Allogeneic Lymphocyte Proliferation Research Articles

Structural characterization and immune modulation activities of Chinese Angelica polysaccharide (CAP) and selenizing CAP (sCAP) on dendritic cells

sCAP was obtained by the nitrate‑sodium selenite method. SEM, molecular weight evaluation, monosaccharide composition, FT-IR and NMR of sCAP were carried out. Compared with CAP, sCAP had a relatively smooth and lamellar sheet morphology with edge folds on the surface, presented molecular weights in range of 0.90–97.08 KDa, and was mainly composed of GalA, Ara and Gal. sCAP had both α and β configurations of the pyranose ring, the characteristic vibrational peak of Se-O-C and the signal of galacturonic acid residue. The phagocytic activity of immature BMDCs, the expression of CD40, CD80, CD86, and MHCII on BMDCs were detected by flow cytometry, the ability of sCAP-treated BMDCs to stimulate the proliferation of allogeneic lymphocytes, presentation of antigens, cytokines in the supernatants and the protein in MyD88/NF-κB signaling pathway were detected. The results showed that the phagocytic activity of immature BMDCs was significantly enhanced when sCAP was at 3.92–1.96 μg·mL−1. The levels of IL-6, TGF-β1, INF-γ, and TNF-α were significantly elevated, IL-1β and MIP-1α were significantly reduced. These results indicate that sCAP could be as a new immunopotentiator by increasing MyD88/NF-κB signaling pathway. This study provides a reference for the research and development of new dosage forms of polysaccharide.

Could Increased Expression of Hsp27, an "Anti-Inflammatory" Chaperone, Contribute to the Monocyte-Derived Dendritic Cell Bias towards Tolerance Induction in Breast Cancer Patients?

Dendritic cells (DCs) are the most efficient antigen-presenting cells and link the innate immune sensing of the environment to the initiation of adaptive immune responses, which may be directed to either acceptance or elimination of the recognized antigen. In cancer patients, though DCs would be expected to present tumor antigens to T lymphocytes and induce tumor-eliminating responses, this is frequently not the case. The complex tumor microenvironment subverts the immune response, blocks some effector mechanisms, and drives others to support tumor growth. Chronic inflammation in a tumor microenvironment is believed to contribute to the induction of such regulatory/tolerogenic response. Among the various mediators of the modulatory switch in chronic inflammation is the “antidanger signal” chaperone, heat shock protein 27 (Hsp27), that has been described, interestingly, to be associated with cell migration and drug resistance of breast cancer cells. Thus, here, we investigated the expression of Hsp27 during the differentiation of monocyte-derived DCs (Mo-DCs) from healthy donors and breast cancer patients and evaluated their surface phenotype, cytokine secretion pattern, and lymphostimulatory activity. Surface phenotype and lymphocyte proliferation were evaluated by flow cytometry, interferon- (IFN-) γ, and interleukin- (IL-) 10 secretion, by ELISA and Hsp27 expression, by quantitative polymerase chain reaction (qPCR). Mo-DCs from cancer patients presented decreased expression of DC maturation markers, decreased ability to induce allogeneic lymphocyte proliferation, and increased IL-10 secretion. In coculture with breast cancer cell lines, healthy donors' Mo-DCs showed phenotype changes similar to those found in patients' cells. Interestingly, patients' monocytes expressed less GM-CSF and IL-4 receptors than healthy donors' monocytes and Hsp27 expression was significantly higher in patients' Mo-DCs (and in tumor samples). Both phenomena could contribute to the phenotypic bias of breast cancer patients' Mo-DCs and might prove potential targets for the development of new immunotherapeutic approaches for breast cancer.

