探討犬瀰漫性B細胞淋巴癌之外泌體對於犬毒殺型T細胞的影響

Abstract

瀰漫型大B細胞淋巴瘤（DLBCL）是犬隻中最普遍的侵襲性淋巴瘤亞型。雖然淋巴瘤可以透過化療得到有效的緩解，但大多數的患者都會面臨復發，並往往伴隨著強烈的抗藥性。在免疫系統中，毒殺型T細胞是最主要能辨識且消滅癌細胞的免疫細胞。藉由毒殺型T細胞所分泌的細胞毒素能夠誘導癌細胞死亡。然而最近有研究強調，腫瘤來源的外泌體是造成免疫抑制的重要原因之一，它會導致毒殺型T細胞的功能被削弱以及抑制，從而減弱整體的抗癌作用。外泌體是胞內起源的細胞外囊泡，會從原始細胞中攜帶各類物質，幫助細胞間的通訊。目前已有多項報告顯示，外泌體會通過複雜的路徑調節腫瘤的發生和轉移。因此在本研究中，將會藉由犬DLBCL細胞株CLBL-1產生之外泌體與健康犬的毒殺型T細胞共培養，以此探討外泌體對免疫細胞整體的影響性。首先利用次世代定序，比對兩組暴露於外泌體下的CD8+ T細胞之基因變化，從差異表達基因的功能分析中發現，下調基因涉及有絲分裂、細胞週期、DNA複製和細胞凋亡等相關路徑，闡明了外泌體可能抑制T細胞的生長、代謝並使其死亡。接著我們利用功能性試驗，研究外泌體對於毒殺型T淋巴細胞的影響。根據體外實驗的結果顯示，CLBL-1之泌體會降低T細胞的生長和增殖，並誘導其凋亡。同時減少干擾素-的分泌，造成T細胞的活性和功能受到抑制及損害。此外，CLBL-1之外泌體會增加免疫抑制標誌物CTLA-4在毒殺型T細胞上的表現。不僅如此，從細胞激素晶片的結果指出，發炎細胞激素和化學趨化因子的表達有明顯變化，說明CLBL-1外泌體可能改動毒殺型T細胞的抗腫瘤免疫反應。綜合上述結果，這項研究首度指出犬瀰漫性B淋巴瘤來源的外泌體對免疫細胞的全面抑製作用，未來將進一步剖析其引發的免疫抑制機制和途徑，為犬淋巴瘤的治療提出新策略及治療選擇。

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma in dogs. Although up to 90% of lymphomas are relieved via chemotherapy, most patients relapse and accompanied by severe chemoresistance. CD8+ cytotoxic T cells (CTLs) can recognize the cancer cells and eliminate tumors by producing cytotoxins. In the tumor microenvironment, tumor-derived exosomes (TDEs) are the critical factors to cause immunosuppression, such as undermining the anti-cancer effects of CTLs. This study aims to investigate the effects the canine DLBCL cell line CLBL-1 derived exosomes on CTLs of healthy dogs. RNA-seq analysis has revealed that CD8+ T cells exposed to exosomes specifically highlighted pathways strongly involved in mitosis, cell cycle, DNA replication, and apoptosis. The results clarified that exosomes possibly regulated cell growth, death, and metabolism of CD8+ T cells. These findings were further validated by in vitro functional assays. CLBL-1 exosomes reduced proliferation ability and triggered apoptosis of CD8+ T cells. Meanwhile, the interferon- were decreased by exosomes, thereby the activation and function of CD8+ T cells were significantly inhibited. Furthermore, CLBL-1 exosomes also promoted the expression of CTLA-4 on CD8+ T cells. The cytokine array analysis also showed the increased proinflammatory cytokines and chemokine production of CTLs treated by exosomes. Collectively, this study firstly indicated that CLBL-1 exosomes could comprehensively suppress the CD8+ T cells. Further studies could be conducted to elucidate the detailed mechanisms of immunosuppression mediated by DLBCL exosomes.