TLR1/2信号促CD8~+T细胞功能及其机制
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摘要
目的:探讨Toll受体1/2(Toll like receptor1/2,TLR1/2)信号对荷瘤小鼠来源的CD8~+T细胞功能的影响及其可能机制。方法:利用小鼠Lewis肺癌细胞株3LL建立小鼠肺癌荷瘤模型,MACS分选小鼠脾CD8~+T细胞;体外经PBS或TLR1/2激动剂BLP刺激后,Real-time PCR和流式细胞术分别从基因和蛋白水平检测CD8~+T细胞的TLR分子表达;用ELISA和流式细胞术检测经PBS或BLP刺激后的CD8~+T细胞分泌细胞因子和增殖的能力,并用关键信号分子抑制剂分析可能的分子机制。结果:与PBS对照组相比,TLR1/2激动剂BLP不但有效上调荷瘤机体CD8~+T细胞TLR1和TLR2分子的基因水平[TLR1:(0.353±0.015)vs(0.101±0.017),P<0.01;TLR2:(0.232±0.031)vs(0.080±0.004),P<0.05]及蛋白水平(P<0.05),而且显著促进CD8~+T细胞分泌功能性细胞因子[IFN-γ:(2 375±305)vs(850±50),P<0.05;IL-2:(1 600±200)vs(350±50),P<0.05]和增殖的能力(P<0.05),这一效应依赖于NF-KB和P38通路。结论:TLR1/2信号直接作用于荷瘤小鼠的CD8~+T细胞并促进其功能,该研究既丰富了TLR的作用范围,也为基于TLR激动剂的肿瘤生物治疗提供了实验依据。
Objective;To investigate the effect of Toll like receptor 1/2(TLR1/2) signaling on CD8~+T cells derived from 3LL tumor-bearing mice and its underlying mechanisms.Methods;3LL Lewis lung carcinoma cell line was used to establish tumor-bearing mice model,of which CD8~ + T cells were purified from the spleen by MACS.CD8~ + T cells were stimulated in vitro with PBS or TLR1/2 agonist,BLP,and then the gene and protein expression levels of TLRs in CD8~ +T cells were measured by Real-time PCR and Flow cytometry.What' s more,cytokine secretion and proliferation of CD8~ +T cells after PBS or BLP stimulation were detected by ELISA and Flow cytometry.The inhibitors of key signal molecules were used to explore the underlying mechanisms of BLP influencing CD8~+ T cells derived from 3LL-bearing mice.Results:Compared with PBS group,BLP not only greatly increased the expressions of TLR1 and TLR2 genes(TLR1;[0.353 ±0.015]vs[0.101 ±0.017],P<0.01;TLR2:([0.232 ±0.031]vs[0.080 ±0.004],P<0.05) and proteins(P <0.05),but also significantly enhanced the functional cytokine secretion(IFN-γ:[2 375 ±305]vs[850 ±50],P < 0.05;IL-2:[1 600 ± 200]vs[350 ± 50],P < 0.05);in addition,it promoted the proliferation of CD8~+ T cells(P<0.05).All of these were dependent on NF-kB and P38 pathways.Conclusion;TLR1/2 signaling could directly promote the functions of CD8~ + T cells derived from 3LL-tumor bearing mice,which might enrich the scope of TLRs and also provide the basis for TLR agonist-based tumor immunotherapy.