The immunoregulatory effect of sulfated Echinacea purpurea polysaccharide on chicken bone marrow-derived dendritic cells

Echinacea purpurea polysaccharide (EPP) was obtained by modern separation technology and sulfated EPP (sEPP) was prepared by sulfation modification. The immunological effects of EPP and sEPP were compared on chicken bone marrow-derived dendritic cells (chBM-DCs). The results showed that the surface marker expression of CD11c and CD80 was increased after chBM-DCs were cultured with three dosage of sEPP, especially in sEPPM group. Three dosage of sEPP, EPPL and LPS could significantly enhance the effects of chBM-DCs on the proliferation of allogenic mixed lymphocytes. After chBM-DCs treatment with EPP or sEPP in vitro, the levels of IL-2 of sEPPH and EPPM groups were significantly higher than those of LPS group (P < 0.05). All sEPP and EPP groups could enhance the level of IFN-γ and down-regulated the level of IL-4 and IL-10. Results indicated that both sEPP and EPP had immunoregulatory effects on chBM-DCs, sEPP possessed better immunoregulatory effects as compared with EPP.

MAP7 and MUCL1 Are Biomarkers of Vitamin D3-Induced Tolerogenic Dendritic Cells in Multiple Sclerosis Patients.

The administration of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases, such as multiple sclerosis (MS). Specifically, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most widely studied approaches, as it has evidenced significant immune regulatory properties, both in vitro and in vivo. In this article, we generated human vitD3-tolDC from monocytes from healthy donors and MS patients, characterized in both cases by a semi-mature phenotype, secretion of IL-10 and inhibition of allogeneic lymphocyte proliferation. Additionally, we studied their transcriptomic profile and selected a number of differentially expressed genes compared to control mature and immature dendritic cells for their analysis. Among them, qPCR results validated CYP24A1, MAP7 and MUCL1 genes as biomarkers of vitD3-tolDC in both healthy donors and MS patients. Furthermore, we constructed a network of protein interactions based on the literature, which manifested that MAP7 and MUCL1 genes are both closely connected between them and involved in immune-related functions. In conclusion, this study evidences that MAP7 and MUCL1 constitute robust and potentially functional biomarkers of the generation of vitD3-tolDC, opening the window for their use as quality controls in clinical trials for MS.

Intrasplenic Transplantation of Cytotoxic T-Lymphocyte Associated Protein 4-Fas Ligand--Modified Hepatic Oval Cells for Acute Liver Injury in Rats

BackgroundIntrasplenic transplantation of xenogeneic hepatic oval cells (HOCs) may provide metabolic support for acute liver injury. However, xenoreactive lymphocyte-mediated immune response hinders HOCs' survival in the xeno-spleen parenchyma. Cytotoxic T-lymphocyte associated protein 4-Fas ligand (CTLA4.FasL), a fusion product integrating 2 inhibitory elements against lymphocytes into 1 molecule, effectively inhibited the proliferation of allogeneic and autoimmune lymphocytes. The purpose of this study was to explore the effect of CTLA4.FasL on the proliferation of xenoreactive lymphocytes and evaluate the therapeutic efficacy of CTLA4.FasL-modified HOC transplantation on acute liver injury in rats. MethodsThe effect of CTLA4.FasL-modified mouse liver epithelial progenitor cells (CTLA4.FasL-LEPCs) on the proliferation of rat lymphocytes in xeno-mixed lymphocyte reaction was investigated. Furthermore, CTLA4.FasL-LEPCs were intrasplenically transplanted in carbon tetrachloride- and partial hepatectomy-treated rats, and the therapeutic effect was evaluated using hematoxylin and eosin staining and alanine aminotransferase and aspartate aminotransferase assays. The hepatocytic differentiation of CTLA4.FasL-LEPCs in xenogeneic spleen was monitored by immunohistochemical staining for albumin. ResultsIn xeno-mixed lymphocyte reaction, CTLA4.FasL-LEPCs substantially inhibited the rat lymphocytes proliferation. CTLA4.FasL-LEPC transplantation significantly ameliorated liver injury compared with mCherry-modified LEPC and LEPC transplantation, as assessed by hematoxylin and eosin staining, alanine aminotransferase, and aspartate aminotransferase assays. Albumin positive cells appeared only in CTLA4.FasL-LEPCs group, but not in the mCherry-modified LEPCs group and LEPCs group. ConclusionsOur results indicate CTLA4.FasL-LEPCs substantially improved liver function and structure in carbon tetrachloride- and partial hepatectomy-induced acute liver injury rats through long-term hepatocytic differentiation.

