1、肿瘤微环境简介肿瘤微环境简介苏州大学免疫学系 居颂光.？.Sid P.Kerkar and Nicholas P.Restifo.Cancer Res 2012;72:3125-3130.肿瘤微环境肿瘤微环境如果仅仅关注肿瘤细胞，那我们在盲人摸象如果仅仅关注肿瘤细胞，那我们在盲人摸象.肿瘤研究肿瘤研究肿瘤细胞研究肿瘤细胞研究一个世纪的观念：肿瘤是局部组织一个世纪的观念：肿瘤是局部组织/器官细胞异常生长形成的疾病器官细胞异常生长形成的疾病 肿瘤是个系统性疾病肿瘤是个系统性疾病 肿瘤是一种组织（间质、血管、微环境），不是一堆肿瘤细胞肿瘤是一种组织（间质、血管、微环境），不是一堆肿瘤细胞 免疫功能失
2、控，肿瘤患者的免疫系统不是简单的低下：免疫功能失控，肿瘤患者的免疫系统不是简单的低下：保护肿瘤生长的免疫细胞：功能增强保护肿瘤生长的免疫细胞：功能增强 抑制肿瘤细胞的免疫细胞：功能低下抑制肿瘤细胞的免疫细胞：功能低下 .肿瘤的免疫编辑肿瘤的免疫编辑免疫成分在肿瘤发生过程中免疫成分在肿瘤发生过程中的作用的作用.Dunn G,et al.Nature immunology.2002.3(11):991Elimination corresponds to immunosurveillianceThe host immune system and any tumor variant that has
3、survived in the elimination process enter into the dynamic equilibrium:*lymphocytes and IFN-exert potent selection pressure.*A tumor bed is containing many genetically unstable and rapidly mutating tumor cells.Survival tumor variants that have acquired insensitivity to immunologic detection and/or e
4、limination begin to expand in uncontrol manner.Dunn G,et al.Nature immunology.2002.3(11):991.Solid tumors reach a certain size:Grow invasively,require an enhanced blood supply.Inflammatory signals leading to recruitment of cells of innate immune system:macrophages,DCs,-T,NK and NKT.Produce IFN-.The
5、initial IFN-starts a cascade of immune response:1.Induction of chemokines:CXCL10,CXCL9,CXCL11.Block neovascularization of tumor,recruit macrophages,DCs,-T,NK and other immune cells.2.Antiproliferation of developing tumor.3.The activation of cytocidal activity in macrophages and NK cells entering the
6、 tumor.Dead tumor cells or tumor cells debris are ingested by DC and are trafficked to the draining lymph node.1.Tumor growth is kept in check by the cytocidal NK and mocrophages.2.CD4+T and CD8+T cells that are specific for tumor antigens are developing in draining lymph node.Tumor specific CD4+T a
7、nd CD8+T cells home to tumor site along a chemokine gradient.Dunn G,et al.Nature immunology.2002.3(11):991Elimination corresponds to immunosurveillianceThe host immune system and any tumor variant that has survived in the elimination process enter into the dynamic equilibrium:*lymphocytes and IFN-ex
8、ert potent selection pressure.*A tumor bed is containing many genetically unstable and rapidly mutating tumor cells.Survival tumor variants that have acquired insensitivity to immunologic detection and/or elimination begin to expand in uncontrol manner.肿瘤细胞肿瘤细胞.肿瘤抗原肿瘤抗原Olivera J.Finn,Ph.D.N Engl J M
9、ed 2008;358:2704-15Olivera J.Finn,Ph.D.N Engl J Med 2008;358:2704-15.肿瘤抗原的加工提呈肿瘤抗原的加工提呈Olivera J.Finn,Ph.D.N Engl J Med 2008;358:2704-15.肿瘤干细胞肿瘤干细胞.Knutson K,et al.Cancer Immunol Immunother 2005,54:721728APCTHTregCytoxic T cellsOthers IFNIL-2IL-12GranzymePerforinAbsothersTumor microenvironment:.N En
10、gl J Med 2006;355:1253 -1261.Cancer stem cellNature Reviews Cancerdoi:10.1038/nrc1232Tumor Heterogeneity and Cancer Stem Cells.肿瘤微环境中的免疫稳态肿瘤微环境中的免疫稳态失衡失衡.Nature.2005,5:263.肿瘤患者的免疫状态是有别于正常人的肿瘤患者的免疫状态是有别于正常人的免疫稳态的。免疫稳态的。.肿瘤微环境中的免疫细胞肿瘤微环境中的免疫细胞.TH17 cells in tumour immunity andImmunotherapy.NATuRE REVI