Objective;To investigate the effect of Toll like receptor 1/2(TLR1/2) signaling on CD8~+T cells derived from 3LL tumor-bearing mice and its underlying mechanisms.Methods;3LL Lewis lung carcinoma cell line was used to establish tumor-bearing mice model,of which CD8~ + T cells were purified from the spleen by MACS.CD8~ + T cells were stimulated in vitro with PBS or TLR1/2 agonist,BLP,and then the gene and protein expression levels of TLRs in CD8~ +T cells were measured by Real-time PCR and Flow cytometry.What' s more,cytokine secretion and proliferation of CD8~ +T cells after PBS or BLP stimulation were detected by ELISA and Flow cytometry.The inhibitors of key signal molecules were used to explore the underlying mechanisms of BLP influencing CD8~+ T cells derived from 3LL-bearing mice.Results:Compared with PBS group,BLP not only greatly increased the expressions of TLR1 and TLR2 genes(TLR1;[0.353 ±0.015]vs[0.101 ±0.017],P<0.01;TLR2:([0.232 ±0.031]vs[0.080 ±0.004],P<0.05) and proteins(P <0.05),but also significantly enhanced the functional cytokine secretion(IFN-γ:[2 375 ±305]vs[850 ±50],P < 0.05;IL-2:[1 600 ± 200]vs[350 ± 50],P < 0.05);in addition,it promoted the proliferation of CD8~+ T cells(P<0.05).All of these were dependent on NF-kB and P38 pathways.Conclusion;TLR1/2 signaling could directly promote the functions of CD8~ + T cells derived from 3LL-tumor bearing mice,which might enrich the scope of TLRs and also provide the basis for TLR agonist-based tumor immunotherapy.
引文
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[21]SABADO R L,PAVLICK A,GNJATIC S,et al.Resiquimod as an immunologic adjuvant for NY-ESO-1 protein vaccination in patients with high-risk melanoma[J].Cancer Immunol Res,2015,3(3):278-287.DOI:10.1158/2326-6066.CIR-14-0202.
[22]YANG G L,ZHANG L H,BO J J,et al.Increased expression of HMGB1 is associated with poor prognosis in human bladder cancer[J].J Surg Oncol,2012,106(1):57-61.DOI:10.1002/jso.23040.
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[2]LIEW F Y,XU D,BRINT E K,et al.Negative regulation of tolllike receptor-mediated immune responses[J].Nat Rev Immunol,2005,5(6):446458.DOI:10.1038/nril630.
[3]ZHANG Y,LUO F,CAI Y,et al.TLR1/TLR2 agonist induces tumor regression by reciprocal modulation of effector and regulatory Tcells[J].J Immunol,2011,186(4):1963-1969.DOI:10.4049/jimmunol.1002320.
[4]LIU H,KOMAI-KOMA M,XU D,et al.Toll-like receptor 2 signaling modulates the functions of CD4~+CD25~+regulatory T cells[J].Proc Nad Acad Sci USA,2006,103(18):7048-7053.DOI:10.1073/pnas.0601554103.
[5]JOSEPH A M,SRTVASTAVA R,ZABALETA J,et al.Cross-talk between 4-1BB and TLR1-TLR2 signaling in CD8~+T cells regulate TLR2's costimulatory effects[J].Cancer Immunol Res,2016,4(8):708-716.DOI:10.1158/2326-6066.CIR-15-0173.
[6]KAWAI T,AKIRA S.Toll-like receptors and their crosstalk with other innate receptors in infection and immunity[J].Immunity,2011,34(5):637-650.DOI:10.1016/j.immuni.2011.05.006.
[7]IWASAKI A,MEDZHITOV R.Toll-like receptor control of the adaptive immune responses[J].Nat Immunol,2004,5(10):987-995.DOI:10.1038/ni1112.
[8]ZHANG M,LUO F,ZHANG Y,et al.Pseudomonas aeruginosa mannose-sensitive hemagglutinin promotes T-cell response via tolllike receptor 4-mediated dendritic cells to slow tumor progression in mice[J].J Pharmacol Exp Ther,2014,349(2):279-287.DOI:10.1124/jpet.113.212316.
[9]ZHANG Y,LUO F,LI A,et al.Systemic injection of TLR1/2agonist improves adoptive antigen-specific T cell therapy in gliomabearingmice[J].Clin Immunol,2014,154(1):26-36.DOI:10.1016/j.dim.2014.06.004.
[10]YUAN Y,JIANG Y C,SUN C K,et al.Role of the tumor microenvironment in tumor progression and the clinical applications(Review)[J].Oncol Rep,2016,35(5):2499-2515.DOI:10.3892/or.2016.4660.