Oral administration of inactivated porcine epidemic diarrhea virus activate DCs in porcine Peyer\u2019s patches

BackgroundPeyer’s patches (PPs) can be considered as the immune site of the intestine. Within PPs, Dendritic cells (DCs) can uptake antigens from the gut lumen by extending dendrites into epithelium, and process it and then present to lymphocytes, which effectively antigen produces an immune response. Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), an acute and highly contagious enteric viral disease. The interaction between inactivated porcine epidemic diarrhea virus and porcine monocyte-derived dendritic cells (Mo-DCs) has been reported. However, little is known about the interaction between inactivated PEDV and DCs in porcine PPs.ResultsIn this study, for the first time we investigated the role of DCs in porcine PPs after oral administration inactivated PEDV. Firstly, a method to isolate DCs from porcine PPs was established, in which the purity of SWC3a+/MHC-II+ DCs was more than 90%. Our findings clearly indicate that DCs in porcine PPs after oral administration of inactivated PEDV not only stimulated the proliferation of allogeneic lymphocytes, but also secreted cytokines (IL-1, IL-4). Furthermore, the number of DCs and IgA+ cells in porcine intestinal mucosal significantly increased and the levels of anti-PEDV specific IgG antibody in the serum and SIgA antibody in the feces increased after oral administration inactivated PEDV.ConclusionsOur findings indicate that oral administration of inactivated PEDV activate DCs in porcine Peyer’s patches and inactivated PEDV may be a useful and safe vaccine to trigger adaptive immunity.

The influence of therapeutic vaccine candidate against HBeAg pEGFP-N1-C (472-507)-ecdCD40L on dendritic cells.

Hepatitis B virus (HBV) infection is a major public health problem and immune tolerance is responsible for persistent HBV infection. HBV therapeutic vaccines targeting HBV e antigen (HBeAg) may have an excellent effect in overcoming HBV immune tolerance. Thus, there is urgency for designing therapeutic vaccine candidates that target HBeAg. In this research, we fused the C (472-507) gene sequence of HBV with the extracellular domain of human CD40 ligand sequence and ligated this fused sequence into the pEGFP-N1 vector to construct the recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L. Then, the dendritic cells (DCs) generated from human peripheral blood were transfected with this recombinant plasmid. After this, the phenotype and function of DCs were assessed. Compared with the three control groups of pEGFP-N1-C (472-507), pEGFP-N1 and phosphate buffered saline (PBS), we found that DCs transfected with the recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L enhanced the expression of costimulatory molecules (CD80, CD86 and HLA-DR) and secretion of cytokine IL-12p70. Furthermore, the capacity of inducing the proliferation of allogeneic lymphocytes was also improved. Our study validated that transfecting DCs with recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L could activate DCs and enhance their functions. Therefore, C (472-507)-ecdCD40L fusion sequence may be a promising vaccine candidate for chronic hepatitis B therapythat targets HBeAg.

