氫氣調控耗竭T細胞PD-1表現之機制探討

Abstract

在正常生理條件下，表現在T細胞上的細胞程式死亡配體1蛋白（Programmed cell death protein 1, PD-1) 可調控體內的免疫反應。但當T細胞處於腫瘤微環境時，因受到抗原不斷的刺激而過度表現PD-1，進而失去對抗腫瘤細胞的能力，這種現象稱為T細胞耗竭（T cell exhaustion）。在臨床上，PD-1免疫療法被用於恢復T細胞能力以對抗癌症，但其治療費用卻十分高昂。過去的臨床研究發現氫氣治療可以減少T細胞上的PD-1表現；但其中的調控機制並未解碼。因此，本研究的目標即是針對氫氣是如何減少T細胞上PD-1的機制進行探討。根據即時定量聚合酶連鎖反應、西方墨點法和流式細胞儀的分析結果顯示，當T細胞生長在富含氫氣的環境中，PD-1的表現量會明顯減少。而氫氣會通過調控細胞質內鈣離子濃度而降低調控PD-1的重要轉錄因子NFATc1活化的數量。另外，我們發現氫氣也會改變T細胞的粒腺體膜電位，且提升細胞的活性。由我們的研究結果可知氫氣是通過調控T細胞內鈣離子濃度進而減少PD-1的表現，這為氫氣在臨床上的應用提供了更有力的證據。此外藉由新發現的調控路徑，我們合理化氫氣可以恢復T細胞活性的機制，也為癌症免疫療法的發展提供新的標的。

Programmed cell death protein 1 (PD-1) is an inhibitory receptor that regulates immune balance in normal physiologic condition. However, T cells in the tumor microenvironment (TME) was found to express high level of PD-1, resulted in a loss of their ability in eliminating cancer. This phenomenon was called T-cell exhaustion. Reversing T-cell exhaustion thus became an inspiring strategy for cancer treatment. PD-1 immunotherapy has been executed for T cell restoration. However the medical cost was too high to be affordable. Previous clinical study has observed the reduced expression of PD-1 on T cells via hydrogen therapy; however, the mechanism underlying remain unclear. We herein aimed to reveal the mechanism on how hydrogen reduce the expression of PD-1 in T cells. We found that hydrogen down-regulated the expression of PD-1 by incubating T cells in hydrogen-rich environment, which was identified by real-time PCR, western blot and flow cytometry. Furthermore, our preliminary data showed that hydrogen suppressed the expression of the activated NFATc1, an important transcription factor for PD-1, via regulating the cellular Ca2+ concentration, as evidenced by decreased amount of activated NFATc1 extracted from cytosol and decreased [Ca2+] in cytosol. In addition, we confirmed that hydrogen exerted an effect on the mitochondrial membrane potential. In summary, we discovered that hydrogen-mediated PD-1 downregulation in T cell was manipulated by Ca2+-mediated pathway, and these results rationalize the application of hydrogen in clinical practice. Moreover, as hydrogen-mediated T cell restoration pathway was uncovered, new targets can be discovered and considered to be used in future development of anti-cancer immunotherapy.