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Objective To investigate the effects of water extracts of Lepidium meyenii walp (LMWE) collected from two different places in Xinjiang on the maturation and function of dendritic cells (DCs) and to evaluate their roles in immunoregulation. Methods Water extracts of LMWE growing in Tashikuergan County (Ta xian) and A La gou of Xinjiang were prepared and named as LMWE-T and LMWE-A,respectively. Bone marrow-derived DCs and splenocytes isolated from C57BL/6 mice were treated with different concentrations of polysaccharide extracts from LMWE-T/A. Effects of LMWE-T/A on the per-centage and apoptosis of DC,expression of co-stimulatory molecules and secretion of cytokines were detected by flow cytometry and ELISA. MTT assay was used to analyze the proliferation of splenocytes. Changes in the functions of DC were evaluated by mixed lymphocyte reaction(MLR). Results LMWE-T/A had no in-fluence on the percentage and viability of DC. Expression of CD40 and CD86,and secretion of TNF-α,IL-12p40 and IFN-γ were significantly increased by LMWE-T/A treatment in a dose-dependent manner. LMWE-T/A treatment enhanced the functions of DCs and also dose-dependently promoted the proliferation of splenocytes. LMWE-A was more effective than LMWE-T in promoting CD86 expression,IFN-γ secretion and splenocyte proliferation. Pretreatment with TAK-242,an inhibitor of Toll-like receptor 4(TLR4),could sig-nificantly inhibit the regulatory effects of LMWE-T/A on CD40 expression and the secretion of TNF-α and IL-12p40. Conclusion This study shows that LMWE could promote the maturation of DC through TLR4 signaling pathway,enhance the functions of DC without side effects on DC survival,and increase the prolif-eration of splenocytes,indicating that LMWE has a potential immunopotentiating effect. LMWE-A has better effects than LMWE-T on immune enhancement.
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Objective:To explore the effects of suppress or enhancer of lin-12-like(Sel1L) on differentiation and function of bone marrow-derived dendritic cells.Methods:To generate conditional knockout mice by the Cre-Loxp recombination system.ELISA and Real-time fluorescence quantitative PCR(RT-PCR) was used for analyzing the protein levels and mRNA levels of IL-6/IL-12 in BMDCs.The protein levels of Sel1L in BMDCs were detected by Western bolt.The expression of CFSE,CD80,CD86,MHC-Ⅰ,MHC-Ⅱon BMDCs and the capability in priming OVA specific CD4+T cells proliferation were analyzed by the flow cytometry.Results:The deficiency of Sel1L decreases the proliferation of DCs during its differentiation,up-regulates the secretion of IL-6,IL-12 and the expression of MHC-Ⅰ.Notably,Sel1L-null DCs was failed to up-regulate MHC-Ⅱexpression and dramatically impaired their ability to prime OVA323-339specific CD4+T cell.Conclusion:The deletion of Sel1L can reduce the proliferation of BMDCs and down-regulate its ability in priming the proliferation of OVA specific CD4+T cells.
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B7-H3,a newly discovered co-stimulatory regulatory protein member of the B7 family.Its mRNA is ubiquitously expressed in a wide spectrum of tissues.As a co-stimulatory or co-inhibitory signal molecule which can regulate immune response,B7-H3 plays an important role in the immune system.Besides,B7-H3 can be also involved in cancer progression via non-immunological.Recently,the aberrant expression of B7-H3 has been described in various malignancies,and significantly correlated with poor prognosis and cancer progression.Therefore,B7-H3 is considered as an early diagnostic and prognostic marker and therapeutic target for tumors,but the specific molecular mechanisms of B7-H3 regulation are poorly understood.The immune and gene therapy of tumor by target B7-H3 has made some progress.
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Objective To investigate the therapeutic effects of rapamycin (RAPA) on concanavalin A (ConA)-induced acute autoimmune hepatitis in a mouse model and to analyze the possible mechanism.Methods Thirty eight-week-old female C57BL/6 mice were randomly divided into three groups including control group,ConA model group and ConA + RAPA treatment group.The levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in serum samples were measured after injection of mice with ConA for 24 hours for assessing the liver function.Hematoxylin-eosin (HE) staining was used to observe the hepatic pathological changes in mice.Splenocytes were harvested 24 h after ConA injection for the detection of the percentages of splenic DCs,CD4+T,CD8+T and CD4+ Foxp3+ Treg cells as well as the expression of co-stimulatory molecules (CD40,CD80 and CD86) on DCs by using flow cytometry.Results The levels of ALT and AST in mice from the RAPA treatment group were significantly lower than those of the ConA model group.Results of the HE staining assay showed that the liver damages in RAPA treated mice were less severe than those in mice from the ConA model group.Compared with mice form the ConA model group,those treated with RAPA showed decreased percentages of splenic CD4+ and CD8+ T cells,inhibited expression of CD80 and CD86 on splenic DCs,but increased percentages of splenic CD4+ Foxp3+ Treg cells.No statistically significant differences in the percentages of splenic DCs and the expression of CD40 were observed between the RAPA treatment group and the ConA model group.Conclusion The immunosuppressive effects of RAPA on mice with ConA-induced hepatitis might be achieved through the regulation of immune cells including DCs and T cells in spleen tissues.This study might pave the way for further investigation on the prevention and treatment of autoimmune hepatitis.
