CCT007093藉由調降淋巴球的抑制性受體來緩解慢性B型肝炎引起的免疫衰竭

CCT007093 alleviates immune exhaustion in chronic hepatitis B infection by downregulation of inhibitory receptors on lymphocytes

游雨璇(Yu-Syuan You)

指導教授 ： 曾賢忠

國立臺灣大學/醫學院/藥理學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603247

摘要

免疫系統在抵抗病源上扮演重要的角色，然而在慢性感染中受病毒感染的細胞可以使淋巴球呈現“免疫衰竭”狀態，逃脫免疫抵禦。免疫衰竭細胞的特徵為細胞表面會同時表現多個抑制性受體而抑制細胞的活化與增生。值得注意的是在慢性B型肝炎的病人中，T細胞的PD-1表達增加。而在人類免疫缺陷病毒感染的病人中，衰竭B細胞的FcγRIIB表達量上升。我們推論用藥物降低抑制性受體表達應能緩解免疫衰竭。CCT007093為Wip1去磷酸酶抑制劑，先前研究可抑制淋巴球細胞株上抑制性受體的表達。本研究探討其機制並進一步檢驗CCT007093是否能改善慢性B型肝炎的老鼠動物模式中的免疫衰竭現象。結果顯示CCT007093可以調降PD-1在Jurkat T細胞的表達，也可以減少PD-1和FcγRIIB在BJAB B細胞的表達。在慢性B型肝炎感染老鼠，CCT007093能減少血清中的HBsAg，顯示感染緩解現象. 而在抑制性受體的表達方面，經過CCT007093的治療後，減少在血液、脾臟以及肝臟中PD-1表達的T細胞的數量。這些組織中表達PD-1以及FcγRIIB的B細胞也呈現下降。此外，未治療的老鼠相比較，接受治療的老鼠的PD-1highCD127low 衰竭T細胞亦顯著減少。更重要的是CCT007093可以促進脾臟和肝臟中T細胞產生IFN-γ以改善T細胞的功能。在作用機制方面，我們發現CCT007093調降PD-1和FcγRIIB與磷酸化的p53 (S15)和NF-κB (S536)有關。此外，藉由PD-1啟動子螢光報導系統發現YY1和NF-κB在CCT007093調降PD-1基因轉錄中扮演角色。Wip1可以和p53結合且CCT007093增加p53 (S15)之磷酸化，Wip1亦可與YY1結合且該結合會受到CCT007093的抑制。YY1可與p53結合，且CCT007093會抑制YY1與p53的結合反應。有報導顯示YY1對p53具有抑制作用。由於在螢光報導的實驗中顯示p53可能對於PD-1的表達為正向的調控，故YY1應為負調控PD-1重要因子。若YY1也抑制其他的抑制性受體的表達，應可考慮為治療免疫衰竭的藥物標的。

關鍵字

免疫衰竭 ； B型肝炎 ； FcγRIIB ； PD-1 ； CCT007093

並列摘要

Host immune system plays a critical role in defending against pathogens. However, in chronic infection virus-infected cells can evade host defense by making lymphocytes in a state of so-called immune exhaustion. Under this circumstance, immune cells are characterized by a concurrent up-regulation of multiple inhibitory receptors on their surface to negatively influence activation and proliferation. Notably, PD-1 is up-regulated on T cells in chronic HBV-infected patients; likewise, the levels of FcγRIIB increase on exhausted B cells in HIV patients. Here, we explore the use of CCT007093, a Wip1 phosphatase inhibitor, to decrease the expression levels of inhibitory receptors on lymphocyte cell lines and examine its underlying mechanisms. We found that CCT007093 down-regulated the expression of PD-1 on Jurkat T cells and also decreased PD-1 and FcγRIIB expression on BJAB B cells. We next determined whether CCT007093 can ameliorate immune exhaustion induced by chronic HBV infection in a mouse model. The CCT007093 reduced the serum HBsAg level in chronic infected mice. The percentages of PD-1+ T cells were decreased in the blood, spleen and liver after CCT007093 treatment. Similarly, PD-1+ and FcγRIIB+ B cells decreased in these compartments in response to CCT007093. Moreover, the percentages of PD-1highCD127low exhausted T cells also significantly decreased in CCT007093-treated mice in comparison to untreated mice. Moreover, CCT007093 enhanced the production of IFN-γ of T cells in the spleen and liver. On the other hand, we found that CCT007093 can down-regulate the gene expression levels of FcγRIIB and PD-1 by up-regulation of phosphorylated p53 (Ser 15) and NF-κB (Ser 536), respectively. We further identified that YY1 and NF-κB binding sites on PD-1 promoter play a crucial role in the inhibition of gene expression by CCT007093. CCT007093 increased the phospho-p53 (S15) by blocking Wip1. Interestingly, the interaction between YY1 and Wip1 was reduced by CCT007093. It is known that YY1 is a negative regulator of p53 and can interact with p53. Because the PD-1 promoter reporter assays indicated that p53 may be a positive regulator in the PD-1 gene expression, YY1 thus might be a key factor to down-regulate the gene expression of PD-1. If YY1 can inhibit the expression of other inhibitory receptors, YY1 might be a specific drug target for the reversal of immune exhaustion.

並列關鍵字

Immune exhaustion ； hepatitis B virus infection ； FcγRIIB ； PD-1 ； CCT007093

參考文獻

Banerjee A,Sindhava V,Vuyyuru R,Jha V,Hodewadekar S,Manser T,Atchison ML. YY1 Is Required for Germinal Center B Cell Development. Plos One. 2016, 11(5): e0155311.

Barber DL,Wherry EJ,Masopust D,Zhu BG,Allison JP,Sharpe AH,Freeman GJ,Ahmed R. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006, 439(7077): 682-687.

Barford D,Das AK,Egloff MP. The structure and mechanism of protein phosphatases: Insights into catalysis and regulation. Annu Rev Bioph Biom. 1998, 27:133-164.

Bengsch B,Seigel B,Ruhl M,Timm J,Kuntz M,Blum HE,Pircher H,Thimme R. Coexpression of PD-1, 2B4, CD160 and KLRG1 on Exhausted HCV-Specific CD8+T Cells Is Linked to Antigen Recognition and T Cell Differentiation. Plos Pathog. 2010, 6(6): e1000947.

Blackburn SD,Shin H,Haining WN,Zou T,Workman CJ,Polley A,Betts MR,Freeman GJ,Vignali DAA,Wherry EJ. Coregulation of CD8(+) T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol. 2009, 10(1): 29-37.