研究背景: 已經有許多研究結果顯示，浸潤在腫瘤微環境 (tumor microenvironment, TME) 的免疫細胞，對於癌症的發展過程中扮演相當重要的腳色。毒殺性CD8+ T细胞是一種在腫瘤微環境中可以攻擊癌細胞的免疫细胞。然而有些機制確會導致CD8+ T细胞失去毒殺癌細胞的能力。程序性细胞死亡蛋白1 (Programmed cell death protein 1, PD-1) 是一種表現在T细胞表面上並造成T细胞功能異常以及在腫瘤微環境中形成免疫抑制的受體。已經有許多文獻探討PD-1的轉錄調控，但是對於PD-1的後轉譯修飾以及是如何被運送到T 細胞表面上目前尚不清楚。
研究目的: 先前本實驗室的研究發現小G蛋白 (small GTPase) Rab37會透過調控不同分子的釋放來影響癌症的轉移，幹性和血管新生。值得注意的是，我們也同時發現在CD8+ T細胞於Rab37基因剔除老鼠 (knockout mice) 其細胞膜上PD-1的表現量有下降的趨勢。因此，本研究探討於肺癌進程中，Rab37是否在T 細胞中調控PD-1的細胞膜運送，並且促進肺癌疾病進程。
研究結果: 根據我們在共聚焦顯微鏡的免疫螢光染色 (immunofluorescence) 結果顯示，Rab37與PD-1在由WT老鼠所分離出的CD8+ T細胞有共定位 (colocalization) 的現象。重要的是T細胞株Jurkat中Rab37以鳥苷核苷酸依賴性的方式 (GTP-dependent manner) 調控PD-1的穿膜表現。另外，為了探討Rab37是否介導PD-1的胞內運輸，我們在經由PMA/Io活化的人類T細胞株Jurkat之囊泡分離 (vesicle isolation) 實驗中，證明PD-1的確存在於Rab37所調控的囊泡中。我們同樣透過穿透式電子顯微鏡、共聚焦縮時攝影成像以及全反射倒立螢光顯像分別顯示了Rab37介導的PD-1囊泡匯集的奈米結構和細胞內動態運輸。我們進一步在蔗糖濃度差離心分離 (sucrose density gradient centrifugation)實驗中，發現具有醣化修飾的野生型PD-1主要分布在細胞膜；相反的，醣化位點突變的PD-1則有些分佈在內體(endosome)之中。另外，我們在流式細胞儀實驗中觀察到，肺癌細胞與大量表現Rab37野生型Jurkat 細胞進行共培養時，Jurkat 細胞的生長以及活化相較於控制組均有下降的情形。相反地，在大量表現Rab37野生型Jurkat 細胞膜表面上，PD-1 + TIM3 +標記共同表達則是有上升的情形，這些標記代表這些Jurkat T細胞呈現“T細胞衰竭”以及低增殖活性。藉由肺癌病患癌組織的免疫組織化學分析 (immunohistochemistry analysis)，我們發現浸潤的CD8 +T細胞中Rab37與PD-1之間的相關性相較於初期的肺癌病人，在末期的肺癌病人有更高的相關性。
研究結論： 本研究提出新穎機制指出T細胞當中的Rab37介導PD-1穿膜運輸進而促進肺癌進程，而腫瘤浸潤T細胞共定位Rab37及PD-1可能為臨床惡化的證據。因此，如果透過檢查T細胞Rab37及PD-1表現程度，可能提供臨床對於免疫標靶治療效果更好的預判。

Background: CD8+ T cells are cytotoxic T cells which secrete cytokines to attack cancer cells in the tumor microenvironment (TME). However, exhaustion of CD8+ T cells cause an immunosuppressive TME in cancer progression. PD-1 is an inhibitory receptor which expresses on the surface of T cells and causes T cell dysfunction and failure of killing cancer cells in the TME. Although a lot of research have pointed out the mechanism of transcription regulation in PD-1, however, the post-transcriptional regulation and trafficking of PD-1 in T cells are still unclear.
Purpose: Our published studies show that Rab37 small GTPase mediates trafficking of cargos to influence cancer metastasis, stemness, and angiogenesis. Notably, we observed a downregulation of PD-1 expression on splenic CD8+ T cells isolated from whole body Rab37 knockout (Rab37 KO) mice compared to those from wild-type (WT) mice. Therefore, our aims are to investigate the role and mechanism of Rab37 in trafficking of PD-1 on the cell membrane of T cells in promoting lung cancer progression.
Results: We found colocalization of Rab37 and PD-1 in splenic CD8+ T cells isolated from Rab37 WT by confocal microscopy, immune-EM and FLOW. Importantly, we found that the level of PD-1 in plasma membrane (PM) presentation on Jurkat T cells was more in Rab37 wild-type (Rab37-WT) and constitutively active (Rab37-Q89L) expressing cells than that in control and dominant negative (Rab37-T43N) expressing Jurkat T cells by confocal microscopy, FLOW and membrane fraction. To investigate whether Rab37 was involved in PD-1 intracellular trafficking, vesicle isolation results showed the presence of PD-1 in Rab37-specific vesicles in PMA/Io treated Jurkat T cells. Moreover, we used immune-EM, time-lapse confocal imaging and total internal reflection fluorescence demonstrated ultrastructure of Rab37-mediated vesicle recruitment of PD-1 and intracellular trafficking dynamic, respectively. Furthermore, we used sucrose density gradient centrifugation and found that glycosylated WT PD-1 proteins were mostly located in membrane compartment, while some of the glycosylation mutant PD-1 proteins were distributed in the endosome compartment. To confirm the effect of Rab37 mediated PD-1 in T cells, we demonstrated that the proliferation and activity were downregulated in Jurkat T cells overexpressing Rab37 in mix-culture with cancer cells. In addition, the PM presentation of exhaustion marker PD-1+ TIM-3+ on Jurkat T cells overexpressing Rab37 increased in mix-culture with cancer cells. Finally, we performed immunohistochemistry analysis on tumor specimens from lung cancer patients to show that the correlation between Rab37 and PD-1 in tumor infiltrated CD8+ T cells were more in tumors derived from advanced stage lung cancer patients than those from early staged patients. Together, these results suggested that Rab37 mediated PD-1 trafficking and membrane presentation characterizing “T cell exhaustion”.
Conclusion: Our results provide first trafficking mode of PD-1 mediated by Rab37 small GTPase and novel evidence of the tumor promoting function of Rab37/PD-1 axis in T cells and its clinical implications.

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