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Objective To investigate the role of regulatory B cells ( Breg ) in the immunological pathogenesis of Kawasaki disease ( KD) .Methods Twenty-eight children with acute KD and twenty-eight age-matched healthy subjects were enrolled in this study .Blood samples were collected before and after the treatment of intravenous immunoglobulin (IVIG).The proportions of CD19+CD24hiCD38hi Breg and CD4+CD25+Foxp3+regulatory T cells (Treg), and the expressions of co-stimulatory molecules (CD80, CD86 and PD-L1) were analyzed by flow cytometry .The concentration of IL-10 protein was measured by enzyme-linked immunosorbent assay .Real-time PCR was performed to evaluate the expressions of Foxp 3, CTLA4 and GITR in CD4+T cells and IL-10 in CD19+B cells at mRNA level.Results (1) The proportions of CD19+CD24hi CD38hi Breg in peripheral blood and the expressions of IL-10 at mRNA level in CD19+B cells from patients with KD were lower than those from healthy controls (P0.05).(3) The proportions of CD4+CD25+Foxp3+Treg and the expressions of Foxp3, CTLA-4 and GITR at mRNA level were significantly decreased in KD group compared with those in control group (P<0.05), but were increased to some extent after IVIG treatment (P<0.05). In addition, a positive correlation between the proportion of CD 19+CD24hiCD38hi Breg and the proportion of CD4+CD25+Foxp3+Treg was found in patient with acute KD (r=0.62, P<0.05).Conclusion Breg cell deficiency and its impaired function might be one of the important factors causing immune dysfunction in pa -tients with acute KD .
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Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6 percent, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4 percent) and CD86 (80.13 ± 2.81 percent)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.
Subject(s)
Animals , Female , Humans , Mice , /genetics , Adenoviridae/genetics , Apoptosis/genetics , Dendritic Cells/virology , Prostate-Specific Antigen/genetics , /immunology , Adenoviridae/immunology , Apoptosis/immunology , Cytotoxicity, Immunologic/immunology , Dendritic Cells/immunology , Dendritic Cells/pathology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , /immunology , /immunology , Phenotype , Prostate-Specific Antigen/immunology , Recombinant Proteins/genetics , Transduction, Genetic/methodsABSTRACT
CD46 is not only identified as a complement regulatory protein which protects host cells from complement attack,but also a new co-stimulatory molecule for human T cells.CD3/CD46 co-stimulation can induce a T-regulatory 1 cell(Tr1)-specific cytokine phenotype in human CD4+T cells.However,the role of CD46 as a co-stimulatory molecule in the modulation of the acquired immunity,such as transplant immunology,remains unclear.In this study,CD4+T cells were isolated from human CD46-transgenic C57BL/6 mice by magnetic-activated cell sorting,and further induced by anti-CD3,anti-CD28 and anti-CD46 antibodies respectively,and anti-CD3/anti-CD28 antibodies,anti-CD3/anti-CD46 antibodies,or the monoclonal antibody panel against CD3/CD28/CD46.The levels of interleukin-2(IL-2),γ-interferon(γ-IFN),interleukin-10(IL-10)and transforming growth factor-β(TGF-β)were detected in the supematants of different groups.Suppression of allogeneic T cell proliferation were assessed by using mixed lymphocyte reaction(MLR)assay,in which monoclonal antibodies against CD46 were added to the culture.The results showed that CD3/CD28,CD3/CD46 and CD3/CD28/CD46 co-stimulation could significantly induce stronger proliferation of T cells than CD3 stimulation(P＜0.05),and CD3/CD28/CD46 co-stimulation significantly increased the proliferation of T cells when compared with CD3/CD28 or CD3/CD46 co-stimulation(P＜0.05 for each).IL-2 and γ-IFN levels were much higher in CD3/CD28 co-stimulation group than in CD3,CD28,CD46 and CD3/CD46 groups(P＜0.05 for each).IL-10 and TGF-β levels were dramatically increased in CD3/CD46 co-stimulation group as compared with those in the CD3,CD28,CD46 and CD3/CD28 groups(P＜0.05 for each).CD3/CD46 co-stimulation significantly inhibited the T cell proliferation and allogenic immune responses through the secretion of IL-10 and TGF-β in MLR (P＜0.05).These results suggested that CD3/CD46 can induce Tr1 cells to modulate allogenic immune responses,and it may become a novel target for the development of new therapeutic approach for T-cell-mediated diseases.CD46 plays an important role in regulating the T cell-mediated immune responses by bridging innate and acquired immunity.