Jie Yang

Fudan University, Shanghai, Shanghai Shi, China

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Publications (20)30.4 Total impact

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    ABSTRACT: This study investigated the priming effect of sphingosine 1-phosphate (S1P) on formyl-Met-Leu-Phe-OH (fMLP)-activated neutrophils, by specific analysis of the neutrophil respiratory burst and the signaling pathway involved in S1P activity. The neutrophil respiratory burst was indirectly detected by the cytochrome c reduction method and the dihydrorhodamine 123 staining method. The signal transduction pathways of neutrophil respiratory burst primed by S1P were detected by Western blotting. Our results showed that the S1P receptors (S1PRs) 1, 4 and 5 were the predominantly expressed neutrophil S1PRs at the cDNA level. After pretreatment with S1P, the fMLP-activated neutrophils released increased levels of superoxide anions. PI3K and Akt proteins were involved in the signaling pathway of the neutrophil respiratory burst primed by S1P. The results indicate that S1P is a new priming reagent for neutrophils and primes the respiratory burst of fMLP-activated neutrophils. S1P interacts with its receptors on neutrophils, resulting in NADPH oxidase activation by the PI3K-Akt cell signaling pathway and induction of the neutrophil respiratory burst. © 2015 S. Karger AG, Basel.
    Acta Haematologica 04/2015; 134(1):49-56. DOI:10.1159/000369291 · 1.12 Impact Factor
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    Jie Yang · Yiming Yang · Huahua Fan · Hejian Zou ·
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    ABSTRACT: TGF-β-induced regulatory T cells (iTregs) retain Foxp3 expression and immune-suppressive activity in collagen-induced arthritis (CIA). However, the mechanisms whereby transferred iTregs suppress immune responses, particularly the interplay between iTregs and dendritic cells (DCs) in vivo, remain incompletely understood. In this study, we found that after treatment with iTregs, splenic CD11c(+)DCs, termed "DCiTreg," expressed tolerogenic phenotypes, secreted high levels of IL-10, TGF-β, and IDO, and showed potent immunosuppressive activity in vitro. After reinfusion with DCiTreg, marked antiarthritic activity improved clinical scores and histological end-points were observed. The serological levels of inflammatory cytokines and anti-CII antibodies were low and TGF-β production was high in the DCiTreg-treated group. DCiTreg also induced new iTregs in vivo. Moreover, the inhibitory activity of DCiTreg on CIA was lost following pretreatment with the inhibitor of indoleamine 2,3-dioxygenase (IDO). Collectively, these findings suggest that transferred iTregs could induce tolerogenic characteristics in splenic DCs and these cells could effectively dampen CIA in an IDO-dependent manner. Thus, the potential therapeutic effects of iTregs in CIA are likely maintained through the generation of tolerogenic DCs in vivo.
    Research Journal of Immunology 10/2014; 2014:831054. DOI:10.1155/2014/831054
  • Jie Sun · Yana Ren · Yiming Yang · Jie Yang · Rufeng Xie · Huahua Fan ·
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    ABSTRACT: Objective: To explore the biological characteristics and the immuno-suppression function of tolerogenic dendritic cells (tDC) induced by tacrolimus. Methods: Human monocytes derived from peripheral blood were cultured in the cGMP-compliant CellGro DC medium supplemented with GM-CSF and IL-4 to obtain dendritic cells (DCs), and 0.1 μmol/L immunosuppressive drug tacrolimus was added to the culture medium at the third and fifth day to obtain tDCs. The molecular markers of them and the livability were assayed by flow cytometry. Then the tolerance functionality of tDCs induced by many agents and these tDCs modulated allogeneic CD4 T cells was determined via CFSE proliferation assay. And the research also analyzed the biological characters and immunosuppression function of tDCs induced by tacrolimus after storing. Results: tDCs induced by tacrolimus exhibit a typical tolerogenic phenotype, whose level of costimulatory molecules CD80, CD83, CD86 and HLA-DR is (2.95 ± 1.32)%, (2.33 ± 1.60)%, (90.02 ± 7.42)% and (91.80 ± 6.18)%, respectively. It's survival rate was (85.2 ± 4.72)%. And immunosuppressive drugs didn't influence the differentiation of tDCs from monocytes. tDCs induced by immunosuppressive drugs dexamethasone, cyclosporin A and tacrolimus had lower immunogenic than control DCs as CD4+ T proliferation rate of tDCs induced by tacrolimus is 0.42% and could not primed allogeneic CD4+ T cells proliferation. Functional analyses showed that tDCs induced by tacrolimus can more effectively suppressed mature DC-induced T cell proliferation than other tDCs, whose inhibition rate can reach (67.01 ± 19.73)%. Importantly, tDCs induced by tacrolimus had phenotypical and functional stability after storing. Conclusion: tDCs induced by tacrolimus with tolerance functionality are a promising cellular therapeutic for immunomodulation.