ICOS Signal Facilitate Foxp3 Transcription To Favor Suppressive Function Of Tregs

BackgroundRegulatory T cells(Tregs)are the important mechanism of peripheral immune tolerance.Tregs play an important role in maintaining immune balance by releasing immunosuppressive cytokines or by inhibiting the activation of T cells.In mice and humans,the lack of Tregs can lead to fatal immune disease,In addition,Tregs function enhanced will lead to the body’s immune tolerance to the tumor.Treg cells play an immunosuppressive function in tumor microenvironment and antitumor immunity.Increased Treg can lead to the development of tumor immune escape,which is not conducive to the immunotherapy of tumor.Therefore,it is very important to explore the regulatory mechanism of Treg cells.Inducible costimulator(ICOS),which is expressed on the surface of activated T cells,is a member of the CD28 family.According to whether they express ICOS or not,Tregs can further divide into ICOS+Tregs and ICOS-Tregs subsets.In including breast cancer,ovarian cancer,stomach cancer,liver cancer,melanoma,Tregs infiltrating in tumor were mostly ICOS+Tregs.But whether there are ICOS+Tregs in prostate cancer has not been reported.In vitro experiments,ICOS+Tregs are characterized of highly suppressive and superior survival properties compared with ICOS-Tregs.Although similar in structure,ICOS and CD28 appear to play different roles in modulating the activation of T cells.ICOS possesses the YMFM motif that lacking of the asparagine disability,can not be recruit Grb2,API cooperative with NFAT to activate T cells.Treg inhibitory function depends on the cooperation of Foxp3 with NFAT.Based on these theories,we postulated that ICOS is insufficient to activate the NFAT/AP-1 site for the activation of T cells due to it fails to recruit Grb2 for the Ras-Erk1/2-AP1 pathway,relatively optimize the interaction of NFAT/Foxp3 that promotes the function of Tregs,ICOS only binding to PI3K for activation of PI3K-Akt pathway to promote cell survival.Objective1.To explore the relationship between ICOS+Tregs cells and prostate cancer.2.To clarify whether ICOS promotes Foxp3 transcription by inhibiting Rp-Erkl/2-AP1 pathway,ultimately promotes Treg function.3.To explore whether ICOS through the activation of PI3K-Akt pathway to promote the survival of Tregs.MethodsIn order to confirm Treg in tumor,Prostate cancer tissue samples were embedded in paraffin for paraffin sections,and then the Foxp3 and ICOS were detected by immunofluorescence double stainingThe erythrocytes were deprived by subjecting the samples to erythrocytolysin treatment and centrifugation.Then CD4+CD25+Tregs were isolated from the murine splenocytes through positive magnetic selection and negative magnetic selection.The CD4+CD25+Tregs were cultured in RPMI 1640 medium containing 10%FBS,at 37℃ under 5%CO2.Anti-CD3 antibody(activated TCR)and anti-CD28 antibody or anti-ICOS antibody were added to stimulate the TregsIn order to examine ICOS on T-regulatory cells survival,apoptosis was detected by flow cytometry.Tregs were cultured upon the stimulation of anti-CD3 antibody(activated TCR)and anti-CD28 antibody or anti-ICOS antibody for four days.Cells were Annexin V-FITC/PI stained then analyzed by flow cytometry.For Western blotting cells were lysed by ice-cold lysis buffer.The proteins were separated by SDS-PAGE with 10%polyacrylamide gel and transferred onto polyvinylidene fluoride membranes.After blocking,the membranes were incubated with anti-Ras antibody;anti-Erk1/2 antibody;anti-phospho-Erkl/2 antibody;anti-Akt antibody;anti-phospho-Akt antibody,anti-Bcl2 antibody.After washing with TBST,the membranes were incubated with HRP-conjugated IGg at room temperature for 1h.Immunoreactive bands were detected using the enhanced chemiluminescence detection kit.For Co-immunoprecipitation analysis,cells were lysed by ice-cold lysis buffer,the proteins immunoprecipitation with NFAT1 antibody,Western bloting detected the protein level of Foxp3 and c-Jun levelsFor reverse transcription real-time polymerase chain reaction(RT-qPCR),total RNA was extracted from each stimulated groups by using RNAiso Plus.RNA synthesised the complementary deoxyribonucleic acid(cDNA).PCR and amplification monitoring were run by using a Applied Biosystems 7500 Real Time PCR System to detect the expression of various cytokines.ResultsImmunofluorescence confirmed that Tregs infiltrating in prostate cancer were mostly ICOS+Tregs.To examine the effect of ICOS on Tregs,Tregs were cultured in the presence of anti-CD3 antibody and anti-CD28 antibody or anti-ICOS antibody,or anti-CD3 antibody(activated TCR)alone to stimulate the cells for 4 days.Apoptosis was detected by flow cytometry.The results show that ICOS is contributes to Tregs survival.In order to determine if ICOS contributes to Tregs function,we examined the protein levels of Ras,Erk1/2,the phosphorylated Erk1/2,Akt,the phospho-Akt,Bcl2,Foxp3 and c-Jun.The immune co-precipitation and Western blotting results showed that the expression of p-Akt and Bcl2 was up-regulated after anti-ICOS antibody was applied to Tregs,indicating that ICOS may be mediated by PI3K-Akt pathway resistance to apoptosis.The expression of Ras was down-regulated,the p-Erk was significantly decreased after anti-ICOS antibody was applied to Tregs,and the c-Jun that co-precipitated with NFAT was also down-regulated,Foxp3 that co-precipitated with NFAT was increased,indicating that ICOS can not intivate the Ras-Erk 1/2-AP1 pathway,thereby promoting the inhibition functions of Tregs.The results of Q-PCR showed that ICOS promoted the secretion of IL-4,IL-10,TGF-β and inhibited the secretion of IL-2 and IL-6 for Treg immunosuppressive function.Conclusions1、Tregs infiltrating in prostate cancer were mostly ICOS+Tregs.ICOS+Tregs were positively correlated with the development of prostate cancer.2、ICOS can not activate Ras-Erkl/2-AP1 pathway,than facilitate the NFAT:Foxp3 over NFAT;AP-1 to favor suppressive function.ICOS mediated the cytokine secretion for the immunesuppressive function of Tregs.3、ICOS through the activation of PI3K-Akt pathway to promote the survival of Tregs.