子宮內膜癌中CD103+ T細胞免疫調控的角色

Abstract

目的: 腫瘤內浸潤淋巴球(tumor infiltrating lymphocyte, TILs)一直被探討是否影響癌症病患本身的免疫反應及疾病的預後，而T細胞在TIL中對腫瘤免疫監督與免疫逃脫上扮演重要的角色。子宮內膜癌(Endometrial cancer, EC) 細胞和TILS之間的相互作用，可能是一個有效的抗癌免疫反應的關鍵。 在我們團隊之前關於腫瘤內浸潤淋巴球(tumor infiltrating lymphocyte, TILs)的研究中，發現CD8+ T淋巴球中CD69, CD152及CD103有高度表現。CD103 屬於αEβ7 integrin中的α subunit，在一些免疫細胞上均發現它的存在。到目前為止，關於CD103+ T細胞在毒殺腫瘤方面扮演的角色及相關的免疫反應仍不清楚。在本次研究中，我們將探討人類子宮內膜癌中的CD103+ T細胞在其上細胞表面標記的表現，及免疫的功能。 方法: 我們共計從37位接受手術的子宮內膜癌病人檢體中，取下新鮮腫瘤組織，並分離出腫瘤內浸潤淋巴球(tumor infiltrating lymphocyte, TILs)，再利用流式細胞儀分析，進一步分析CD103+ T細胞上的表面標記(phenotype)的表現，及胞內細胞激素(cytokines)的含量表現。另外，我們藉由同種抗原(alloantigen)及TGF-β，成功刺激CD8+ T細胞表面表現CD103，並同時分析其細胞表面之活化相關之標記(activation marker)，及記憶相關之標記(memory marker)。 結果: 在腫瘤浸潤淋巴球中，CD103+ T細胞有高達83.7%亦表現CD8，與我們之前的研究相互呼應，並有83.5%的CD103+ T細胞表現CD69，反映出一個CD69+CD103+的重要T細胞族群---組織定居記憶性T細胞(Tissue-resident memory T cells)。另外，在記憶相關之標記(memory marker)探討中，CD103+ T細胞具有高度CD45RO表現(表現比例80.7%)，及低度CD62L(表現比例25.2%)和CCR7(表現比例18.5%)的表現，符合效應T細胞之特徵。在細胞激素分析實驗中，相較於CD103- T細胞，我們發現CD103+ T細胞含有顯著較多的細胞毒殺激素(cytotoxic cytokines)，即perforin (表現比例CD103+ T細胞為60%, CD103- T 細胞為1.7%，p=0.03)和granzyme B (表現比例CD103+ T細胞為69.2%, CD103- T 細胞為18.0%，p=0.02)。另外，有46.7%的CD103+ T細胞表現了FoxP3，此群FoxP3+CD103+細胞可能為較多的抗發炎細胞激素IL-10 (21.4% vs. 1.7%，p=0.03)及TGF-β (49.1% vs. 19.0%, p=0.01)之來源。在CD103+ T細胞上也發現免疫檢查點標記(immune checkpoint markers)，包括CD152，GITR及LAG-3的高度表現。在CD8+ T細胞經過同種抗原(alloantigen)及TGF-β刺激後，其表面CD103及FoxP3的表現有顯著上升。在腫瘤內浸潤淋巴球中，有較高度表現CD103的病人族群，有較好的無復發存活率。 結論: 在子宮內膜癌的腫瘤內浸潤淋巴球中，有相當大部分的CD3+CD103+ T 細胞屬於組織定居記憶性T細胞(Tissue-resident memory T cells)。藉由同種抗原(alloantigen)及TGF-β刺激實驗，合理說明了在TGF-β含量豐富的腫瘤微環境(tumor microenvironment)下，組織定居記憶性T細胞之表面標記的產生。未來可期待的是，有高度CD3+CD103+ T細胞含量的子宮內膜癌中，針對CD8+FoxP3+調節T細胞(regulatory T cells)或免疫檢查點標記(immune checkpoint markers)進行調控，或許可成為治療的方向，並增加病人存活率。

Purpose: In our previous studies, we found that CD8+ T cells derived from human tumor-infiltrating lymphocytes (TILs) expressed elevated levels of CD69, CD152, and CD103. The CD103 is a cluster of epitopes in the E region of the α subunit as a member of the integrin family αEβ7, and is expressed in several immune cell types. So far, the role of the most enigmatic integrin, αE(CD103) β7 in cytotoxic lymphocyte (CTL)-mediated killing and antitumor immune response remains poorly understood. In the present study, we will explore the phenotypic and functional characteristics of CD103+ TILs in human endometrial cancer (EC). Methods: TILs were isolated from freshly excised surgical tumor specimen in a cohort of 37 endometrial cancer patients. The phenotypic expressions and intracellular cytokines staining of CD103+ TILs were analyzed by flow cytometry. The kinetic phenotypic expressions of activation and memory markers on CD8+ T cells were further analyzed through alloantigen CD103 induction. Results: 83.7% and 83.5% of CD3+CD103+ T cells in TILs derived from human EC highly expressed CD8 and CD69, which is compatible with the phenotype of tissue-resident memory (TRM) T cells. The expression ratio of memory T cells markers including CD45RO (mean 80.7%), CD62L (mean 62.6%), and CCR7 (mean 18.7%) is compatible with the characteristics of effector memory T cells. The CD3+CD103+ T cells expressed significant elevated intracellular perforin and granzyme B. 46.7% of FoxP3+ T cells were identified in these CD3+CD103+ T cells with elevated intracellular cytokines of IL-10 and TGF-β. The immune checkpoint markers including CD152, GITR and LAG-3 were highly expressed on CD3+CD103+ TILs. Up-regulation of CD103 and FoxP3 on CD8+ T cells can be induced through in vitro kinetic alloantigen and TGF-β stimulation. Furthermore, High percentage of CD3+CD103+ T cells in TILs was correlated with a better survival for patients through 5-year survival analysis. (high CD103+ group vs. low CD103+ group, p=0.183) Conclusions: Significant proportion of the CD3+CD103+ TILs derived human EC expressed the markers of TRM cells. Kinetic alloantigen and TGF-β stimulation illustrated the plausible phenotypical expression of TRM under TGF-β abundant tumor microenvironment. The high percentage of CD3+CD103+ T cells in TIL with immune-modulation of CD8+FoxP3+ Tregs and immune checkpoint receptors CD152, GITR or LAG-3 can be relevant to a better clinical survival in EC.