[The role of third-party tolerogenic dendritic cells in the prevention of acute graft-versus-host-disease following allogeneic bone marrow transplantation in mice].

OBJECTIVE: 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. METHODS: tDC from bone marrow cells of D1 mice was cultured with low doses of GM-CSF, IL-10 and TGF-ß1D1. 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. RESULTS: 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-ß, 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. CONCLUSION: 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-ß1 could significantly prolong the survival of recipients and prevent aGVHD in allogeneic BMT.

[Effect of sphingosine 1-phosphate/sphingosine 1-phosphate receptor signal pathway on function of neutrophils].

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 Rhodamine 123 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-transmitting signals through NADPH oxidase, finally results in the respiratory burst.

[A preliminary study on the influence of human plasma exosomes-like vesicles on macrophage Wnt5A-Ca²+ pathway].

OBJECTIVE: To study the influence of human plasma exosomes-like vesicles on the regulatory function of macrophages. METHODS: 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. RESULTS: 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-1ß 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. CONCLUSIONS: 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.

A combination of exosomes carrying TSA derived from HLA-A2-positive human white buffy coat and polyI:C for use as a subcellular antitumor vaccination.

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.

[A proliminary study on the regulatory function of human plasma exosomes-like vesicles].

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 ß-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 ß-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.

The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function.

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.

[Immunity mechanism of exosomes derived from dendritic cells].

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.

[Immune tolerance induced by exosomes derived from regulatory dendritic cells of mice].

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.

[A preliminary study on the biological characteristics and function of exosomes derived from dendritic cells].

OBJECTIVE: To establish a method for isolating exosomes from dendritic cells (DC), and to analyse its biological characteristics and function in antitumor immunity. METHODS: 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. RESULTS: 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. CONCLUSION: A simple and quick method to isolate and analyse exosomes is established. The exosomes can induce antitumor immunity respond.