摘要
背景和立题依据：由病原微生物感染引起的传染病是人类健康和生命的主要威胁之一。目前发现,90%以上的人类传染病经由黏膜途径感染。然而,黏膜抗感染免疫机制至今不明。黏膜抗感染免疫不同于系统免疫,与系统免疫系统相比,黏膜处于一个长期带菌的环境,接触不同来源和性质的抗原,这就要求黏膜免疫系统对有害抗原和无害抗原具有区别能力,并反馈为不同的免疫反应。对于食物,正常菌群等无害抗原,肠道黏膜免疫系统表现为免疫耐受,避免发生超敏反应和自身免疫性疾病,而与无害抗原相反,肠道内的病原体等有害抗原可以被肠道黏膜免疫系统识别,并引起免疫应答。然而,黏膜免疫的机制至今不明。肠道黏膜免疫系统是机体免疫系统中最大也是最为复杂的部分,也最具有代表性。大量研究数据揭示,肠道黏膜免疫系统的免疫耐受机制主要有调节性T细胞(regulatory T cells,Treg)介导,而诱导性Treg的产生与肠道黏膜微环境密不可分,这种特殊联系来源于肠道黏膜免疫系统的几种细胞或细胞亚群分泌的维甲酸。上述资料提示,肠道具有分泌维甲酸能力的细胞或细胞亚群可能在肠道免疫耐受机制中发挥了重要作用。
研究内容及意义：本文通过研究肠道派氏结中不同树突状细胞亚群维甲酸合成限速酶——醛脱氢酶活性来筛选诱导肠道免疫耐受的免疫细胞,研究过程中,发现了一群高表达醛脱氢酶活性的未知细胞,鉴定结果是嗜酸性粒细胞,并定量检测这群肠道嗜酸性粒细胞维甲酸的分泌量,肠道嗜酸性粒细胞Treg诱导相关的细胞因子—-TGF-β和IL-2分泌量,以及肠道嗜酸性粒细胞与初始型T细胞共培养后,T细胞的分化方向。为了检测非黏膜来源的嗜酸性粒细胞是否具有诱导初始型T细胞向Treg分化的能力,分离纯化外周血嗜酸性粒细胞,鉴定外周血嗜酸性粒细胞的表型,外周血嗜酸性粒细胞的维甲酸分泌量,以及外周血嗜酸性粒细胞与初始型T细胞共培养后,Treg的表达情况。以期待揭示嗜酸性粒细胞在肠道黏膜免疫中的功能和机制。
研究方法：本文采用美天尼磁珠分选和流式细胞分选来收集和纯化野生型小鼠肠道黏膜中的树突状细胞以及肠黏膜和外周血中嗜酸性粒细胞；流式细胞仪检测醛脱氢酶活性检测试剂标记的细胞的醛脱氢酶活性；流式细胞检测细胞表型鉴定和瑞氏染色形态学鉴定高表达醛脱氢酶活性细胞的细胞类型；液相色谱-质谱联用定量检测肠道黏膜以及外周血来源的嗜酸性粒细胞的维甲酸分泌量；ELISA检测肠道黏膜以及外周血来源的嗜酸性粒细胞单独培养上清中TGF-β和IL-2的浓度；纯化的肠道黏膜或外周血来源的嗜酸性粒细胞与纯化的CD4+CD62L+OT-II CD4+初始型T细胞共培养数天后,流式细胞仪检测CD4+细胞中CD25+Foxp3+Treg细胞的比例,ELISA检测培养上清中IL-4, IL-17, IFN-γ, TGF-β and IL-2的浓度。
实验结果：实验结果表明,肠道派氏结中,存在一群高表达醛脱氢酶的细胞,经表型和形态学鉴定确认为嗜酸性粒细胞,经液相色谱质谱联用定量检测肠道嗜酸性粒细胞培养上清,发现这群肠道嗜酸性粒细胞分泌高浓度维甲酸,采用ELISA检测上清,证明这群肠道嗜酸性粒细胞同时也分泌TGF-p。将肠道嗜酸性粒细胞与初始型T细胞共培养后,流式细胞检测发现,6.52%CD4+T细胞表达CD25+Foxp3+,说明初始型T细胞向Treg方向分化。为了验证非黏膜来源的嗜酸性粒细胞是否具备诱导Treg的功能,分离和纯化小鼠外周血嗜酸性粒细胞,采用流式鉴定细胞表型,结果显示,肠道嗜酸性粒细胞与外周血嗜酸性粒细胞表型有差异,肠道嗜酸性粒细胞表达CD80和CDllc,而外周血嗜酸性粒细胞不表达。同时,外周血来源的嗜酸性粒细胞不能分泌维甲酸。外周血嗜酸性粒细胞与初始型T细胞共培养后,并不诱导CD4+CD25+Foxp3+T细胞的产生。
结论：肠道黏膜来源嗜酸性粒细胞高表达醛脱氢酶活性,分泌高浓度维甲酸和TGF-β。肠道黏膜来源嗜酸性粒细胞诱导初始型T细胞向Treg方向分化,抑制初始型T细胞向Th1/Th17方向分化。非黏膜来源的嗜酸性粒细胞——外周血嗜酸性粒细胞表型与肠道嗜酸性粒细胞不同,不能分泌维甲酸,也不能诱导初始型T细胞向Treg方向分化。
Background:Infectious diseases caused by pathogenic microorganism are one of the main threat to human health and life. It is well known that the mucosa is the largest immune organ in the body, and it is generally believed that almost all infectious diseases are initiated at mucosal surface. However, little is known about mucosal immunity against pathogens infection.
