||Rab37 mediates exocytosis of IL-6 and enhances PD-1 expression in CD8+ T cells to promote lung cancer progression
||Department of Pharmacology
CD8+ T 細胞
CD8+ T cells
研究背景: 許多的證據顯示浸潤的免疫細胞扮演促進腫瘤進程的角色，腫瘤微環境 (tumor microenvironment) 當中的複雜性造成免疫細胞分泌細胞激素的失調，並被教育成具有促進腫瘤生長能力的族群。先前本實驗室的研究發現小G蛋白 (small GTPase) Rab37調控癌細胞的胞吐作用 (exocytosis)；然而，Rab37蛋白在CD8+ T細胞所調控的胞吐作用在腫瘤微環境的角色以及其調控功能目前仍未知。
研究目的: 由於先導實驗發現CD8+ T細胞於Rab37基因剔除老鼠 (knockout mice) 其細胞激素：白細胞介白素-6 (IL-6) 蛋白分泌量與免疫抑制因子：程序性死亡受體-1 (PD-1) 分子的細胞膜表現量呈正相關，因此本研究探討Rab37是否介導IL-6蛋白的分泌路徑來促進CD8+ T 細胞上PD-1分子的表現進而影響肺癌進程，並且探討結合阻斷IL-6以及免疫抑制因子CTLA-4對於肺癌的治療效果。
研究結果: 從Lewis lung carcinoma (LLC) 皮下腫瘤的動物模式發現，Rab37 knockout (KO) 老鼠中，腫瘤的生長有受抑制的現象，並且進行流式細胞儀的分析後發現PD-1分子在浸潤CD8+ T 細胞的表現有下降的趨勢；除此之外，在經過LLC培養液 (conditioned medium, CM) 處理過後，Rab37 KO 老鼠的splenic CD8+ T細胞上的PD-1相較wild-type (WT) 組別有下降的趨勢，屬於免疫抑制族群的調節T細胞也有降低，指出Rab37在CD8+ T細胞中為一促進腫瘤的因子。接著我們利用細胞激素/趨化激素陣列 (cytokines/chemokines array) 分析，篩選出IL-6在Rab37 KO CD8+ T細胞CM培養液的分泌量相較於Rab37 WT 組別有降低的現象；透過酶聯免疫吸附實驗 (ELISA) 以及西方點墨法證實IL-6在T細胞CM培養液的表現量與Rab37的表現量呈正相關，且利用免疫螢光染色與全反射倒立螢光影像證實Rab37在細胞內與IL-6分佈的情形與位置一致 (colocalization)，並調控IL-6運輸 (trafficking and exocytosis) 的過程。西方點墨法與反轉錄聚合酵素連續反應實驗證明磷酸化STAT-3的蛋白表現量以及PD-1的mRNA表現量在表現Rab37-WT 以及Rab37-Q89L 活化態 (GTP active mutant) 時增加，但Rab37-T43N 去活化態 (GDP inactive mutant) 時下降，顯示IL-6可能透過活化STAT-3路徑增加PD-1基因的表現。而在後續的動物實驗也觀察到聯合阻斷IL-6以及免疫抑制因子CTLA-4在LLC皮下腫瘤模式以及原位腫瘤模式提供更佳的腫瘤抑制效果，並且增加浸潤免疫細胞在腫瘤當中免疫活化的特性。
研究結論：本研究提出新穎機制指出Rab37在腫瘤微環境當中的CD8+ T細胞當中，藉由調控IL-6之分泌來增加腫瘤微環境當中CD8+ T細胞PD-1的表現進而促進肺癌進程。因此，透過同時針對IL-6以及另外一個免疫抑制分子CTLA-4的阻斷抗體，可能提供病人更好的治療效果。
Background: Emerging evidence indicates that tumor-infiltrating immune cells play important roles in tumor progression. The complexity of tumor microenvironment leads to the dysregulation of cytokine production in immune cells to shift themselves to have pro-tumor functions. We previously identified that Rab37 small GTPase mediates exocytosis in cancer epithelial cells. However, the role of Rab37 in tumor microenvironmental cells such as CD8+ T cells is not well understood.
Purpose: Our pilot tests showed a concordant reduction of interleukin-6 (IL-6) secretion and PD-1 membrane presentation on splenic CD8+ T cells derived from Rab37 knockout (KO) mice compared to those from wild-type (WT) mice. We therefore aimed to investigate the trafficking pathway of Rab37-mediated IL-6 secretion to promote PD-1 expression and examine the therapeutic efficacy of combined blockade of IL-6 and immunoinhibitory receptor CTLA-4 in animal models.
Results: Our in vivo results showed that Rab37 KO displayed an anti-tumor effect to inhibit Lewis lung carcinoma (LLC) tumor growth and decreased PD-1 presentation on infiltrated CD8+ T cells. Moreover, the percentage of PD-1 on splenic CD8+ T cells was reduced in Rab37 KO mice compared to that in WT mice, suggesting that Rab37 may play a pro-tumor role in tumor microenvironmental cells. We then collected the conditioned media (CM) of splenic CD8+ T cells from WT and Rab37 KO mice to identify the Rab37-mediated secretory factor(s) that regulated the function of CD8+ T cells using cytokines/chemokines array. The results showed that IL-6 was the potential exocytic cargo mediated by Rab37. Immunofluorescence images demonstrated that Rab37 colocalized with IL-6 in splenic CD8+ as well as mouse EL4 T cells and human Jurkat T cells expressing the Rab37-WT and Rab37-Q89L GTP-bound active mutant, but not in Rab37-T43N GDP-bound inactive mutant. Importantly, our total internal reflection fluorescence data demonstrated the dynamic movement of IL-6 mediated by Rab37, confirming that the IL-6 was the cargo mediated by Rab37. In addition, the protein levels of phosphorylated STAT-3 and the mRNA of PD-1 gene increased upon Rab37 expression in a GTPase nucleotide-dependent manner, indicating that IL-6 may regulate the transcription of PD-1 gene through STAT-3 signal. Furthermore, combination treatment of anti-IL-6 and anti-CTLA-4 inhibited tumor growth and decreased the percentage of CTLA4+PD-1+ on infiltrated CD4+ T cells and immunosuppressive Tregs population in subcutaneous model. In orthotopic model, combined treatment prolonged survival of treated mice and changed immune cell profiling such as the localization of M1/M2 macrophages and CD8+ T cells infiltrating in LLC tumors.
