2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657766摘要：子宮内膜癌是女性生殖系統最常見的侵襲癌，自1950年以來，雖然子宮内膜癌的死亡率降低超過60%，其發生率卻明顯增加，而且年齡層有逐漸降低的趨勢。我們知道腫瘤細胞在癌化過程中，一方面需要大量繁殖增生，另一方面則需對抗來自宿主體內免疫系統的清除作用，才能達到其侵入深部組織與蔓延的目的。腫瘤內浸潤淋巴球(tumor infiltrating lymphocyte, TILs)一直被探討是否影響癌症病患本身的免疫反應及疾病的預後，而T細胞在TIL中對腫瘤免疫監督與免疫逃脫上扮演一重要的角色。抗原呈現細胞（antigen presenting cell, APC）與T細胞的活化有關，APC上許多配體(ligand)的訊號表達，可以是刺激性訊號或是抑制性訊號，而抑制性訊號可能產生一種免疫抑制網絡，以促進腫瘤進展和免疫逃避。程序性細胞死亡蛋白（Programmed cell death 1，PD1）是免疫檢查點的受體，會限制T細胞在末梢組織中對感染發炎性反應的時間，並限制自身免疫，以避免自體免疫疾病。活化的T細胞可以表現PD1受體，其可以與PD-L1（B7-H1）和PD-L2（B7-DC）結合，PD-L1可經由誘導表現在各種免疫細胞，而PD-L2主要表現在活化的巨噬細胞和樹突狀細胞。PD-1/ PD-L1的相互作用顯示出廣泛的免疫抑制，包括對抗病原體，腫瘤，和自身抗原的免疫反應。PD-L1除了表現在活化的免疫細胞，也被發現表現在人類癌症的細胞表面，而且癌症表現PD-L1與臨床預後差有相關。吾人之前的研究(NSC91-2314-B002-302, NSC91-2314-B002-397, NSC92-2314-B002-307)已證實人類癌症細胞會影響癌組織附近免疫細胞的組成，也發現腫瘤內浸潤淋巴球(TILs)有Th2/Tc2模式。吾人證實了HLA基因的突變造成癌細胞上HLA抗原的表現的減少，也發現免疫細胞抑制受體CD94/NKG2A明顯在TILs毒殺性T細胞的表現增加。吾人的研究(98-2314-B-002-106-MY3)也證明了子宮内膜癌病患TILs中Treg與CD8+記憶T細胞及effector cell的消長與疾病的關係。子宮内膜癌病患的腫瘤浸潤淋巴細胞與周邊血液細胞和正常子宮内膜組織的淋巴細胞確有相關分子型態表現差異，尤其是調節性淋巴細胞(CD4+CD25+)比例的增加。CD8+淋巴細胞在腫瘤中其細胞活化的分子(CD45RO)表現雖然增加，但毒殺作用的分子(granzyme B and perforin)卻是減少。經由這個研究，我們可以知道子宮内膜癌病患其腫瘤內PD-L1的表現造成TILs的功能失調。進一步吾人欲了解PD-1/PD-L1在腫瘤微環境中對TILs的免疫調控。吾人用免疫化學組織染色分析子宮内膜癌患者PD-L1和PD-L2的表現。吾人欲利用機械式研磨萃取法(mechanical dispersal technique)來獲得子宮内膜癌之腫瘤內浸潤淋巴球，探討TILs中CD4+，CD8+ T細胞及CD4+CD25+調節T細胞其上PD-1的表現，另外分析抗原呈現細胞單核淋巴球上PD-L1的表現，進一步探討單核淋巴球分泌的cytokine(TNF-α，IL-10，or IL-6)對PD-L1表現的影響，最後探討單核淋巴球上PD-L1的表現對CD4+，CD8+T細胞及CD4+CD25+調節T細胞免疫反應之影響。第一年之研究利用免疫化學組織染色分析子宮内膜癌患者PD-L1和PD-L2的表現，進一步利用吾人所發展出的機械式研磨萃取法分離腫瘤內浸潤淋巴球，以流體細胞儀分析CD4+，CD8+ T細胞及CD4+CD25+調節T細胞其上PD-1的表現，另外分析腫瘤微環境中單核淋巴球上PD-L1，CD80，CD86，和HLA-DR的表現。第二年之研究我們將分離周邊血液單核淋巴球與腫瘤上清液一起培養，並以流體細胞儀分析活化的單核淋巴球上PD-L1，CD80，CD86，和HLA-DR的表現，以ELISA檢測活化的單核淋巴球TNF-α，IL-10，和IL-6的分泌狀態。第三年之研究主要建立T細胞與活化的單核淋巴球之增殖模式(Proliferation Assay)，來測定PD-1的表現與PD-L1的相互作用對CD4+，CD8+ T細胞及CD4+CD25+調節T細胞免疫反應之影響。吾人預期可找出子宮内膜癌之腫瘤細胞與宿主免疫系統間之互動關係，進而了解免疫細胞於微環境中所產生腫瘤抑制現象之機轉。我們的研究目的在於找出免疫細胞抑制子宮内膜癌細胞的免疫監控，進一步希望對於未來發展免疫調節治療有所幫助。<br> Abstract: Cancer cells can modulate adjacent stroma to generate a supportive microenvironment,including altering the ratios of effector to regulatory T (Tregs) cells, affecting the functions ofantigen-presenting cells (APCs) and the expression of cosignaling molecules, which in turncreates an immunosuppressive network to promote tumor progression and immune evasion.Programmed cell death 1 (PD1) is an immune- checkpoint receptor and limit the activity of Tcells in peripheral tissues at the time of an inflammatory response to infection and to limitautoimmunity. PD-1/ PD-L1 interactions have been shown to inhibit a wide range of immuneresponses against pathogen, tumor, and self-antigens. In addition to being expressed onactivated immune cells, PD-L1 is found to express on cell surface in human cancers, and