Within the immune system, a series of anatomically distinct compartments can be distinguished, each of which is specially adapted to generate a response to pathogens present in a particular set of body tissues. The previous part of the chapter illustrated the general principles underlying the initiation of an adaptive immune response in the compartment comprising the peripheral lymph nodes and spleen. This is the compartment that responds to antigens that have entered the tissues or spread into the blood. A second compartment of the adaptive immune system of equal size to this, and located near the surfaces where most pathogens invade, is the mucosal immune system.
The gastrointestinal tract is most classic component of the body's mucosal immune system. In fact, the intestine possesses the largest mass of lymphoid tissue in the human body. The intestinal immune system must constantly maintains immunological tolerance to harmless food antigens and commensal bacteria yet recognizes harmful pathogens and responses to eliminate them. The mechanisms that maintain this balance of intestinal immune homeostasis are poorly understood. Gut-associated lymphatic tissue (GALT) maintain the symbiosis between commensal bacteria and the gut for the fine balance of intestinal immune homeostasis. When the GALT receives signals from the intestinal flora or food antigens, it must limit the magnitude of effector responses and allow the establishment of immunological tolerance. Regulatory T cells (Treg) play an indispensable role in maintaining self tolerance.
Although a role for Treg in the maintenance of immune tolerance has been demonstrated in both humans and mice, the origin of these cells is still not completely understood.
Tregs play an indispensable role in maintaining self tolerance, aside from Tregs arise in the thymus, peripheral conversion of Tregs occurs primarily in the GALT, suggested that the GALT microenviroment is particularly well suited for peripheral conversion of Treg. It has been proved that retinoic acid promote de novo generation of Foxp3+Treg cells via retinoic acid (RA)
The vitamin A metabolite RA is a lipophilic molecule that controls the activity of a constellation of genes via binding to nuclear receptors. Vitamin A is derived from the diet, and the liver constitutes a large reservoir of vitamin A in the form of retinyl esters. Retinyl esters are hydrolyzed to retinol and released into the blood. Once retinol enters cells expressing appropriate enzymes, it is converted successively into retinal and RA. The first step of the conversion is catalyzed by alcohol dehydrogenases and by microsomal retinol dehydrogenases that are expressed by most cells, including dendritic cells (DCs). The second step consists of the oxidation of retinal into RA and is catalyzed by3aldehyde dehydrogenases (ALDHs), known as RALDH1,2, and3and encoded by the Aldhlal,-2, and-3genes, respectively. RALDH expression is limited to certain cell types and, despite the widespread availability of retinol, only cells expressing one of the RALDHs can oxidize retinaldehyde to RA.
Recently, DCs that are located in GALT and express Aldhla2have gained considerable attention because of their ability to produce RA. On migration to mesenteric lymph nodes (MLNs), this exclusive property allows them to promote the expression of the gut-tropic α4β7integrin and CCR9chemokine receptor on antigen-responsive T cells and in turn confer them gut-seeking properties. Importantly, RA production by GALT-associated DCs is also involved in the generation of induced Foxp3+Treg(iTreg). Treg can be distinguished from "naturally occurring" Foxp3+Treg (iTreg) on the basis of their development. Whereas nTreg develop in the thymus, iTreg develop de novo in secondary lymphoid organs from conventional, naive CD4+T cells. This conversion that is triggered by DCs requires submitogenic dose of antigen and low costimulation, high levels of transforming growth factor-β (TGF-β) and is greatly enhanced by the presence of RA. The exact mechanism through which DC-produced RA impacts on the generation of iTreg is still a matter of debate. For instance, RA has been proposed to enhance the TGF-β-dependent differentiation of naive CD4+T cells into Foxp3+iTreg by blocking their differentiation into proinflammatory T cells. Alternatively, RA may indirectly affects iTreg generation by preventing memory CD4+T cells from producing cytokines (interleukin-4[IL-4], IL-21, and interferon-y), which inhibit the differentiation of iTreg. RA production by GALT-associated DCs has been proposed to maintain the balance between effector and Treg in the gastrointestinal tract and to constitute a major mechanism underlying oral tolerance. The production of RA by gut DCs is restricted to mDCs expressing the integrin αE chain CD103and requires the presence of both granulocyte-macrophage colony stimulating factor (GM-CSF) and RA in the lamina propria (LP).
