口腔鱗狀上皮細胞癌中的CD8 T細胞亞群對人類與細菌α-烯醇酶的交互反應

Abstract

在台灣，口腔癌位列國人十大癌症第六名，而口腔鱗狀細胞癌（Oral Squamous cell carcinoma，OSCC）則是最常見的口腔癌類型。而α-烯醇酶（α-Enolase）是糖解作用中的一環，在大多數組織中皆會表達。過去有許多研究指出，包含口腔癌在內，α-烯醇酶會在許多不同癌症中的有表現增加的情形。在癌症中的角色可能作為糖解酶或是纖溶酶原受體，會促使癌症侵襲，生長和轉移。 之前在本實驗室的研究中，我們將人和細菌α-烯醇化酶的氨基酸序列進行了比對，然後將這些序列分成3群：特屬於人類的序列，特屬於細菌的序列，和人類與細菌具有高度相似的序列。我們從健康人與OSCC的患者身上採集血液，分離周血單核球細胞（Peripheral blood mononuclear cells，PBMCs），也從OSCC患者切除的腫瘤中，分離腫瘤浸潤淋巴細胞（Tumor infiltrating Lymphocytes，TILs），然後以前述所提過的3群氨基酸序列進行刺激。實驗結果顯示，不論是在健康人與OSCC患者的PBMCs，或者是OSCC患者的TILs中，都能夠產生針對α-烯醇化酶的免疫反應。我們針對CD8+ T 細胞進行分析，發現這群細胞在刺激後會產生如干擾素γ（Interferon-γ IFN-γ）和腫瘤壞死因子α（Tumor necrosis factor，TNF-α）等免疫反應。 為了分析這些不同免疫反應在腫瘤中的角色。分析這些α-烯醇酶反應性CD8 + T細胞的作用有助於腫瘤進展，我們從美國NIH tetramer core facility獲得了MHC-表位肽四聚體 (peptide-MHC Tetramer)，並成功找出了α-烯醇酶特異性CD8 + T細胞。接著，我們發現針對人類與細菌相似的序列的T細胞中，有較多細胞表現PD-1，以及共同表現CD39與CD103，是潛在作為腫瘤特異性細胞的特徵。而我們進一步比較疾病的臨床資訊，發現針對人類與細菌相似的序列，晚期的OSCC患者相比於早期患者，其所產生免疫反應較差。更進一步證實針對人類與細菌相似的序列的T細胞可能存在的抗腫瘤作用。此外，我們利用細菌α-烯醇化酶的氨基酸序列刺激，並在體外培養T細胞。這群細胞中我們也成功找到針對人類與細菌相似的序列的T細胞，並證明其具有細胞毒殺的能力。這個結果反應了免疫交互作用對於腫瘤的影響。

In Taiwan, oral cancer ranks as the 5th highest incidence of cancer in general population. Characterized by high metastatic potency, oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Alpha-enolase (α-enolase) is a glycolytic enzyme expressed in most tissues. Many researches have pointed out the over-expression of α-enolase in various cancer type, including oral cancer. Its role as glycolytic enzyme and plasminogen receptor were reported to promote cancer invasion, proliferation, and metastasis. Our previous works revealed that both healthy people and OSCC patients were able to produce immune response against α-enolase. We aligned the sequence of human and bacterial α-enolase, and later divided those sequence into 3 pools of peptides -- human-specific pool, bacteria-specific pool, and shared pool, defined by level of homology between human and bacteria sequences. CD8+ T cells from peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) were detected producing cytokines like interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) upon stimulation by these 3 peptide pools. Aiming to analyze the effect of these α-enolase responsive CD8+ T cells contributes to tumor progression, we have acquired the peptide-MHC tetramers from NIH tetramer core facility, and successfully identified α-enolase specific CD8+ T cells. Combining results of α-enolase -reactive/specific cells, we found that T cells recognized shared group peptide express a relative higher potential in activation and tumor reactivity, with evidence of higher expression of PD-1 and co-expression of CD39 and CD103 in TILs. Analysis the immune response of three pools with clinical disease status gave us a further surprising outcome. OSCC patients in late stage are found out with lower IFN-γ production against peptides of shared pool, suggesting an anti-tumor effect in T cells recognized shared peptide pool. The result might contribute to the idea of cross-reactivity. As we further successfully expanded T cells recognized shared peptide pool by using bacteria-derived peptide and prove their cytokine function as well as the ability in cytotoxicity.