The effects of granulocyte colony-stimulation-factor (G-CSF) on stem cell mobilization and its impact on the amplification of myeloid-derived suppressor cells (MDSCs) of donor mice were examined. A mouse model of stem cell mobilization was established by consecutive subcutaneous injection of 100 μg/kg G-CSF for 5 days. The blood from the donor mice was routinely examined during mobilization. Stem cells and MDSCs were analyzed by flow cytometry. The immunosuppressive molecules derived from MDSCs in serum and spleen, including hydrogen dioxide (H2O2) and nitric oxide (NO), and the activity of nitric oxide synthase (NOS) were determined during the mobilization. Apoptosis of T lymphocytes was assessed by using Annexin-V/PI. During stem cell mobilization, the number of lymphocytes and white blood cells in the peripheral blood was increased, and peaked on the 4th day. The number of stem cells in G-CSF-treated mice was significantly greater than that in controls (P<0.01). The expansions of MSDCs were also observed after G-CSF mobilization, with a more notable rate of growth in the peripheral blood than in the spleen. The activity of NOS and the production of NO were increased in the donor mice, and the serum H2O2 levels were approximately 4-fold greater than the controls. Consequently, apoptosis of T lymphocytes was increased and showed a positive correlation with the elevated percentage of MDSCs. It was concluded that G-CSF could provide sufficient peripheral blood stem cells for transplantation. Exogenous administration of G-CSF caused the accumulation of MDSCs in the peripheral blood and the spleen, which could lead to apoptosis of T lymphocytes and may offer a new strategy for the prevention and treatment of graft versus host disease.
Animals ; Apoptosis ; drug effects ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Hydrogen Peroxide ; metabolism ; Mice ; Mice, Inbred C57BL ; Myeloid Progenitor Cells ; cytology ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; T-Lymphocytes ; cytology ; drug effects ; metabolism

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide-induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4+CD8+ double-positive (DP) thymocytes. Furthermore, the CD4loCD8lo, CD4+CD8lo and CD4loCD8+ thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4+CD8+ DP thymocytes present during thymus regeneration were TCR(hi) and CD69+ unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCRhi/int and CD69+/int phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4+CD8+ DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.
4-1BB Ligand/genetics/*metabolism ; Animals ; Antigens, CD137/genetics/*metabolism ; CD4-Positive T-Lymphocytes/cytology/metabolism ; CD8-Positive T-Lymphocytes/cytology/metabolism ; Cell Adhesion ; Cell Differentiation ; Cell Line ; Cells, Cultured ; Cyclophosphamide/pharmacology ; Epithelial Cells/cytology ; Gene Expression Regulation ; Immunosuppressive Agents/pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; RNA, Messenger/genetics ; *Regeneration ; T-Lymphocytes/*cytology/metabolism ; Thymus Gland/*cytology/drug effects/*physiology

The aim of this study was to compare cell proliferation and function of the T cells acquired under various culture conditions for establishing a simple, safe and efficient cell expansion protocol in vitro. The peripheral blood mononuclear cells (PBMNC) were isolated and stimulated with autologous dendritic cells (DC) and EBV-transformed B lymphoblastoid cell line (BLCL) weekly. The cell proliferation test, flow cytometry with PI and Annexin V double staining, Cr release test and ELISPOT test were used to detect the cell expansion level, frequency of IFN-γ producing T cells, killing activity of antigen-specific T cells, cell apoptotic status and cell differentiation potential, respectively. The results indicated that use of IL-2 combined with IL-7 and IL-15 resulted in the highest cell expansion comparing to the use of IL-2 alone and the use of CD3/28 Microbeads. Also the cells obtained under cultivating with IL-2, IL-7 and IL-15 together showed high frequency of IFN-γ producing cells, strong killing activity, high viability and high differentiation potential with large portion of CD3(+)CD8(+) population among the T cells. It is concluded that a protocol is established in which the use of IL-2 combined with IL-7 and IL-15 induces the biggest cell expansion, expanded cells show high viability, strong differentiation potential, high frequency of IFN-γ producing cells and strong killing activity.
Cell Line, Transformed ; Cell Proliferation ; Cell Separation ; Dendritic Cells ; cytology ; metabolism ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-7 ; pharmacology ; T-Lymphocytes ; cytology ; drug effects ; metabolism

OBJECTIVEDendritic cells play an important role in the pathogenesis of atherosclerosis. To explore the effects of hyperglycemia on the maturation and immune function of human monocyte derived dendritic cells (MDCs).