Considering that, under physiologic conditions, ALDH expression constitutes the only parameter that limits RA production, we used a flowcytometry-based assay to measure ALDH activity at the single-cell level and performed a comprehensive analysis of the RA-producing cell populations present in Peyer's patches under steady-state conditions.
Methods:Isolation of cells with microbeads on LS MACS columns and FACS sorting. To identified the cells with high ALDH activity, we employed flowcytometry-based assay to measure phenotype and May-Grunwald-Giemsa staining to observe morphology. LC (liquid chromatography)/MS/MS assay was used to detect the RA secreting by intestinal eosinophil(Eos), the intestinal cells with high ALDH activity. ELISA assay was performed to test the secretion of TGF-P and IL-2by intestinal Eos. For in vitro stimulation, purified peripheral blood Eos or intestinal Eos were cultured together with unlabeled naive CD4+CD62L+OT-II CD4+T cells, Foxp3expression in CD4+T cells was evaluated using the Foxp3staining set. ELISA assay was performed to test the concentration of IL-4, IL-17, IFN-γ, TGF-β and IL-2in the co-culture supernatant.
Results:There is no significant difference among the ALDH activity of three distinct DC in Peyer's patches. However, a group of cells(CD11c+CD11b+CD8-) in intestinal expressed high level of ALDH activity. CD11c+CD11b+CD8-intestinal cells had moderate expression of Siglec-F+CCR3+, which indicated a Eos character. The image show that the CD11c+CD11b+CD8-intestinal cells are Eos with uniquely shaped nuclei and eosinophilic granules. The RA and TGF-β secreting ability of intestinal eosinophils were demonstrated by culture supernatant quantification. After coculture with naive CD4+CD62L+OT-II CD4+T cells in the presence of antigen and TGF-β, approximately6.5%of cells expressed the marker Foxp3and CD25. After coculture with naive CD4+CD62L+OT-II CD4+T cells in the presence of antigen and TGF-β, approximately6.5%of cells expressed the marker Foxp3and CD25. Culture with intestinal eosinophils lead to strong reduction of IFN-y and IL-17, and correlated with increased TGF-β production. The expression of phenotype is different between intestinal and peripheral blood Eos, which is no expression of CD11c and CD80. Peripheral blood Eos have low level of ALDH activity and no RA secreting. After coculture with naive T cells in the presence of antigen and TGF-β, almost no cells expressed the marker Foxp3and CD25.
Conclusion:Intestinal Eos inducing the differentiation of naive T cell into regulatory T cell, and inhibiting the differentiation of Th1and Th17. The mechanism is cause via RA and TGF-β. The expression of phenotype is different between intestinal and peripheral blood Eos. Peripheral blood Eos cannot induce the differentiation of naive T cell into regulatory T cell.
引文
[1]Romero E. C., Pimenta F., Diament D. Neglected infectious diseases:Mechanism of pathogenesis, diagnosis, and immune response [J]. Interdiscip Perspect Infect Dis,2012,2012:701648.
[2]Valdenegro-Vega V. A., Crosbie P., Vincent B., etc. Effect of immunization route on mucosal and systemic immune response in atlantic salmon (salmo salar) [J]. Vet Immunol Immunopathol,2013,151(1-2):113-23.
[3]Acevedo R., Callico A., del Campo J., etc. Intranasal administration of proteoliposome-derived cochleates from vibrio cholerae o1 induce mucosal and systemic immune responses in mice [J]. Methods,2009,49(4):309-15.
[4]Hansel T. T., Johnston S. L., Openshaw P. J. Microbes and mucosal immune responses in asthma [J]. Lancet,2013.
[5]Amuguni H., Lee S., Kerstein K., etc. Sublingual immunization with an engineered bacillus subtilis strain expressing tetanus toxin fragment c induces systemic and mucosal immune responses in piglets [J]. Microbes Infect,2012, 14(5):447-56.
[6]Bordon Y. Mucosal immunology:A wee immune response [J]. Nat Rev Immunol, 2013,13(4):220-1.
[7]Himi T., Takano K., Ogasawara N., etc. Mucosal immune barrier and antigen-presenting system in human nasal epithelial cells [J]. Adv Otorhinolaryngol,2011,72:28-30.
[8]Ciccone E. J., Greenwald J. H., Lee P. I., etc. Cd4+ t cells, including th17 and cycling subsets, are intact in the gut mucosa of hiv-1-infected long-term nonprogressors [J]. J Virol,2011,85(12):5880-8.
