B-1與B-2細胞誘導調節性T細胞之功能性探討

Abstract

研究背景: 先前研究指出，B細胞在免疫系統中能發揮雙向調控的作用。近年許多文獻報告將傳統B-2細胞與T細胞共同培養以誘發調節性T細胞的產生。相對於B-2細胞，B-1細胞是B細胞族群中另一亞群，因能製造大量抑制性的介白素-10 (interleukin-10, IL-10) 在免疫系統中有其獨特的角色。已知在誘導Tr1或Tr1-like調節性T細胞生成的過程中需要介白素-10的參與，但 B-1細胞是否能藉由分泌介白素-10誘發調節性T細胞的生成仍不清楚。本研究欲透過分析B-1與B-2細胞在誘發調節性T細胞生成上的能力，進而闡述B-1與B-2細胞各自透過何種機制達到免疫調控。此外由B-1與B-2細胞所誘導出的T細胞，其特性和抑制免疫反應的機制也會一併釐清。 實驗方法: 於卵蛋白胜肽片段（OVA323-339）加上抗原呈現細胞的抗原特異性刺激系統或anti-CD3/CD28單株抗體刺激系統中，我們將分離出的B-1與B-2細胞分別和CD4+CD25－ T細胞共同培養。3天後重新分離B-1與B-2細胞所誘導出出CD4+ T細胞並分析其是否具有抑制能力和免疫特性。我們利用Foxp3-GFP × DO11.10基因轉殖鼠來研究轉錄因子Foxp3於B細胞所誘導出的T細胞上的表達水平；介白素-10基因剔除鼠則被用來研究介白素-10在誘導調節性T細胞或調節性T細胞本身抑制機轉中的作用。 實驗結果: 在所使用的兩種刺激系統中，B-1與B-2細胞均能誘導出具抑制能力的調節性T細胞，並證實誘導過程中不需要介白素-10的參與。我們將B-1細胞與B-2細胞誘導出的調節性T細胞分別命名為“Treg-of-B1”與“Treg-of-B2”細胞。接著，我們發現不同於CD4+CD25+ 天然調節性T細胞 (nTreg)，Treg-of-B1與 Treg-of-B2細胞皆不表現Foxp3並各自具有獨特的細胞表面標誌和細胞激素分泌。更進一步地，我們證明Treg-of-B1細胞的抑制機制主要是通過介白素-10之外的其它分泌型因子，而Treg-of-B2細胞的抑制機制則是需要細胞與細胞接觸。 結論: 本篇的實驗結果支持了B-1與B-2細胞可能藉由誘導調節性T細胞產生達到負向調控免疫反應的作用。我們首先釐清了B-1細胞並非藉由分泌介白素-10來誘導Tr1細胞產生。更進一步分析Treg-of-B1與Treg-of-B2細胞的細胞表面標記跟細胞激素分泌情形，顯示由B-1與B-2細胞所誘導出的這兩群細胞分屬兩群獨特的調節性T細胞群，也不同於nTreg或Tr1細胞。此外，這兩群調節性T細胞是藉由截然不同的機制達到抑制CD4+ T細胞的作用。這些研究結果使我們更加了解B-1與B-2細胞如何透過誘導出特性跟抑制機轉不相同的調節性T細胞，反映出各自在免疫調節上所扮演的角色。

Background: B cells have been found to induce both effective as well as tolergenic immune responses. Conventional B-2 cells have been shown to execute immune modulation through induction of Treg cells. The B-1 cell, belongs to another subset of B cell population, is specific and functionally important subset which exert their regulatory role through the production of IL-10. IL-10 has been correlated with the induction of Tr1 or Tr1-like cells, however, whether IL-10 producing B-1 cells are able to induce the generation of Treg cells especially the Tr1 lineage remain poorly understood. In this study, we aimed to study the effect of B-1 and B-2 cells on the inducting ability of Treg cells. Moreover, the properties as well as the effector function of B-1 and B-2 cell-induced Treg cells will be clarified. Methods: To induce Treg cells, B-1 cells and B-2 cells were isolated and co-cultured with naive CD4+CD25－ T cells under OVA323-339 pulsed-APCs or anti-CD3/CD28 antibodies stimulation. After 3 days, CD4+ T cells in the co-cultures were purified and their suppressive function was examined. The requirement of IL-10 during the induction or suppression process was investigated using IL-10 knockout mice, and the Foxp3-GFP × DO11.10 mice was used to trace the Foxp3 expression of induced Treg cells. Results: In our two induction systems, we found that B-1 and B-2 cells were both able to convert naive T cells into Treg cell population, which we called “Treg-of-B1 cells” and “Treg-of-B2 cells”. More importantly, dissimilar to nTreg cells, we indentified Treg-of-B cells did not necessarily express Foxp3 and acquired unique surface molecule expression as well as cytokine profile. Finally, we showed that the suppressive function of Treg-of-B2 cells required cell-cell interaction whereas Treg-of-B1 cells mainly relied on IL-10-independent but soluble factors mediated suppression. Conclusions: Our results supported the idea that B-1 and B-2 cells might modulate immune responses via the induction of Treg cells. Also, we further clarified that IL-10 producing B-1 cells do not induce Tr1 cells via IL-10 but to induce Treg-of-B1 cells thorough IL-10-independent mechanism. Moreover, we demonstrated that Treg-of-B1 and Treg-of-B2 cells were two unique Treg populations and they interacted with CD4+ T cells through distinct mechanisms. Taken together, these findings might help us in further understanding the role of B-1 and B-2 cells in immune regulation.