FcγRIIB和PD-1在B細胞之基因表達及抑制性功能之研究

Studies on the gene expression and inhibitory function of FcγRIIB and PD-1 in B cells

李宛諭(Wan-Yu Li)

指導教授 ： 曾賢忠

國立臺灣大學/醫學院/藥理學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10691

摘要

FcγRIIB (又名CD32B)是抑制性的Fc受體，是IgG的受體之一。在B細胞，FcγRIIB的主要作用是經由免疫複合體 (immune complex)去抑制與抗原結合的B細胞抗原受體 (B-cell antigen receptor, BCR)，藉以阻斷B細胞的活化和增殖。另一方面，FcγRIIB也可以自體聚集 (homo-aggregation)促使B細胞凋亡，與B細胞的體內平衡 (homeostasis)有關。不過，其抑制性訊息傳遞較不清楚。我們從人類周邊血分離B細胞來探討其機制。人類周邊血B細胞可依CD27和CD38表面標記分成初始B細胞 (na iuml;ve B cells，CD27-CD38-)、記憶B細胞 (memory B cells，CD27+CD38-)和漿細胞 (plasma cells，CD27+CD38+)。當給予非抗原專一性免疫複合體去活化FcγRIIB時，三群B細胞均呈現凋亡，尤以漿細胞最顯著，這與漿細胞最高表達FcγRIIB吻合。此外，三種B細胞凋亡均可被BTK和p38激酶抑制劑 (kinase inhibitor)阻斷。有趣的是，只有初始B細胞和漿細胞凋亡亦可被c-Abl激酶抑制劑逆轉。初步結果顯示c-Abl應是BTK下游訊息分子，但需在三種細胞進一步確認。由於FcγRIIB是抑制B細胞功能最重要的受體，而FcγRIIB基因剔除鼠呈現B細胞異常增生、過度活化以及製造過量自體免疫抗體，最終導致紅斑性狼瘡。在人類紅斑性狼瘡患者，周邊血記憶B細胞和漿細胞的FcγRIIB表達均較正常個體低。我們先前的研究發現白藜蘆醇可促進FcγRIIB基因轉錄，進而緩解紅斑性狼瘡鼠病徵。因此，我們接著研究白藜蘆醇調節FcγRIIB基因轉錄的機制。首先採用克隆的3.3 kb 和其5’端基因刪除片段FcγRIIB基因啟動子(promoter)輔以冷螢光酶報導系統，在人類BJAB B細胞株分析關鍵片段區域。結果發現基因啟動子-600到-471 bp和-372到-331 bp這兩個片段是重要區域。NF-κB在-600到-471 bp的片段區域最重要，並受白藜蘆醇乙醯化作用調控。而在-372到-331 bp的片段區域中，則是轉錄因子YY-1和AP-4較具調節潛力。由於2 kb PD-1 (programmed cell death 1，又名CD279)基因啟動子亦受白藜蘆醇類似調節，我們也做了初步分析。綜合上述，我們認為透過研究增強FcγRIIB抑制性訊息傳遞和提升FcγRIIB轉錄的分子機制，應可找出最合適的分子作為藥物標靶來應用於治療自體免疫疾病。

關鍵字

免疫抑制性受體 ； B細胞

並列摘要

FcγRIIB (a.k.a. CD32B) is a sole inhibitory Fc receptor. When it is co-ligated with B-cell antigen receptor (BCR), it blocks BCR signaling for activation and proliferation of B cells. In contrast, cross-linking FcγRIIB without coengagement of BCR triggers an apoptotic response. The inhibitory signaling, however, through FcγRIIB homo-aggregation remains largely unknown. To investigate apoptotic signals transduced through FcγRIIB in human B cells, we purified na iuml;ve B cells (CD27-CD38-), memory B cells (CD27+CD38-) and plasma cells (PCs, CD27+CD38+) from peripheral blood and observed apoptosis induced by non-cognate immune complexes (ICs) in all three subsets. The apoptotic response was most significant in PCs, consistent with its highest surface level of FcγRIIB. Moreover, FcγRIIB mediated apoptosis in all three subsets of B cells through BTK- and p38-dependent pathways as judged by specific kinase inhibitors. Interestingly, c-Abl was also involved in apoptosis by FcγRIIB activation in na iuml;ve B cells and PCs but not memory B cells. Preliminary data showed that c-Abl was likely downstream of BTK. The importance of the inhibitory role of FcγRIIB is most evident from FcγRIIB deficient mice, which manifest uncontrolled B-cell expansion and massive autoantibody production, leading to lupus-like disease. In contrast, overexpression of FcγRIIB on B cells confers protection in lupus-prone mice. Similarly, patients with SLE exhibit a decrease of FcγRIIB’s expression on memory B cells and PCs. We previously showed that resveratrol could upregulate the gene transcription of FcγRIIB on B cells and ameliorate lupus symptoms of lupus prone mice. To investigate the underlying mechanism, we analyzed the 5’-deletion of 3.3 kb FCGR2B promoter constructs by luciferase reporter gene assay and found that the region of between 600 to 471 bp and between 372 to 331 bp upstream to FCGR2B start codon were critical for the effect of resveratrol. At the -600 to -471 bp region, reduced binding of acetylated NF-κB to the promoter appeared to confer increased transcription of FcγRIIB. Moreover, binding of YY-1 and AP-4 at -372 to -331 bp region was likely critical to the resveratrol-mediated up-regulation of FcγRIIB. Based on these findings, the identification of the key intermediaries of pro-apoptosis signaling by FcγRIIB or the key transcriptional regulator of FcγRIIB gene to enhance or to restore inhibitory function of FcγRIIB may be able to select a druggable target to eliminate over-expanded and hyperactive B cells in patients with autoimmune diseases. Thus, pharmacological augmentation of inhibitory function of FcγRIIB may become a new strategy for the treatment of SLE.

並列關鍵字

immune inhibitory receptor ； B cell

參考文獻

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