[9]Mavigner M., Cazabat M., Dubois M., etc. Altered cd4+ t cell homing to the gut impairs mucosal immune reconstitution in treated hiv-infected individuals [J]. J Clin Invest,2012,122(1):62-9.
[10]Zeng R., Oderup C., Yuan R., etc. Retinoic acid regulates the development of a gut-homing precursor for intestinal dendritic cells [J]. Mucosal Immunol,2012.
[11]Jonker M. A., Hermsen J. L., Sano Y., etc. Small intestine mucosal immune system response to injury and the impact of parenteral nutrition [J]. Surgery,2012, 151(2):278-86.
[12]Taschuk R., Griebel P. J. Commensal microbiome effects on mucosal immune system development in the ruminant gastrointestinal tract [J]. Anim Health Res Rev,2012,13(1):129-41.
[13]Sanz Y., De Palma G. Gut microbiota and probiotics in modulation of epithelium and gut-associated lymphoid tissue function [J]. Int Rev Immunol,2009,28(6): 397-413.
[14]Sato K., Takahashi K., Tohno M., etc. Immunomodulation in gut-associated lymphoid tissue of neonatal chicks by immunobiotic diets [J]. Poult Sci,2009, 88(12):2532-8.
[15]Koboziev I., Karlsson F., Grisham M. B. Gut-associated lymphoid tissue, t cell trafficking, and chronic intestinal inflammation [J]. Ann N Y Acad Sci,2010, 1207 Suppl 1:E86-93.
[16]Casteleyn C., Doom M., Lambrechts E., etc. Locations of gut-associated lymphoid tissue in the 3-month-old chicken:A review [J]. Avian Pathol,2010, 39(3):143-50.
[17]Rottiers P., De Smedt T., Steidler L. Modulation of gut-associated lymphoid tissue functions with genetically modified lactococcus lactis [J]. Int Rev Immunol,2009, 28(6):465-86.
[18]Luongo D., D'Arienzo R., Bergamo P., etc. Immunomodulation of gut-associated lymphoid tissue:Current perspectives [J]. Int Rev Immunol,2009,28(6):446-64.
[19]Park S. G., Mathur R., Long M., etc. T regulatory cells maintain intestinal homeostasis by suppressing gammadelta t cells [J]. Immunity,2010,33(5): 791-803.
[20]Balogh A., Persa E., Bogdandi E. N., etc. The effect of ionizing radiation on the homeostasis and functional integrity of murine splenic regulatory t cells [J]. Inflamm Res,2013,62(2):201-12.
[21]Peterson R. A. Regulatory t-cells:Diverse phenotypes integral to immune homeostasis and suppression [J]. Toxicol Pathol,2012,40(2):186-204.
[22]Guilliams M., Crozat K., Henri S., etc. Skin-draining lymph nodes contain dermis-derived cd103(-) dendritic cells that constitutively produce retinoic acid and induce foxp3(+) regulatory t cells [J]. Blood,2010,115(10):1958-68.
[23]Xiao S., Jin H., Korn T., etc. Retinoic acid increases foxp3+ regulatory t cells and inhibits development of th17 cells by enhancing tgf-beta-driven smad3 signaling and inhibiting il-6 and il-23 receptor expression [J]. J Immunol,2008,181(4): 2277-84.
[24]Shi Q., Cao H., Liu J., etc. Cd4+ foxp3+ regulatory t cells induced by tgf-beta, il-2 and all-trans retinoic acid attenuate obliterative bronchiolitis in rat trachea transplantation [J]. Int Immunopharmacol,2011,11(11):1887-94.
[25]Maynard C. L., Hatton R. D., Helms W. S., etc. Contrasting roles for all-trans retinoic acid in tgf-beta-mediated induction of foxp3 and il10 genes in developing regulatory t cells [J]. J Exp Med,2009,206(2):343-57.
[26]Cha H. R., Chang S. Y., Chang J. H., etc. Downregulation of th17 cells in the small intestine by disruption of gut flora in the absence of retinoic acid [J]. J Immunol,2010,184(12):6799-806.
[27]Elias K. M., Laurence A., Davidson T. S., etc. Retinoic acid inhibits th17 polarization and enhances foxp3 expression through a stat-3/stat-5 independent signaling pathway [J]. Blood,2008,111(3):1013-20.
[28]Bai A., Lu N., Guo Y., etc. All-trans retinoic acid down-regulates inflammatory responses by shifting the treg/th17 profile in human ulcerative and murine colitis [J]. J Leukoc Biol,2009,86(4):959-69.
[29]Hammerschmidt S. I., Friedrichsen M., Boelter J., etc. Retinoic acid induces homing of protective t and b cells to the gut after subcutaneous immunization in mice [J]. J Clin Invest,2011,121(8):3051-61.