Conclusion: Together, these results provide a new insight that Rab37 plays a pro-tumor role to increase IL-6 secretion and PD-1 presentation on CD8+ T cells in the tumor microenvironment to promote lung cancer progression. Targeting IL-6 with immunoinhibitory receptor CTLA-4 may be helpful to improve patient outcome.
I. Lung cancer 1
(A) Epidemiology of lung cancer 1
(B) Therapeutic strategies in lung cancer 1
II. Tumor microenvironment (TME) 2
(A) Role of immune cells in tumor microenvironment 2
(B) Current immunotherapies in lung cancer 4
III. Cytokines in the tumor microenvironment 6
(A) The overviews of cytokines in the immune system 6
(B) The signaling of interleukin 6 (IL-6) in the TME and its therapeutic potential 7
(C) Transcription regulation of immunosuppressive molecule programmed cell death protein 1 (PD-1) 9
IV. Rab GTPases in vesicle trafficking and tumor progression 10
(A) Role of Rab family in vesicle trafficking 10
(B) Rab GTPase and diseases 11
(C) Our previous findings on Rab37 12
(D) Rab proteins in immune cells 13
Study basis and specific aims 15
Materials and methods 17
1. Animal studies 17
2. Cell lines and culture condition 18
3. CD4+ and CD8+ T cells isolation and activation 18
4. Cytokines and chemokines array 19
5. Plasmid, RNAi and transfection 19
6. Conditioned medium preparation, collection and treatment 20
7. Membrane fraction and Western blot analysis 21
8. Vesicle isolation and immunoprecipitation 22
9. Confocal microscopy 22
10. Total internal reflection fluorescence microscopy 23
11. FLOW cytometry 24
12. Immunohistochemistry assay (IHC) and immunofluorescence-IHC 24
13. Enzyme-linked immunosorbent assay 25
14. Statistical analysis 26
1. Rab37-/- knockout (KO) mice provided an anti-tumor microenvironment to inhibit tumor growth. 27
2. IL-6 secretion and PD-1 membrane presentation were regulated by Rab37-mediated exocytosis. 28
3. Immunofluorescence showed that Rab37 colocalized with IL-6 in a GTP-dependent manner. 29
4. The dynamics of Rab37-regulated IL-6 trafficking in 293T cell line. 31
5. Overexpression of Rab37 and IL-6 increased STAT-3 phosphorylation and PD-1 mRNA and protein expression. 31
6. Combination of anti-CTLA-4 and anti-IL-6 further inhibited tumor growth and prolonged overall survival. 32
7. Combined treatment exchanged the distribution of immune cells. 33
Appendix Tables and Figures 73
Figure 1. Rab37 deficiency possessed an anti-tumor microenvironment in part by increasing the anti-tumor activity of CD8+ T cells. 54
Figure 2. Identification and validation of Rab37-mediated IL-6 secretion in relation to PD-1 membrane presentation. 56
Figure 3. Rab37 mediated the exocytosis of IL-6 in a GTP-dependent manner. 58
Figure 4. The dynamics of Rab37-mediated IL-6 intracellular trafficking. 59
Figure 5. Rab37 regulated PD-1 transcription and membrane presentation via IL-6/STAT3 signal in CD8+ T cells. 60
Figure 6. Combined blockade of CTLA-4 and IL-6 provided synergistic anti-tumor immune responses. 61
Figure 7 Combined treatment of -IL-6 and -CTLA-4 changed the distribution of infiltrated immune cells. 63
Figure 8. The schematic model of Rab37/IL-6/STAT3/PD-1 axis in CD8+ T cell in regulation of lung tumor growth ------------------------------------------------------------------ 64
Table 1. Current clinical studies using anti-IL-6 or anti-IL-6R antibodies as therapeutic strategies. 66
Table 2. Current clinical trials which use immune checkpoint inhibitors to measure the levels of IL-6 in patients’ plasma. 67
Table 3. The plasmids and their characteristics used in the current study 68
Table 4. The primers used in the current study 69
Table 5. Antibodies and their reaction conditions used in the current study 70
Table 6. List of the cytokines/chemokines that were downregulated in CD8+ T cells from Rab37 KO mice compared to wild-type mice 72
Appendix Figure 1. Sequential steps of Rab-mediated vesicle trafficking. 74
Appendix Figure 2. Localization of Rab proteins and their functions. 75
Appendix Figure 3. Circulating IL-6 level is a prognostic marker for advanced non-small cell lung cancer (NSCLC) patients treated with chemotherapy. 76
Appendix Figure 4. IL-6 mRNA overexpression in the non-responders of NSCLC patients received anti-PD-1 therapy. 77
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