- 學位論文
乳癌腫瘤浸潤淋巴細胞分析:比較調節性T細胞、CD8+T細胞與相關免疫標記的表現
Expression and Cell Markers of Regulatory T Cells and CD8+ T Cells in Tumor-Infiltrating Lymphocytes of Human Breast Cancer
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摘要
背景與目的 腫瘤細胞的生長需對抗來自宿主免疫系統的清除作用，在腫瘤組織微環境中有大量的浸潤淋巴球聚集（tumor infiltrating lymphocytes，TILs）。其中有一群 T 細胞具有調節免疫作用，具有 CD4+CD25+ 表現特徵，稱為調節性 T 細胞。調節性 T 細胞於細胞內高度表現 FOXP3 轉錄分子，其負向調節作用需靠細胞與細胞接觸的機制。許多的研究指出腫瘤細胞的免疫逃脫機制有調節性 T 細胞參與。T 細胞經過與抗原表現細胞接觸後受到刺激，naive cell 會轉化成記憶 T 細胞，之後若經一定刺激可以再度被活化。記憶 T 細胞（memory T lymphocyte）以返回次級淋巴器官的能力與 effector 的功能區分為兩種類型：central memory cell（TCM）及 effector memory cell（TEM）。依據之前本實驗室對乳癌病患腫瘤浸潤淋巴球的測定，發現 TIL 中的 CD8+ T 細胞的比例會增加，而 CD4+ T 細胞的比例會減少。而增加的 CD8+ T 細胞顯著與乳癌疾病期別進展有相關。CD8+ T 細胞具有毒殺腫瘤細胞能力，但於腫瘤微環境中由於免疫功能被抑制，其毒殺腫瘤細胞能力亦被抑制。我們欲了解乳癌病患 TILs 中調節性 T 細胞與 CD8+ T 細胞其免疫標記表現與細胞毒殺能力的關係，尤其是記憶細胞是否能經刺激後再具免疫功能，希望能對未來發展對腫瘤的免疫治療有所幫助。 材料與方法 實驗標本的收集：臨床診斷第一至第三期乳癌的病患，依疾病狀況接受手術治療。病患乳癌腫瘤組織（註記為 TIL）將會被收集成實驗組。同時收集病患的血液（註記為 PBL）為對照組。依據病理報告的結果，記錄每個病例的臨床病理特徵。在分離純化浸潤淋巴細胞與分離出周邊血液中的單核細胞後對細胞表面抗原與細胞內作用分子作測定，運用流式細胞儀分析。探討 TIL 中CD4+CD25+ 調節性 T 細胞及其上 FOXP3、GITR、CD103 和 CD152（CTLA-4） 的表現，及探討調節性 T 細胞分泌 cytokine 的表現，最後探討調節性 T 細胞對 CD8+ T 細胞毒殺能力之影響。也分析調節性 T 細胞與 CD8+記憶 T 細胞及 effector cell 表現之相關。然後我們將檢測 CD4+CD25+ 調節性 T 細胞分泌 Th1 cytokines（IFN-γ、IL-12 和 TNF-α）與 Th2 cytokines（IL-4 和IL-10）的狀態，也對細胞內毒殺顆粒（包括 granzyme B 及 perforin）的表現作分析。 結果 共有30位病患列入分析，性別均為女性，平均年齡為56.8歲（28歲至89歲）。CD4+CD25+ 調節性 T 細胞在腫瘤中的比例與血液相比為增加（11.6
並列摘要
Background: To determine the functional attributes of CD4+CD25+regulatory T cells (Tregs) in cancer microenvironment . Material and Methods: Triple-color flow cytometry was utilized to study the phenotype expression of CD4+CD25+Tregs and CD8+T-cell in the peripheral blood lymphocytes (PBLs) and tumor infiltrating lymphocytes (TILs) of 30 stage I to III breast cancer. Results: The prevalence of CD4+CD25+T cells was significantly higher in the TILs than PBLs. The expression of FOXP3, CD103 and GITR on CD4+CD25+Tregs was lower in PBLs than TILs. Most tumor-infiltrating CD8+T cells were CD28-CD45RA-CD45RO+CCR7-, suggesting good terminal differentiation. Most of them had an activated role with CD69+CD103+CD152+. Functionally, both granzyme B and perforin were scarcely expressed in peripheral Tregs but were highly expressed in Treg cells in the tumor micro-environment. On the contrary, CD8+cytotoxic T cells derived from PBLs expressed both granzyme B and perforin, and were significantly higher than those in TILs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules can be synchronously up-regulated in CD8+cytotoxic T cells. Conclusions: Tregs in the tumor microenvironment may abrogate CD8+T cell cytotoxicity in a granzyme B-and perforin-dependent conduit. Decreases in both Th1 cytokines and cytotoxic enzymes are relevant for Treg-mediated restraint of tumor clearance in vivo. Of clinical significance, the expression of Tregs in TILs may mediate T-cell immune repression within cancer milieu.
參考文獻
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