effects of WEE1 silencing, combination of this approach with immune co-stimulation could not boost the reactivity of cultured T cells against the tested breast cancer cells.

Differential induction of IL-17, IL-10, and IL-9 in human T helper cells by B7h and B7.1.

ICOS and CD28 are expressed by T cells and are involved in costimulation of cytokine production in T helper (TH) cells. ICOS binds B7h expressed by several cell types, whereas CD28 binds B7.1 and B7.2 expressed by activated antigen presenting cells. This work investigated the role of B7h and B7.1 in TH17 and TH9 cell differentiation by assessing activity of recombinant B7h-Fc and B7.1-Fc on human naïve TH cells activated in the presence of different combinations of exogenous cytokines. In the presence of TGF-ß1 and IL-1ß (TH17 promoting condition), B7h-Fc was more effective than B7.1-Fc in inducing IL-17A and IL-10 secretion, whereas B7.1-Fc was more effective in inducing IL-17F. Dual costimulation with B7h-Fc and B7.1-Fc displayed an intermediate pattern with predominance of IL-17F over IL-17A, secretion of high levels of IL-10, and secretion of IL-9 levels lower than those induced by B7.1-Fc alone. In the presence of TGF-ß1 and IL-4 (TH9 promoting condition), B7h-Fc induced IL-17A only, whereas B7.1-Fc induced also IL-17F, IL-10, and high levels of IL-9. Experiments on memory TH cells showed that B7h-Fc mainly supported secretion of IL-17A and IL-10, whereas B7.1-Fc supported secretion of IL-17A, IL-17F, IL-10, and IL-9. These data indicate that B7h and B7.1 play different roles in modulation of TH17 and TH9 differentiation. This plasticity might be important in the immune response to pathogens and tumors, and in the development of autoimmune diseases, and should be taken in consideration in designing of immunotherapeutic protocols triggering ICOS or CD28.

Programmed death-1/B7-H1 negative costimulation protects mouse liver against ischemia and reperfusion injury.

UNLABELLED: Programmed death-1 (PD-1)/B7-H1 costimulation acts as a negative regulator of host alloimmune responses. Although CD4 T cells mediate innate immunity-dominated ischemia and reperfusion injury (IRI) in the liver, the underlying mechanisms remain to be elucidated. This study focused on the role of PD-1/B7-H1 negative signaling in liver IRI. We used an established mouse model of partial liver warm ischemia (90 minutes) followed by reperfusion (6 hours). Although disruption of PD-1 signaling after anti-B7-H1 monoclonal antibody treatment augmented hepatocellular damage, its stimulation following B7-H1 immunoglobulin (B7-H1Ig) fusion protected livers from IRI, as evidenced by low serum alanine aminotransferase levels and well-preserved liver architecture. The therapeutic potential of B7-H1 engagement was evident by diminished intrahepatic T lymphocyte, neutrophil, and macrophage infiltration/activation; reduced cell necrosis/apoptosis but enhanced anti-necrotic/apoptotic Bcl-2/Bcl-xl; and decreased proinflammatory chemokine/cytokine gene expression in parallel with selectively increased interleukin (IL)-10. Neutralization of IL-10 re-created liver IRI and rendered B7-H1Ig-treated hosts susceptible to IRI. These findings were confirmed in T cell-macrophage in vitro coculture in which B7-H1Ig diminished tumor necrosis factor-α/IL-6 levels in an IL-10-dependent manner. Our novel findings document the essential role of the PD-1/B7-H1 pathway in liver IRI. CONCLUSION: This study is the first to demonstrate that stimulating PD-1 signals ameliorated liver IRI by inhibiting T cell activation and Kupffer cell/macrophage function. Harnessing mechanisms of negative costimulation by PD-1 upon T cell-Kupffer cell cross-talk may be instrumental in the maintenance of hepatic homeostasis by minimizing organ damage and promoting IL-10-dependent cytoprotection.

Programmed death-1 receptor negatively regulates LPS-mediated IL-12 production and differentiation of murine macrophage RAW264.7 cells.