[30]Mora J. R., von Andrian U. H. Role of retinoic acid in the imprinting of gut-homing iga-secreting cells [J]. Semin Immunol,2009,21(1):28-35.
[31]Iwata M., Hirakiyama A., Eshima Y., etc. Retinoic acid imprints gut-homing specificity on t cells [J]. Immunity,2004,21(4):527-38.
[32]Menning A., Loddenkemper C., Westendorf A. M., etc. Retinoic acid-induced gut tropism improves the protective capacity of treg in acute but not in chronic gut inflammation [J]. Eur J Immunol,2010,40(9):2539-48.
[33]Van Y. H., Lee W. H., Ortiz S., etc. All-trans retinoic acid inhibits type 1 diabetes by t regulatory (treg)-dependent suppression of interferon-gamma-producing t-cells without affecting th17 cells [J]. Diabetes,2009,58(1):146-55.
[34]Dudziak D., Kamphorst A. O., Heidkamp G. F., etc. Differential antigen processing by dendritic cell subsets in vivo [J]. Science,2007,315(5808):107-11.
[35]Rescigno M. Ccr6(+) dendritic cells:The gut tactical-response unit [J]. Immunity, 2006,24(5):508-10.
[36]Salazar-Gonzalez R. M., Niess J. H., Zammit D. J., etc. Ccr6-mediated dendritic cell activation of pathogen-specific t cells in peyer's patches [J]. Immunity,2006, 24(5):623-32.
[37]Bochner B. S. Siglec-8 on human eosinophils and mast cells, and siglec-f on murine eosinophils, are functionally related inhibitory receptors [J]. Clin Exp Allergy,2009,39(3):317-24.
[38]Fulkerson P. C., Fischetti C. A., Rothenberg M. E. Eosinophils and ccr3 regulate interleukin-13 transgene-induced pulmonary remodeling [J]. Am J Pathol,2006, 169(6):2117-26.
[39]Bain B. J. Review:Eosinophils and eosinophilic leukemia [J]. Clin Adv Hematol Oncol,2010,8(12):901-3.
[40]McEwen B. J. Eosinophils:A review [J]. Vet Res Commun,1992,16(1):11-44.
[41]Wang A., Fernando M., Leung G., etc. Exacerbation of oxazolone colitis by infection with the helminth hymenolepis diminuta:Involvement of il-5 and eosinophils [J]. Am J Pathol,2010,177(6):2850-9.
[42]Klion A. D., Nutman T. B. The role of eosinophils in host defense against helminth parasites [J]. J Allergy Clin Immunol,2004,113(1):30-7.
[43]Balla K. M., Lugo-Villarino G., Spitsbergen J. M., etc. Eosinophils in the zebrafish:Prospective isolation, characterization, and eosinophilia induction by helminth determinants [J]. Blood,2010,116(19):3944-54.
[44]Min D. Y., Lee Y. A., Ryu J. S., etc. Caspase-3-mediated apoptosis of human eosinophils by the tissue-invading helminth paragonimus westermani [J]. Int Arch Allergy Immunol,2004,133(4):357-64.
[45]Padigel U. M., Hess J. A., Lee J. J., etc. Eosinophils act as antigen-presenting cells to induce immunity to strongyloides stercoralis in mice [J]. J Infect Dis,2007, 196(12):1844-51.
[46]Padigel U. M., Lee J. J., Nolan T. J., etc. Eosinophils can function as antigen-presenting cells to induce primary and secondary immune responses to strongyloides stercoralis [J]. Infect Immun,2006,74(6):3232-8.
[47]Svensson-Frej M. Immunobiology of intestinal eosinophils-a dogma in the changing? [J]. J Innate Immun,2011,3(6):565-76.
[48]Hogan S. P., Waddell A., Fulkerson P. C. Eosinophils in infection and intestinal immunity [J]. Curr Opin Gastroenterol,2013,29(1):7-14.
[49]Kane M. A., Chen N., Sparks S., etc. Quantification of endogenous retinoic acid in limited biological samples by lc/ms/ms [J]. Biochem J,2005,388(Pt 1):363-9.
[50]Bempong D. K., Honigberg I. L., Meltzer N. M. Normal phase lc-ms determination of retinoic acid degradation products [J]. J Pharm Biomed Anal, 1995,13(3):285-91.
[51]Yang H., Cheng E. Y, Sharma V. K., etc. Dendritic cells with tgf-betal and il-2 differentiate naive cd4+ t cells into alloantigen-specific and allograft protective foxp3+ regulatory t cells [J]. Transplantation,2012,93(6):580-8.
[52]Fattouh R., Jordana M. Tgf-beta, eosinophils and il-13 in allergic airway remodeling:A critical appraisal with therapeutic considerations [J]. Inflamm Allergy Drug Targets,2008,7(4):224-36.