Human Amniotic Mesenchymal Stem Cells Exhibit Similar Immunosuppressive Ability with Bone Marrow Mesenchymal Stem Cells and Possess a Higher Proliferation Activity and Clearer Stem Cell Properties in vitro

Objectives: Human bone marrow mesenchymal stem cells (hBMSCs) have been used for the prevention and treatment of acute graft-versus-host disease (aGVHD) in post-hematopoietic stem cell transplant patients. In this study, we compared the biological characteristics and immunosuppressive activity of human amniotic mesenchymal stem cells (hAMSCs) and hBMSCs to provide experimental evidence for the potential use of hAMSCs for treating aGVHD, thus solving the problem of insufficient hBMSCs sources. Methods: HAMSCs were isolated by enzymatic digestion. hBMSCs were isolated using Ficoll-Hypaque density gradients. The biological characteristics of both stem cell types were compared by morphological analysis, analysis of cell growth, cell cycle profiling, immunophenotyping, and immunofluorescence assays. An in vitro co-culture of MSCs and peripheral blood mononuclear cells (PBMCs) was performed and lymphocyte proliferation measured using the Cell Counting Kit-8(CCK-8) assay. IFN-γ production was determined in the co-culture supernatant using enzyme linked immunosorbent assay (ELISA). Results: Both hAMSCs and hBMSCs had fibroblast-like morphology. hAMSCs could be maintained for at least 15 culture passages, but hBMSCs started to show signs of aging and remarkably reduced proliferation at 6-7 passages. There was no significant difference in the proportion of cells in G2/M phase between hAMSCs and hBMSCs (P>0.05). Immunophenotyping revealed positive expression of CD105, CD90, and CD73 and negative expression of CD34, CD45, CD11b, CD19, and HLA-DR on the surface of both hAMSCs and hBMSCs. hAMSCs were positive for Oct-3/4, but hBMSCs were not. Both hAMSCs and hBMSCs expressed vimentin. PHA-stimulated PBMCs proliferation was inhibited by hAMSCs and hBMSCs. This inhibition was stronger, as the proportion of MSCs increased. There were not significant differences between the inhibitory effects of the two MSCs types on PBMCs proliferation (P>0.05). Interferon-γ (IFN-γ) production was lower when PBMCs were co-cultured with either hAMSCs or hBMSCs than when they were cultured alone (P 0.05). Conclusion: The results of this study demonstrated that hAMSCs have higher proliferation activity and clearer stem cell properties than hBMSCs. Both hAMSCs and hBMSCs were able to suppress the proliferation of allogeneic peripheral blood lymphocytes and reduce IFN-γ secretion stimulated by PHA in vitro.

Immunologic properties of differentiated and undifferentiated mesenchymal stem cells derived from umbilical cord blood.

The expression of immunogenic markers after differentiation of umbilical cord blood (UCB)-derived mesenchymal stem cells (MSC) has been poorly investigated and requires extensive in vitro and in vivo testing for clinical application. The expression of human leukocyte antigen (HLA) classes on UCB-derived MSC was tested by Fluorescence-activated cell sorting analysis and immunocytochemical staining. The undifferentiated MSC were moderately positive for HLA-ABC, but almost completely negative for HLA-DR. The MSC differentiated to chondrocytes expressed neither HLA-ABC nor HLA-DR. The proliferation of MSC was not significantly affected by the allogeneic lymphocytes stimulated with concanavalin A. The responder lymphocytes showed no significant decrease in proliferation in the presence of the MSC, but the apoptosis rate of the lymphocytes was increased in the presence of MSC. Taken together, these findings indicate that UCB-derived MSC differentiated to chondrocytes expressed less HLA class I and no class II antigens. The MSC showed an immunomodulatory effect on the proliferation and apoptosis of allogeneic lymphocytes. These data suggest that the differentiated and undifferentiated allogeneic MSC derived from umbilical cord blood can be a useful candidate for allogeneic cell therapy and transplantation without a major risk of rejection.

CXCR4 Antagonist TG-0054 Mobilizes Mesenchymal Stem Cells, Attenuates Inflammation, and Preserves Cardiac Systolic Function in a Porcine Model of Myocardial Infarction.