While programmed death-1 (PD-1), a co-inhibitory member of CD28 immunoglobulin superfamily plays negative roles in effector functions of T cells and B cells, little is known about the function of PD-1 expressed on innate immune cells. In this study, we demonstrate that IL-12 production was greatly suppressed in LPS-stimulated RAW264.7 cells upon PD-1 engagement with B7-H1.Fc fusion protein, and was restored in the presence of antagonistic anti-PD-1 mAb. PD-1-mediated suppression of IL-12 production in LPS-stimulated RAW264.7 cells was mediated by inhibition of Janus N-terminal-linked kinase (JNK) signaling pathway, and to a lesser extent, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway through the recruitment of SHP-2 to PD-1 cytoplasmic tail. B7-H1.Fc-mediated PD-1 engagement also downregulates the expression of co-stimulatory molecules such as CD80, CD86, MHC class I and II proteins in LPS-stimulated RAW264.7 cells. Furthermore, the endocytic activity is enhanced but the allostimulatory capacity is suppressed in LPS-treated RAW264.7 cells upon PD-1 engagement. Taken together, our results reveal a novel function of macrophage PD-1 in the negative regulation of IL-12 synthesis and differentiation into dendritic cell-like cells.

Use of B7 costimulatory molecules as adjuvants in a prime-boost vaccination against Visna/Maedi ovine lentivirus.

RNA transcripts of the B7 family molecule (CD80) are diminished in blood leukocytes from animals clinically affected with Visna/Maedi virus (VMV) infection. This work investigates whether the use of B7 genes enhances immune responses and protection in immunization-challenge approaches. Sheep were primed by particle-mediated epidermal bombardment with VMV gag and env gene recombinant plasmids together with plasmids encoding both CD80 and CD86 or CD80 alone, boosted with gag and env gene recombinant modified vaccinia Ankara virus and challenged intratracheally with VMV. Immunization in the presence of one or both of the B7 genes resulted in CD4+ T cell activation and antibody production (before and after challenge, respectively), but only immunization with CD80 and CD86 genes together, and not CD80 alone, resulted in a reduced number of infected animals and increased early transient cytotoxic T lymphocytes (CTL) responses. Post-mortem analysis showed an immune activation of lymphoid tissue in challenge-target organs in those animals that had received B7 genes compared to unvaccinated animals. Thus, the inclusion of B7 genes helped to enhance early cellular responses and protection (diminished proportion of infected animals) against VMV infection.

Defining dose-response relationships in the therapeutic blockade of B7RP-1-dependent immune responses.

The ICOS (Inducible T cell Co-Stimulator)/B7RP-1 (B7-related protein 1) interaction is critical for the proper activation of a T lymphocyte. In this manuscript we describe a systematic in vivo approach to determine the level of blockade required to impair the generation of a T cell-dependent antibody response. We have developed an overall strategy for correlating drug exposure, target saturation, and efficacy in a biological response that can be generalized for most protein therapeutics. Using this strategy, we determined that low levels of B7RP-1 blockade are still sufficient to inhibit the immune response. These data suggest that contact between the T cell and the antigen-presenting cell during antigen presentation is much more sensitive to inhibition than previously believed and that ICOS/B7RP-1 blockade may be efficacious in the treatment of autoimmune diseases.

B7 blockade alters the balance between regulatory T cells and tumor-reactive T cells for immunotherapy of cancer.

PURPOSE: In prostate cancer-bearing host, regulatory T (Treg) cells restrain activity of tumor antigen-specific T cells. Because B7:CD28 interactions are needed for both function of CD4+CD25+ Treg cells and CD8+ effective T cells, targeting this pathway may help to overcome the immunotherapy barriers. EXPERIMENTAL DESIGN: The anti-B7-1/B7-2 monoclonal antibodies were administered to a transgenic mouse model of prostate cancer (TRAMP) ectopically expressing SV40 large T antigen in different tumor development stages for prevention and therapy of prostate cancer. The treatment was also tested in treating transplanted MC38 colon adenocarcinoma in mice. RESULTS: Here, we showed that short-term administration of anti-B7-1/B7-2 monoclonal antibodies in TRAMP mice leads to significant inhibited primary tumor growth and the size of metastatic lesions. The treatment is effective to inhibit MC38 colon cancer growth. Correspondingly, this treatment results in a transient reduction of Treg in both thymus and the periphery. In vivo cytotoxicity assay revealed T antigen-specific CTL effectors in anti-B7-treated but not control IgG-treated TRAMP mice. CONCLUSIONS: Transient blockade of B7-1/B7-2 alters the balance between Treg and cancer-reactive T cells to enhance cancer immunotherapy.

