研究B細胞誘導的調節性CD8+ T細胞特徵及其在小鼠發炎疾病模型中的免疫調節功能

Abstract

調節性T細胞（Regulatory T cells, Tregs）是一群具有抑制功能的淋巴細胞，在維持體內免疫反應中扮演重要角色。先前我們實驗室發現B細胞可誘導CD4+CD25- T細胞轉變為一群CD25+FOXP3-調節性T細胞（Treg-of-B cell），在自體免疫疾病與過敏模型中皆證明其免疫抑制功能。除了典型的CD4+調節性T細胞，許多研究也顯示CD8+ T細胞中也存在具有免疫抑制活性的族群。因此本研究的目的為探討B細胞誘導產生的CD8+調節性T細胞（CD8+ Treg-of-B cell）的特徵，並進一步研究其在小鼠發炎性疾病模型中的免疫調節作用。 此一研究結果發現，CD8+ Treg-of-B細胞表現CD25、ICOS、LAG3、OX40、GITR、PD-1、CTLA-4等分子，但並不表達轉錄因子Foxp3。與CD8+CD25- T細胞相比，活化後的CD8+ Treg-of-B細胞可分泌更多的IL-10、IFN-γ和TNF-α。此外CD8+ Treg-of-B細胞需透過細胞接觸機制對CD4+和CD8+ T細胞發揮抑制能力。為了探討CD8+ Treg-of-B細胞在體內的免疫調節功能，我們透過葡聚糖硫酸鈉（dextran sulfate sodium salt, DSS）與CD4+CD45RBhi T細胞建立小鼠結腸炎模式，以及利用卵清蛋白（ovalbumin, OVA）誘發小鼠氣喘模型。在發炎性腸道疾病模型中，CD8+ Treg-of-B細胞降低促發炎細胞因子IL-1β與IL-17的表現，並減輕DSS結腸炎的病理特徵。在CD4+CD45RBhi T細胞誘導的結腸炎中，接受CD8+ Treg-of-B細胞治療的小鼠也降低其結腸組織中IFN-γ的表現。此外CD8+ Treg-of-B細胞在體外也抑制第二型輔助性T細胞（T helper 2 cells, Th2）增生，並降低Th2細胞因子IL-4、IL-5和IL-13的產生。在OVA引發的氣喘小鼠中，以 CD8+ Treg-of-B細胞治療顯著下調呼吸道過度反應，並減少支氣管肺泡灌洗液中嗜酸性白血球的浸潤。此外，CD8+ Treg-of-B細胞與CD8+CD122+調節性T細胞具有相似表型特徵，且比起天然CD8+CD122+調節性T細胞表達更高水平的IL-10。 總結以上結果，我們的研究為CD8+調節性T細胞領域提供了新的發現。CD8+ Treg-of-B細胞是一種新型的CD8+調節性T細胞，在體外透過細胞接觸發揮抑制效果。我們也證明了CD8+ Treg-of-B細胞能減緩小鼠實驗性結腸炎與呼吸道發炎。這些數據可能支持CD8+ Treg-of-B細胞在發炎疾病中的治療潛力。

Regulatory T cells (Tregs) play a crucial role in maintaining homeostasis and mediating suppressive functions. In the previous studies, our laboratory found that naïve B cells facilitate the conversion of CD4+CD25- T cells into forkhead box protein 3 (Foxp3)-CD25+ regulatory T cells, named Treg-of-B cells. The immunosuppressive function of Treg-of-B cells has been demonstrated in a variety of allergic and autoimmune disease models. In addition to classical CD4+ Tregs, many studies have also revealed suppressive functions in CD8+ T cells. Hence, this study aimed to characterize CD8+ regulatory T cells induced by B cells (CD8+ Treg-of-B cells) and investigate their regulatory effects in murine models of inflammatory disease. In this study, CD8+ Treg-of-B cells were characterized by expression of CD25, lymphocyte activation gene 3 (LAG3), inducible co-stimulator (ICOS), tumor necrosis factor receptor superfamily member 4 (OX40), glucocorticoid-induced TNFR-related protein (GITR), programmed cell death protein 1 (PD-1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), but did not express Foxp3. Activated-CD8+ Treg-of-B cells secreted higher levels of interleukin (IL)-10, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) compared to CD8+CD25- T cells. In addition, CD8+ Treg-of-B cells exerted suppressive ability on both CD4+ and CD8+ T cells through a cell-cell contact-dependent mechanism in vitro. To further investigate the immunomodulatory function of CD8+ Treg-of-B cells in vivo, chronic dextran sulfate sodium salt (DSS)-induced colitis, CD4+CD45RBhi T cell transfer colitis, and ovalbumin (OVA)-induced asthma models were used. Significant reductions in the levels of pro-inflammatory cytokine IL-1β and IL-17 were observed following treatment with CD8+ Treg-of-B cells, which also attenuated the pathological features of DSS-induced colitis. In addition, the level of IFN-γ was decreased in the colonic tissue of T cell transfer colitis mice. Moreover, CD8+ Treg-of-B cells effectively inhibited the proliferation and cytokine secretion of IL-4, IL-5, and IL-13 by T helper (Th) 2 cells in vitro. In asthmatic mice, CD8+ Treg-of-B cells down-regulated airway hyperresponsiveness and decreased eosinophilic infiltration in bronchoalveolar lavage. In addition, CD8+ Treg-of-B cells shared a similar phenotype with CD8+CD122+ Tregs and expressed a higher level of IL-10 than CD8+CD122+ Tregs. In conclusion, our study provided new insight into the characterization of CD8+ Tregs. CD8+ Treg-of-B cell is a novel Foxp3-CD8+ Treg subset with suppressive effects through cell contact in vitro. We also demonstrated that CD8+ Treg-of-B cells attenuated the severity of experimental colitis and airway inflammation in vivo. These data might suggest the therapeutic potential of CD8+ Treg-of-B cells in inflammatory diseases.