METHODSImmature MDCs were cultured in RPMI1640 medium with either 5.5 mmol/L D-glucose (NG), 25 mmol/L D-glucose (HG) or 5.5 mmol/L D-glucose + 19.5 mmol/L mannitol (HM) in the absence or presence of 30 mmol/L N-acetylcysteine [NAC, a reactive oxygen species inhibitor (ROS)] for 48 hours. FACS was used to investigate the MDCs immunophenotypic expression. Immune function was evaluated by allogeneic mixed T lymphocyte reaction and measurement of cytokine levels from culture supernatants. Intracellular ROS production in MDCs was also measured by 2', 7'-dichlorodihydrofluorescein (DCF, 10 micromol/L) fluorescence using confocal laser-scanning microscopy techniques.

RESULTSCompared with NG and HM treated MDCs, the expression of maturation markers such as CD1a, HLA-DR, CD83, CD86 were significantly upregulated, allogeneic T cells proliferation as well as the cytokines secretions (IL-2, IL-12, IL-10 and IFN-gamma) significantly increased in HG treated MDCs. Intracellular ROS production in MDCs was also significantly increased and all these stimulatory effects of HG could be partially attenuated by NAC.

CONCLUSIONHigh glucose promote the maturation of MDCs and augment their capacity to stimulate T-cell proliferation and cytokine secretions at least in part through enhancing intracellular ROS generation. These stimulating effects of high glucose on MDCs maturation may be one of the mechanisms of accelerated atherosclerosis found in patients with diabetes.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; Cytokines ; biosynthesis ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Glucose ; adverse effects ; pharmacology ; Humans ; Immunophenotyping ; Monocytes ; cytology ; Reactive Oxygen Species ; metabolism ; T-Lymphocytes ; cytology

OBJECTIVETo study the effect of Chinese drugs for invigorating qi and tonifying Shen (IQTS) on expression of CD4+CD25+ regulatory T cells in spleen and maternal-fetal interface of abortion-prone mice during pregnancy.

METHODSCBA female mice were mated with DBA/2 male mice to establish abortion-prone models, which were randomly divided into 4 groups, the negative control group (fed with normal saline), the positive control group (treated with CsA), the Chinese medicine group (treated with IQTS), and the Chinese and Western medicine group (treated with IQTS+CsA). Mice were sacrificed in batches on the 9th and the 14th day of gestation, their splenic and decidual tissues were taken out to analyse CD4+CD25+ regulatory T-cell expression by flow cytometry.

RESULTSCompared with the negative control group, the expression of splenic CD4+CD25+ regulatory T all significantly increased on the 9th day of gestation (P < 0.01 or P < 0.05). There was no statistical difference in intergroup comparison of the three treatment groups (P > 0.05). Compared with the negative control group, the expression of splenic CD4+CD25+ regulatory T all significantly increased on the 14th day of gestation (P < 0.01 or P < 0.05). Of them, its expression was the highest in the Chinese and Western medicine group, showing significant difference from that in the Chinese medicine group and the positive group (P < 0.01). The difference between the Chinese medicine group and the positive group was insignificant (P > 0.05). On day 9 of gestation, compared with the negative control group, the expressions of CD4+CD25+ regulatory T in maternal-fetal interface increased in the three treated groups, showing no statistical significance (P > 0.05). Its expression was ordered from high to low in sequence as the Chinese and Western medicine group, the positive control group, the Chinese medicine group, and the negative control group. On day 14 its expression was obviously enhanced in the Chinese and Western medicine group, showing statistical difference from that in the negative control group (P < 0.05). But its expression was obviously enhanced in the Chinese medicine group and the positive group, showing insignificant difference from that in the negative group. The same sequence was found in the percentage of CD4+CD25+ T cells in CD4+ T cells.