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 06/2014; 35(6):533-6. DOI:10.3760/cma.j.issn.0253-2727.2014.06.013
  • Yana Ren · Yiming Yang · Jie Yang · Rufeng Xie · Huahua Fan ·
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    ABSTRACT: Background: Tolerogenic dendritic cells (tDCs) can be generated in vitro by a variety of methods, including genetic or pharmacological modification. DCs that were modified by the immunosuppressive drug tacrolimus were considered to be endowed with tolerogenic functions. Study design and methods: DCs derived from human monocytes were induced in vitro by GM-CSF/IL-4 with tacrolimus. The phenotype and production of cytokines in these DCs were analyzed. The functionality of tDCs modified by tacrolimus was subsequently determined via a CFSE proliferation assay. The severity of arthritis was monitored in CIA mice after treatment with tDCs modified by tacrolimus. Results: tDCs that were modified by tacrolimus exhibited an immature phenotype. The expression of mRNA encoding IL-10 and TGF-β increased after 12h of tacrolimus stimulation, with the strongest responses being observed after 24h. The mRNA was further upregulated after tDCs were treated with LPS and IFN-γ. tDCs secreted more IL-10 and less TNF-α and had a reduced ability to activate allo-CD4(+)CD25(-) T cells. These cells suppressed mDC-induced-proliferation of CD4(+)CD25(-) T cells and produced less TNF-α and IFN-γ but increased the level of IL-10 than imDCs. Treatment of arthritic mice with tDCs modified by tacrolimus significantly inhibited the severity and progression of the disease. tDC treatment also altered the proportion of the Th1 and Th17 populations in the spleen. Conclusions: tDCs modified by tacrolimus suppressed CD4(+) T cell proliferation and inhibited collagen-induced arthritis. These results suggest the potential use of tDCs as a therapeutic approach for autoimmune arthritis.
    International Immunopharmacology 05/2014; 21(1). DOI:10.1016/j.intimp.2014.05.004 · 2.47 Impact Factor
  • Yiming Yang · Yan Yang · Jie Yang · Rufeng Xie · Yana Ren · Huahua Fan ·
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    ABSTRACT: Objectives: To determine the effect of nicotine stimulation on collagen-induced arthritis (CIA), especially on Th17 cells, and the influence of activated acetylcholine receptor signaling on the induction and function of in vitro-cultured Th17 cells. Methods: Mice were divided into control and experimental (nicotine) group, and PBS or nicotine-PBS was orally administered from Day 21 to Day 28. Phenotypic changes in spleen CD4(+) cells were measured by flow cytometry. α7nAChR expression in Th17 cells was detected using flow cytometry, western blotting and real-time PCR. Purified Th17 cells were further stimulated with nicotine. The cytometric bead array (CBA) assay was employed to measure TNF-α levels in mice serum and IL-17A levels in the supernatants of nicotine-treated cell cultures. Results: Compared with their counterparts, mice receiving oral nicotine showed a delayed progress of arthritis and more attenuated signs of histological changes. Moreover, serum TNFα levels were lower in the nicotine-treated group. Spleen IL-17 level of nicotine-treated mice was lower than that of the control group, and the mRNA expression of pro-inflammatory cytokines (IL-17A and IL-6) in splenocytes were also lower than that of the control group. α7nAChR expression was detected on in vitro-cultured IL-17A(+) cells. Cells treated with 10 (- 6) M nicotine expressed lower IL-17A levels. Similarly, supernatants from nicotine-treated cell cultures also showed lower IL-17A levels. Conclusions: Nicotine stimulation attenuated signs and severity of arthritis in mice. Activation of nicotine acetylcholine receptors on in vitro-cultured Th17 cells decreased their pro-inflammatory function, which may play a potential role in alleviating arthritis in mice.
