2013-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/649667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(tumorinfiltrating 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。EC細胞和TILS粘合之間的相互作用，可能是一個有效的抗癌免疫反應的關鍵。現在已知ICAM- 1和LFA-1之間的粘附相互作用對T淋巴細胞的活化的重要性，而且LFA- 1和ICAM- 1的相互作用可以增加T淋巴細胞的細胞毒殺作用，引起細胞毒性顆粒分泌。然而最近發現T淋巴細胞上一種和粘合作用相關的分子αEβ7(CD103)，其和對於腫瘤的相互作用對T淋巴細胞的活化、毒殺及影響卻鮮為人知。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CD4+CD25+ Tregs造成CD8+毒殺細胞的功能失調。進一步吾人欲了解CD103在腫瘤微環境中對毒殺性T細胞的免疫調控。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及癌細胞，探討TILs中CD8+CD103+ T細胞及其上CD4, CD8,CD56, CD103, CD25, CD28, CD11a (LFA-1), HLA-DR, CD45RO, CCR7 和62L的表現，另外分析癌細胞上E-cadherin和ICAM-1的表現，進一步探討CD8+CD103+ T細胞分泌的cytokine表現，最後探討CD103的表現對毒殺性T細胞其毒殺力之影響。第一年之研究主要利用吾人所發展出的mechanical dispersal technique分離腫瘤內浸潤淋巴球，以流體細胞儀分析CD8+CD103+ T細胞及其上CD4, CD8, CD56, CD103, CD25,CD28, CD11a (LFA-1), HLA-DR, CD45RO, CCR7 和62L的表現，另外也會分析癌細胞上E-cadherin和ICAM-1的表現。第二年之研究我們將以ELISA檢測CD8+CD103+ T細胞分泌Th1 cytokines (IFN-γ、IL-12和TNF-α)和Th2 cytokines (IL-4和IL-10)的狀態，進一步以流體細胞儀分析CD103與毒殺性T細胞內毒殺顆粒（包括granzyme B及perforin）的表現。第三年之研究主要建立腫瘤細胞與自體免疫細胞之毒殺模式(Cytotoxicity Assay)，來測定CD103的表現與E-cadherin的相互作用對毒殺性T細胞其毒殺力之影響，以期能找出免疫細胞抑制癌細胞之途徑，可用於了解腫瘤癌細胞與免疫細胞間的互動關係。以進行腫瘤免疫調控(immuno -regulation)之研究。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: CD8+ T cells (cytotoxic T lymphocytes, CTLs) play a critical role in antitumor immuneresponse. Previously, we have demonstrated that human cancer cells may alter the functionalcomposition of anti-tumor effector cells, including CD8+ cytotoxic T cells, within the tumormilieu. We defined predominant Th2/Tc2 patterns of cytokine expression in CD3+CD8+ T-cellsubsets of tumor-infiltrating lymphocytes (TILs) in the human cancer microenvironment(3).We have further illustrated that cancer-derived mediators such as metalloproteinase areresponsible for the immunosuppressive conditions of TILs in human cancer, which arestrongly associated with clinical prognosis. We also demonstrate the global HLA class I genemutations in human cervical cancer, which can lead to general down-regulated expression ofHLA class I molecules. Furthermore, we previously demonstrated that increased expressionof CD94/NKG2A restricted to tumor-infiltrating CD8+ T cell subsets may shape thecytotoxic responses, which indicate a possible role of tumor escape from host immunity inhuman endometrial cancer (EC). Thereafter, we also reported the immunoregulatory effectsof T regulatory cells (Tregs) in human EC. Tregs in the tumor microenvironment mayabrogate CD8+T cell cytotoxicity in a granzyme B-and perforin-dependent conduit.Decreases in both Th1 cytokines and cytotoxic enzymes are relevant for Treg-mediatedrestraint of tumor clearance in vivo. In contrast, many TILs have been described in EC,suggesting that they may contribute to control of the tumor.Since little is known about the actual function ofαE(CD103)β7 integrin on Tlymphocytes in human EC, our further studies try to establish a novel costimulatory role ofCD103 in tumor-specific CTL activation. We propose that the selective expression of integrinαE(CD103)β7 by the TILs was crucial for directional cytotoxic action in the E-cadherin+tumor leading to cell death. This study will analyze the immune status of EC patients in detailto obtain a clear idea of their innate, humoral and cellular immune functions.In the first year project, we will investigate the expression of CD4, CD8, CD56, CD103,CD25, CD28, CD11a (LFA-1), HLA-DR, CD45RO, CCR7 and 62L on CD8+CD103+ Tcells from PBMC and TILs in human EC. Moreover, the expression of E-cadherin andICAM-1 on EC cells will be also studied. In the second year project, we will assess thecytokine secretory status of CD8+CD103+ T cells from PBMCs and TILs of Th1 cytokines(IFN-γ, IL-12 and TNF-α) and Th2 cytokines (IL-4 and IL-10). In addition, theintracellular cytotoxic molecules, perforin and granzyme B, will be assessed. In the thirdyear project, we will determine whether the CD8+CD103+ T cells from EC patients arefunctional CTLs. The correlation between prevalence of CD8+CD103+ T cells and clinicaloutcome will be clarified for the patients.毒殺性T細胞子宮内膜癌CD3+CD103+T細胞腫瘤內浸潤淋巴球αEβ7 intergrinE-cadherincytotoxic T lymphocytesCD8+CD103+tumor-infiltrating lymphocytesendometrial cancerαE(CD103)β7 integrinE-cadherin