Wip1在B淋巴球調控 FcγRIIB 表現之研究

Abstract

抑制性接受器在免疫細胞扮演著負調節功能的重要角色。我們先前篩選到CCT007093這個藥物可以抑制FcγRIIB和PD-1基因促進子 (promoter)的活性。以淋巴球而言，FcγRIIB僅表達於B淋巴球，而PD-1則是B和T淋巴球均有表達。在研究過程，我首先用3.3 kb的FcγRIIB促進子連結螢光報導基因系統驗證了CCT007093確實隨劑量增加而增強抑制效果。接著利用BJAB 和Raji兩人類B細胞株，看CCT007093對內源性抑制性接受器表達的影響，發現除了FcγRIIB和PD-1之外，CCT007093還可以抑制CD72和LILRB2。這抑制基因轉錄作用在8小時後最為明顯。由於CCT007093顯然能夠在B細胞抑制多個抑制性接受器，我們接著想瞭解這樣的調節是否能直接影響細胞的功能?我以細胞分裂作為功能性探討。因為臨床上許多慢性感染症和腫瘤會伴隨著免疫系統失調，最主要的病徵是T細胞及B細胞呈現所謂的「衰竭」現象，也就是細胞表面的抑制性接受器全面表達升高，導致細胞分裂和毒殺能力下降，以及細胞激素和抗體分泌減少，最終可導致細胞凋亡。我們先前發現白藜蘆醇能夠促進B細胞上的抑制性接受器上升，所以我們先檢驗白藜蘆醇是否能模仿細胞衰竭現象，結果確實抑制了B和T細胞株分裂。相反地，CCT007093能夠促進細胞增殖。甚至，CC007093能夠反轉白藜蘆醇對細胞分裂的抑制效果。此外，我們分離了正常人周邊血液的淋巴球，發現CCT007093同樣能促進B淋巴球的分裂。綜合上述，我們發現CCT007093應可以進一步在慢性感染和腫瘤動物模式進行療效驗證。另一方面，由於CCT007093是p53的促進劑，而Wip1抑制p53，p53又能促進Wip1基因轉錄，再者CCT007093還影響NF-κB、p38MAPK、c-Jun和CREB等轉錄因子。對於目前免疫細胞衰竭導致抑制型接受器表達普遍上升的分子機制應予以詳加探討，並在動物實驗驗證，冀能提供更好的藥物標靶和治療方式。

Inhibitory receptors on immune cells play a crucial role in negative control of cell activation. We previously identified CCT007093 as a negative transcriptional regulator of FcγRIIB and PD-1 during a drug screen. In this study, I first confirmed that CCT007093 could inhibit 3.3 kb promoter of FcγRIIB linked to reporter genes in BJAB B cells in a dose-dependent manner. Moreover, CCT007093 could down-regulate the transcription of endogenous FcγRIIB, PD-1, CD72 and LILRB2 genes as well as the surface expression level of FcγRIIB and PD-1. Since CCT007093 appears to negatively affect multiple inhibitory receptors, I next investigated whether this would cause functional consequences. I examined the effects of CCT007093 on cell proliferation found that CCT007093 could not only promote cell proliferation but also reverse growth inhibition induced by resveratrol, which increased the expression of multiple inhibitory receptors in Raji B and Jurkat T cells. Similarly, the effect of CCT007093 on proliferation was recapitulated in purified human B lymphocytes. Taken together, these data strongly suggest CCT007093 as a potential pan-inhibitor of the expression of inhibitory receptors of immune cells. Since global up-regulation of inhibitory receptors is a signature of immune cell exhaustion in chronic infection and in tumor, CCT007093 may be an ideal candidate for therapy. In addition, CCT007093 is a Wip1 inhibitor that can predominantly up-regulate p53 expression. Other than p53, CCT007093 also regulates NF-κB, p38MAPK, c-Jun and CREB. In summary, my results have provided evidence that Wip1 inhibits expression of multiple inhibitory receptors, e.g. FcγRIIB and PD-1. To further underscore the importance of these findings, how Wip1 plays a role in promoting the expression of inhibitory receptors and whether Wip1 inhibitor might effectively relieve immune cells exhaustion in chronically infected mice are under investigation.