    Modern Rheumatology 12/2013; 24(5). DOI:10.3109/14397595.2013.862352 · 2.40 Impact Factor
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    Jie Yang · Yiming Yang · Yana Ren · Rufeng Xie · Hejian Zou · Huahua Fan ·
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    ABSTRACT: Tolerogenic dendritic cells (tDCs) are immunosuppressive cells with potent tolerogenic ability and are promising immunotherapeutic tools for treating rheumatoid arthritis (RA). However, it is currently unknown whether allogeneic tDCs (allo-tDCs) induce tolerance in RA, and whether the numbers of adoptively transferred allo-tDCs, or the requirement for pulsing with relevant auto-antigens are important. tDCs were derived from bone marrow precursors of C57BL/B6 mice, which were induced in vitro by GM-CSF, IL-10 and TGF-β1. Collagen-induced arthritis (CIA) was modeled in D1 mice by immunization with type II collagen (CII) to test the therapeutic ability of allo-tDCs against CIA. Clinical and histopathologic scores, arthritic incidence, cytokine and anti-CII antibody secretion, and CD4(+)Th subsets were analyzed. tDCs were characterized in vitro by a stable immature phonotype and a potent immunosuppressive ability. Following adoptive transfer of low doses (5×10(5)) of CII-loaded allo-tDCs, a remarkable anti-arthritic activity, improved clinical scores and histological end-points were found. Serological levels of inflammatory cytokines and anti-CII antibodies were also significantly lower in CIA mice treated with CII-pulsed allo-tDCs as compared with allo-tDCs. Moreover, treatment with allo-tDCs altered the proportion of Treg/Th17 cells. These findings suggested that allo-tDCs, especially following antigen loading, reduced the severity of CIA in a dose-dependent manner. The dampening of CIA was associated with modulated cytokine secretion, Treg/Th17 polarization and inhibition of anti-CII secretion. This study highlights the potential therapeutic utility of allo-tDCs in autoimmune arthritis and should facilitate the future design of allo-tDC immunotherapeutic strategies against RA.
    PLoS ONE 10/2013; 8(10):e77729. DOI:10.1371/journal.pone.0077729 · 3.23 Impact Factor
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    ABSTRACT: The aim of this study was to examine the priming effect of sphingosine 1-phosphate (S1P) on fMLP-activated neutrophils, mainly to detect the neutrophil respiratory burst products, and to investigate the signaling pathway involved in S1P activity. Flow cytometry was used to evaluate the new isolated neutrophil; the superoxide anion output was detected indirectly by cytochrome C reduction in respiratory burst; the dihydro-rhodamine 123 was used to detect the intensity of respiratory burst; the signal transduction pathways of neutrophil respiratory burst were explored by Western blot. The results showed that after pretreated with S1P, the level of superoxide anion released by fMLP-activated neutrophils significantly increased; the Rhodamine123 mean fluorescence intensity in S1P primed fMLP-activated neutrophils group was significantly higher than that in fMLP treatment group; PI3K and Akt proteins involved in the signal pathway of neutrophil respiratory burst. It is concluded that S1P is a new priming reagent, which primes respiratory burst of fMLP-activated neutrophils; this signal pathway may be that S1P interacts with its receptor, activates PI3K, then activates Akt-transmiting signals through NADPH oxidase, finally results in the respiratory burst.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2012; 20(4):989-94.
