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中文摘要
鼻咽癌 (nasopharyngeal carcinoma, NPC) 和EB病毒 (Epstein-Barr virus, EBV) 感染有密切的相關性。就發病人數比例看來，中國人遠超過其他國家，而居世界首位，所以可說是中國人特有的疾病。鼻咽癌組織中浸潤有許多的淋巴球 (tumor infiltration lymphocyte, TIL)，但這些免疫細胞卻仍然不能有效的清除腫瘤細胞。為了了解TIL是否因為活化不完全，而造成免疫反應的缺失，我們利用免疫化學染色，觀察石蠟切片中TIL細胞上與活化有關的表面分子的表現。結果在腫瘤組織中的細胞，可以染到CD3，CD4，CD8，CD28，HLA-DR，CD25，CD45RO，以及B7.1的表現，但是CD25+的細胞非常的少。接著，我們分離鼻咽癌腫瘤部位的TIL，以PMA/ionomycin刺激病人的TIL和PBMC並且和健康人的PBMC相互比較。結果發現TIL受刺激後的活化能力不同於病人或健康人的PBMC。另外，我們也證實鼻咽癌病人經放射線治療後，其PBMC經PMA/ionomycin處理過，細胞上CD3的表現會明顯的下降。除此之外，在體外培養的條件下，病人或健康人的血清對Jurkat細胞的活化沒有影響。
人類細胞激素IL-10 (Interleukin-10, IL-10) 與EB病毒的病毒IL-10在結構或功能上非常相似。IL-10對於淋巴細胞的調控是重要的因子。在鼻咽癌病人的新鮮TIL及PBMC當中，均有IL-10的表現，尤其是TIL的表現很強。為了了解鼻咽癌病人的PBMC或血清是否會影響T細胞或B細胞表現IL-10，我們應用一套IL-10啟動子 (promoter) 帶有加強型綠色螢光 (enhanced green fluorescence protein, EGFP) 的報告基因系統來觀察。結果病人或健康人的血清或PBMC對穩定細胞株IL-10 promoter activity的調控沒有影響。
綜合以上結果：具有IL-10高量表現的TIL可能只有少部分處於活化狀態。而且TIL與周邊的PBMC活化能力不同，顯示TIL在腫瘤局部可能有不同的免疫反應化狀態，這可能是TIL無法有效清除癌細胞的原因。
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Abstract
Nasopharyngeal carcinoma (NPC), highly incident in Southeast Asia, is a malignant epithelial cancer associated with Epstein-Barr virus infection. The NPC tumor biopsies are characterized by abundant tumor infiltrating lymphocytes (TILs), some of them are T cells. In the microenvironment of NPC, the interaction between the tumor cells and TILs is not well defined. Our purpose is to understand the reason why TILs do not kill the tumor cells. We propose that there is an improper activation signaling in TILs. In this study, we applied immunohistochemical stain (IHC) to determine the expressions of surface stimulatory molecules in TILs. CD3, HLA-DR, CD4, CD8, CD45RO, CD28, and B7.1 were detected in TILs, while they expressed few CD25. The levels of CD3, CD25, and CD28 in TILs and the peripheral blood mononuclear cells (PBMC) were confirmed by flow cytometric analysis. After treating with PMA/ionomycin, the different activation is observed between TILs and PBMC of the patients and the healthy people. The expression of CD3 was decreased in PBMC of the NPC patients after radiotherapy. Besides, the sera of the NPC patients did not alter the expression of CD3, CD25, and CD28 in Jurkat cells treated with or without PMA/ionomycin.
Interleukin-10 (IL-10) has been implicated as an important modulator of lymphoid cells, and the DNA sequence of human IL-10 is highly homologous to vIL-10 (viral IL-10) gene in the EBV genome. IL-10 has been suspected to decrease the host immune response towards tumor cells in the NPC patients. IL-10 mRNA was detected by RT-PCR in TILs and PBMC from the NPC patients. To determine whether factors of PBMC or serum in NPC patients can alter the IL-10 expression, we applied a reporter gene system, in which IL-10 promoter regulates the expression of enhanced green fluorescent protein. The activities of IL-10 promoter were not altered in Jurkat- or BJAB-derived cell lines no matter treated with serum or co-cultured with PBMC from the NPC patients.
In conclusion: few TILs, that may express high level of IL-10, are in activated state.
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