腺病毒介导反义CⅡTA基因对实验性自身免疫性心肌炎的治疗

目的　研究反义CⅡTA基因对心肌肌凝蛋白诱导的实验性自身免疫性心肌炎 (experi mentalautoimmunemyocarditis,EAM)的治疗作用。方法　以携带反义CⅡTA基因的腺病毒载体对肌凝蛋白免疫后的小鼠进行静脉注射 ,同时设立空载病毒组与单纯免疫组作为对照 ,以多种手段观察反义CⅡTA基因对EAM的治疗效果。结果　治疗组与两对照组相比 ,心肌病理损伤程度减轻 ;血浆抗心肌肌凝蛋白抗体滴度及cTnⅠ浓度明显下降 (P <0 .0 5 ) ,流式细胞术检测外周血与脾脏T细胞I Ad表达量明显下降 (P <0 .0 5 )。结论　反义CⅡTA基因对心肌肌凝蛋白诱导的EAM具有较好的治疗作用。     

MHC I类抗原多肽致敏IL-2基因修饰的树突状细胞对小鼠 引流淋巴结T细胞亚群的影响及其免疫保护作用

目的探讨MHCI类分子限制性肿瘤抗原多肽Mutl体外冲击白细胞介素2(Interleukin-2,IL-2)基因修饰的树突状细胞(Dendrticcells,DC)对小鼠体内特异性免疫的活化机制。方法用腺病毒作为载体介导小鼠IL-2基因修饰DC,用小鼠Lewis肺癌3LL细胞株MHCI类分子限制性八肽Mutl冲击IL-2基因修饰的DC(DC-IL-2-Mutl)免疫小鼠,用流式细胞术(FACS)分析免疫保护小鼠接受3LL细胞再攻击后引流淋巴结内T细胞亚群的比例变化。结果用Mutl冲击的DC免疫的小鼠抵抗3LL细胞再攻击时,引流淋巴结内CD8     

IL-2基因修饰对树突状细胞的生物学特征和功能的影响

目的:观察白细胞介素2(IL-2)基因修饰对树突状细胞(DC)的生物学特征和功能的影响,探讨用IL-2基因修饰DC,增强DC介导特异性抗肿瘤免疫的机制。方法:IL－2基因修饰小鼠骨髓来源的DC后,用扫描电镜观察其表面形态的变化,FACS分析IL-2基因修饰对DC表面免疫分子表达的影响,RT-PCR方法检测DC中 IFN-γ mRNA表达。用3H-TdR掺入法检测IL-2基因修饰后,DC对同种异体T淋巴细胞的刺激作用和对肿瘤抗原的特异性提呈功能。结果:经IL-2基因修饰后,DC表面的伪足增多、变长;其表面与抗原提呈相关的免疫分子Ia、B7-1、B7-2和CD40的表达明显上调;il-2基因修饰的DC(DC-IL-2)中表达IFN-γ mRNA;CD-IL-2不但对同种异体T淋巴细胞有较强的促增殖作用,而且对肿瘤抗原的特异性提呈功能亦明显增强。结论:IL-2基因修饰DC,能促进DC的发育,上调DC表面与抗原提呈相关的免疫分子,增强了DC的生物活性。     

罗格列酮通过抑制IL-17表达改善自身免疫性心肌炎小鼠心肌损伤

目的:探讨过氧化物酶体增殖物激活受体γ激动剂罗格列酮对小鼠实验性自身免疫性心肌炎的干预作用及其作用机制。方法:70只BALB/c小鼠随机分为4组:罗格列酮组(A组)、罗格列酮+GW9662组(B组)、心肌炎组(C组)以及正常对照组(D组)。应用猪心肌肌凝蛋白分别对A、B、C 3组小鼠进行皮下注射,构建实验性自身免疫性心肌炎模型。所有小鼠在实验的第25天处死。分别采用酶联免疫吸附法(ELISA)、实时荧光定量PCR(real-time PCR)、免疫组织化学染色、苏木素-伊红(HE)染色对各组小鼠外周血以及心肌组织进行检测。结果:与C组小鼠相比,A组小鼠外周血IL-17水平、心肌IL-17m RNA相对表达量、心肌组织IL-17的表达以及心肌组织中炎症细胞数均显著降低;而B组小鼠与C组小鼠相比较上述指标差异不显著。结论:应用罗格列酮激活PPARγ可以明显减轻小鼠实验性自身免疫性心肌炎病情,抑制心肌炎症反应。罗格列酮的这种作用可能是通过减少IL-17的表达实现的。     

