黏膜B細胞誘發調節性T細胞緩解過敏性呼吸道發炎之研究

Abstract

氣喘是一種常見的慢性呼吸道發炎疾病。透過黏膜給予過敏原引起的免疫耐受性，已經被用來當做治療過敏疾病的方法。研究發現，皮耶氏體中最主要的細胞群─B細胞，可以促使調節性T細胞的生成。本篇研究即是針對皮耶氏體的B細胞，在產生調節性T細胞中扮演的角色，以及這些調節性T細胞的功能。利用體外抑制細胞增生實驗及測量細胞激素方法來確定這些由皮耶氏體B細胞刺激產生的調節性T細胞〈在此稱為Treg-of-B(P)細胞〉。對於測量 Treg-of-B(P)細胞的生理功能，我們利用細胞移植實驗將Treg-of-B(P)細胞注入用OVA蛋白致敏的小鼠體內，藉由偵測肺沖洗液中OVA特異性免疫球E的濃度，第二型T細胞激素，嗜伊紅性白血球增多現象，以及組織切片染色法確定Treg-of-B(P)細胞療效。 首先，我們證明連續五天口服餵食小鼠OVA 0.5毫克，可以降低肺沖洗液中第二型T細胞反應。我們也發現，高劑量的OVA〈20毫克〉會降低CD4 T細胞的數量。反之，低劑量餵食過敏原可以幫助生成調節性T細胞。試管外實驗證明Treg-of-B細胞具有抑制T細胞增生的能力。相較於脾臟B細胞，從皮耶氏體分出來的B細胞，在接觸過抗原之後，呈現較不活化的狀態。此外，藉由近距離的細胞接觸，以及介白素-10分泌促成此Treg-of-B細胞的抑制作用。利用螢光流式細胞儀分析發現，Treg-of-B(P)細胞會表現CTLA4、ICOS、OX40、PD-1和TNFRII。分泌的細胞激素方面，Treg-of-B(P)細胞會分泌低量的介白素-2，高量的介白素-10，甚至比自然產生的調節性T細胞還高。我們進一步的分析發現，介白素-10，GITR及PD-1都參與調控Treg-of-B(P)細胞的抑制作用中。除此之外，本篇實驗也比較了不同B細胞產生調節性T細胞的能力。在脾臟及皮耶氏體中的FOB及MZB細胞，可以將初始T細胞(naïve T cell)轉變為調節性T細胞。並且，皮耶氏體中的B細胞比起一般認為的專業的抗原呈現細胞，在此為樹突細胞，在生成調節性T細胞的能力更為明顯。為了確定此Treg-of-B(P)細胞的生理活性，我們將此調節細胞打入氣喘小鼠體內，發現不論在小鼠致敏前或致敏後打入細胞，都可以有效的抑制第二型T細胞激素產生，嗜伊紅性細胞浸潤，及緩解呼吸道阻力的症狀。 總合上述的發現，本篇研究證明在腸胃道的B細胞具有生成調節性T細胞的能力，並且藉此參與構成口服耐受性特性的過程，進而緩解氣喘疾病的症狀。

Asthma is one of the most common chronic airway inflammatory diseases. Induction of immunologic tolerance via mucosa has been used for treating allergic diseases. B cells, the major cell population in Peyer’s patches, have been shown to induce the development of Treg cells. This study aimed to investigate the role of B cells in Peyer’s patches to tolerance induction and on Treg cell functions. In vitro suppressive assay and ELISA were used to evaluate the function of T cells stimulated by Peyer’s patches B cells (Treg-of-B(P) cells). The therapeutic potential of Treg-of-B(P) cells on ovalbumin (OVA)-induced murine asthma is evaluated by measuring OVA-specific IgE, Th2 cytokine production, eosinophilia in Bronchoalveolar lavage fluid (BALF) and histopathological analysis. First, we demonstrated that oral fed mice with OVA 0.5 mg for five days could diminish Th2 responses in BALF. High dose antigen administration (20mg) results in decreased CD4 T cell number, in contrast, low dose antigen helped Foxp3+ or IL-10 producing T cells induction. In vitro assay showed that Treg-of-B cells were found to exert suppressive function on T cell proliferation. Antigen-loaded B cells isolated from Peyer’s patches were more tolerogenic, compared with splenic B cells, and had the potential to generate more suppressive Treg-of-B cells via IL-10 production and cell-cell contract. Treg-of-B cells expressed CTLA4, ICOS, OX40, PD-1, and TNFRII and produced lower IL-2 and higher IL-10. Blocking experiment shows that IL-10, GITR and PD-1 mediate the suppressive effect of Treg-of-B(P) cells. Furthermore, we identify that FOB and MZB cells are capable of Treg cells generation. In addition, Peyer’s patches B cells are much better than Peyer’s patches DCs on generating Treg cells. In the murine model of asthma, an adoptive transfer of Treg-of-B(P) cells pre- or post- immunization could sufficiently suppress Th2 cytokine production and eosinophil infiltration, and alleviate asthmatic symptoms. B cells isolated from gut-associated lymphoid tissues, GALT, can generate regulatory T cells that may be important in oral tolerance and be applicable for alleviating allergic symptoms.