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    ABSTRACT: To explore the biological characteristic of third-party-derived tolerogenic DC(tDC) and the influence of third-party-derived tDC on acute graft-versus-host-disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) in mice. tDC from bone marrow cells of D1 mice was cultured with low doses of GM-CSF, IL-10 and TGF-β1D1. The phenotype, expression of cytokines and function associated molecules were identified with FACS and RT-PCR. Mixed lymphocyte reaction was applied to analyze the influence of third-party-derived tDC on allo-CD4(+)T cells proliferation in vitro. Different doses of D1-tDC were adoptive transferred in the aGVHD model in allogeneic BMT which B6 mice as donors and D2 mice as recipients. Survival time, clinical GVHD score and the levels of Th1/2 cytokines in serum were monitored after allo-BMT using the aGVHD model as control. tDC expressed lower levels of MHC II and co-stimulatory molecules, such as CD80, CD86 and CD40, even when stimulated by LPS. The results by RT-PCR indicated that tDC expressed low levels of IL-12p40 and high levels of immunosuppressive molecules, such as IL-10, TGF-β, Fas Ligand, indoleamine 2, 3-dioxygenase (IDO) and arginase. In the allogeneic MLR, third-party tDC suppressed allo-CD4(+)T cells proliferation, which was relative to the dose of tDC. In the B6→D2 mouse model, all aGVHD mice died within 18 days. Remarkably, if 10(4) third-party tDC were transferred, 60% mice survived at least 60 days. When the doses of tDC were reduced to 10(3) cells, only 20% of mice survived day 60, and when increased tDC to 10(5), all of the mice died within day 37 after allo-BMT. The cytokine levels in serum indicated that 10(4) tDC-treated mice secreted in vivo high level of IL-10 21d after BMT (P < 0.05), the levels of IL-10 in 10(3), 10(4) and 10(5) tDC-treated mice were (114.23 ± 7.78), (646.18 ± 212.02), (121.97 ± 10.47) ng/L, respectively. Third-party tDC could suppress allo-CD4(+)T cells proliferation in vitro and prevent aGVHD in allogeneic BMT mode, which may be mediated by modulating tolerogenic cytokines secretion, such as IL-10. And this effect was associated with the dose of tDC. Adoptive therapy by transfusing third-party tDC cultured with low doses of GM-CSF, IL-10 and TGF-β1 could significantly prolong the survival of recipients and prevent aGVHD in allogeneic BMT.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 06/2012; 33(6):461-6.
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    ABSTRACT: In this study, we expanded regulatory T cells (Tregs) ex vivo from CD4(+) CD25(+) T cells from cord blood (CB) and CD4(+) CD25(+) CD127(-) T cells from adult peripheral blood (APB) and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. Cell yield after Treg expansion with polyclonal stimulation was greater than that of Tregs expanded with combined alloantigen and polyclonal stimulation. The expanded Tregs expressed high levels of Foxp3, CD39 and cytotoxic T-lymphocyte antigen-4 and low levels of CD127, interleukin-2 and interferon-γ. After two cycles of expansion, the CB Tregs maintained expression of the GARP gene and showed greater suppressive function than APB Tregs. The CB Tregs that were expanded with two cycles of polyclonal stimulation suppressed not only the polyclonal antigen-driven responder T (T(resp)) cell proliferation but also the HLA mismatched dendritic cell-driven T(resp) cell proliferation. When CB and APB Tregs were expanded with a primary alloantigen stimulus followed by a secondary polyclonal stimulus, the Tregs showed a potent, antigen-specific suppressive capacity. The Tregs expanded with two cycles of polyclonal stimulation from both CB and APB alleviated acute graft-versus-host disease symptoms and prolonged survival in a murine model of graft-versus-host disease. In conclusion, CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs. It is feasible to obtain human functional alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers.
    Immunology 02/2012; 136(2):218-30. DOI:10.1111/j.1365-2567.2012.03573.x · 3.80 Impact Factor
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    ABSTRACT: Human cord blood (CB) is a superior source of regulatory T cells (Tregs) compared with peripheral blood. Initial studies have shown that CB-derived Tregs can be effectively expanded ex vivo. However, in vitro suppressor activity of expanded CB-Tregs and their efficacy in the prevention of acute graft-versus-host disease (aGVHD) in vivo are poorly understood. In vitro, human CB CD4+CD25+ T cells expanded with anti-CD3/CD28 beads plus interleukin (IL)-2 and the phenotypes, expression of cytokines, and suppression of expanded cells were analyzed after two cycles of stimulation. In vivo, the addition of human CB-Tregs was transferred in the major histocompatibility complex-mismatched aGVHD mouse model. Survival, body weight, GVHD scoring, histopathologic specimens, serum cytokines, and Th subsets were analyzed in CB-Treg-treated mice and untreated controls. After being expanded ex vivo, human CB-derived Tregs with potent suppressor function could meet clinical demands. Up to 85% of mice with CB-Tregs treatment survived beyond Day 63 after bone marrow transplantation; however, all aGVHD mice died within 18 days. In the serum of the CB-Treg-treated mice, the production of transforming growth factor-β increased continuously, as opposed to IL-17, which decreased quickly. Consistent with the changes of cytokines, the percentage of mouse CD4+ forkhead box protein 3+ Tregs increased while that of Th17 cells decreased. Ex vivo expanded human CB-Tregs significantly prevented allogeneic aGVHD in vivo by modulating various cytokine secretion and polarizing the Treg/Th17 balance toward Treg, which suggests the potential use of expanded CB-Tregs as a therapeutic approach for GVHD.
