Thymosin α-1 Regulates The Tumor Cells-derived Apoptotic Bodies Antigen Presentation In Dendritic Cells

Thymosin alpha 1(Thymosin α-1,Tα-1)is a small peptides molecules.Tα-1 is often used in combination with chemotherapy in the treatment of tumor patients.However,the mechanisms have not been fully elucidated.In our previous study we found that apoptotic tumor cells in large amounts are induced by chemotherapeutic drugs(Paclitaxel,Cisplatin,Bortezomib,ect).Macrophages activate TBK1 signaling pathway,secrete IL-10 and lead to M2 polarization during the efferocytosis.Tα-1binds with surface PS on apoptotic cells,reverses M2 polarization by TLR7-SHIP1 signaling pathway and enhances the activity of tumor infiltrating lymphocytes(TILs)to upregrate anti-tumor effects of chemotherapy.apoptotic bodies(ABs)are breakages of apoptotic tumor cells.ABs also have large amounts of surface PS.Antigen presentation in dendritic cells(DC)is depressed after apoptotic bodies engulfment.Therefore,one question still to be studied is whether Tα-1binds with ABs,upregulates the apoptotic bodies antigen presentation in dendritic cells,or enhances tumor specific T cell response and amounts of TILs.Objectives:To study whether Tα-1 regulates the tumor cells-drived apoptotic bodies antigen presentation in dendritic cells and its mechanism.To study the mechanism of antitumor immunity induced by Tα-1.Methods:1.Thymosin α-1 regulates the function of dendritic cells after apoptotic bodies engulfment and and its mechanism1.1 Thymosin α-1 regulates the function of dendritic cells after apoptotic bodies engulfment4T1 were induced apoptosis by 100 n M Bortezomib.Apoptotic bodies were extracted by differential centrifugation.Flow cytometry and transmission electron microscopy were used to identify apoptotic bodies.Bone Marrow-Derived Dendritic Cells(BMDC)were generated and co-cultured with apoptotic bodies incubated with Tα-1(ABs/Tα-1,100μg/ml),Tα-1(100μg/ml),and ABs(100μg/ml)for 24 hours.IL-12p70 in supernatants was detected by ELISA,and CD86 and MHC Ⅱ expression in BMDC were detected by flow cytometry.1.2 The mechanism of Tα-1 regulating the function of dendritic cells after apoptotic bodies engulfmentApoptotic bodies were incubated with Tα-1.And then the PS of ABs were labeled with Annexin V-FITC.The propotion of apoptotic bodies binded with Tα-1 was detected by flow cytometry.Apoptotic bodies were incubated with Tα-1 linked with CY5(Tα-1-CY5).Then ABs/Tα-1-CY5 were labeled with Annexin V-FITC and co-cultured with DC for 6 hours.Next,DC lysosomes were labeled with Lyso-Tracker Red.Finally,the proportion of apoptotic bodies binded with Tα-1-CY5 were detected by flow cytometry.ABs/Tα-1-CY5 were detected by confocal scanning laser microscopy.2.Thymosin α-1 regulates the apoptotic bodies antigen presentation in dendritic cells2.1 Thymosin α-1 regulates the apoptotic bodies antigen presentation in dendritic cells in vitroMice were immunized subcutaneously with alpha-alumina nanoparticles coupled with OVA(α-Al2O3-OVA).Mice spleen cells were stimulated with different concentrations of OVA.IFN-γ in supernatants were detected by ELISA.Apoptotic bodies from OVA+B16F10(OVA+ABs),OVA+/ABs incubated with Tα-1(OVA+ABs/ Tα-1)and Tα-1 were co-cultured with BMDC for 24 hours.Then those prepared BMDC were co-cultured with spleen cells from OVA specific response mice for 24 hours.Finally,the proportion of IFN-γ+ CD3+ CD8-and IFN-γ+ CD3+ CD8+ cells was detected by flow cytometry.IFN-γ in supernatants was detected by ELISA.2.2 Thymosin α-1 enhances the chemotherapy efficacy in the treatment of melanomaMice were immunized subcutaneously with OVA+B16F10.Then,those mice were divided into 4 groups: only Tα-1 group,only DDP group,Tα-1 and DDP group,Saline group(NS);The tumour growth curve were made on the basis of tumour size;In the next day after the last therapy,proportion of DC and CD8α+ DC cells and their CD86,MHC Ⅱ expression in lymph nose cells were detected by flow cytometry;proportion of IFN-γ+ CD3+ CD8-and IFN-γ+CD3+CD8+cells in spleen cells and Lymph node cells were detected by flow cytometry;Secretion IFN-γ of tumor infiltrating lymphocyte(TILs)was detected by ELISA.Results:1.Thymosin α-1 regulates the function of dendritic cells after apoptotic bodies engulfment and and its mechanism1.1 Thymosin α-1 regulates the function of dendritic cells after apoptotic bodies engulfmentOver 90% purity apoptotic bodies were extracted by differential centrifugation from apoptotic 4T1.Tα-1 have no modulation of IL-12p70 secretion,MHC Ⅱ and CD86 expression of dendritic cells.Dendritic cells after apoptotic bodies engulfment showed a downregulation of IL-12p70 secretion,MHC Ⅱ and CD86 expression.Tα-1 can enhance the IL-12p70 secretion,MHC Ⅱ and CD86 expression of dendritic cells after apoptotic bodies engulfment.1.2 The mechanism of Tα-1 regulating the function of dendritic cells after apoptotic bodies engulfmentMore the 90% apopototic bodies were binded with Tα-1 by the surface PS.DC2.4 engulfed apopototic bodies and Tα-1 binded with apoptotic bodies;ABs/Tα-1 were associated with DC2.4 phagosome-lysosome.2.Thymosin α-1 regulates the apoptotic bodies antigen presentation in dendritic cells2.1 Thymosin α-1 regulates the apoptotic bodies antigen presentation in dendritic cells in vitroSpleen cells of the mouse had a dose-dependent up-dependent secretion of IFN-γ.OVA specific response T cells had been induced.Dendritic cells after apoptotic bodies engulfment showed a downregulated antigen presentation: decreased proportion of IFN-γ+CD3+CD8-and IFN-γ+CD3+CD8+ cells in spleen cells.Tα-1 upregulated antigen presentation in DC: increased proportion of IFN-γ+CD3+CD8-and IFN-γ+CD3+ CD8+ cells in spleen cells and secretion of IFN-γ.2.2 Thymosin α-1 enhances the chemotherapy efficacy in the treatment of melanomaOnly Tα-1 cannot depress the tumour growth.But Low-Dose DDP can depress the growth.However,Low-Dose DDP and Tα-1 have a significant depression on tomour growth.Compared with other three groups,DDP and Tα-1 group showed the highest proportion of DC and CD8α+DC cells in lymph node cells;the highest expression of CD86 and MHC Ⅱ;the highest proportion of IFN-γ+ CD3+ CD8-and IFN-γ+ CD3+ CD8+ cells in spleens cells and lymph nodes;the highest secretion of IFN-γ from TILs.Conclusion:1.Thymosin α-1 bind with surface PS on apoptotic bodies is engulfed by dendritic cells.Thymosin α-1 enhances the IL-12 secretion,CD86 and MHCII expression of dendritic cells after apoprtotic bodies engulfment;2.Thymosin α-1 upregulates the antigen presentation in dendritic cells after apoprtotic bodies engulfment,and has anti-tumour effects with DDP;...