CONCLUSIONSChinese drugs for IQTS could up-regulate the expression of CD4+CD25+ regulatory T in spleen of abortion-prone mice in the early and late pregnancy stages. When combined with CsA, it also could up-regulate its expression in maternal-fetal interface in the mid and late pregnancy stages, suggesting that Chinese drugs for IQTS are facilitate to maintain the immune tolerance state in mice during pregnancy.

Abortion, Spontaneous ; drug therapy ; immunology ; metabolism ; Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Male ; Mice ; Mice, Inbred CBA ; Phytotherapy ; Pregnancy ; Spleen ; cytology ; drug effects ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; metabolism

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism

OBJECTIVETo investigate the specific antitumor immunity induced by antigen peptide mixture prepared from different T lymphocytic leukaemia cells and the cross-reaction among the mixtures of different cell lines.

METHODSAntigen peptide mixtures were prepared from different leukaemia cell lines and then bound with Hsp70 in vitro. The activation and proliferation of peripheral blood mononuclear cell (PBMC) were observed after the stimulation by different Hsp70-peptide complexes. The cytotoxicity of such activated PBMCs to different target cells was assayed.

RESULTSThe antigen peptides from different leukaemia cell lines were mixed ones, which could activate PBMC effectively with Hsp70 and stimulate the activated PBMC to proliferate. The proliferative PBMC had specific cytotoxicity to the corresponding leukaemia cells. To Hut-78 cell, Molt-4 cell and Jurkat cell, the cytotoxicity of PBMC activated by either Hut78-peptides or Molt-4-peptides was significantly stronger than that of PBMC activated by HL-60-peptides (P < 0.05). The cytotoxicity to Jurkat cell of PBMC activated by Hut78/Molt-4-peptides was significantly stronger than that of PBMC activated by Hut78-peptides or Molt-4-peptides alone (P < 0.05).

CONCLUSIONAntigen peptide mixture from T lymphocytic leukaemia cells is able to induce specific antitumor immunity. There is a cross-reactivity among antigen peptide mixtures from different T lymphocytic leukaemia cell lines, with the more crossed antigen peptides obtained from the mixtures of different antigen peptides from different T lymphocytic leukaemia cell lines, which suggests that the antigen peptide mixture with broad antigenic spectrum could possibly be prepared by using multiple leukaemia cell lines.

Antigens, Neoplasm ; metabolism ; pharmacology ; Cross Reactions ; HL-60 Cells ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Leukemia, T-Cell ; immunology ; Leukocytes, Mononuclear ; cytology ; drug effects ; Peptides ; pharmacology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Tumor Cells, Cultured ; chemistry ; immunology

The OPG/RANKL/RANK system plays an important role in osteoclastogenesis and represents a great progress in bone biology. RANKL, which expresses on the surface of osteoblast/stromal cells and activated T cells, binds to RANK on the osteoclastic precursors or mature osteoclasts, and promotes osteoclastogenesis and bone resorption. While osteoprotegerin (OPG), which is expressed by osteoblasts/stromal cells, strongly inhibits bone resorption by binding to its ligand RANKL and thereby blocks the interaction between BANKL and RANK. A number of cytokines and hormones exert their effects on bone metabolism by regulating the OPG/RANKL ratio in the bone marrow microenvironment. RANK is also expressed on mammary epithelial cells and RANKL expression in these cells is induced by pregnancy hormones, RANKL and RANK are essential for the formation of the lactating mammary gland and the transmission of maternal calcium to neonates in mammalian species. Modulation of these systems provides a unique opportunity to develop novel therapeutics to inhibit bone loss in osteoporosis, rheumatoid arthritis, and bone metastasis of cancer. Further research should be focused on the cooperation of OPG/RANKL/RANK system with other signal pathways and the interactions among bone remodeling, immune system and endocrinology system. Currently, the development of OPG analogues or compounds which may stimulate OPG expression is becoming an attractive industry which may be profitable to both patients and manufacturers.
Animals ; Bone Resorption ; immunology ; metabolism ; Humans ; Osteoclasts ; cytology ; metabolism ; pathology ; Osteogenesis ; drug effects ; genetics ; immunology ; Osteoprotegerin ; metabolism ; physiology ; RANK Ligand ; metabolism ; physiology ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; pharmacology ; physiology ; T-Lymphocytes ; drug effects ; immunology