Interaction between chemokine stromal cell-derived factor 1 and the CXC chemokine receptor 4 (CXCR4) governs the sequestration and mobilization of bone marrow stem cells. We investigated the therapeutic potential of TG-0054, a novel CXCR4 antagonist, in attenuating cardiac dysfunction after myocardial infarction (MI). In miniature pigs (minipigs), TG-0054 mobilized CD34(+)CXCR4(+), CD133(+)CXCR4(+), and CD271(+)CXCR4(+) cells into peripheral circulation. After isolation and expansion, TG-0054-mobilized CD271(+) cells were proved to be mesenchymal stem cells (designated CD271-MSCs) since they had trilineage differentiation potential, surface markers of MSCs, and immunosuppressive effects on allogeneic lymphocyte proliferation. MI was induced in 22 minipigs using balloon occlusion of the left anterior descending coronary artery, followed by intravenous injections of 2.85 mg/kg of TG-0054 or saline at 3 days and 7 days post-MI. Serial MRI analyses revealed that TG-0054 treatment prevented left ventricular (LV) dysfunction at 12 weeks after MI (change of LV ejection fraction from baseline, -1.0 ± 6.2% in the TG-0054 group versus -7.9 ± 5.8% in the controls). The preserved cardiac function was accompanied by a significant decrease in the myocardial expression of TNF-α, IL-1β, and IL-6 at 7 days post-MI. Moreover, the plasma levels of TNF-α, IL-1β, and IL-6 were persistently suppressed by the TG-0054 treatment. Infusion of TG-0054-mobilized CD271-MSCs reduced both myocardial and plasma cytokine levels in a pattern, which is temporally correlated with TG-0054 treatment. This study demonstrated that TG-0054 improves the impaired LV contractility following MI, at least in part, by mobilizing MSCs to attenuate the postinfarction inflammation. This insight may facilitate exploring novel stem cell-based therapy for treating post-MI heart failure.

Magnesium corrosion particles do not interfere with the immune function of primary human and murine macrophages.

Magnesium is currently under investigation as a prospective biodegradable implant material. Biodegradation of magnesium causes a release of magnesium, hydroxide ions and hydrogen gas but it can also lead to the formation of particulate debris. Implant-derived particles may have immunotoxic effects. To investigate the influence of magnesium-derived particles on the immune functions of primary macrophages, up to 500 μg/ml magnesium or magnesium corrosion particles were added to the cell culture medium. No major effects were observed on cell viability and on the release of the proinflammatory cytokine tumor necrosis factor (TNF)α. In addition, the ability of macrophages to stimulate proliferation of allogenic lymphocytes in a mixed leukocyte reaction remained unaffected. When macrophages were incubated with magnesium particles and then infected with the apathogenic Mycobacterium smegmatis, infection-induced TNFα secretion from murine macrophages was inhibited but not from human macrophages. However, the bactericidal activity of either cell type was not influenced. In conclusion, magnesium-related particles did not restrict the immune function of macrophages, suggesting that magnesium implants and corrosion particles derived thereof are highly biocompatible and have a low inflammatory potential.Electronic supplementary materialThe online version of this article (doi:10.1007/s40204-014-0032-9) contains supplementary material, which is available to authorized users.

Effects of TLR4 on β2-glycoprotein I-induced bone marrow-derived dendritic cells maturation

Our previous study has demonstrated that Toll-like receptor 4 (TLR4) can contribute to anti-β2-glycoprotein I/β2-glycoprotein I (anti-β2GPI/β2GPI)-induced tissue factor (TF) expression in human acute monocytic leukemia cell line THP-1. However, the role of TLR4 in the activation of autoimmune response in antiphospholipid syndrome (APS) has rarely been reported. In this study, we focused on the role of TLR4 in β2GPI-induced maturation of bone marrow-derived dendritic cells (BMDCs). iDCs from C3H/HeN mice stimulated with β2GPI were more mature than that from C3H/HeJ mice, yields of CD11c+MHCII+DCs, CD11c+CD80+DCs and CD11c+CD86+DCs and production of some pro-inflammatory cytokines in iDCs from C3H/HeN were higher than those from C3H/HeJ (p<0.05). Moreover, the ability of β2GPI-treated iDCs from C3H/HeJ to stimulate proliferation of allogeneic mixed lymphocytes was lower than that of iDCs from C3H/HeN. In conclusion, our results indicate that TLR4 may play a significant role in β2-glycoprotein I-induced BMDCs maturation.

