2017-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/706909摘要：調節性T 細胞對於免疫系統的衡定有重大的作用，它經由細胞表面的分子與抗原呈現細胞表面的co-stimulatory 分子作用，可抑制T 細胞的增生，同時它也可以經由細胞激素來抑制T 細胞的增生。調節性T 細胞包括自然存在型及誘發型的調節性T 細胞，二者都可以抑制T 細胞的增生，其中最為人所熟知的就是自然存在型調節性T 細胞，它來自於胸腺表現Foxp3。而誘發型調節性T 細胞可由naïve T 細胞經細胞接觸或是細胞激素的影響而產生，因為可以自體外增生，因此被認為是細胞治療免疫相關疾病的重要細胞群。顆粒酶(granzyme) B 是一種毒殺性T 細胞和自然殺手細胞用以殺死細胞的主要顆粒，同時在調節性T 細胞中也扮演著重要角色。granzyme B 在自然存在型的調節性T 細胞，會影響其藉由細胞接觸產生的抑制作用。而本計畫中採用的疾病模式是發炎性腸炎的小鼠模式， dextran sulfate sodium(DSS)-induced colitis。此疾病被認為在腸道局部的調節性T 細胞功能不佳，而在TNF 拮抗劑的治療下會增加腸道局部調節性T 細胞的數量。我們之前利用naïve 脾臟B 細胞發展出一種LAG3+ Treg-of-B 細胞，除了具有抑制T 細胞增生的功能外，利用細胞轉移至關節炎的小鼠模式中也可以緩解關節炎的症狀，確認了此群細胞在小鼠體內的抑制功能。另人驚訝的是，經由我們初步的研究發現granzyme B 在Treg-of-B 細胞中具有較高的表現量，自Gzmb-/-小鼠培養出的Treg-of-B 細胞具有較差的抑制功能，因此，我們推測granzyme B 在Treg-of-B 細胞的抑制功能扮演著重要的角色。由於我們發展出的Treg-of-B 細胞可以在體外增生，是未來細胞治療的重要細胞群，因此我們想要更進一步了解它的機轉。本計畫為了要證實上述推測，預計分三年進行下列實驗：第一年：分析Treg-of-B 的細胞特性和免疫抑制功能的相關因子。第二年：利用Gzmb 基因剔除小鼠研究granzyme B 在Treg-of-B 細胞的影響 (in vitro 研究)。第三年：利用細胞治療發炎性腸炎的小鼠模式來證實granzyme B 在Treg-of-B 細胞抑制功能的影響 (in vivo 研究)。希望經由此研究我們可以更了解Treg-of-B 的特性及granzyme B 在其中所扮演的角色，對於未來利用誘發型調節性T 細胞的細胞治療來治療免疫疾病具有重大影響。<br> Abstract: Regulatory T cells (Tregs) maintain self-tolerance and immune homeostasis in the immunesystem. Treg cells express inhibitory markers on cell surface and interact with the co-stimulatorymolecules that presented on antigen presenting cells (APCs), thus restrain effector T-cellproliferation. They also secrete cytokines to repress T-cell proliferation.Tregs are heterogeneous and can be classified into naturally occurring Tregs (nTregs) andinduced Tregs (iTregs) according to their origin. nTregs are generated in the thymus andcharacterized by the expression of CD4, CD25, and Foxp3. iTregs can be produced from naive CD4+T cells in the presence of specific antigens through cell contact or immunomodulatory cytokines(e.g., IL-10, TGF-b) in vitro. Cell therapy with iTregs has emerged as a promising approach fortreating immune-mediated diseases.Granzyme B represent a major constituent of the granules of cytotoxic cells, includingcytotoxic T lymphocyte (CTL) and natural killer (NK) cells. Besides, granzyme B is one of the keymechanisms through which CD4+CD25+ Treg induce cell contact-mediated suppression.The mice model of inflammatory bowel disease is dextran sulfate sodium (DSS)-inducedcolitis. The immunopathogenesis of IBD is due partly to insufficient suppressor function andanti-TNF treatment increases the number of Foxp3+ Treg cells in the mucosa of patients.Previously we generated a novel iTreg subset induced by naïve splenic B cells, named LAG3+Treg-of-B cells. This cell population had cytokine profiles similar to those of Tregs and suppressedthe proliferation of Tresp cells. Adoptive transfer of LAG3+ Treg-of-B cells in mice ameliorated theinflammation and clinical severity of CIA. Our findings provide a new approach for controlling RAand other autoimmune diseases by using LAG3+ Treg-of-B cells.Interestingly, we found that these Treg-of-B cells express CD25 but not Foxp3, and high levelof granzyme B. Meanwhile, Gzmb-/- Treg-of-B cells have minor effect in inhibiting the proliferationof Tresp cells (preliminary data). Therefore, we speculated that granzyme B plays an importantrole in the inhibitory function of Treg-of-B cells.Our aim of this study is to clarify the role of granzyme B in the suppressive function ofTreg-of-B cells with working plans as followings:First Year: To generate Treg-of-B cells and study the characteristics and the inhibitory mechanism.Second Year: To evaluate the role of granzyme B in the inhibitory function of Treg-of-B cells invitro by comparing Treg-of-B cells from WT and Gzmb-/- mice.Third Year: To evaluate the role of granzyme B in the inhibitory function of Treg-of-B cells in vivoby adoptive transfer of Treg-of-B cells in dextran sulfate sodium (DSS)-induced colitis model.Through this study, we hope to study the features of the Treg-of-B subset and the contributionof granzyme B in the inhibitory functions of Treg-of-B cells in vitro and in vivo. The mechanismelucidated will give us insight of cell therapy with iTregs as a promising approach for treatingimmune-mediated diseases.