人CD4~+CD25~-CD45RA~+T细胞和CD4~+CD25~-T细胞诱导获得iTreg细胞的方法学研究
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摘要
目的:分析比较CD4~+CD25~-CD45RA~+T细胞和CD4~+CD25~-T细胞在普通/炎性(加入IL-1β和IL-6)条件下,由TGF-β、IL-2和CD3/CD28磁珠共刺激诱导生成i Treg细胞的效率,并对比各组i Treg细胞相关蛋白和细胞因子表达水平变化及其免疫抑制能力的差异。方法:通过Ficoll细胞分离液分离出淋巴细胞,利用MACS分选机,结合CD4阴选磁珠筛出CD4阳性的T细胞,最后再提取其中CD45RA~+和CD25~-T细胞。体外实验中,在普通/炎性条件下诱导i Treg生成,3、6 d通过流式观察各组i Treg细胞的诱导情况及相关分子的表达变化趋势。体内实验分为:CD4~+CD25~-CD45RA~+T细胞诱导的i Treg组、CD4~+CD25~-T细胞诱导的i Treg组和空白对照组,通过观察i Treg对GVHD小鼠模型的免疫调节,分析其免疫抑制能力的差异。结果:CD4~+CD25~-CD45RA~+T细胞组和CD4~+CD25~-T细胞组普通条件下i Treg产率分别为(96. 00±0. 21)%和(60. 23±0. 50)%,炎性环境下为(81. 97±1. 29)%和(56. 80±0. 43)%,前者都显著高于后者(P<0. 001)。各组i Treg细胞中CTLA-4、TGF-β和IL-10的表达未见明显差异,而炎性因子IL-2、IL-17前者均低于后者(P<0. 001),前者IFN-γ也低于后者(P<0. 05)。体内实验表明i Treg均具有免疫调节能力,但CD4~+CD25~-CD45RA~+T细胞组诱导的i Treg能力强于后者。结论:CD4~+CD25~-CD45RA~+T细胞组和CD4~+CD25~-T细胞组都可以成功诱导i Treg细胞,但是前者在普通和炎性微环境下诱导效率高于后者,免疫调节能力也优于后者。
Objective: To analyse the yield rate of i Treg induced from human CD4~+CD25~-CD45 RA~+T cells and CD4~+CD25~-T cells in normal or inflammatory( add IL-1β/6) conditions under co-stimulation of TGF-β,IL-2 and CD3/CD28 beads,compare the difference on immunosuppressive ability and expression levels of i Treg associated functional proteins or cytokines among groups. Methods: Lymphocytes were obtained through Ficoll lymphocyte separation medium,CD4~+CD25~-CD45 RA~+T cells and CD4~+CD25~-T cells were isolated by MACS after co-incubation with microbeads. In vitro,yield of i Treg and expression levels of related molecule/cytokines were detected by Flow cytometry on 3 and 6 day respectively. In vivo,GVHD mice were applied to exam the immunosuppressive ability of i Treg,three groups were designed: CD4~+CD25~-CD45 RA~+T cells derived i Treg group,CD4~+CD25~-T cells derived i Treg group and PBS group. Results: The i Treg yield rate were( 96. 00±0. 21) %,( 60. 23±0. 50) % in normal conditions and( 81. 97±1. 29) %,( 56. 80±0. 43) % in inflammatory conditions,the former was significantly higher than the latter( P<0. 001). CTLA-4,TGF-β and IL-10 didn' t show obvious difference while the secretion of pro-inflammatory cytokines such as IL-2,IL-17 and IFN-γwere significantly improved in latter( P <0. 05). Conclusion: Study indicated that all i Treg induced in vitro exhibited immunosuppressive ability,what's more,CD4~+CD25~-CD45 RA~+T cells derived i Tregs were stronger in both normal and inflammatory environment.
