The Regulation Of Multi-walled Carbon Nanotubes On The Function Of Dendritic Cells And T Cells And Its Mechanism

Background:Antigen presenting cells(APCs)process exogenous antigens into antigenic peptides,presented to the cell surface as MHC class-peptide complexes,and recognized by the T cell receptor(TCR)in CD4+ T cells.Upon TCR-mediated cell activation,naive CD4+ T cells can differentiate into at least four major lineages,Th1,Th2,Th17 and regulatory T cells,all of which participate in different types of immune responses.The Thl cells produce IFN-y,IL-2 and so on,which can promote cellular immune response.Th2 cells secrete IL-4 and IL-10 and are often associated with the humoral immune response.Under normal circumstances,the Thl and Th2 cells are in balance.The Thl/Th2 imbalance is closely related to the development,treatment,and prognosis of many diseases.Therefore,it is important to maintain the Thl/Th2 balance.Dendritic cells are powerful APC,which plays an central role in the banlance of Th1/Th2.Antigens can induce specific immune response and influence the differentiation of Th cells.With unique and fascinating nanostructure,carbon nanotubes are currently applied and investigated as new tools for biomedical applications,and have been used as nanocarriers to transport anticancer drugs,genes and proteins for chemotherapy.Recent studies have shown that carbon nanotubes can affect Thl/Th2 balance.A few studies found that carbon nanotubes could facilitate Th2 differeniation and induced higher humoral immune response.The specific mechanism is still unclear.In this study,the interation of oxidized multi-walled carbon nanotubes(CNT)with dendritic cells and its effect on Th differentiation were investigated.Methods:(1)Bone marrow cells were induced to dendritic cells(BMDCs)in vitro.The morphology of BMDCs were analyzed by wright giemsa staining and transmission electron microscope(TEM).The cell viability was evaluated by flow cytometry.The BMDCs activation,function and uptake of CNT were analyzed by flow cytometry,TEM,and enzyme-linked immuno sorbent assay(ELISA).The antigen presenting ability of BMDCs treated with CNT,CNT/O-2 and CNT/O-3 were analyzed.The activation and proliferation of T cells treated with CNT,CNT/O-2 and CNT/O-3 were analyzed by mixed lymphocyte responses.(2)The Jurkat cells were treated with CNT,PMA(phorbol 12-myristate 13-acetate)and CNT/PMA,Cell viability was determined by cell counting kit-8(CCK-8).The uptake of CNT by Jurkat cells were analyzed by TEM.The expression of IL-10 and IFN-y were measured using ELISA and real time-polymerase chain reaction(RT-PCR).The activation of transcription factors AP-1 and NF-?B were examined by western blot analysis.PLC-specific inhibitor U-73122(U-7)was applied to further study the mechanism of CNT effect on Th differentiation,which were anlyzed by expression of IL-10 and IFN-y.Choosing 4T1 as target cells,the cytotoxicity mediated by lymphocytes treated with CNT,PMA and CNT/PMA were measured.Results:(1)The percentage of CD11c+ cells with typical spikes was about 80%.CNT was largely engulfed by BMDCs.The viability of BMDCs was only affected slightly by the highest concentration of CNT(0.1 mg/mL).The MHCI,MHCII,CD86,CD80 and TNF-a expression of BMDCs were unchanged with the treatment of CNT.BMDCs treated with CNT/O-2 complex did not affect the proliferation of CD8+ T cells,while treated with CNT/O-3 could induce more CD8+ T cell proliferation and the expression of IL-4,IL-10 and IL-13 were significantly inhibited by CNT treated-BMDCs excepting for the concentration of 0.1 mg/mL.(2)The CNT with different concentration applied in our work did not significantly affect the viability of Jurkat cells and a few of them adhered to the cell memberane.The expression of IFN-y was increased with the treatment of CNT.However,more CNT were found in Jurkat cells after treated with CNT/PMA.More importantly,compared to CNT and PMA,CNT/PMA could further increase the level of IFN-y and decreased the expression of IL-10,which facilitated Thl polarization.In addition,CNT/PMA complex significantly enhanced the phosphorylation level of AP-1.PLC inhibitor U-7 could reverse the effect of CNT by suppressing the production of IFN-y and boosting the the level of IL-10.CNT/PMA complex significantly enhanced lymphocyte-mediated cytotoxicity against 4T1 tumor cells.Conclusions:(1)Though CNT did not activate BMDCs,it increased O-3 antigen presenting.CNT/O-3-exposed BMDCs induced more CD8+ T cell proliferation,facilitating Thl polarization.(2)CNT/PMA synergized together to increase IFN-y production,which was transducted through the PLC and intracellular AP-1 signal pathway.The CNT/PMA complex was capable of enhancing the anti-cancer immune reaction by lymphocyte in vitro.