11、EWS|Immunology.doi:10.1038/nri2742Differentiation of helper T cell subsets.TH17 cells and antitumour immunity.肿瘤微环境中的抑制性成分Treg细胞.Treg细胞定义 1995由Sakaguchi等发现了机体重要的负性免疫调节成分调节性CD4+CD25+T细胞（regulatory T cell,Treg）。随后的研究根据细胞内标记分子Foxp3和细胞膜分子CD127表达与否，将经典的Treg细胞定义为CD4+CD25+Foxp3+CD127low/neg T细胞。通过细胞间接触依赖机制
12、发挥作用或分泌IL-10和TGF-等细胞因子，Treg细胞抑制CD4+25-T细胞、CD8+T细胞和树突状细胞等其他免疫细胞的活性和功能。.大量的研究报道表明:CD4+CD25+Treg细胞是抑制机体抗肿瘤免疫应答的关键成分之一，例如：Woo等发现在非小细胞肺癌和卵巢癌患者的肿瘤浸润淋巴细胞中Treg的含量明显增多；在胃肠道肿瘤患者中CD4+CD25-T细胞明显减少而Treg细胞数量增多，而且Treg细胞数量的增高与较差的预后和较低的生存率高度相关；Curiel 等报道，在卵巢癌中Treg细胞能抑制T细胞介导的特异性抗肿瘤免疫应答，并且有利于肿瘤的生长；因此研究者希望通过减少Treg细胞的数量
13、或降低其活性来开辟治疗肿瘤免疫治疗的新途径。例如，以Foxp3为靶点清除荷瘤小鼠体内的Treg细胞后肿瘤会消退。.Treg细胞的作用机制.非小细胞性肺癌（NSCLC）与Treg新亚群.Fig 1.Fig.2.Fig 3.Fig 4.CD8+TCD8+T细胞在肿瘤免疫中的作用细胞在肿瘤免疫中的作用肿瘤微环境中的肿瘤微环境中的“正性正性”免疫成分免疫成分.Gadd45-/-mice or Het/WT mice were injected into intradermally of flank skin with 2.5X104 B16 cells/mouse.Tumor sizes were m
14、easured every 2 days.Tumor inject siteGadd45b KO mice were challenged with melanoma cells:B16 Freeze down the tissue of tumor site.Fig 4 Gadd45-/-mice or WT mice were injected subcutaneously with 2.5X104 B16 cells/mouse,tumor sizes were measured every 2 days.These data are from 3 separated experimen
15、ts.5.Both WT and Gadd45b KO T cells are able to infiltrate into tumor sites.Gadd45-/-mice or WT mice were injected intradermally of flank skin with 5X104 B16 cells/site/20ul PBSSkin of injected sites or opposite sites was resected and freeze down immediatelySpleen Cells Adoptive Transfer and B16 ino
16、culationTumor inject siteCheck tumor formation from injected sites500 RadWTKO500 RadDonorHost.6.Gadd45 is important for lymphocytes in tumor surveillanceP0.05*.CD4+T Cells Adoptive Transfer and B16 inoculation500 RadWT CD4+TKO CD4+T500 RadDonor HostWT splenocytes depleted CD4+TWT splenocytes deplete
17、 CD4+T+.7.Gadd45b KO does not affect function of CD4+T cells in tumor surveillanceKO:Transfer with CD4+T cell depleted spleen cells from WT mice +CD4+T cells from Gadd45b KO micePBS:Only transfer PBSWT:Transfer with CD4+T cell depleted spleen cells from WT mice +CD4+T cells from Gadd45b Het or WT mi
18、ce.CD8+T Cells Adoptive Transfer and B16 inoculation500 RadWT CD8+TKO CD8+T500 RadDonor HostWT splenocytes depleted CD8+TWT splenocytes deplete CD8+T+.8.Gadd45b KO causes function defect of CD8+T cells in tumor surveillanceKO:Transfer with CD8+T cell depleted spleen cells from WT mice +CD8+T cells f
19、rom Gadd45b KO micePBS:Only transfer PBSWT:Transfer with CD8+T cell depleted spleen cells from WT mice +CD8+T cells from Gadd45b Het or WT miceP0.05*.Gadd45b is expressed in CD8+T cells during their activationFigure 1 Gadd45b message is induced by TCR signaling and costimulation.Naive CD8+T cells we