Objective: To analyse the yield rate of i Treg induced from human CD4~+CD25~-CD45 RA~+T cells and CD4~+CD25~-T cells in normal or inflammatory( add IL-1β/6) conditions under co-stimulation of TGF-β,IL-2 and CD3/CD28 beads,compare the difference on immunosuppressive ability and expression levels of i Treg associated functional proteins or cytokines among groups. Methods: Lymphocytes were obtained through Ficoll lymphocyte separation medium,CD4~+CD25~-CD45 RA~+T cells and CD4~+CD25~-T cells were isolated by MACS after co-incubation with microbeads. In vitro,yield of i Treg and expression levels of related molecule/cytokines were detected by Flow cytometry on 3 and 6 day respectively. In vivo,GVHD mice were applied to exam the immunosuppressive ability of i Treg,three groups were designed: CD4~+CD25~-CD45 RA~+T cells derived i Treg group,CD4~+CD25~-T cells derived i Treg group and PBS group. Results: The i Treg yield rate were( 96. 00±0. 21) %,( 60. 23±0. 50) % in normal conditions and( 81. 97±1. 29) %,( 56. 80±0. 43) % in inflammatory conditions,the former was significantly higher than the latter( P<0. 001). CTLA-4,TGF-β and IL-10 didn' t show obvious difference while the secretion of pro-inflammatory cytokines such as IL-2,IL-17 and IFN-γwere significantly improved in latter( P <0. 05). Conclusion: Study indicated that all i Treg induced in vitro exhibited immunosuppressive ability,what's more,CD4~+CD25~-CD45 RA~+T cells derived i Tregs were stronger in both normal and inflammatory environment.
引文
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[11] Xia Y,Zhuo H,Lu Y,et al. Glycogen synthase kinase 3beta inhibition promotes human i Treg differentiation and suppressive function[J]. Immunol Res,2015,62(1):60-70.
[12] Tang Q,Adams JY,Penaranda C,et al. Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction[J]. Immunity,2008,28(5):687-697.
[13] Wan YY,Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression[J]. Nature,2007,445(7129):766-770.
[14] Bluestone JA,Trotta E,Xu D. The therapeutic potential of regulatory T cells for the treatment of autoimmune disease[J].Expert Opin Ther Targets,2015,19(8):1091-1103.
[15] Lu L,Barbi J,Pan F. The regulation of immune tolerance by FOXP3[J]. Nat Rev Immunol,2017,17(11):703-717.
[16] Kolar P,Knieke K,Hegel J K,et al. CTLA-4(CD152)controls homeostasis and suppressive capacity of regulatory T cells in mice[J]. Arthritis Rheum,2009,60(1):123-132.
[17] Sadhu S,Khaitan BK,Joshi B,et al. Reciprocity between Regulatory T cells and Th17 cells:relevance to polarized immunity in leprosy[J]. PLo S Negl Trop Dis,2016,10(1):e4338.
[18] Miyara M,Yoshioka Y,Kitoh A,et al. Functional delineation and differentiation dynamics of human CD4+T cells expressing the Fox P3 transcription factor[J]. Immunity,2009,30(6):899-911.
[19]王海灏,仰霈雯,雷家慧.两种多克隆刺激条件下人诱导性Treg表型的多样性变化[J].中国免疫学杂志,2012,28(9):769-774.Wang HH,Yang PW,Lei JH,et al. Plasticity of human induced regulatory T cells during two different polyclonal stimulations[J].Chin J Immunol,2012,28(9):769-774.
[20] Daniel V,Naujokat C,Sadeghi M,et al. Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN-gamma+blood lymphocytes in kidney transplant recipients with good long-term graft outcome[J]. Transpl Int,2008,21(7):646-660.
[2] Hori S,Nomura T,Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3[J]. Science,2003,299(5609):1057-1061.
[3] Kitazawa Y,Li XK,Liu Z,et al. Prevention of graft-versus-host diseases by in vivo supCD28mAb-expanded antigen-specific n Treg cells[J]. Cell Transplant,2010,19(6):765-774.