Genomic ablation of HMGB1 in dendritic cells mitigates their capacity to both promote carcinogenesis and effectively confer anti-tumor immunity as vaccines (TUM4P.919)

Abstract HMGB1 is the prototypical damage associated molecular pattern (DAMP) molecule. It plays a critical role in many aspects of DC biology. To study the role of DC HMGB1 in tumor immunity, DC-specific HMGB1-/- mice (DCH) were generated. In both subcutaneous and liver metastasis models of pancreatic and colorectal cancer respectively, DCH mice exhibited inhibited tumor growth. DC HMGB1 is also critical for carcinogenesis; bone marrow from DCH mice was transplanted into mice expressing pancreas-specific oncogenic Kras (KC). KC mice given DCH bone marrow exhibited lower frequencies of malignant pancreatic and less pro-tumor inflammatory infiltrate. Genomic loss of HMGB1 also negatively impacted other aspects of DC functionality. Mice inoculated subcutaneously with pancreatic and melanoma cancer cell lines and then vaccinated with bone-marrow derived KO DCs pulsed with tumor antigen were unable to achieve therapeutic protection in both models. Although both WT and KO DCs were able to stimulate allogeneic lymphocyte proliferation, KO DCs skewed responses away from Th1/Th2 phenotypes, and favored Treg/Th17 polarization. KO DCs also expressed lower levels of both RAGE and TLR4, suggesting these signaling pathways may be impaired in DCH mice, thus accounting for our observations.

FOXP3 is a direct target of miR15a/16 in umbilical cord blood regulatory T cells.

Exact mechanism of action of umbilical cord blood (CB)-derived regulatory T cells (Tregs) in the prevention of GVHD remains unclear. On the basis of selective overexpression of peptidase inhibitor 16 in CB Tregs, we explored the related p53 pathway, which has been shown to negatively regulate miR15a/16 expression. Significantly lower levels of miR15a/16 were observed in CB Tregs when compared with conventional CB T cells (Tcons). In a xenogeneic GVHD mouse model, lower levels of miR15a/16 were also found in Treg recipients, which correlated with a better GVHD score. Forced overexpression of miR15a/16 in CB Tregs led to inhibition of FOXP3 and CTLA4 expression and partial reversal of Treg-mediated suppression in an allogeneic mixed lymphocyte reaction that correlated with the reversal of FOXP3 demethylation in CB Tregs. On the other hand, miR15a/16 knockdown in CB Tcons led to expression of FOXP3 and CTLA4 and suppression of allogeneic lymphocyte proliferation. Using a luciferase-based mutagenesis assay, FOXP3 was determined to be a direct target of miR15a and miR16. We propose that miR15a/16 has an important role in mediating the suppressive function of CB Tregs and these microRNAs may have a 'toggle-switch' function in Treg/Tcon plasticity.

Immature Dendritic Cells Generated from Cryopreserved Human Monocytes Show Impaired Ability to Respond to LPS and to Induce Allogeneic Lymphocyte Proliferation

Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.