[11]YANG Z Z,NOVAK A J,STENSON M J,et al.Intratumoral CD4~+CD25~+regulatory T-cell-mediated suppression of infiltrating CD4~+T cells in B-cell non-Hodgkin lymphoma[J].Blood,2006,107(9):3639-3646.DOI:10.1182/blood-2005-08-3376.
[12]BEDOGNETTI D,MACCALLI C,BADER S B,et al.Checkpoint inhibitors and their application in breast cancer[J].Breast Care(Basel),2016,11(2):108-115.DOI:10.1159/000445335.
[13]BARRETT D M,GRUPP S A,JUNE C H.Chimeric antigen receptor-and TCR-modified T cells enter main street and wall street[J].J Immunol,2015,195(3):755-761.DOI:10.4049/jimmunol.1500751.
[14]MAUS M V,JUNE C H.Making better chimeric antigen receptors for adoptive T-cell therapy[J].Clin Cancer Res,2016,22(8):1875-1884.DOI:10.1158/1078-0432.CCR-15-1433.
[15]TEL J,BENITEZ-RIBAS D,JANSSEN E M,et al.Dendritic cells as vaccines:key regulators of tolerance and immunity[J/OL].Mediators Inflamm,2016,2016:5789725[2016-08-20].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906213/.DOI:10.1155/2016/5789725.
[16]MANRIQUE S Z,DOMINGUEZ A L,MIRZA N,et al.Definitive activation of endogenous antitumor immunity by repetitive cycles of cyclophosphamide with interspersed Toll-like receptor agonists[J].Oncotarget,2016,7(28):42919-42942.DOI:10.18632/oncotarget.10190.
[17]HARTMAN L L,CRAWFORD J R,MAKALE M T,et al.Pediatric phaseⅡtrials of poly-ICLC in the management of newly diagnosed and recurrent brain tumors[J].J Pediatr Hematol Oncol,2014,36(6):451457.DOI:10.1097/MPH.0000000000000047.
[18]ROSENFELD M R,CHAMBERLAIN M C,GROSSMAN S A,et al.A multi-institution phaseⅡstudy of poly-ICLC and radiotherapy with concurrent and adjuvant temozolomide in adults with newly diagnosed glioblastoma[J].Neuro Oncol,2010,12(10):1071-1077.DOI:10.1093/neuonc/noq071.
[19]MICALI G,LACARRUBBA F,NASCA M R,et al.Topical pharmacotherapy for skin cancer:part A.Clinical applications[J/OL].J Am Acad Dermatol,2014,70(6):979[2016-08-20].http://www.sciencedirect.com/science/article/pii/S0190962214010020.DOI:10.1016/j.jaad.2013.12.037.
[20]DIETSCH G N,RANDALL TD,GOTTARDO R,et al.Late-stage cancer patients remain highly responsive to immune activation by the selective TLR8 agonist motolimod(VTX-2337)[J].Clin Cancer Res,2015,21(24):5445-5452.DOI:10.1158/1078-0432.CCR-15-0578.
[21]SABADO R L,PAVLICK A,GNJATIC S,et al.Resiquimod as an immunologic adjuvant for NY-ESO-1 protein vaccination in patients with high-risk melanoma[J].Cancer Immunol Res,2015,3(3):278-287.DOI:10.1158/2326-6066.CIR-14-0202.
[22]YANG G L,ZHANG L H,BO J J,et al.Increased expression of HMGB1 is associated with poor prognosis in human bladder cancer[J].J Surg Oncol,2012,106(1):57-61.DOI:10.1002/jso.23040.
[23]Of,WU K,ZHAO E,et al.HMGB1 recruits myeloid derived suppressor cells to promote peritoneal dissemination of colon cancer after resection[J].Biochem Biophys Res Commun,2013,436(2):156-161.DOI:10.1016/j.bbrc.2013.04.109.