thisexpression was correlated with poor clinical prognosis.We have further illustrated that cancer-derived mediators such as metalloproteinase areresponsible for the immunosuppressive conditions of tumor-infiltrating lymphocytes (TILs)in human cancer, which are strongly associated with clinical prognosis. We also demonstratethe global HLA class I gene mutations in human cervical cancer, which can lead to generaldown-regulated expression of HLA class I molecules. Furthermore, we previouslydemonstrated that increased expression of CD94/NKG2A restricted to tumor-infiltratingCD8+ T cell subsets may shape the cytotoxic responses, which indicate a possible role oftumor escape from host immunity in human endometrial cancer (EC). Thereafter, we alsoreported the immunoregulatory effects of Tregs in human EC. Tregs in the tumormicroenvironment may abrogate CD8+T cell cytotoxicity in a granzyme B-andperforin-dependent conduit. Decreases in both Th1 cytokines and cytotoxic enzymes arerelevant for Treg-mediated restraint of tumor clearance in vivo. In contrast, many TILs havebeen described in EC, suggesting that they may contribute to control of the tumor.Since little is known about the expression and function of PD-L1 on APCs in theinflammatory activated stroma of human EC in situ and the actual interaction between PD1and PD-L1 on T lymphocytes. Our further studies try to establish a novel coinhibitory role ofPD1/PD-L1 in TILs of EC. We will study the expression of PD-L1 in monocytes/ M andtumor microenvironment in EC patients. We will demonstrate a major role of activatedmonocytes in EC microenvironment expressing PD-L1 molecules to suppress tumor-specificT cell function.In the first year project, we will use immunohistochemistry to assess the expression ofB7-H1 and B7-DC in the EC patients. We will use flow cytometry to investigate theexpression of PD-L1, CD80, CD86, or HLA-DR on monocytes and expression of CD25,PD-1, FoxP3, CD152 on T cells. In the second year project, we will use ELISA toevaluate the effects of autocrine cytokines (TNF-α, IL-10, or IL-6) from activatedmonocytes co-cultured with tumor supernatants. In addition, we will use flow cytometryto measure the expression of PD-L1, CD80, CD86, or HLA-DR on activated monocytes.In the third year project, we will use ELISA and BrdU proliferation assay to elucidatethe effect of PD-L1+ monocytes on T cell immunity. We will study immune status of ECpatients in detail to obtain a clear idea of their innate, humoral and cellular immunefunctions. Finally, the correlation between prevalence of PD-L1 expression ontumor-infiltrating monocytes and clinical outcome will be clarified for the EC patients.單核細胞子宮内膜癌調節T細胞腫瘤內浸潤淋巴球PD-1PD-L1endometrial cancermonocytesPD-1PDL1regulatory T celltumor-infiltrating lymphocytes