2014-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/649324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。我們在第一年的研究計畫中主要是要研究黏膜分離出來的B細胞是否能夠誘發出具有調節能力的Treg/B細胞，可以應用到氣喘小鼠模式的治療上。同時，我們也持續進行第二年的研究計畫內容，如研究不同的B細胞來誘發調節性T細胞的能力。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。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。在此一計畫中，我們發現由黏膜系統分離出的B細胞與T細胞一道培養後可以誘發出具有調節能力的T細胞。我們計畫研究這群由B細胞所誘發出的調節性T細胞(Treg/B cells)的特性和基因表現，也要進一步將這些Treg/B細胞轉送入氣喘的小鼠，以了解是否能夠抑制過敏免疫反應。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。<br> 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. ResultsTreg-of-B (P) cells express LAG3Similar to the previous study, Treg-of-B (P) cells, which are generated by Peyer’s patch B cells cultured with naïve CD4+ CD25- T cells in the presence of OVA323-339, suppressed the proliferation of responder T cells. In contrast to nTreg cells, Treg-of-B (P) cells secreted higher levels of IL-10 and exerted a suppressive effect in the absence of Foxp3 expression. Compared with nTreg cells, Treg-of-B (P) cells expressed a higher level of LAG3, and this result was confirmed by real-time PCR. In the present study, our data showed that a greater number of LAG3+ Foxp3- T cells were present in Peyer’s patches. In contrast to the number of Foxp3- LAG3+ CD4+ T cells in the spleen, the number of Foxp3- LAG3+ CD4+ T cells in Peyer’s patches increased after the administration of OVA 0.5 mg for five days, implying that naïve T cells exposed to OVA presented by Peyer’s patch B cells can become LAG3+ T cells.Treg-of-B (P) cells generated by follicular B cells in Peyer’s patches have better suppressive functionB cells play an important role in mucosal tolerance induction and maintain the microenvironment in the intestine. Different subsets of B cells express CD23 and CD21, including follicular B (FOB) cells and marginal zone B (MZB) cellsIn this study, we found that B cells in Peyer’s patches, in contrast to B cells in the spleen, comprise FOB cells and CD23lo CD21lo B cells, and FOB cells accounted for the largest cell population. To determine which B cell subset was able to generate Treg-of-B (P) cells, FOB and CD23lo CD21lo B cells were sorted and cultured with naïve T cells. The cytokine profile showed that Treg cells induced by CD23lo CD21lo B cells secreted more IL-4, possibly leading to less effective suppression.LAG3 participates in the suppressive function of Treg-of-B (P) cellsBecause the level of LAG3 expression was elevated in Treg-of-B (P) cells, we determined whether LAG3 was required for the regulatory function of Treg-of-B (P) cells. After three days of co-culturing, CD4+ LAG3+ Treg-of-B (P) cells were sorted and used in the suppressive function assay in the presence of anti-LAG3 antibodies. Our previous study showed that Treg-of-B (P) cells also express CTLA4 and ICOS. This result suggests that LAG3 is not only a marker of Treg-of-B (P) cells but also involved in their function.Foxp3- LAG3+ Treg-of-B (P) cells decrease OVA-specific IgE and Th2 cytokine production and alleviate asthmatic symptomsAn OVA-immunized murine asthma model was used to determine the biological effect of Foxp3- LAG3+ Treg-of-B (P) cells in vivo. Foxp3- LAG3+ Treg-of-B (P) cells were sorted and adoptively transferred into mice on day -1. This result suggests that LAG3+ Treg-of-B (P) cells have a regulatory effect on systemic Th2 responses, including antibody class-switch and cytokine production. This mild reaction might result from decreased eosinophil infiltration due to the diminished IL-5 and eotaxin production in the lungs. In addition, these results indicate that Foxp3- LAG3+ Treg-of-B cells generated by Peyer’s patch B cells were capable of alleviating allergic airway inflammation.ConclusionIn this study, we found that naïve CD4+ T cells stimulated by Peyer’s patch B cells became Treg-of-B cells and expressed higher LAG3 levels according to the microarray analysis and fluorescent antibody staining. It has been reported that compared with the spleen, Peyer’s patches are enriched in CD4+ LAG3+ T cells, (approximately 8%). This T cell population is hypo-proliferative and is able to inhibit the induction of colitis. Therefore, we speculated that LAG3+ T cells might play a role in oral tolerance. Similar to the results of a previous study, higher numbers of LAG3+ T cells were observed in Peyer’s patches than in the spleen in the present study. Furthermore, after the oral administration of OVA for five days, the proportion of LAG3+ CD4+ T cells was increased in Peyer’s patches (approximately 15%), although this proportion was not different from that in the spleen. These data imply that when antigens enter the intestines, Peyer’s patch B cells to naïve T cells might present them and induce naïve T cells to become LAG3+ Foxp3- regulatory T cells.In the present study, we further investigated the ability of different subsets of Peyer’s patch B cells to induce the production of Treg cells. B220+ splenic B cells include MZB, FOB and CD23- CD21- B cells. Due to their anatomical site, splenic MZB cells are considered to be the first line of protection against blood borne pathogensis. The main function of FOB cells is to differentiate into antibody-secreting cells in response to TD and TI antigens. In this study, we found that compared with CD23lo CD21lo B cells, Peyer’s patch FOB cells can be good antigen-presenting cells to generate Treg-of-B (P) cells. It is suggested that Peyer’s patch B cells, which generate Treg cells, might exert a better therapeutic effect on treating airway inflammation than Peyer’s patch DCs. With the ability to produce high level of regulatory cytokines IL-10, CD5+ B-1a cells are considered as B cells with regulatory function. Among diverse Treg populations, Tr1 cells specially arise from naïve precursors in the presence of IL-10. Therefore, the natural ability to produce IL-10 is of great interest for us to examine the ability of B-1 cells to promote the induction of Tr1 cells directly through IL-10 secretion. In this study, we demonstrated that B-1 cells are potent regulatory cells to convert CD4+ T cells into T cells with suppressive function. To the best of our knowledge, this is the first report to show that peritoneal isolated B-1 cells were able to induce Treg cells during in vitro co-cultures without the prescence of any additional cytikines such as IL-10. This discovery prompted us to extend the role of B-1 cells in the induction of Treg cells in futher studies. By far, one of the major differences noted between Treg-of-B1 and Treg-of-B2 cells is the suppressive mechanism. The suppressive ability of Treg-of-B2 cells was potentiated by contact with Teff cells, which is consistent with results of Reichardt et al, but the suppressive ability of Treg-of-B1 cells could solely be facilitated by soluble factors. This finding indicated that both B-1 and B-2 cell subsets acquired immune regulatory functions to induce Treg cells. However, the distinct properties of B-1 and B-2 cell subsets might result in inducing Treg cells with different suppressive mechanism.In conclusion, in this study, we found that the oral administration of OVA could increase the number of LAG3+ CD4+ T cells in Peyer’s patches. Peyer’s patch B cells convert naïve CD4+ Foxp3- LAG3- T cells into Foxp3- LAG3+ T cells, which have a regulatory function. These Treg-of-B (P) cells have the biological effect of relieving airway inflammation. Further, we also clarify the immune regulatory role of B-1 cells by understanding how they negatively regulate T cell response through the induction of Treg cells. In the future, more studies are needed to fully characterize which molecules regulate the effector functions of B-1 induce Treg cells. The in vivo function of induced Treg-of-B1 cells will be examined using disease model, therefore, using B-1 to generate large numbers of functional suppressive Treg cells may facilitate the Treg-based immunotherapy in the treatment of autoimmune diseases, infection or organ transplantation in the future.氣喘調節性T細胞mucosal B cellsregulatory T cells induced by B cells