[53]Levi-Schaffer F., Garbuzenko E., Rubin A., etc. Human eosinophils regulate human lung- and skin-derived fibroblast properties in vitro:A role for transforming growth factor beta (tgf-beta) [J]. Proc Natl Acad Sci U S A,1999, 96(17):9660-5.
[54]Ohno I., Nitta Y, Yamauchi K., etc. Transforming growth factor beta 1 (tgf beta 1) gene expression by eosinophils in asthmatic airway inflammation [J]. Am J Respir Cell Mol Biol,1996,15(3):404-9.
[55]Matteoli G, Mazzini E., Iliev I. D., etc. Gut cd103+ dendritic cells express indoleamine 2,3-dioxygenase which influences t regulatory/t effector cell balance and oral tolerance induction [J]. Gut,2010,59(5):595-604.
[56]Siddiqui K. R., Powrie F. Cd103+ galt dcs promote foxp3+ regulatory t cells [J]. Mucosal Immunol,2008,1 Suppl 1:S34-8.
[57]Iliev I. D., Mileti E., Matteoli G, etc. Intestinal epithelial cells promote colitis-protective regulatory t-cell differentiation through dendritic cell conditioning [J]. Mucosal Immunol,2009,2(4):340-50.
[58]Wallon C., Persborn M., Jonsson M., etc. Eosinophils express muscarinic receptors and corticotropin-releasing factor to disrupt the mucosal barrier in ulcerative colitis [J]. Gastroenterology,2011,140(5):1597-607.
[59]Myller J. P., Toppila-Salmi S. K., Toppila E. M., etc. Mucosal eosinophils and 1-selectin ligands are associated with invasive and noninvasive sinus surgery outcomes [J]. Am J Rhinol Allergy,2009,23(1):21-7.
[60]Rotting A. K., Freeman D. E., Eurell J. A., etc. Effects of acetylcysteine and migration of resident eosinophils in an in vitro model of mucosal injury and restitution in equine right dorsal colon [J]. Am J Vet Res,2003,64(10):1205-12.
[61]Sakaguchi S., Yamaguchi T., Nomura T., etc. Regulatory t cells and immune tolerance [J]. Cell,2008,133(5):775-87.
[62]Romagnani S. Regulation of the t cell response [J]. Clin Exp Allergy,2006,36(11): 1357-66.
[63]Kobayashi T., Nakatsuka K., Shimizu M., etc. Ribavirin modulates the conversion of human cd4(+) cd25(-) t cell to cd4(+) cd25(+) foxp3(+) t cell via suppressing interleukin-10-producing regulatory t cell [J]. Immunology,2012,137(3):259-70.
[64]Martinez R. J., Zhang N., Thomas S. R., etc. Arthritogenic self-reactive cd4+ t cells acquire an fr4hicd73hi anergic state in the presence of foxp3+ regulatory t cells [J]. J Immunol,2012,188(1):170-81.
[65]Ryba M., Mysliwska J. [cd4+cd25+foxp3+ t lymphocytes:Naturally occuring regulatory t cells] [J]. Pediatr Endocrinol Diabetes Metab,2010,16(4):289-94.
[66]Park Y. H., Koo S. K., Kim Y, etc. Effect of in vitroexpanded cd4(+)cd25(+)foxp3(+) regulatory t cell therapy combined with lymphodepletion in murine skin allotransplantation [J]. Clin Immunol,2010,135(1):43-54.
[67]Fragale A., Gabriele L., Stellacci E., etc. Ifn regulatory factor-1 negatively regulates cd4+ cd25+ regulatory t cell differentiation by repressing foxp3 expression [J]. J Immunol,2008,181(3):1673-82.
[68]Kang J., Huddleston S. J., Fraser J. M., etc. De novo induction of antigen-specific cd4+cd25+foxp3+ regulatory t cells in vivo following systemic antigen administration accompanied by blockade of mtor [J]. J Leukoc Biol,2008,83(5): 1230-9.
[69]Fu S., Zhang N., Yopp A. C., etc. Tgf-beta induces foxp3+ t-regulatory cells from cd4+cd25-precursors [J]. Am J Transplant,2004,4(10):1614-27.
[70]Collins C. B., Aherne C. M., McNamee E. N., etc. Flt3 ligand expands cd103(+) dendritic cells and foxp3(+) t regulatory cells, and attenuates crohn's-like murine ileitis [J]. Gut,2012,61(8):1154-62.
[71]Ishimaru N., Yamada A., Kohashi M., etc. Development of inflammatory bowel disease in long-evans cinnamon rats based on cd4+cd25+foxp3+ regulatory t cell dysfunction [J]. J Immunol,2008,180(10):6997-7008.
