探討介白素4在B細胞誘導調節性T細胞的發育與功能之角色

Abstract

B細胞可作為抗原呈現細胞誘導出⼀群Foxp3-的調節性T細胞,稱為B細胞誘發的調節性T細胞 (Treg-of-B cells),B細胞需通過與T細胞接觸的方式進⾏誘導,非以分泌介白素10 (IL-10) 的機制,⽽B細胞誘發的調節性T細胞也是透過緊密的細胞接觸來執行抑制的功能,因此相較於天然調節性T細胞 (nTreg) 及第⼀型調節性T細胞 (Tr1),B細胞誘發的調節性T細胞是不同的族群。 目前的研究指出B細胞誘發的調節性T細胞可發展成治療移植排斥和風溼性疾病的⽅方式之⼀,然⽽在我們的研究中發現B細胞誘發的調節性T細胞的分化過程中,有訊息傳遞轉錄活化因⼦子6 (STAT6)的磷酸化和介白素4 (IL-4)分泌的現象,因此本研究將探討介白素4的訊息傳遞路徑在B細胞誘發的調節性T細胞中扮演的角色,我們透過中和介白素4來探討B細胞在誘發調節性T細胞對於其發育或細胞激 素分泌的影響;而實驗結果發現,實驗組與對照組的抑制能⼒力並無顯著性的差異,但實驗組分泌較少的介白素4和介白素10; 另外也利用介白素4基因剔除鼠進行研究,探討介白素4在誘發調節性T細胞其發育及功能上所扮演的角色,實驗結果發現,介白素4基因剔除的B細胞誘發的調節性T細胞依然保有抑制能力但分泌較少的介白素10,此結果顯示,介白素4可能未影響B細胞誘發的調節性T細胞的誘導及免疫調控能⼒; 先前研究指出介白素4能促使許多免疫細胞的⽣長與存活,因此,我們同時也探討介白素4是否會影響B細胞誘發的調節性T細胞的⽣存,藉由誘發細胞凋亡後,以Annexin V/ PI染⾊色的⽅方式,比較實驗組與對照組在 細胞凋亡比例上的差異,或分析與細胞凋亡相關基因的表現,實驗結果顯示,中和介白素4及介白素4基因剔除的B細胞誘發的調節性T細胞有較多的細胞凋亡,儘管外加介白素2和介白素4可減少對照組的細胞凋亡,⽽實驗組在外加介白素2也有部分降低細胞凋亡的效果,但兩個細胞激素結合給予可提升實驗組與對照組比列上差距,另外,分析B細胞淋巴瘤-2 (B-cell lymphoma 2, bcl-2) 基因家族的抗細胞凋亡基因bcl-x和bcl-w,或促細胞凋亡基因bax的表現,實驗結果顯示,中和介白素4實驗組的bcl-x基因有些微降低,在介白素4基因剔除的B細胞誘發的調節性T 細胞有較明顯的的差異,bcl-x和bcl-w基因表現皆下降,因此結果暗示,介白素4可能會維持B細胞誘發的調節性T細胞的生存,若細胞的微環境中缺乏介白素4,則可能會促使B細胞誘發的調節性T細胞更傾向細胞凋亡。 總結來說,我們的研究結果顯示介白素4對於B細胞誘發的調節性T細胞的發育及功能沒有顯著的影響,然⽽在缺乏介白素4的情況下,會促使B細胞誘發的調 節性T細胞更傾向細胞凋亡,因此介白素4可能參與在維持B細胞誘發的調節性T細 胞的⽣存。在未來的研究,可持續探討介白素4是否透過轉錄活化因⼦子6的訊息傳遞路徑來調控細胞激素的分泌或細胞凋亡。

Naïve B cells could act as antigen-presenting cells to induce a subpopulation of Foxp3- regulatory T cells, called Treg-of-B cells. Naïve B-cell-primed T cells is through cell-cell contact and independent of IL-10. The suppressive function of Treg-of-B cells partly requires close cell-cell proximity. Therefore, Treg-of-B cells is a population different from natural Treg cells (nTreg) and Type 1 regulatory T cells (Tr1). Recent studies had demonstrated that Treg-of-B cells could be developed as a therapeutic approach against transplant rejection, allergy and rheumatological diseases. However, our studies showed that the differentiation of Treg-of-B cells involved STAT6 phosphorylation and IL-4 secretion. Hence, IL-4 might play a role in induction and/or function of Treg-of-B cells. This study was performed to examine the role of IL-4 signaling pathway in the development and function of Treg-of-B cells. We blocked IL-4 by anti-IL4 antibodies to study the suppressive function and cytokines profile of Treg-of-B cells. However, the result showed no significant difference between experimental group and control group in the suppressive ability, but the levels of IL-4 and IL-10 were lower in neutralized group. We next used Il4-/- mice for further investigation by suppressive function analysis, cytokines profile comparison. the result showed nosignificant difference between WT, Il4-/- Treg-of-B cells in suppressive ability but the level of IL-10 was lower in Il4-/- Treg-of-B(WT) cells. These results might indicate that IL-4 did not affect the induction and function of Treg-of-B cells. Previous study had demonstrated that IL-4 control several immune cell growth and survival. Therefore, we also examined whether IL-4 might maintain the survival of Treg-of-B cells. After induced cell death, we compared the cell death rate experimental group and control group by Annexin V/ PI staining or analyzed apoptosis-related gene expression. The results showed that more cell death induced in IL-4 blocking Treg-of-B cells and Il4-/- Treg-of-B cells. Although IL-2 and IL-4 could rescue the cell death in control group, IL- 2 also partially rescued the cell death of experimental group, but the combination of these two cytokines might enlarged the gap of cell death rate between IL-4 neutralization or not. Furthermore, we analyzed bcl-2 gene family, including anti- apoptotic gene bcl-x, bcl-w and pro-apoptotic gene bax. The results showed that IL-4 blocking Treg-of-B cells expressed slightly lower level of bcl-x. Il4-/- Treg-of-B cells showed more significant difference with lower levels of bcl-x and bcl-w gene expression. The results hinted that IL-4 might involve in maintaining Treg-of-B cells survival. In the deficient of IL-4, more apoptosis might be induced on Treg-of-B cells. In summary, our studies suggested that IL-4 might not affect the induction and function of Treg-of-B cells. In the deficient of IL-4, Treg-of-B cells might tend to go on apoptosis. Therefore, IL-4 might maintain the survival of Treg-of-B cells. In the future, the regulatory mechanism of cytokines production and apoptosis by IL-4-STAT6 signaling pathway might require further researches.