20、re stimulated with platebound anti-CD3(-CD3)plus-CD28 and Th1 condition for 0h,1 h,12h,24h,48h and 96h.The Gadd45b message was detected with RT-PCR.2.Gadd45 is important for the function of CD8+T cells.IL12/IL18-CD3.MAP kinase profile activated in CD8+T cells-CD3(10 g/ml)IL-12(10 ng/ml)+IL-18(30 ng/
21、ml)Figure 3 Gadd45 mediates p38 activation by TCR signaling or IL-12 plus IL-18 in CD8+T cells.(a)Effector CD8+T cells were stimulated(times,above lanes)with plate-bound anti-CD3(-CD3;10 g/ml)or(b)IL-12(10 ng/ml)and IL-18(30 ng/ml).Cell extracts were analyzed by immunoblot with anti-phospho-ERK(-pho
22、spho-ERK),-phospho-JNK and-phospho-p38.p-,phosphorylated.Gadd45b KO causes function defect of tumor infiltrating CD8+T cellsFig 5 Gadd45 KO cause tumor infiltrating CD8+T cell function defect in vivo.Tumor mass was removed at day 13 after B16 F0 cells inoculation and digested with collagenase I.Cell
23、s were stimulated with phorbol 12-myristate 13-acetate(50 ng/ml)and ionomycin(500 ng/ml)for 6 h and incubated for the last 3 h with Brefeldin A.Cells producing IFN-was identified with intracellular cytokine staining.Type,Density,and Location of Immune Cells Within Human Colorectal Tumors Predict Cli
24、nical OutcomeGalon J,et al.29 SEPTEMBER 2006 VOL 313 SCIENCE.Type,Density,and Location of Immune Cells Within Human Colorectal Tumors Predict Clinical OutcomeGalon J,et al.29 SEPTEMBER 2006 VOL 313 SCIENCE(B)Comparison of the mean of immune cell densities in the CT and IM from patients with tumor re
25、currence(black bars)or without tumor recurrence(white bars).肿瘤微环境中的免疫分子肿瘤微环境中的免疫分子.肿瘤微环境中的Immune checkpointsNATURE REVIEWS|CANCER 2012 doi:10.1038/nrc3239.Multiple co-stimulatory and inhibitory interactions regulate T cell responses.Immune checkpoints regulate different components in the evolution o
26、f an immune response.Two general mechanisms of expression of immune-checkpoint ligands on tumour cells.Implications of the adaptive immune resistance mechanism for combinatorial immunotherapy of cancer.Clinical responses and immune-mediated toxicities on antibody blockade of the CTLA4 mediated immun
27、e checkpoint.Blockade of PD-1 or CTLA-4 Signaling in Tumor Immunotherapy.10.1056/nejme1205943.肿瘤微环境中的免疫成分正与负的肿瘤微环境中的免疫成分正与负的相对性相对性.SGN-40 CP870,893 CHIR-12.12B lymphomaSolid tumorMultiple myelomaThree CD40mAbs in preclinical trials.CD40 signaling regulates angiogenesis.Giving blood:a new role for CD
28、40 in tumorigenesis Bergmann S,Pandolfi PP.J Exp Med.2006 Oct 30;203(11):2409-12.Epub 2006 Oct 16.Growth factorsTumorvasculatureDisseminatingtumor cellsInvasionCell cycle controlProliferatingtumor cellsAngiogenesisSignal transductionDouble-edged weapon single-edged for tumor?.Knutson K,et al.Cancer Immunol Immunother 2005,54:721728APCTHTregCytoxic T cellsOthers IFNIL-2IL-12GranzymePerforinAbsothersTumor microenvironment:.谢谢 谢！谢！.