    Transfusion 11/2011; 52(6):1333-47. DOI:10.1111/j.1537-2995.2011.03448.x · 3.23 Impact Factor
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    ABSTRACT: To improve its antitumor effect, we used human leukocyte antigen -A2 (HLA-A2)-positive human dendritic cell (DC)-derived DEXs (DC-derived exosomes) to support NY-ESO-1 antigen and polyI:C, with the aim of increasing the proliferation of specific cytotoxic T lymphocytes (CTL) in transgenic mice. Mature dendritic cells derived from peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adults with positive HLA-2A. Using centrifuge and membrane ultrafiltration, EXO (exosomes) were extracted from the supernatant of DCs secretions. Transgenic C57 mice were immunized with human-derived tumor testis antigen NY-ESO-1/EXO, with or without polyI:C. Mice were sacrificed four weeks after immunization, and spleen cells were isolated and tested for function. The experiments included antigen-specific CTL proliferation, as tested by dimerization and antitumor effects for K562 cells as well as melanoma, tested at different ratios of effected cells:target cells (0:1, 10:1, 50:1, and 100:1). Dimerization experiments indicated that the effect of DEX/TSA (tumor specific antigens) + PolyI:C was 2.36 ± 1.10% and the control was 0.38 ± 0.31%, while the effect of DEX/TSA was 1.97 ± 0.63% and the control was 0.36 ± 0.07%. Antitumor effects by DEX/TSA: PolyI:C for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 11.14 ± 1.36%, 14.17 ± 0.62%, 15.71 ± 2.48%, and 24.31 ± 2.91%, respectively, for K562 cells. The antitumor effects for DEX/TSA for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 12.23 ± 2.25%, 13.10 ± 1.57%, 15.27 ± 2.93%, and 19.87 ± 2.72%, respectively, for K562 cells. With ratios of 10:1 and 100:1, the antitumor effects of DEX/TSA + PolyI:C were better than for the DEX/TSA group (P < 0.05). However, higher ratios of effecter cells to target cells increased, and there were no significant improvements in antitumor effect for control cells. Combining PolyI:C with DEX/TSA derived from healthy human blood positive for HLA-A2 is a promising strategy for developing new subcellular antitumor vaccination.
    Journal of Immunoassay and Immunochemistry 07/2011; 32(3):207-18. DOI:10.1080/15321819.2011.559295 · 0.73 Impact Factor
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    ABSTRACT: To study the influence of human plasma exosomes-like vesicles on the regulatory function of macrophages. The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugation and ultrafiltration. Macrophages were derived from cultured human blood monocytes. The molecular markers of macrophages were assayed by FACS. After cultured with exosomes-like vesicles, the changes of macrophages cytoplasma Ca(2+), and related genes and proteins were assayed by FACS, RT-PCR and Western Blot, respectively. After cultured with exosomes-like vesicles, mean fluorescent intensity (MFI) of macrophages cytoplasma Ca(2+) was increased. The vesicles enhanced macrophages to express cytokines genes, the expression of IL-1β and TNF-α genes being increased by 0.85 and 1.69 times respectively at 2 h, and that of IL-6 gene 3.7 times compared with the control at 8 h. However, the vesicles inhibited the expression of macrophages IL-10 gene, had no influence on the Frizzled5 receptor expression and could induce CaMKII phosphorylation. Exosomes-like vesicles can up-regulat macrophages expression of inflammatory cytokines genes, and increase the secretion of inflammatory cytokines by activating the Wnt5A-Ca(2+) signaling pathway.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 06/2011; 32(6):404-7.