[4] Kitagawa Y,Sakaguchi S. Molecular control of regulatory T cell development and function[J]. Curr Opin Immunol,2017,49:64-70.
[5] Mao X,Wu Y,Diao H,et al. Interleukin-6 promotes systemic lupus erythematosus progression with Treg suppression approach in a murine systemic lupus erythematosus model[J]. Clin Rheumatol,2014,33(11):1585-1593.
[6] Lu L,Ma J,Li Z,et al. All-trans retinoic acid promotes TGF-betainduced Tregs via histone modification but not DNA demethylation on Foxp3 gene locus[J]. PLo S One,2011,6(9):e24590.
[7] Lu Y,Wang X,Gu J,et al. i Treg induced from CD39(+)naive T cells demonstrate enhanced proliferate and suppressive ability[J].Int Immunopharmacol,2015,28(2):925-930.
[8] Zhou X,Kong N,Zou H,et al. Therapeutic potential of TGF-betainduced CD4(+)Foxp3(+)regulatory T cells in autoimmune diseases[J]. Autoimmunity,2011,44(1):43-50.
[9]杨洁,孔宁,杨懿铭,等.耐受型DC/TGF-β联合诱导的i Treg细胞能有效抑制自身免疫性关节炎[J].安徽医科大学学报,2017,52(1):66-72.Yang J,Kong N,Yang YM,et al. i Tregs induced by tolerogenic DCs plus TGF-βcould effectively suppress autoimmune arthritis[J]. Acta J Univ Med Anhui,2017,52(1):66-72.
[10]汤庆,章文,周春雷,等. TGF-β诱导及病毒感染获得i Treg细胞的方法学研究[J].天津医科大学学报,2013,19(5):368-372.Tang Q,Zhang W,Zhou CL,et al. A comparative study of TGF-Binduced method and Viral infection method to obtain the i Treg cells[J]. J Tianjin Med Univ,2013,19(5):368-372.
[11] Xia Y,Zhuo H,Lu Y,et al. Glycogen synthase kinase 3beta inhibition promotes human i Treg differentiation and suppressive function[J]. Immunol Res,2015,62(1):60-70.
[12] Tang Q,Adams JY,Penaranda C,et al. Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction[J]. Immunity,2008,28(5):687-697.
[13] Wan YY,Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression[J]. Nature,2007,445(7129):766-770.
[14] Bluestone JA,Trotta E,Xu D. The therapeutic potential of regulatory T cells for the treatment of autoimmune disease[J].Expert Opin Ther Targets,2015,19(8):1091-1103.
[15] Lu L,Barbi J,Pan F. The regulation of immune tolerance by FOXP3[J]. Nat Rev Immunol,2017,17(11):703-717.
[16] Kolar P,Knieke K,Hegel J K,et al. CTLA-4(CD152)controls homeostasis and suppressive capacity of regulatory T cells in mice[J]. Arthritis Rheum,2009,60(1):123-132.
[17] Sadhu S,Khaitan BK,Joshi B,et al. Reciprocity between Regulatory T cells and Th17 cells:relevance to polarized immunity in leprosy[J]. PLo S Negl Trop Dis,2016,10(1):e4338.
[18] Miyara M,Yoshioka Y,Kitoh A,et al. Functional delineation and differentiation dynamics of human CD4+T cells expressing the Fox P3 transcription factor[J]. Immunity,2009,30(6):899-911.
[19]王海灏,仰霈雯,雷家慧.两种多克隆刺激条件下人诱导性Treg表型的多样性变化[J].中国免疫学杂志,2012,28(9):769-774.Wang HH,Yang PW,Lei JH,et al. Plasticity of human induced regulatory T cells during two different polyclonal stimulations[J].Chin J Immunol,2012,28(9):769-774.
[20] Daniel V,Naujokat C,Sadeghi M,et al. Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN-gamma+blood lymphocytes in kidney transplant recipients with good long-term graft outcome[J]. Transpl Int,2008,21(7):646-660.
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