Multipotent Human Mesenchymal Stromal Cells Mediate Expansion of Myeloid-Derived Suppressor Cells via Hepatocyte Growth Factor/c-Met and STAT3

SummaryMesenchymal stromal cells (MSCs) are multilineage progenitors with immunomodulatory properties, including expansion of immunomodulatory leukocytes such as regulatory T lymphocytes (Tregs) and tolerogenic dendritic cells. We report that human MSCs can expand CD14−CD11b+CD33+ human myeloid-derived suppressor cells (MDSCs). MSC-expanded MDSCs suppress allogeneic lymphocyte proliferation, express arginase-1 and inducible nitric oxide synthase, and increase the number of Tregs. This expansion occurs through the secretion of hepatocyte growth factor (HGF), with effects replicated by adding HGF singly and abrogated by HGF knockdown in MSCs. In wild-type mice, the liver, which secretes high levels of HGF, contains high numbers of Gr-1+CD11b+ MDSCs, and injection of HGF into mice significantly increases the number of MDSCs. Expansion of MDSCs by MSC-secreted HGF involves c-Met (its receptor) and downstream phosphorylation of STAT3, a key factor in MDSC expansion. Our data further support the strong immunomodulatory nature of MSCs and demonstrate the role of HGF, a mitogenic molecule, in the expansion of MDSCs.

Granulocyte Colony-Stimulating Factor-Mediated Immunoregulation Via Inducing Blood Cells Expressing Human Leucocyte Antigen-G.

Abstract 3051 BackgroundPeripheral blood stem cells (PBSCs) obtained from granulocyte colony-stimulating factor (G-CSF)-mobilized donors have been used more frequently than bone marrow stem cells as the source of cells in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although G-CSF–mobilized PBSC grafts contain more mature T cells than bone marrow cell grafts, the incidence and severity of graft-versus-host disease (GVHD) were not elevated. G-CSF-induced immune tolerance might be via inducing T helper type 2 (Th2) polarization, the promotion of regulatory T cell and tolerogenic dendritic cell (DC) differentiation. However, these mechanisms are not fully understood. Human leucocyte antigen-G (HLA-G) is a tolerogenic molecule which participates in the regulation of immune response. In this study, to explore the mechanisms of G-CSF-induced immunoregulation, the effect of G-CSF on blood cells expressing HLA-G was studied. MethodsMembrane-bound HLA-G (mHLA-G) was detected using flow cytometry analysis; soluble HLA-G (sHLA-G), interferon gamma (IFN-γ) and interleukin 10 (IL-10) were determined by enzyme-linked immunosorbent assay (ELISA); HLA-Gpos cells were isolated using flow cytometry, and mixed leukocytes reaction (MLR) was carried out to assess the suppressive effect of HLA-Gpos cells. ResultsCD3+CD4+ T cells, CD3+CD8+ T cells, CD19+ cells and CD14+ cells all expressed mHLA-G in peripheral blood (PB) and bone marrow (BM) from 18 healthy donors before and after G-CSF mobilization. The levels of sHLA-G and mHLA-G on these cells in PB and BM all increased significantly after G-CSF mobilization (all P<0.05). The levels of sHLA-G and mHLA-G on these cells in BM were all higher than that in PB, including before and after G-CSF mobilization (all P<0.05). Bone marrow mononuclear cells (BMMCs) were stimulated with G-CSF in vitro, and the levels of mHLA-G on these cells and sHLA-G in culture supernatant all increased significantly after BMMCs cultivated with G-CSF for 24 hours (all P<0.001). In addition, the levels of IFN-γ and IL-10 also elevated in culture supernatant (both P<0.05). Antibodies blocking experiments for IL-10 and IFN-γ showed that IL-10 and IFN-γ were not necessary for G-CSF-induced HLA-G expression of blood cells. The results of MLR showed that HLA-Gpos cells were able to suppress the proliferation of allogeneic lymphocytes. ConclusionG-CSF could directly induce blood cells expressing HLA-G, which might be another mechanism of G-CSF-mediated immunoregulation in G-CSF–mobilized PBSC transplants. Disclosures:No relevant conflicts of interest to declare.

25(OH)D(3) affects the maturation and function of mouse bone marrow-derived dendritic cells stimulated by Mycobacterium bovis BCG.