CD28 ligation in the absence of TCR promotes RelA/NF-kappaB recruitment and trans-activation of the HIV-1 LTR.

CD28 is one of the most important co-stimulatory receptors necessary for full T lymphocyte activation. CD28 can act as a TCR-independent signalling unit by delivering specific signals which may induce HIV transcription and replication. However, the mechanisms by which CD28 regulates HIV expression remain largely unknown. Here we show that the TCR-independent CD28 signals lead to the trans-activation of HIV-1 LTR in an NF-kappaB-dependent manner. In particular, we found that CD28 engagement by B7 induces the specific recruitment of RelA/NF-kappaB subunit to the HIV-1 LTR promoter both in vitro and in ex vivo infected cells. The results obtained by mutating specific tyrosine residues within the CD28 cytoplasmic tail as well as by using LY294002 inhibitory drug evidenced that the recruitment and activation of the phosphatidylinositol 3-kinase/Akt signalling pathway is crucial in mediating CD28-induced HIV transcription through RelA/NF-kappaB.

Expression of fusion IL2-B7.1(IgV+C) and effects on T lymphocytes.

The search for an effective immunotherapeutic treatment for tumors is an important area of cancer research. To prepare a more effective form of the bifunctional fusion protein IL2-B7.1(IgV+C) and analyze its effect on the stimulation of T lymphocyte proliferation, we used DNAStar 5.03 software to predict the structural diversity and biochemical character of IL2-B7.1(IgV+C). We then prepared fusion protein IL2-B7.1(IgV+C) by establishing its prokaryotic expression system, and tested its effect on the stimulation of T lymphocytes in vitro. The results indicated that IL2-B7.1(IgV+C) correctly formed a secondary structure in which both IL2 and B7.1(IgV+C) maintained their original hydrophilicity and epitopes. Western blot analysis revealed that IL2-B7.1(IgV+C) was efficiently expressed. Our analysis of CTLL-2 and T-cell proliferation showed that recombinant human (rh) IL2-B7.1(IgV+C) exerted the combined stimulating effects of both rhIL2 and rh B7.1(IgV+C) on cell proliferation, and that these effects could be blocked by adding either anti-IL2 or anti-B7.1 monoclonal antibodies. A >2-fold increase in [3H]TdR incorporation compared with that of cells treated with recombinant protein IL2, or B7.1(IgV+C) alone, revealed that rhIL2-B7.1(IgV+C) had dose-dependent synergetic effects on T-cell activation in the presence of anti-CD3 monoclonal antibody. We concluded that the augmented potency of rhIL2-B7.1(IgV+C) resulted in a stronger stimulation of T-cell proliferation than either rhB7.1(IgV+C) or rhIL2 alone.

Adenoviral B7-H3 therapy induces tumor specific immune responses and reduces secondary metastasis in a murine model of colon cancer.

Current cancer gene therapies aim at the induction of systemic antitumor immune responses. Tumors may deliver antigens to T-cells, but may lack the costimulatory signals necessary for mounting an effective response. The purpose of this study was to evaluate the efficacy of an adenoviral delivery of the B7-H3 costimulatory molecule in mice to induce antitumor immune responses. Colon cancers were established by orthotopic injection of syngeneic colon cancer cells into the cecum on Balb/c mice. After two weeks, these mice were treated by intratumoral injection of an adenovirus expressing mouse B7-H3 (Ad-B7-H3-GFP) or a control virus (Ad-GFP). Ad-B7-H3-GFP treatment resulted in a reduction of tumor size compared to the controls. In addition, the occurrence of secondary metastasis was significantly reduced in B7-H3 treated mice compared to control animals (lymph node 7/10 vs. 10/10; liver 2/10 vs. 8/10, p

IL-21 promotes differentiation of naive CD8 T cells to a unique effector phenotype.