Triptolide( TP) is isolated from the traditional Chinese medicine Tripterygium wilfordii,which exhibits notable immuneregulative effect. Th17 cells involve in inflammatory response and Treg cells contribute to immune tolerance. They both play an important role in immune response. Previous studies have investigated that TP induced hepatic Th17/Treg imbalance. However,the effect of TP on spleen Th17/Treg cells remains unclear. Therefore,the aim of present study was to investigate the effect of TP on Th17/Treg cells in spleen. In this study,the effect of TP on the proliferation of splenic lymphocyte was detected by cytotoxicity test in vitro. After different concentrations of TP( 2. 5,5,20,40 nmol·L~(-1)) were given to splenic lymphocyte,cytokines secreted from the supernatant of splenic lymphocyte were detected by cytometric bead array,and the expression of suppressor of cytokine signaling( SOCS) mRNA was detected by qRT-PCR. Female C57 BL/6 mice were continuously observed for 24 h after treatment of 500 μg·kg-1 TP. The effects of TP on the splenic tissue structure and the percentage of Th17/Treg cells were examined. The results showed that the IC50 of TP was19. 6 nmol·L~(-1) in spleen lymphocytes. TP inhibited the secretion of IL-2 and IL-10 and induced the expression of SOCS-1/3 mRNA in spleen lymphocytes at the dosage of 2. 5 and 5 nmol·L~(-1) after 24 h in vitro. Administration of TP at dosage of 500 μg·kg-1 had no significant spleen toxicity in vivo. TP treatment increased the percentage of Th17 cells after 12 h and inhibited the proportion of Treg cells after 12 and 24 h. In conclusion,TP reduced the secretion of IL-2 and IL-10 through SOCS-1/3 signaling pathway,thereby induced the percentage of Th17 cells and inhibited the percentage of Treg cells.
Animals ; Cytokines ; metabolism ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Female ; Mice ; Mice, Inbred C57BL ; Phenanthrenes ; pharmacology ; Signal Transduction ; Spleen ; cytology ; drug effects ; Suppressor of Cytokine Signaling 1 Protein ; metabolism ; Suppressor of Cytokine Signaling 3 Protein ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

OBJECTIVETo study the mechanism underlying the inhibitory effect of the anti-HIV peptide VIR576 on antigen-specific T cell activation.

METHODSCCK-8 assay was used to investigate the effect of VIR576 on the proliferation of splenocytes of OVA-specific DO11.10 Tg mice in response to chicken OVA. Hemolysis test, hemolysis inhibition assay and fluorescence binding assay were used to investigate the interaction of VIR576 with the transmembrane domain (TMD) of the T cell receptor (TCR).

RESULTSVIR576 inhibited HIV glycoprotein gp41 fusion peptide-mediated antigen specific T cell activation, and VIR576 itself also inhibited splenocyte proliferation in responses to OVA (P<0.05). Hemolysis test, hemolysis inhibition assay and fluorescence binding assay demonstrated that VIR576 suppressed TCR-TMD-mediated hemolysis and competitively inhibited Rho-VIR576 binding to TCR-TMD peptide.

CONCLUSIONVIR576 is effective in suppressing the antigen-specific T cell activation via TCR and can interact with TCR-TMD. VIR576 may serve as a potent microbicide candidate to block sexual transmission of HIV due to of its inhibitory effect on both HIV entry and antigen-specific T cell activation.

Animals ; Anti-HIV Agents ; pharmacology ; Cell Membrane ; metabolism ; HIV Infections ; prevention & control ; Humans ; Lymphocyte Activation ; drug effects ; Mice ; Receptors, Antigen, T-Cell ; immunology ; Sincalide ; analysis ; Spleen ; cytology ; immunology ; T-Lymphocytes ; immunology ; Virus Internalization ; drug effects