口腔鱗狀細胞癌病患週邊血液中調節性T細胞的變化

Abstract

在口腔中約90%的惡性腫瘤為口腔鱗狀細胞癌，而在台灣是一個前六大男性常見侵襲癌症死因，此外台灣主要是和嚼食檳榔、五年的存活率不佳以及高度死亡率著稱。在頭頸部腫瘤中，許多腫瘤產生之免疫抑制性機轉被提出，但是否在口腔鱗狀細胞癌中有類似的現象仍不清楚。在本篇研究中，針對治療前後，CD4+CD25+ 調節性T細胞在周邊血液的分布調、CD8+T淋巴球中自然殺手細胞抑制受體 (inhibitory natural killer cell receptors，iNKRs)，以及其他不同種類T淋巴球的表現。實驗中，我們針對30個口腔鱗狀細胞癌的病患 (其TNM分期為第一至四期) 以及20個年齡相符的健康正常血液捐贈者取其血液中之單核球利用單株抗體和三或四色之流式細胞儀進行分析。我們發現在口腔鱗狀細胞癌病患之周邊血液中，比正常對照組有較高之CD4+/CD8+比例，而主要是由於 CD8+ T cells下降的關係；而在深入分析CD3+CD4+之淋巴球中CD25+ 之調節性T淋巴球在口腔鱗狀細胞癌病患(5.86±5.23%)之週邊血液相對於正常捐贈組(1.68±0.95%)有統計學上意義(p<0.001)；再者，將上述明顯表現的CD4+CD25+調節性T淋巴球藉由流式細胞儀再依據細胞的分布來進一步分析細胞的表現，CD4+ CD25+高度表現群在口腔鱗狀上皮細胞癌病患(1.84±1.74%) 週邊血液相較於正常捐贈組(0.43±0.40%)是具有統計學上意義的(p<0.005)；更者，我們以細胞內染色來標定Foxp3，利用它為一重要之轉錄因子以及為調節性T細胞之專一分子標定記號來做進一步確認CD4+ CD25+高度表現群與低度表現群為調節性T淋巴球。而我們也發現CD4+ CD25+高度表現群和TNM分期和癌症發生之位置並無明顯相關。在病患接受治療後，在口腔鱗狀細胞癌中CD4+ CD25+之比例也較術前減少 (2.87±2.69 %, p<0.05)。然而在CD8+ T淋巴球中自然殺手細胞抑制受體，無論是NKG2A或NKG2D和正常對照組並無明顯差異。但在CD4+/CD8+比例以及治療前發現減少之CD8+數量在接受治療後皆有發現逐步恢復正常的情況。這些結果顯示在循循環血液中之調節性T細胞和疾病之嚴重度以及CD8+數量之下降量有直接的關係。而調節性T細胞對癌症病患體中所造成之效應可以幫助我們未來在口腔鱗狀細胞癌治療之方向。

Oral squamous cell carcinoma (OSCC) accounts for more than 95% of all malignant neoplasms in the oral cavity, and ranks as the sixth major cause of cancer mortality in Taiwan. Here, OSCC is associated with the areca nut chewing and characterized by an unsatisfactory 5-year survival rate after treatment. Emerging evidence suggested that head and neck carcinoma exhibits multiple immunosuppressive characteristics, but whether OSCC displays similar phenomenon is still not clear. In the present study, the distribution of CD4+CD25+ regulatory T cells (Treg) in the peripheral blood before or after treatment and the change in ratio or iNKR markers of CD8+T lymphocytes were investigated. we also examined the expression of various on T lymphocytes (especially). A total of 30 OSCC patients (TNM Stage I to IV) and 20 aged-match healthy donors were enrolled and their peripheral blood mononuclear cells (PBMCs) were analyzed by triple-color and quadruple flow cytometry using a panel of specific monoclonal antibodies. Patients with OSCC had significantly higher CD4+/CD8+ ratio than normal controls attributable to a decrease in CD8+ T cells. Among the CD3+CD4+ subsets, CD25+ Treg represented 5.86±5.23 % in OSCC patients or 1.68±0.95 % in normal controls (p < 0.001). Furthermore, Mean percentage of CD4+CD25high Treg, which exhibited higher mean flurescence intensity of CD25+ expression, of OSCC patients (1.84±1.74%) is also significantly higher than in healthy donors (0.43±0.40 %, p< 0.005). Identity of Treg in both CD4+CD25high or CD4+CD25low subsets were confirmed by a positive intra-cellular staining of Foxp3+, an important transcription factor and the most specific molecular marker for regulatory T cells. The increase in CD25high Treg was related to TNM stage, but not site of cancer occurrence. After treatment, the percentage of CD4+CD25+ Treg in the OSCC patients decreased significantly (2.87±2.69 %, p<0.05) to the level comparable to (before) normal controls. There was no significant difference in the expression of iNKRs, NKG2A or NKG2D, on circulating CD8+ T cells from OSCC patients compared to normal controls. But the increased CD4+/CD8+ ratio or the decreased CD8 number in OSCC patients converted to normal level after treatment. These results suggested that the frequency of circulating Treg correlated directly with the severity of disease and the decreased circulating CD8+ count in OSCC patients, and conversion of circulating Treg might reflect efficacy of cancer treatment.