摘要:過敏疾病在過去幾年來可以說是逐年增加,已經成為所有國民健康上的一個重要問題,所以如何更進一步了解過敏疾病的發生機轉和研發出更有效的治療方法是一個刻不容緩的研究課題。目前已經知道過敏疾病的主要免疫調節是跟第一型與第二型T輔助細胞的調控有關,第二型T輔助細胞所分泌的IL-4和IL-5會分別刺激IgE抗體及嗜伊紅性白血球的產生而導致發炎反應。所以,研究出更有效的治療方法和了解這些治療方法的機轉將有助於我們在未來研發出更有效的治療方法。而在我們之前的研究中發現利用口服方式給予過敏原對降低呼吸道的發炎反應有很重要的影響,因此我們進一步研究黏膜免疫系統中的細胞在口服耐受性中所扮演的角色。此外,我們發現如果利用B細胞與T細胞一道培養後可以有效地誘發出具有調節能力的T細胞。我們在此一計畫中要來進一步研究這些由B細胞誘發出來的調節性T細胞(Treg/B cells)在黏膜免疫中所扮演的角色,同時也要研究這些Treg/B細胞的功能和基因表現,未來如何來誘發這一群細胞能夠應用到免疫疾病的調控上。在第一年的計畫中,我們計畫進一步研究是否能夠利用B細胞來誘發出具有調節能力的T細胞,再研究這些T細胞的特性和表面標記的表現。此外,我們也將分離不同的抗原呈現細胞與T細胞一道培養,以了解這些調節性T細胞是否也能夠像B細胞一樣誘發具有調節能力的細胞。我們在第一年的計畫中也分別利用黏膜B細胞、樹突細胞和巨噬細胞與T細胞一道培養,結果發現利用黏膜B細胞來誘發的T細胞有最好的調節能力。我們也進一步研究Treg/B細胞表現各種不同的細胞表面分子,同時也進行當初在計畫書中所提到的利用microarray來看看是否能夠找到較新的分子,其中我們發現PD-1和CTLA-4的表現較高,也進一步瞭解是否能夠阻斷這些細胞表面分子而達到改變此一調節性T細胞的抑制功能。同時,我們也將這些Treg/B細胞注射入氣喘的小鼠體內,以了解是否能夠改善氣喘小鼠的呼吸道發炎,尤其是降低過敏原特異性抗體和改善呼吸道發炎反應。此外,我們也將分離出不同活化和分化階段的B細胞與T細胞一道培養,如B-1和B-2細胞,活化和未活化的B細胞,記憶性B細胞,以了解哪些B細胞能夠誘發出具有調節能力的T細胞。我們在這一年的計畫中也已經初步完成B-1和B-2細胞誘發T細胞,我們的研究結果也發現不論是B-1或是B-2細胞的確都有誘發出調節性T細胞的能力,但是兩者之間的機轉似乎還是有些不同。我們在前兩年的研究中已經對這群由B細胞誘發出的調節性T細胞有了更清楚的研究,而且也已經找到一些新的研究方向。我們的研究成果包括:一. 我們在第一年的研究結果中除了證明由黏膜分離出來的B細胞,與樹突細胞和巨噬細胞相較之下會誘發出具有調節能力的Treg細胞。而且利用microarray的方法分析也發現在這些Treg/B細胞上會表現較高量的LAG-3分子,除了利用microarray的方法來測定外,也進一步利用real-time PCR的方法來加以確定LAG-3在這些Treg/B細胞的確有較高的表現。二. 我們的研究也發現由Peyer’s patches所分離出來的濾泡B細胞可以誘發出最具有調控能力的Treg/B細胞。三. 在此一研究計畫成果中我們進一步研究LAG-3是否參與在Treg/B細胞的功能中,所以我們利用抗LAG-3抗體來加入培養中,可以發現抗LAG-3抗體能夠有效地將Treg/B細胞抑制T細胞增殖能力的反應回復,顯示LAG-3分子的確在這些Treg/B細胞的功能中扮演一個重要的角色。四. 為了進一步了解這些LAG-3陽性Foxp-3陰性的Treg/B細胞是否能夠在氣喘的動物模式具有改善呼吸道發炎的效果,我們進一步分離出這些Treg/B細胞注射入小鼠體內。結果發現這些LAG-3陽性的Treg/B細胞的確能夠有效地抑制過敏抗體的產生和降低呼吸道的發炎情形。五. 我們也進行分離出B-1和B-2細胞來與T細胞一道培養,再研究這些不同來源的B細胞是否能夠誘發出具有調節能力的Treg/B細胞。我們進一步研究發現這些Treg/B細胞都具有免疫調節的效果,也針對其可能的機轉做了更進一步的研究和探討。六. 為了進一步了解這群Treg/B細胞的特性,我們進一步研究IL-10在這群Treg/B細胞所扮演的角色。研究結果發現IL-10在誘發和作用的機轉上並不是扮演一個重要的角色。我們在此一研究計畫中我們主要是要研究利用B細胞誘發出的Treg/B細胞的特性和功能,同時進一步將這些Treg/B細胞應用到各種不同免疫疾病的治療上。我們已經完成了第一年計畫的研究內容,我們已經完成這些相關的研究,包括這些Treg/B細胞的表面標記和細胞激素製造的情形,其中發現LAG-3的Treg/B細胞在功能上最有其意義。此外,針對這群Treg/B細胞的細胞激素分泌情形也已經有分析清楚,而且應用到氣喘的小鼠模式也發現改善呼吸道的發炎。我們也比較了不同抗原呈現細胞如樹突細胞和巨噬細胞對誘發Treg細胞的能力,結果發現B細胞的能力還是最好。在過去兩年的研究中,我們已經建立了Treg/B細胞培養的方法,而且證明可以在小鼠的氣喘動物模式來改善呼吸道的發炎。這群由B細胞所誘導出來的調節性T細胞可能在整個免疫反應的回饋性調控上也扮演著一個重要的角色,因此更值得加以研究。我們發現這些Treg/B細胞在發育和作用上並不需要如IL-10的細胞激素,這點與傳統的第一型調節性T細胞(Tr1)不同。此外,我們也發現這些細胞中會表現較高量的LAG-3分子,這些Foxp-3- LAG-3+的Treg/B細胞在氣喘的小鼠動物模式也有改善呼吸道發炎的效果。我們相信這群新的調節性T細胞如果能夠研究地更清楚,將有助於未來進一步將這些細胞應用到臨床免疫疾病的治療上。
Abstract: Mucosal tolerance, which induces immunologic tolerance to non-pathogenic antigens in the mucosa of the respiratory, gastrointestinal, and urogenital tracts, has been used in humans for the treatment of allergic diseases for a century. In addition, both oral and nasal tolerance are used to treat several inflammatory diseases, including experimental autoimmune encephalomyelitis, arthritis, and food allergies. It has been proposed that clonal deletion due to high-dose antigen exposure and Treg cell production or anergy due to low-dose antigen exposure result in the induction of mucosal tolerance.Our previous study demonstrated that mucosal B cells have a better ability to convert naïve T cells into Treg cells, so-called Treg-of-B (P) cells. These Treg-of-B (P) cells, which produce more IL-10 and express cytotoxic T lymphocyte antigen 4 (CLTA4), inducible costimulator (ICOS), OX40 (CD134), programmed death-1 (PD-1) and TNF-RII, alleviate allergic airway inflammation.Recently, lymphocyte activation gene 3 (LAG3) has been identified as a marker of regulatory T (Treg) cells. LAG3 mRNA is selectively expressed by naturally occurring Treg (nTreg) cells and is not found in CD4+ CD25- T cells. In addition to modulating Treg cell function in vitro and in vivo, the ectopic expression of LAG3 confers a regulatory function to CD4+ T cells. A clinical study demonstrated that one particular Treg cell population, CD4+ CD25hi Foxp3+ LAG3+ cells, preferentially expanded in peripheral blood monocytic cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in cancer patients and might contribute to tolerance at tumor sites. In Peyer’s patches, CD4+ LAG3+ T cells are enriched to approximately 8%, compared with 2% in the spleen, implying that LAG3 might participate in mucosal tolerance. In the present study, our data showed that LAG3 was expressed in Treg-of-B (P) cells and modulated the suppressive function of these cells. The number of LAG3+ CD4+ T cells in the Peyer’s patches increased after the oral administration of OVA. We also demonstrated that follicular B (FOB) cells in Peyer’s patches had a better ability to generate Treg-of-B (P) cells compared with CD23lo CD21lo B cells. Finally, the results showed that sorted LAG3+ Foxp3- Treg-of-B (P) cells could alleviate allergic airway inflammation and hypersensitivity. Treg cells play a critical role in the maintenance of tolerance. It has been reported that conventional B-2 cells are able to achieve immune modulation via the induction of Treg cells. While IL-10 has been correlated with the induction of type 1 regulatory T cells (Tr1) or Tr1-like cells, whether IL-10-producing B-1a cells are able to induce Treg cells, especially the Tr1 lineage has not been clearly defined. We here demonstrated that similar to B-2 cells, B-1a cells were able to convert naïve CD4+CD25- T cells into a subset of T cells with suppressive function, which we called “Treg-of-B1” cells. We characterized Treg-of-B1 cells by the surface markers OX40, PD-1, ICOS and IL-10R expression. Interestingly, the induced Treg-of-B1 cells resembled Tr1 cells in several ways; they did not express Foxp3 and produced high level of IFN-γ and IL-10 but minimal amounts of IL-4. However, utilizing IL-10-/- mice, we found that IL-10 was neither required for the induction nor the suppression of Treg-of-B1 cells. Moreover, Treg-of-B1 cells mediated soluble factor(s)-dependent suppression whereas Treg cells induced by conventional B-2 cells mediated the suppression required cell-cell contact. Therefore, the results suggested that B-1a cells induced a functionally and phenotypically distinct Treg population dissimilar to reported Foxp3+ Treg or Tr1 cells. Together, these findings might contribute to a better understanding of the role of B-1a cell subset in immune regulation.In summary, our team has finished most of the experiments mentioned in the second year’s project. We have already submitted two manuscripts related to the project for publication. One of them has been accepted and published, the other one now is under revised. We believe that more interesting results will be generated in the third year’s project.