Jagged-1对骨髓来源树突状细胞生成细胞因子的影响

目的:探讨可溶性Jagged-1/Fe嵌合蛋白(Jagged-1)对重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和白细胞介素4(rmIL-4)体外诱导小鼠骨髓来源树突状细胞(DC)产生细胞凶子的影响.方法:建立rmCM-CSF和rmIL-4体外诱导DC的模型,观察Jagged-1/Fc对DC分化的形态学影响.通过Luminex蛋白液相芯片技术和ELlSA检测其细胞因子的表达水平,藉MTT法测定可溶性Jagged-1/Fc诱导的DC对同种异基因淋巴细胞增殖的刺激能力.结果:除了TGF-β,Jag-ged-1/Fc诱导的DC与细菌脂多糖(LPS)或酵母聚糖A诱导的DC不同,表现为生成TNF-α的水平明显降低,IL-4显著增高,而IL-10、IL-6、IL-2、IL-12和IFN-γ的水平与对照组无明显差异.γ分泌酶抑制剂DAFT能逆转Jagged-1/Fc抑制DC生成TNF-α.混合淋巴细胞反应显示Jagged-1/Fc诱导的DC对T细胞增殖的刺激能力最弱,LPS诱导的DC的刺激能力最强.结论:Jagged-1/Fc诱导的DC倾向介导免疫耐受,指导初始T细胞向Th2细胞偏离.     

诱导实验性肌炎模型并观察调节性T细胞的表达及意义

目的:观察实验性自身免疫性肌炎(EAM)小鼠脾细胞中调节性T细胞(Tregs)表达情况,并探讨相关免疫学机制。方法:雌性BALB/c小鼠随机分为肌球蛋白免疫的EAM模型组和生理盐水对照组,采用苏木精-伊红染色(HE)进行组织病理学评分,使用流式细胞术(FCM)检测小鼠脾细胞中CD4+CD25+Foxp3+Treg细胞表达比例。结果:EAM模型组小鼠肌组织出现大量炎细胞浸润;FCM检测EAM模型组小鼠脾细胞中CD4+CD25+Foxp3+Treg较对照组明显升高(P<0.01)。结论:CD4+CD25+Foxp3+Treg参与调控EAM的病理过程。     

小鼠自身免疫性心肌炎发病过程中4-1BB/4-1BBL和IL-15的表达及意义

目的： 探讨在小鼠自身免疫性心肌炎发病过程中4-1BB/4-1BBL、IL-15的表达和变化,及其免疫学活性对心肌炎的影响。方法：将提纯的猪心肌肌球蛋白和完全弗氏佐剂等体积混合成乳浊液在1 d、8 d及30 d免疫具有遗传易感性的BALB/c小鼠,建立实验性自身免疫性心肌炎（EAM）模型。对照组小鼠仅用完全弗氏佐剂皮下注射。分别于初次免疫后21 d、80 d进行心肌炎症评分及血清肌钙蛋白I(cTnI)测定,免疫组化检测心肌淋巴细胞活化诱导受体配体（4-1BBL）的表达,ELISA法检测血清白细胞介素-15(IL-15)的浓度,RT-PCR技术检测4-1BB/4-1BBL和IL-15 mRNA在小鼠心肌组织中的表达。结果：在急性期21 d,EAM组小鼠心肌组织见不同程度的炎性细胞浸润和心肌细胞变性坏死、血清cTnI水平升高（P<0.05）;80 d EAM组小鼠心肌组织炎症减弱伴有纤维化出现、cTnI较前期降低;心肌4-1BBL和血清IL-15在EAM组中表达明显,在对照组中少量表达（P<0.05）;21 d EAM组小鼠心肌组织中4-1BB/4-1BBL和IL-15基因表达水平均高于对照组(P<0.01),且与心肌炎症呈明显的正相关,80 d时表达仍然升高(P<0.05)。结论：4-1BB/4-1BBL和IL-15在EAM小鼠发病过程中的表达上调,4-1BB/4-1BBL共刺激通路和IL-15可能协同参与了自身免疫性心肌炎的发生发展过程。     

抗CD4单抗诱导免疫耐受防治自身免疫性心肌炎的研究

目的探讨体内注射CD4单克隆抗体诱导大鼠对猪心肌肌凝蛋白产生免疫耐受及这种免疫耐受对自身免疫性心肌炎大鼠的治疗作用。方法分别于第0、7天给予Lewis大鼠体内注射猪心肌肌凝蛋白诱导自身免疫性心肌炎的产生。分别于第-2、-1、0、1天注射CD4单克隆抗体诱导免疫耐受。初次免疫后18d,检测免疫耐受的诱导情况及其对心肌炎大鼠的作用。结果同非治疗组鼠相比较,抗体治疗组鼠心功能明显改善;没有明显的心肌变性、坏死、炎症细胞浸润及纤维化;抗原特异性淋巴细胞增殖反应明显下降;血清抗心肌肌凝蛋白自身抗体明显降低;血清T_H1细胞因子IFN-γ、1L-2的水平显著下调;T_H2细胞因子IL-6、IL-10的水平没有改变或者被上调。结论CD4单抗能够诱导机体对猪心肌肌凝蛋白产生免疫耐受,通过免疫耐受的诱导阻止了自身免疫性心肌炎大鼠心功能的紊乱和心肌的损伤。     

