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Objective@#To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice.@*Methods@#Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand (rmFlt3-L) in vitro, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ (MHC-Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin-12 (IL-12), interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA).@*Results@#The lymphocyte proliferation in mice stimulated with mDC+ pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively (P<0.05). When the ratio of effector cells: target cells (E∶T) was 10∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E∶T was 20∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 54.77%±3.28%, significantly higher than 35.25%±1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E∶T was 40∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 73.01%±0.91%, significantly higher than 51.36%±0.58% of mDC group and 22.65%±1.28% of pDC group, respectively (P<0.05). With the rate of E∶T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC-Ⅱ of mDC: pDC=1 group pulsed with heat-treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL-6 cytokine concentrations of mDC+ pDC group, mDC group and pDC group loaded with heat-treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67±16.07) pg/ml, respectively. The concentrations of IL-12 in each group were (2 568.75±119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF-α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL-6, IL-12 and TNF-α secreted by mDC+ pDC group were much higher than those of mDC group and pDC group under the same culture conditions (P<0.05).@*Conclusions@#The mDCs and pDCs combined vaccines pulsed with heat-treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti-tumor effect is related with up-regulation of co-stimulatory molecules and increased secretion of cytokines.
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Objective:To investigate the effect of IL-34 on the phenotype of monocyte derived dendritic cells in RA,and to speculate the role of IL-34 in the differentiation of myeloid dendritic cells.Methods:The peripheral blood of RA patients was collected to harvest PBMC by Ficoll density gradient centrifugation and cultured for 4h.Adherent cells were stimulated with GM-CSF+IL-4,IL-4, IL-4+IL-34 for 3 days,and then the expression of CD83,CD86 and CD14 was tested by flow cytometry.In addition,the cells stimulated by GM-CSF and IL-4 were added by TNF-α with or without IL-34 for another four days.The expression of CD83,CD86 and/or CD14 was detected by flow cytometry.Results:(1)The expression of CD83 and CD86 on immature DC induced by IL-34+IL-4 was upregulated compared with IL-4 alone(P<0.01),but no difference of the CD14 levels between the two groups(P>0.05).The levels of CD86 and CD14 induced by IL-34+IL-4 were slightly decreased compared with GM-CSF+IL-4 stimulation(P<0.05),but no difference of CD83 expression between the two groups(P>0.05).(2)The expression of CD83 and CD86 stimulated by GM-CSF+IL-4+IL-34 was lower than the GM-CSF+IL-4+TNF-α group(P<0.05),but no difference compared with GM-CSF+IL-4 group(P>0.05). (3)The CD83 expression induced by GM-CSF+IL-4+TNF-α+IL-34 was lower than GM-CSF+IL-4+TNF-α group(P<0.05),but there was no difference of CD86 and CD14 expression between the two groups(P>0.05).Conclusion:IL-34 played roles in the process of immature DC differentiation,but the effect was slightly weaker than that of GM-CSF.IL-34 did not effect the phenotype change of mature DC,but involved in the maintainence of immature DC.
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AIM:To investigate the maturation of mice immature myeloid dendritic cells (mDCs) induced by antigen(Ag)85B of mycobacterium tuberculosis, and the expression of TSLPR and OX40L mediated by TSLP in vitro. METHODS:Recombinant mouse GM-CSF ( rmGM-CSF) and rmIL-4 were used to induce bone marrow precursor cells of C57BL/6 mice to differentiate into immature mDCs in vitro.mDCs were identified followed by purification using CD 11c binding magnetic beads .The morphological characteristic of mDCs was observed under inverted phase-contrast microscope and scanning electron microscope .The surface phenotypes of mDCs were determined by flow cytometry .To obtain the opti-mal concentrations of Ag85B and TSLP, immature mDCs were cultured with different concentrations of Ag 85B or TSLP at 0 (control group), 50, 100 and 200 μg/L for 24 h, and the expression of cell surface molecules CD 80, CD86, TSLPR and OX40L was detected by flow cytometry.In addition, the expression of TSLPR and OX40L in Ag85B and TSLP-co-stimula-ted mDCs was determined by flow cytometry .RESULTS:After 7 d of culture in vitro, the cells showed irregular dendritic protrusions under the inverted-phase contrast microscope , and had wrinkles and dendritic splits under scanning electron mi-croscope , conformed to the morphological characteristics of immature mDCs .The mDCs cells expressed higher level of spe-cific marker CD11c, lower level of co-stimulatory molecules CD80 and CD86, which conformed to the phenotype of imma-ture mDCs.The CD80 +and CD86 +cell ratios of mDCs displayed significant increases in 50, 100 and 200μg/L Ag85B or TSLP groups compared with control group (P<0.05).The ratios of TSLPR +and OX40L+cells did not differ among dif-ferent concentrations of Ag 85B groups.The ratios of TSLPR +and OX40L+cells were significantly increased in 100 μg/L and 200μg/L TSLP groups compared with control group and 50μg/L TSLP group (P<0.05).Under the circumstance of optimal Ag85B or TSLP treatment concentration at 200 μg/L, there was significantly decreased in TSLPR and OX 40L cell ratio of mDCs in Ag85B group or Ag85B combined with TSLP group when compared with TSLP group (P<0.05), and no significant difference among Ag85B group, Ag85B combined with TSLP group and control group was observed .CONCLU-SION: Ag85B enhances mDCs maturation by up-regulating the expression of co-stimulatory molecules CD80 and CD86, and inhibit the expression of pro-inflammatory specific molecules TSLPR and OX40L on TSLP-activated mDCs, indicating that Ag85B modifies the development of asthmatic airway inflammation through the pathway of TSLP -activated mDCs.
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Objective To explore the frequencies and phenotype of myeloid and plasmacytoid dendritic cells (mDC and pDC) in chronic HCV infection and to investigate the relationships between DC frequencies and HCV viral load and serum ALT level. Methods PBMC were isolated from chronic HCV infected patients and healthy control. Multi-color flow cytometry was used to analyze the frequencies and surface marker expression on mDC and pDC. The relationship between DC frequencies and viral load and ALT level was also calculated. Results In comparison with healthy control, frequencies of mDC and pDC in chronic HCV infection were significantly decreased (0. 37 ± 0. 19 and 0. 19 ± 0. 12 vs 0. 51 ± 0. 18 and 0. 29 ± 0.13, P＜0.05). The frequency of mDC was negatively correlated with HCV viral load (r= -0.5878, P ＜ 0. 0001 ) and serum ALT level ( r = - 0. 4628 , P = 0. 003 ). Both costimulatory markers ( HLA-DR, CD83, CD86, and CD40) and coinhibitory marker (PD-L1) expression on mDC and pDC in HCV infection were increased (P＜0.01 for costimulatory marker, P＜0.05 or F＜0.01 for coinhibitory marker). Conclusion The frequencies of mDC and pDC in chronic HCV infection were decreased, while the expression of costimulatory markers and coinhibitory marker were increased or not decreased in HCV infection. The decreased frequency of mDC was probably related to persistance of HCV infection.