從口腔癌分離或共同培養誘發之產生介白素-17之調節性T細胞之特性分析

Abstract

口腔癌近年來在台灣癌症排名中，從過去的十名之外大幅提昇到第六名，在男性癌症致死率的排名更是高居第四，對國民健康造成重大的威脅。口腔癌中以口腔鱗狀細胞癌最為常見，佔口腔癌的90%以上，在口腔癌研究中本實驗室先前研究發現調節性T細胞與Th17細胞在口腔麟狀上皮細胞癌病患的腫瘤浸潤細胞中都有高量比例之分佈，並且也發現一群特殊的細胞群同時表現IL-17+Foxp3+，然而此群細胞在口腔癌微環境扮演的角色仍不清楚，且在口腔癌症微環境中，口腔癌症細胞是如何逃避免疫細胞的辨認和攻擊，仍需進一步的探討。本研究將腫瘤浸潤細胞從口腔癌組織中分離，將其染色後利用流式細胞儀分析，發現IL-17+Foxp3+與IL-17-Foxp3+ 調節性T細胞均表現高量的CTLA-4，但都不會分泌IFN,IL-2和IL-22。為了進一步研究IL-17-Foxp3+調節性T細胞在癌症環境中的功能，本研究利用與Th17相關的化學趨化因子受體CCR6作為標記，分離出CCR6+CD25+和CCR6-CD25+兩群調節性T細胞，確認CCR6+CD25+放出較高量的IL-17，且在體外實驗中具有較強的免疫抑制能力。而在CD4+ T細胞與口腔麟狀上皮細胞癌細胞株SAS的共同培養系統中，進一步的確認本實驗室先前的發現SAS細胞與CD4+ T細胞共同培養誘發Foxp3+調節性T細胞，主要是來自記憶性T細胞(CD45RO+)，並具有體外的免疫抑制能力的。共同培養系統中也發現癌細胞株能夠直接抑制CD4+ T細胞的增生及釋放IFN和IL-2。利用1-methyl-tryptophan (1-MT)更進一步發現SAS細胞可能是透過indoleamine 2,3-dioxygenase (IDO) 去抑制CD4+ T細胞而與B7H1無關。總結本研究認為IL-17+Foxp3+調節性T細胞在口腔癌癌環境中扮演了較強免疫抑制的角色；而在與SAS細胞共同培養實驗中，則發現口腔癌細胞可以直接抑制免疫細胞的活性與增生。

Oral cancer is one of the major health issues in Taiwan. Around 90% of oral cancer is oral squamous cells carcinoma (OSCC). Our laboratory previously demonstrated that in OSCC, Th17 cells and Treg cells are increased in tumor infiltrating lymphocytes (TILs). Most interestingly, a specific subset of T cells, IL-17+Foxp3+, is also increased in TILs. However, the function and phenotype of IL-17+Foxp3+ Treg cells and the immunosuppressive mechanisms of oral cancer cell are not clear. Therefore the main purposes of this study are to delineate the functional characteristics of IL-17+ FoxP3+ CD4+ T cells and the interaction between oral cancer SAS cells and CD4+ T cells in vitro. TILs were stained by intracellular staining and analyzed by flow cytometry. The IL-17+ Treg cells produced a significantly higher level of IL-10 than IL-17- Treg cells, but the production of IL-2, IFN-γ, CTLA-4 or IL-22 were similar. The IL-17+ or IL-17- Treg cells were purified based on the expression of CCR6 by cell sorting. The expression level of il17 or rorc was significantly higher in CD4+CD25+CCR6+ than CCR6- Treg cells. Moreover, IL-17+ Treg cells displayed higher suppressive activity than IL-17- Treg cells in vitro. When co-cultured with SAS cells, purified memory (CD45RO+), but not naive (CD45RO-), CD4+ T cells exhibited enhanced expression of FoxP3 and suppressive activity with down-regulation of IFN-γ and IL-2. The SAS-mediated inhibition of T cells is reversed by the inhibitor of indoleamine 2,3-dioxygenase (IDO) and is independent of B7H1. Taken together, these results suggested that IL-17+ Treg cells may exhibit a higher suppressive activity in the oral cancer microenvironment, and the cancer cell alone is sufficient to inhibit directly the T cell activation and proliferation.