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    ABSTRACT: Mesenchymal stem cells (MSCs) hold great promise for treating immune disorders owing to their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of human umbilical cord mesenchymal stem cell (HUCMSC)-mediated immunosuppression involves TGF-β and indoleamine 2,3-dioxygenase (IDO). In this study, we investigated the influence of xenogeneic HUCMSCs on acute graft-versus-host disease (aGVHD) in murine allogeneic bone marrow transplantation (BMT). In the HUCMSC-treated group, lethally irradiated DBA/2(H-2Kd) mice were adoptively transferred with expanded HUCMSCs, bone marrow (BM), and splenocytes (SCs) from C57BL/6 (H-2Kb) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and aGVHD scoring. In the HUCMSC-treated group, 60% of the mice survived past day 30 after BMT, but in the control group, all mice died within 18 d. The mice treated with HUCMSCs exhibited light symptoms of aGVHD after day 30. The results suggest that xenogeneic HUCMSCs could alleviate aGVHD symptoms and prolong survival after allogeneic BMT. Our study suggests that in vitro expanded HUCMSCs might be used to inhibit severe aGVHD effectively in allogeneic hematopoietic cell transplantation clinically.
    European Journal Of Haematology 04/2011; 87(3):235-43. DOI:10.1111/j.1600-0609.2011.01635.x · 2.07 Impact Factor
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    ABSTRACT: Exosomes are small membrane vesicles that are secreted from many cell types into various body fluids. These vesicles are thought to play a role in cell-cell interactions. Vesicles were isolated from human plasma of healthy donors by differential ultracentrifugation and ultrafiltration. The vesicles were identified by transmission electron microscopy, and their biochemical characteristics were analyzed by Western blot and flow cytometry. The immune-modulatory ability of exosomal-like vesicles was examined by incubating them with CD4+ T cells for CD4+ T-cell proliferation and apoptosis assays in vitro. Vesicles purified from human plasma displayed shapes and sizes similar to those of previously described exosomes and contained exosomes marker proteins CD63 and CD81. They also expressed molecules such as MHC Class II molecules, CD80, CD86, and the cell signal transduction molecules Wnt3a, Wnt5a, and FasL. Furthermore, functional analysis showed that allogeneic plasma exosomes restrained the survival of CD4+ T cells. Plasma exosomes can induce dose-dependent suppression of proliferation of activated CD4+ T cells, with the strongest responses induced by 500 µg/mL exosomes in vitro. Antibodies against exosomes FasL can block the activity of exosomes on CD4+ T-cell apoptosis. Moreover, three different concentrations of CD4+ T cells were inhibited by plasma exosomes and the suppressive function was not dependent on interleukin-2. Exosomes present in human plasma contain immunity-associated molecules and can induce CD4+ T-cell apoptosis in vitro. Plasma exosomes have the capacity to influence immune responses.
    Transfusion 11/2010; 51(5):1002-11. DOI:10.1111/j.1537-2995.2010.02909.x · 3.23 Impact Factor
  • Yue-E Ma · Ya-Na Ren · Jie Yang · Dan Li · Chen-Zhi Guo · Juan Guo · Bing Ni · Hua-Hua Fan ·
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    ABSTRACT: OBJECTIVE: To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect. METHODS: The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration. Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry. CD4(+)T cells and CD4(+)CD25(+)CD127low Treg cells were purified from peripheral blood mononuclear cells (PBMCs) by Magnetic cell sorting. After exosomes-like vesicles cultured with CD4(+)T cells or CD4(+)CD25(+)CD127low Treg cells, cell proliferation and apoptosis were assayed. Phosphorylated β-catenin level in Wnt signaling by phosflow. RESULTS: Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-II molecule, costimulatory molecules CD86 etc. After co-cultured with CD4(+) T cells, exosomes-like vesicles inhibited the proliferation of the T cells in a dose-dependent manner. After Treg cells cultured with exosomes-like vesicles for 14 days, the survival rate of the Treg cells was 57.07%, while that of the control Treg was 30.91%. Frizzled receptors 2, 3, 4and LRP6 gene mRNA expressed (the relative gray value was 48.50, 34.84, 23.85, 49.73) in the Treg cells by RT-PCR, and Wnt molecular expressed in exosomes-like vesicles. After Treg cells co-cultured with exosomes-like vesicles, the MFI of phosphorylated β-catenin decreased (from 20.06 ± 2.99 to 12.41 ± 2.08), and the expression of Bcl-2 mRNA was upregulated significantly (the relative gray value from 0.45 to 84.97). CONCLUSIONS: Exosomes-like vesicles existed in human plasma and express immune regulatory molecules. They can suppress the proliferation of activated CD4(+) T cells induce their apoptosis and pro-long the survival of natural Treg cells via Wnt signaling pathway.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 06/2010; 31(6):376-380.