IL-21, the most recently described member of the common gamma-chain cytokine family, is produced by activated CD4 T cells, whereas CD8 T cells express the IL-21 receptor. To investigate a possible role for IL-21 in the priming of naive CD8 T cells, we examined responses of highly purified naive OT-I CD8 T cells to artificial APCs displaying Ag and B7-1 on their surface. We found that IL-21 enhanced OT-I clonal expansion and supported development of cytotoxic effector function. High levels of IL-2 did not support development of effector functions, but IL-2 was required for optimal responses in the presence of IL-21. IL-12 and IFN-alpha have previously been shown to support naive CD8 T cell differentiation and acquisition of effector functions through a STAT4-dependent mechanism. Here, we show that IL-21 does not require STAT4 to stimulate development of cytolytic activity. Furthermore, IL-21 fails to induce IFN-gamma or IL-4 production and can partially block IL-12 induction of IFN-gamma production. CD8 T cells that differentiate in response to IL-21 have a distinct surface marker expression pattern and are characterized as CD44(high), PD-1(low), CD25(low), CD134(low), and CD137(low). Thus, IL-21 can provide a signal required by naive CD8 T cells to differentiate in response to Ag and costimulation, and the resulting effector cells represent a unique effector phenotype with highly effective cytolytic activity, but deficient capacity to secrete IFN-gamma.

Characterization of B7S3 as a novel negative regulator of T cells.

T cell activation by APCs is regulated by B7-like costimulatory molecules. In this study, we describe a new B7 superfamily member, B7S3, with two differentially spliced isoforms expressed in lymphoid and nonlymphoid tissues. A soluble B7S3-Ig protein bound to professional APC constitutively as well as to activated but not naive T cells. B7S3-Ig treatment greatly inhibited T cell proliferation and IL-2 production. B7S3-Ig also reduced cytokine production by effector T cells. Interestingly, although human genome appears to contain a single-copy B7S3 homolog, the mouse B7S3 gene has 10 relatives within a 2-Mb region constituting a B7S3 gene family. This study identifies B7S3 as a novel negative regulator of T cells, and suggests evolutionarily divergent T cell regulation mechanisms in mammals.

VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.

T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family-related protein V-set and Ig domain-containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell-dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues.

Induction of a gene expression program in dendritic cells with a cross-linking IgM antibody to the co-stimulatory molecule B7-DC.

While bivalent antibodies can block ligand-receptor interactions, IgM pentamers efficiently cross-link cell surface targets and evoke physiological responses. We have described one such interaction between an IgM antibody (Ab) and the B7-DC costimulatory molecule expressed by dendritic cells that induces strong antitumor immunity and modulates pathogenic responses associated with allergic asthma. Progressive changes in gene expression in dendritic cells activated by an IgM B7-DC cross-linking Ab resulted in the increased expression in 350 genes and decreased expression of more than 200 genes over the course of 24 h following Ab treatment. In particular, up-regulation of the caspase inhibitor FLIP and the chemokine receptor CCR7, and the down-regulation of the CXCR4 receptor provide a mechanistic basis of Ab-induced survival and enhanced migration into draining lymph nodes. Increased expression of both cell surface and secreted molecules known to be mediators of the immunomodulatory properties of dendritic cells was detected at both the levels of RNA and protein expression. This analysis documents the ability of IgM Ab to activate a gene expression cascade leading to important biological changes in cellular function and provides mechanistic insight into the potent immunomodulatory properties attributed to this Ab.

Recombinant human B7-H4 expressed in Escherichia coli inhibits T lymphocyte proliferation and IL-2 secretion in vitro.

AIM: To explore the biofunctions of human B7-H4 generated from prokaryotic system. METHODS: The gene of human B7-H4 extracellular region (IgV-like and IgC-like domains) was obtained by PCR from human cDNA FLJ22418 and then inserted into the prokaryotic expression vector pGEX-5X-3 expressing glutathione s-transferase (GST) fusion protein. After being identified by restriction enzyme digestion and sequencing, the recombinant vector was transferred into host strain E coli BL21-RIL(DE3). A 47 kDa fusion protein (GST/hB7-H4) was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by standard methods reported in the prokaryotic system. The inhibitory effect of GST/hB7-H4 on proliferation of T cells was observed in vitro by CD3mAb activated T-cell culturing system and [(3)H]-thymidine incorporation assay. The concentrations of interleukin-2 and iterferon-g in the supernatants of T cells were determined by ELISA. RESULTS: We successfully constructed the method for high-level expression and purification of the hB7-H4 extracellular domain as GST fusion protein from E coli. The GST/hB7-H4 fusion protein produced in bacteria had obvious biological activity to inhibit T-lymphocyte proliferation and IL-2 secretion. CONCLUSION: The prokaryote expression system could be used to generate hB7-H4 protein with natural spatial conformations and biological functions, which provided an efficient and economical way for the preparation of this target protein.