探討細胞失能相關因子對B細胞誘導調節性T細胞的發育及功能所扮演的腳色

Abstract

B細胞可以做為抗原呈現細胞誘導出一群不表達Foxp3的調節性T細胞，將其命名為B細胞誘發的調節性T細胞(Treg-of-B),然而不同於天然調節性T細胞與第一型調節性T細胞，這群B細胞誘發調節性T細胞是藉由緊密的細胞接觸來執行其抑制的功能，與其他兩群調節性細胞有所分別。數篇研究指出在一些自體免疫疾病的動物模式中，這群B細胞誘發調節性T細胞可以減緩其發炎的程度。細胞失能是周邊耐受性的機制之一，為較弱或是缺少共同刺激分子刺激下所造成的細胞不反應現象。而天然調節性T細胞被認為是自然狀態下細胞失能型態的細胞，而這些細胞失能型態的特色不只是調節性細胞的表徵特色，同時也有文獻指出這些細胞失能相關基因對於調節性T細胞的發展與抑制功能有相關。 在本篇研究中，首先確認B細胞誘發的調節性T細胞具有細胞失能型態，其次觀察其細胞失能相關基因的表現。同時我們設計各種不同B細胞誘發的調節性T細胞誘發條件包含抗原專一性B細胞與不同強度的CD28刺激，再進而探討白介素2抑制因子-Aiolos在B細胞誘發的調節性T細胞中的不同表現與其對於誘導與功能扮演的相關性。實驗結果發現，B細胞誘發的調節性T細胞呈現一個細胞失能的型態，包括刺激後細胞不增殖，且較低的白介素2分泌，但B細胞誘發的調節性T細胞卻不表達NFAT相關的細胞失能的相關基因，藉由此，我們再深入了探討白介素2抑制因子-Aiolos在B細胞誘發的調節性T細胞中的不同表現，發現Aiolos在B細胞誘發的調節性T細胞量介於Foxp3+自然調節性T 細胞與初始T細胞之間。由此我們延伸到探討調節性T細胞的誘導活化過程，我們利用兩種不同的活化條件，分別是不同強度的CD28刺激與卵清蛋白專一性B細胞所誘導出調節性T細胞。此篇研究的結果發現較高量的CD28刺激，或者是運用抗原特異性的B細胞進行誘導，並不會影響其細胞失能的表徵與相關基因表現，但卻會增加其B細胞誘發的調節性T細胞在再次刺激後的增值能力，除此之外，其抑制作用型T細胞的能力也有下降的趨勢，我們進而檢測其Aiolos的表現，發現在兩個變因下，其Aiolos的表現都相較於控制組低，且其抑制相關的分子表現也有下降的趨勢。 本篇研究發現了Aiolos與B細胞誘發的調節性T細胞活化與失能間的相關性，但Aiolos實際在發育與功能上的腳色還需要進一步的研究。此外不同誘導條件，會影響其抑制功能與細胞增殖的能力，也暗示了在誘導過程中適當程度的活化，對於其功能與發育的影響。

B cells are considered to be poor antigen presenting cells which tend to convert CD4+CD25- naïve T cells into CD25+Foxp3- regulatory T cells, which are termed Treg-of-B cells. Treg-of-B cells suppressed effector T cells by cell-cell contact manner which is different from the other two groups of regulatory T cells, natural regulatory T cells and type 1 regulatory T cells. In several studies of autoimmune disease animal models, Treg-of-B cells have been able to alleviate the level of inflammation. Anergy is one of the mechanisms of peripheral tolerance which is caused by weak or non-costimulatory signal result in unresponsiveness. Regulatory T cells are considered to be naturally anergic because of its hypoproliferate characters and low IL-2 production after stimulation, the cell with these characteristics are considered to have anergic phenotype. In addition to this anergic phenotype, several studies indicated that anergy associated factors take part in Treg cells development and suppressive function. In this study, we first characterized the anergic phenotype if Treg-of-B cell, second to analyze the anergy associated gene expression. We designed several induction systems to discuss how antigen specificity and co-stimulatory signal affecting the development and the function of Treg-of-B cell. Finally, we analyzed the IL-2 suppressor – Aiolos expression of Treg-of-B cells in a variety of induction systems. The results showed that Treg-of-B cells exerted anergic phenotype including hypoproliferate and low IL-2 production but they did not have the significant expression of NFAT related anergy associated gene. Hence, we further examed the IL-2 regulator Aiolos, we found that the Aiolos expression of Treg-of-B cells were somewhere in between the expression of nTreg cells and Naïve T cells. We then extend the experiment to the different process of the induction of Treg-of-B cells, one is the different strengths of CD28 mAb stimulation, we also used OVA-specific B cell to perform the B cell- T cell co-culture. We discovered that stronger CD28 stimulation and the involvement of antigen-specific B cell—OB1 B cell, did not alter the anergic phenotype of Treg-of-B cell or T-of-OB1 cells, but somehow increased their ability to proliferate, and dreaded the suppression capacity and the molecules involved in the suppressive function compared to the control Treg-of-B cells. In this study, we observed that there is an association between the activation/anergy status of Treg-of-B cells, but the actual role of Aiolos in the development and function of Treg-of-B cells still need further studies. We also found that different activation condition during the induction of Treg-of-B cells would affect their suppressive capacity and the proliferation ability, which suggest that the appropriate level of activation is crucial for the development and function of Treg-of-B cells.