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    ABSTRACT: LPS is an immunostimulatory component of Gram-negative bacteria. Acting on the immune system in a systemic fashion, LPS exposes the body to the hazard of septic shock. In this study we report that cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2/Crispld2; human and mouse/rat versions, respectively), expressed by multitissues and leukocytes, is a novel LPS-binding protein. As a serum protein, median CRISPLD2 concentrations in health volunteers and umbilical cord blood samples are 607 microg/ml and 290 microg/ml, respectively. Human peripheral blood granulocytes and mononuclear cells including monocytes, NK cells, and T cells spontaneously release CRISPLD2 (range, 0.2-0.9 microg/ml) and enhance CRISPLD2 secretion (range, 1.5-4.2 microg/ml) in response to stimulation of both LPS and humanized anti-human TLR4-IgA Ab in vitro. CRISPLD2 exhibits significant LPS binding affinity similar to that of soluble CD14, prevents LPS binding to target cells, reduces LPS-induced TNF-alpha and IL-6 production, and protects mice against endotoxin shock. In in vivo experiments, serum Crispld2 concentrations increased in response to a nontoxic dose of LPS and correlated negatively with LPS lethality, suggesting that CRISPLD2 serum concentrations not only are indicators of the degree of a body's exposure to LPS but also reflect an individual's LPS sensitivity.
    The Journal of Immunology 11/2009; 183(10):6646-56. DOI:10.4049/jimmunol.0802348 · 4.92 Impact Factor
  • Jie Yang · Li Gao · Yan Liu · Yana Ren · Rufeng Xie · Huahua Fan · Kaicheng Qian ·
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    ABSTRACT: Invariant natural killer T cells (iNKT cells) may suppress graft-versus-host disease (GVHD) after allogeneic transplantation. The purpose of this study was to investigate the therapeutic potential of iNKT cells from major histocompatibility complex (MHC)-mismatched donors for preventing GVHD after allogeneic bone marrow transplantation (BMT). In vitro, mouse iNKT cells were expanded with alpha-galactosylceramide and interleukin (IL)-2 treatment. In the NKT-treated group, lethally irradiated DBA/2(H-2K(d)) mice were adoptively transferred with expanded iNKT, bone marrow (BM), and spleen cells (SCs) from C57BL/6 (H-2K(b)) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and serum cytokine analysis. In the iNKT-treated group, 80% of mice survived past Day 60 after BMT, but all died within 38 days in the control group. The mice treated with iNKT did not exhibit signs of GVHD after Day 42 except for a change in fur color. There were higher IL-4 levels by Day 7 in serum of mice that received iNKT compared to those without iNKT treatment, while the interferon-gamma levels showed no significant difference between two groups. Levels of IL-2 and IL-5 increased by Day 21 only in iNKT-treated mice. The results suggest that donor iNKT cells could alleviate GVHD symptoms and prolong survival after MHC-mismatched allogeneic BMT, which may be associated with the maintenance in IL-4 levels. These findings indicate that the therapy based on iNKT cells from MHC-mismatched donors has great potential in protection against GVHD after allogeneic hematopoietic stem cell transplantation.
    Transfusion 09/2009; 50(2):407-17. DOI:10.1111/j.1537-2995.2009.02395.x · 3.23 Impact Factor
  • Ya-Na Ren · Hua-Hua Fan · Xiao-Xuan Nie · Li Gao · Jie Yang · Yan Liu · Feng Gao ·
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    ABSTRACT: To confirm the mechanism of exosomes as tumor vaccines inducing immunity response, dendritic cells (DCs) were induced from human peripheral blood mononuclear cells, while exosomes were isolated from DC loaded tumor antigen. The effect of exosomes on priming T cell proliferation was analysed under conditions with or without DCs, or DCs at different mature stages. The function of exosomes in immunity was detected through block test after blocking some molecules (CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83). The effect of DCs on embedded exosomes was observed by confocal microscopy, the effect of blocking surface molecules on exosomes on DC-embedding exosomes was assayed by flow cytometry. The results indicated that both exosomes derived from imDC (imDex) and exosomes derived from mDC (mDex) could not prime T cells without DC or with imDC. The exosomes derived from mDC induced with different cytokines (LPS, TNF-alpha, CpG, CD40L) were no significant difference in concentrations but were different in effect. The immunity function of exosomes depended on CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83 molecules, the effect of priming T cells is reduced when these molecules were blocked. Confocal microscopy and FACS assay showed that blocking CD11a and CD54 could inhibit exosome-targeted DC and DC-embedded exosomes. It is concluded that the exosomes target DCs through their surface molecules, therefore results in immune response of T cells.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2008; 16(3):650-4.