It has been shown that vitamin D deficiency increases an individual’s susceptibility to tuberculosis (TB). However, very little is known about the effect of vitamin D on the immune response to Mycobacterium tuberculosis (M. tb) in dendritic cells (DCs). Because DCs play an important role in TB infection, we investigated the phenotypic characteristics and functional capabilities of mouse bone marrow-derived dendritic cells (BMDCs) after stimulation with Bacillus Calmette-Guérin (BCG) in the presence or absence of 25(OH)D3(100 nM). Bone marrow cells from mice were cultured with GM-CSF (20 ng/ml) and were then treated with 25(OH)D3 for 7 days. On day 6, 5 µg/ml of BCG (≥1.0×106 CFU/mg) was added to the cells for 24 hours, and on day 7, the non-adherent cells were harvested for phenotypic and functional analyses. After incubation with 25(OH)D3, the expression levels of MHC-II and CD86 on the surface of the dendritic cells (DCs) and the ability of the DCs to stimulate proliferation of allogeneic mixed lymphocytes were lower than control cells (p<0.05). Furthermore, the level of Interleukin (IL) -4 secreted by the BMDCs in the 25(OH)D3 culture was lower than that in the control culture (p<0.01). However, the BMDCs cultured with 25(OH)D3 produced significantly higher levels of IL-2, IL-6, IL-10 and interferon gamma(IFN-γ) than those in the control culture (p<0.05). These findings suggest that 25(OH)D3 modulates the immune response during mycobacterial infection by affecting the maturation and function of DCs.

Effects of simvastatin on the osteogenic differentiation and immunomodulation of bone marrow mesenchymal stem cells.

The present study aimed to investigate the effects of simvastatin on the bone differentiation capacity and immunological characteristics of bone marrow mesenchymal stem cells (BMSCs). BMSCs were isolated and cultured in medium containing 1.0µmol/mlsimvastatin. The alkaline phosphatase activity, mRNA expression levels of osteocalcin and bone sialoprotein, and calcium nodule formation were assessed to determine the osteogenic differentiation capability of BMSCs. To investigate alterations in the immunological properties of simvastation‑treated BMSCs, the immunogenicity of these cells and the effect of BMSCs on phytohemagglutinin‑stimulated lymphocyte proliferation were also assessed. Following treatment with simvastatin, the alkaline phosphatase activity, and mRNA expression levels of osteocalcin and bone sialoprotein were increased significantly in the BMSCs. In addition, von Kossa staining revealed a brown calcium‑positive reaction zone in simvastatin‑treated cells. Simvastatin‑induced BMSCs revealed no affect on the proliferation of allogeneic lymphocytes, however, inhibited phytohemagglutinin‑induced lymphocyte proliferation. Collectively, simvastatin promoted the osteogenic differentiation of BMSCs significantly without affecting their immunosuppressive properties.

Indoleamine 2,3‐dioxygenase (IDO) induced by Leishmania infection of human dendritic cells

Dendritic cells (DC) play a pivotal role in regulating immunity, establishing immunologically privileged tissue microenvironments and maintaining homoeostasis. It is becoming increasingly clear that one key mechanism that mediates many DC functions is production of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). For pathogens that cause chronic infection, exploitation of host DCs is a solution to establish and persist within a host. Leishmania parasites cause a range of clinical manifestations, all involving chronic infection, and are proficient at avoiding immune responses. We demonstrate here that infection of human myeloid-derived DC with L.major and L.donovani induces IDO expression using a mechanism that involves autocrine or paracrine stimulation with a DC-secreted factor. Leishmania-induced IDO suppresses allogeneic and tetanus toxoid-specific lymphocyte proliferation, an inhibition that is reversed with the IDO inhibitor, 1-methyl tryptophan (1-MT). Furthermore, IDO expression by human DC does not require live Leishmania infection, as parasite lysates also up-regulate IDO mRNA production. Our data suggest that one mechanism Leishmania parasites utilize to circumvent immune clearance may be to promote the induction of IDO among host DC within the infection microenvironment.