Ag85B lentivirus inhibits expression of TSLP and TSLP receptor in human bronchial epithelial cells

AIM: To investigate the effect of Mycobacterium tubeculosisantigen （Ag85B） lentivirus on the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in cultured normal human bronchial epithelial cells (NHBEC) in vitro. METHODS: Ag85Bgene was subcloned into 293T cells with recombinant plasmid to yield the Ag85B lentivirus. The NHBEC were cultured and transfected with Ag85B lentivirus, NS control and empty lentivirus control using Lipofectamine 2000. The expression of Ag85B, TSLP and TSLPR in NHBEC at mRNA and protein levels was confirmed by real-time PCR and Western blotting.RESULTS: Ag85B lentivirus was constructed with a viral titer of 2×1011 TU/L and the Ag85B expression was detected. As compared with control group and empty lentivirus group, the mRNA expression of TSLP in Ag85B lentivirus group was found to slightly decreased, and the mRNA expression of TSLPR and the protein expression of TSLP and TSLPR were significantly decreased in Ag85B lentivirus group (P<0.05). CONCLUSION: The constructed Ag85B lentivirus antagonizes the expression of TSLP and TSLPR in cultured NHBEC, suggesting that Ag85B inhibits the initiation of asthmatic inflammation by blockage of TSLP-TSLPR pathway.     

Antigen-loaded dendritic cells and CD40L triggers the killing effects of cytotoxic T lymphocytes on K562 cells in vitro

AIM:To investigate the anti-tumor effects of special cytotoxic T lymphocytes (CTLs) activated by dendritic cells (DCs) loaded with antigens and CD40L in vitro.METHODS:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation from normal human heparinized blood.The adherent cells were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF),interleukin-4 (IL-4),alpha tumor necrosis factor (TNF-α),DCs were co-cultured with frozen-thawed antigen of K562 cells and CD40L,then triggered T cells into specific CTLs.RESULTS:Most suspended cells exhibited distinctive morphological features of DCs which expressed CD40 96%,CD86 97%,CD80 77%,CD1a 69%,and gained the powerful capacity to stimulate proliferation of allogenic lymphocytes.Under the effector∶target ratio of 20∶1,CTLs derived from cultures with DCs and frozen-thawed antigen of K562 cells were showed 71.3% cytotoxicities against K562 cells.CTLs derived from cultures with DCs loaded with frozen-thawed antigen and CD40L were showed 86.9% cytotoxicities against K562 cells.Cytotoxicities by CTLs derived from cultures with unloaded DCs against K562 cells were 37.6% and cytotoxicities by monocytes were 21.1%.Cytotoxicities by CTLs derived from experiment groups were stronger than control groups (P<0.05).CONCLUSIONS:The tumor antigen-pulsed DCs induces efficient and specific anti-tumor immunity,CTLs derived from cultures containing DCs pulsed with CD40L show the strongest cytolytic activities on K562 cells.     

Influence of stimulation by LPS and CD40 ligandization in vitro on the TLR4-MD2 expression and IL-12 production in DCs modified by sCD40 gene

AIM: To study the influence of stimulation by LPS and CD40 ligandization in vitro on the TLR4-MD2 expression and IL-12 production in dendritic cells (DCs) modified by sCD40 gene and provide the experimental clues of inducing dornor-specific immune tolerance.METHODS: Plasmid pEGFP-N1/sCD40 and pEGFP-N1 was transfected into DC2.4 cell line with lipofectamine.After 6 h of treatment with LPS and anti-CD40mAb,the expression of TLR4-MD2 on DCs was determined with flow cytometry (FCM) and RT-PCR.IL-12p70 protein was detected by ELISA.RESULTS: LPS treatment of DCs down-regulated surface expression of TLR4-MD2,LPS treatment along with anti-CD40mAb significantly up-regulated TLR4-MD2 surface expression.CD40 ligandization did not affect TLR4-MD2 mRNA expression in DCs but partly increased its low level induced by LPS and markly enhanced IL-12p70 secretion after LPS stimulation.DCs modified by sCD40 gene inhibited the above effect.CONCLUSION: After treatment with LPS and anti-CD40mAb,DCs modified by sCD40 gene down-regulate surface expression of TLR4-MD2 and IL-12p70 secretion decreases significantly,which might be linked with the interruption of TLR4-MD2 transportation from cytoplasm.