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    ABSTRACT: The study was aimed to explore the roles of exosomes derived from regulatory dendritic cells of mice in the induction of immune tolerance. Immature DC (iDC) from mouse bone marrow cells and regulatory DCs (rDC) were induced by treating iDC with TGF-beta1 and IL-10. The phenotype of regulatory DCs and normal DCs were assayed by flow cytometry. Exosomes from immature DCs (iDex) and regulatory DCs (rDex) were isolated by ultracentrifugation and ultrafiltration. A skin transplantation model was established with the recipients BALB/c mice and the donor C57BL/6 mice. Recipients were divided into PBS control group, iDex group (injection 10 microg iDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation), rDex group (injection 10 microg rDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation). The capacity of the donor mice and the unrelated allogeneic donor mice to stimulate allogeneic T lymphocyte proliferation was examined by mixed lymphocyte culture (MLR). The results showed that TGF-beta1 and IL-10 could down-regulate the expressions of costimulatory molecules, including CD80, CD86 and CD40. The graft mean survival time (MST) in control group, iDex group and rDex group was 7.8, 10.7 and 18.8 days, respectively. There was significant difference in MST between iDex group and control group (p<0.05), and between rDex group and iDex group (p<0.01). The results of MLR assays indicated donor-specific hyporeactivity especially in rDex group, while the tolerant B/C mice were still immunocompetent to unrelated allogeneic DBA mouse. It is concluded that injection iDex or rDex of donor mice via tail vein before skin transplantation induces immunotolerance, and the effect of rDex is more significant.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 04/2008; 16(2):406-10.
  • Ya-na Ren · Hua-hua Fan · Li Gao · Xiao-xuan Nie · Jie Yang · Yan Liu · Feng Gao ·
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    ABSTRACT: To establish a method for isolating exosomes from dendritic cells (DC), and to analyse its biological characteristics and function in antitumor immunity. Immature DCs (im-DC) from human peripheral blood mononuclear cells were loaded with the antigen of K562 tumor cells, then exosomes were secreted from imDC and lipopolysaccharide (LPS) induced mature DC (mDC). The exosomes from imDC and mDC were isolated separately by ultracentrifugation and ultrafiltration. The exosomes diameter was determined, their profile was observed by electron microscope, and the surface molecules were detected by Western blot. To analyse the effect of exosomes on antitumor immunity, the proliferation, IFN-gamma expression, CD69 up-regulation and cytotoxicity of antigen-specific T cells were measured. Exosomes were small flattened sphere vesicles with an average diameter of 72.3 nm and expressed CD80, CD86, HLA-DR, FasL, CD54 and MFG-E8 molecules. As compared to immature exosomes, exosomes from mDC were proved to express more CD80 and less MFG-E8, to be more potent for inducing antigen-specific T cells proliferation and immunity respond in vitro: at its optimum concentration, the absorption value of T cell proliferation test was 0.50 +/- 0.01, CD69 was up-regulated and (13.4 +/- 5.8)% of T cells was in proliferating, (22.8 +/-2.4)% of T cells expressed IFN-gamma, and (21.3 +/-8.6)% of tumor cells were killed. A simple and quick method to isolate and analyse exosomes is established. The exosomes can induce antitumor immunity respond.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 06/2007; 28(5):298-302.

Publication Stats

90 Citations
30.40 Total Impact Points


  • 2010-2014
    • Fudan University
      Shanghai, Shanghai Shi, China
  • 2011-2012
    • East China Normal University
      • Institute of Biomedical Sciences and School of Life Sciences
      Shanghai, Shanghai Shi, China