[72]Su L., Creusot R. J., Gallo E. M., etc. Murine cd4+cd25+ regulatory t cells fail to undergo chromatin remodeling across the proximal promoter region of the il-2 gene [J]. J Immunol,2004,173(8):4994-5001.
[73]Thornton A. M. Signal transduction in cd4+cd25+ regulatory t cells:Cd25 and il-2 [J]. Front Biosci,2006,11:921-7.
[74]Kolar P., Knieke K., Hegel J. K., etc. Ctla-4 (cd152) controls homeostasis and suppressive capacity of regulatory t cells in mice [J]. Arthritis Rheum,2009,60(1): 123-32.
[75]Sakurai J., Ohata J., Saito K., etc. Blockade of ctla-4 signals inhibits th2-mediated murine chronic graft-versus-host disease by an enhanced expansion of regulatory cd8+ t cells [J]. J Immunol,2000,164(2):664-9.
[76]Manzotti C. N., Tipping H., Perry L. C., etc. Inhibition of human t cell proliferation by ctla-4 utilizes cd80 and requires cd25+ regulatory t cells [J]. Eur J Immunol,2002,32(10):2888-96.
[77]Sugita S., Ng T. F., Lucas P. J., etc. B7+ iris pigment epithelium induce cd8+ t regulatory cells; both suppress ctla-4+ t cells [J]. J Immunol,2006,176(1): 118-27.
[78]Tai X., Van Laethem F., Pobezinsky L., etc. Basis of ctla-4 function in regulatory and conventional cd4(+) t cells [J]. Blood,2012,119(22):5155-63.
[79]Frydecka D., Beszlej A., Karabon L., etc. The role of genetic variations of immune system regulatory molecules cd28 and ctla-4 in schizophrenia [J]. Psychiatry Res,2013.
[80]Poirier N., Azimzadeh A. M., Zhang T., etc. Inducing ctla-4-dependent immune regulation by selective cd28 blockade promotes regulatory t cells in organ transplantation [J]. Sci Transl Med,2010,2(17):17ra10.
[81]Sansom D. M., Walker L. S. The role of cd28 and cytotoxic t-lymphocyte antigen-4 (ctla-4) in regulatory t-cell biology [J]. Immunol Rev,2006,212: 131-48.
[82]Jonson C. O., Hedman M., Karlsson Faresjo M., etc. The association of ctla-4 and hla class ii autoimmune risk genotype with regulatory t cell marker expression in 5-year-old children [J]. Clin Exp Immunol,2006,145(1):48-55.
[83]Katoh H., Zheng P., Liu Y. Foxp3:Genetic and epigenetic implications for autoimmunity [J]. J Autoimmun,2013.
[84]Zhang Y., Bandala-Sanchez E., Harrison L. C. Revisiting regulatory t cells in type 1 diabetes [J]. Curr Opin Endocrinol Diabetes Obes,2012,19(4):271-8.
[85]Lowther D. E., Hafler D. A. Regulatory t cells in the central nervous system [J]. Immunol Rev,2012,248(1):156-69.
[86]Bilate A. M., Lafaille J. J. Induced cd4+foxp3+ regulatory t cells in immune tolerance [J]. Annu Rev Immunol,2012,30:733-58.
[87]Hoglund P. Induced peripheral regulatory t cells:The family grows larger [J]. Eur J Immunol,2006,36(2):264-6.
[88]Wahl S. M., Chen W. Transforming growth factor-beta-induced regulatory t cells referee inflammatory and autoimmune diseases [J]. Arthritis Res Ther,2005,7(2): 62-8.
[89]Moore C., Fuentes C., Sauma D., etc. Retinoic acid generates regulatory t cells in experimental transplantation [J]. Transplant Proc,2011,43(6):2334-7.
[90]Beyersdorf N., Hanke T., Kerkau T., etc. Superagonistic anti-cd28 antibodies: Potent activators of regulatory t cells for the therapy of autoimmune diseases [J]. Ann Rheum Dis,2005,64 Suppl 4:iv91-5.
[91]Cai X. Y., Luo M., Lin X. J., etc. [expression and significance of th17 and treg cells in peripheral blood of patients with systemic lupus erythematosus] [J]. Zhonghua Yi Xue Za Zhi,2012,92(7):460-3.
[92]Coombes J. L., Siddiqui K. R., Arancibia-Carcamo C. V., etc. A functionally specialized population of mucosal cd103+ dcs induces foxp3+ regulatory t cells via a tgf-beta and retinoic acid-dependent mechanism [J]. J Exp Med,2007, 204(8):1757-64.
[93]Li J., Lai X., Liao W., etc. The dynamic changes of th17/treg cytokines in rat liver transplant rejection and tolerance [J]. Int Immunopharmacol,2011,11(8):962-7.
