2017-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/649603摘要：癌症免疫治療的發展已經進入臨床應用，而目前CTLA-4抗體在腫瘤免疫調控，也由試驗階段開始運用於癌症患者的治療上。然而，現階段對於細胞激素(cytokines)與T細胞活化受體(T cell receptor, TCR)，相關的共同刺激活化分子與受體(TCR-related co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2), CTLA-4 CD28 and receptors)共同作用下的免疫調控並不清楚。尤其在癌腫瘤浸潤淋巴球（tumor-infiltrating lymphocytes, TILs）之研究中，癌細胞如何分泌免疫抑制物質，來調控免疫細胞分化與生長最重要的IL-2細胞激素受體CD25 (IL-2Rα)，與逃避TCR相關的共同刺激活化分子與受體共同作用下的免疫毒殺調控，仍需進一步釐清。我們延續先前在腫瘤微環境中，子宮癌病患TILs中CD4+CD25+ FoxP3+免疫調控細胞TREG的改變、與免疫調控CD8+T細胞的毒殺作用的相關研究，針對目前癌症免疫治療的盲點，深入探討IL-2細胞激素群(IL-2 family cytokines, IL-2, -7, -15, and -21)與TCR相關的共同刺激活化分子與受體共同作用下，對腫瘤中TREG細胞相關調控的研究。第一年計劃主要利用淋巴球細胞表面免疫抗原三重螢光染色技術與流式細胞儀(flowcytometry)，利用先前已建立的機械式研磨萃取法(mechanical dispersal technique)來獲得子宮內膜癌之癌腫瘤浸潤淋巴球，深入探討癌腫瘤組織浸潤淋巴球與周邊血液細胞調控型細胞CD4+CD25+ FoxP3 TREG細胞免疫型別(immuno-phenotype) TCR共同刺激活化分子與受體的表現。第二年計劃再經由先前建立的體外腫瘤細胞與自體免疫細胞之混合培養模式(mixed lymphocytes-tumor cells coculture, MLTC) ，進一步探討腫瘤產生的細胞激素與IL-2細胞激素群對於B7 (CD80, CD86), CTLA-4, CD28等TCR相關的共同刺激活化分子與受體共同作用下，分析癌細胞如何利用TREG抑制細胞毒殺等免疫反應之可能原因，藉由細胞激素對TREG受體影響之測定，對於免疫細胞在腫瘤中受抑制之現象做進一步的分析研究。第三年計劃將進一步研究腫瘤微環境中TREG功能性變化的可能機轉，利用IL-2細胞激素群抑制抗體，深入探討不同的IL-2 family cytokines細胞生長激素如何在腫瘤存在的環境中，在TCR相關的共同刺激活化分子與受體共同作用下，進行動態的TREG免疫調控。本研究預期能在細胞激素對TREG影響之測定上、以及腫瘤微環境中，細胞激素調控細胞TREG以及共同刺激活化分子與抑制受體與免疫細胞功能性變化能有所突破，進一步應用於未來婦癌症臨床免疫治療的發展。<br> Abstract: Cancer immunotherapy become possible for the treatment of patients in the clinical applications. Immunologic modulation with monoclonal antibody against CTLA-4 may shade new lights on the restriction of human cancers. However, in some circumstance, cancer spread occurs despite the apparent cellular immune response of tumor-infiltrating lymphocytes (TILs). At present, little is known about the cytokines-mediated homeostasis and expression of CTLA-4 in TCR and CD28 engagement, especially in CD80 (B7-1) and CD86 (B7-2) on tumor-infiltrating CD4+CD25+ FoxP3 TREG in the cancer microenvironment of human endometrial cancer. The effect of IL-2 family cytokines (IL-2, IL-7, -15, and -21) on TREG was unclear and remained to be stratified especially on the actual interactions between TCR-related activation of CTLA-4 and CD28 co-stimulatory molecules. Also the effector functions of activated CD4+CD25+ FoxP3 TREG in cancer milieu are essential for understanding their roles and potential mechanisms in tumor immuno-pathogenesis. The present study will extend our previous findings and explore kinetic regulation of IL-2 family cytokines on the expressions of CTLA-4 in TCR and CD28 engagement, with functional changes of FoxP3+ tumor-infiltrating TREG cells in the human uterine endometrial cancer microenvironment. In the first year project, we will analyze the immuno-phenotypes and expression ratio of IL-2 family cytokine receptors on fresh-isolated TREG cells by flowcytometry. Our focus is concentrated on the expression of activation markers on CD3+CD25+ FoxP3+ TILs. The basic activation analyses of CD4+ T cells, CD8+ T cells, and NK-T cells will be illustrated between normal and cancer groups. In the second year project, we will investigate the homeostasis and functional expressions of CTLA-4 in TCR and CD28 engagement of up-regulated FoxP3 in CD4+ T cells, CD8+T cells by utilizing our previously established models of mixed lymphocytes and tumor cells coculture (MLTC). We will define the possible cancer-derived essential role of IL-2 family cytokines in MLTC to stratify the functional roles in correlated expression of cytokine receptors on immuno-sorted subsets of TREG. The kinetic activity with concordant expression of CTLA-4 in TCR and CD28 engagement of on the cancer-encountered CTLs & TREG cells will be measured through intracellular staining and flow-cytometric analyses. In the third year project, we will investigate the regulatory roles of IL-2 family cytokines (IL-2, IL-7, -15, and -21) on the kinetic modulations of possible TREG mediated cancer-cytotoxicity in MLTC. Kinetic activity with concordant expressions of certain TCR-related co-stimulatory B7, CTLA-4 CD28 and receptors on the TREG cells will be measured with both intracellular perforin staining and cytotoxicity assays. The functional change of TREG cells will be evaluated with cytokine receptor recombinant antibodies in the well-established MLTC model. With accumulated clinical data, further extended studies will be linked the immuno-phenotypic functional assays with the clinical prognosis in subjects with cancers.癌症細胞激素腫瘤內浸潤淋巴球調控T細胞IL-2B7CTLA-4TCR相關的共同刺激活化分子cancertumor-infiltrating lymphocytesregulatory T cellIL-2B7CTLA-4TCR