[94]Yang J., Chu Y., Yang X., etc. Th17 and natural treg cell population dynamics in systemic lupus erythematosus [J]. Arthritis Rheum,2009,60(5):1472-83.
[95]Takahashi T., Sakaguchi S. The role of regulatory t cells in controlling immunologic self-tolerance [J]. Int Rev Cytol,2003,225:1-32.
[96]Annoni A., Brown B. D., Cantore A., etc. In vivo delivery of a microrna-regulated transgene induces antigen-specific regulatory t cells and promotes immunologic tolerance [J]. Blood,2009,114(25):5152-61.
[97]Takahashi T., Tagami T., Yamazaki S., etc. Immunologic self-tolerance maintained by cd25(+)cd4(+) regulatory t cells constitutively expressing cytotoxic t lymphocyte-associated antigen 4 [J]. J Exp Med,2000,192(2):303-10.
[98]Sakaguchi S. Immunologic tolerance maintained by regulatory t cells: Implications for autoimmunity, tumor immunity and transplantation tolerance [J]. Vox Sang,2002,83 Suppl 1:151-3.
[99]Nishimura E., Sakihama T., Setoguchi R., etc. Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising foxp3+cd25+cd4+ regulatory t cells [J]. Int Immunol,2004, 16(8):1189-201.
[100]Larousserie F., Bardel E., Pflanz S., etc. Analysis of interleukin-27 (ebi3/p28) expression in epstein-barr virus- and human t-cell leukemia virus type 1-associated lymphomas:Heterogeneous expression of ebi3 subunit by tumoral cells [J]. Am J Pathol,2005,166(4):1217-28.
[101]Wang J., Lu Z. H., Gabius H. J., etc. Cross-linking of gml ganglioside by galectin-1 mediates regulatory t cell activity involving trpc5 channel activation: Possible role in suppressing experimental autoimmune encephalomyelitis [J]. J Immunol,2009,182(7):4036-45.
[102]Kubach J., Lutter P., Bopp T., etc. Human cd4+cd25+ regulatory t cells:Proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function [J]. Blood,2007,110(5):1550-8.
[103]Omoto Y., Yamanaka K., Tokime K., etc. Granzyme b is a novel interleukin-18 converting enzyme [J]. J Dermatol Sci,2010,59(2):129-35.
[104]Cai S. F., Fehniger T. A., Cao X., etc. Differential expression of granzyme b and c in murine cytotoxic lymphocytes [J]. J Immunol,2009,182(10):6287-97.
[105]Cao X., Cai S. F., Fehniger T. A., etc. Granzyme b and perforin are important for regulatory t cell-mediated suppression of tumor clearance [J]. Immunity,2007, 27(4):635-46.
[106]Onishi Y., Fehervari Z., Yamaguchi T., etc. Foxp3+ natural regulatory t cells preferentially form aggregates on dendritic cells in vitro and actively inhibit their maturation [J]. Proc Natl Acad Sci U S A,2008,105(29):10113-8.
[107]de Luca A., Bozza S., Zelante T., etc. Non-hematopoietic cells contribute to protective tolerance to aspergillus fumigatus via a trif pathway converging on ido [J]. Cell Mol Immunol,2010,7(6):459-70.
[108]Baban B., Chandler P. R., Johnson B. A.,3rd, etc. Physiologic control of ido competence in splenic dendritic cells [J]. J Immunol,2011,187(5):2329-35.
[109]Iype T., Sankarshanan M., Mauldin I. S., etc. The protein tyrosine phosphatase shp-1 modulates the suppressive activity of regulatory t cells [J]. J Immunol,2010, 185(10):6115-27.
[110]Borsellino G., Kleinewietfeld M., Di Mitri D., etc. Expression of ectonucleotidase cd39 by foxp3+ treg cells:Hydrolysis of extracellular atp and immune suppression [J]. Blood,2007,110(4):1225-32.
[111]Serra S., Horenstein A. L., Vaisitti T., etc. Cd73-generated extracellular adenosine in chronic lymphocytic leukemia creates local conditions counteracting drug-induced cell death [J]. Blood,2011,118(23):6141-52.
[112]Deaglio S., Dwyer K. M., Gao W., etc. Adenosine generation catalyzed by cd39 and cd73 expressed on regulatory t cells mediates immune suppression [J]. J Exp Med,2007,204(6):1257-65.
[113]Shalev I., Liu H., Koscik C., etc. Targeted deletion of fgl2 leads to impaired regulatory t cell activity and development of autoimmune glomerulonephritis [J]. J Immunol,2008,180(1):249-60.
[114]Piechnik A., Dmoszynska A., Omiotek M., etc. The vegf receptor, neuropilin-1 (nrp1) represents a promising novel target for chronic lymphocytic leukemia patients [J]. Int J Cancer,2013.