IL-12基因修饰DC中SOCS1基因沉默对体外诱导特异性CTL的影响

利用重组腺病毒载体pAd CMV/V5-DEST-IL-12转染小鼠骨髓来源的树突状细胞(IL-12/DC),探讨SOCS1基因沉默IL-12/DC在体外诱导细胞毒性T淋巴细胞(CTL)的效能,及其免疫杀伤肺癌细胞株LLC的能力。采用重组腺病毒介导IL-12基因和SOCS1SiRNA基因共同修饰C57BL/6小鼠骨髓来源的DC,经反复冻融法提取LLC抗原,致敏基因修饰的DC;用ELISA法检测各组DC分泌IL-12和IL-10的水平,及各组DC刺激后的T细胞分泌IFN-γ的水平;MTT法检测DC刺激同源小鼠T细胞的增殖能力,微量细胞毒法检测CTL的活性并收集刺激后的T细胞,流式细胞术分析CD8+/CD4+比例和CD4+CD25+Treg的水平;统计学分析各组间的差异。SOCS1SiRNA和IL-12基因共同修饰能有效下调DC中SOCS1蛋白的表达并上调IL-12蛋白的表达;IL-12的分泌水平也明显高于SOCS1SiRNA或IL-12单基因转染组;基因共同修饰的DC表型更加成熟,能明显促进CTL的增殖和活化,减少Treg的生成;CTL分泌高水平的IFN-γ,产生对LLC特异性的细胞免疫。     

调节性T细胞与Th17细胞相关因子在实验性自身免疫性肌炎小鼠淋巴结中的表达及意义

目的研究调节性T细胞(Treg)、Th17细胞的相关细胞因子在实验性自身免疫性肌炎小鼠淋巴结中表达的作用及意义。方法雌性BALB/c小鼠12只,随机分为正常对照组和模型组,模型组采用肌球蛋白诱导实验性自身免疫性肌炎(EAM)。采用实时荧光定量PCR(qRT-PCR)检测各组小鼠淋巴结中与Treg和Th17细胞分化及功能相关的细胞因子Foxp3、IL-10、TGF-β、RORγt、IL-6、IL-17、IL-23的mRNA表达。结果与正常小鼠相比,肌炎组小鼠淋巴结中Treg的转录因子Foxp3表达降低,相关的细胞因子IL-10、TGF-βmRNA表达水平增高;Th17的转录因子RORγt及相关的细胞因子IL-6、IL-17、IL-23 mRNA表达有不同程度的升高(P0.05)。结论实验性自身免疫性肌炎中Treg表达降低,而Th17及其相关因子表达升高,提示Treg和Th17之间的失衡在肌炎发病机制中起一定作用。     

Protection against experimental autoimmune myocarditis is mediated by interleukin-10-producing T cells that are controlled by dendritic cells

Experimental autoimmune myocarditis (EAM) can be induced in the Lewis rat by cardiac myosin or its cryptic S2-16 peptide epitope (amino acids 1052 to 1076). To investigate cellular mechanisms and the role of antigen-presenting cells in regulation of myocarditis, we induced protection against EAM in Lewis rats by administration of S2-16 peptide in incomplete Freund's adjuvant (IFA). Protection to EAM was associated with activation of S2-16-reactive splenocytes secreting high levels of interleukin (IL)-10 and reduced levels of interferon-gamma and IL-2. Adoptive transfer of S2-16:IFA-induced splenocytes producing IL-10 suppressed myocarditis induction in syngeneic recipients, suggesting their regulatory cell nature. However, exposure of S2-16:IFA-induced cells to inflammatory cytokine IL-12 converted them to Th1 effectors that transferred EAM. Differentiated function of S2-16-reactive T cells in protected rats resulted from increased IL-10 production by dendritic cells (DCs). Purified DCs from S2-16:IFA-treated rats promoted S2-16-reactive CD4+ T cells to produce increased IL-10 and reduced interferon-gamma. In addition, adoptive transfer of IL-10-producing DCs from S2-16:IFA-treated rats also induced protection to EAM in recipient rats. These studies demonstrated DCs and key cytokines, such as IL-10 and IL-12, regulated the fate of T cells in myocarditis development in the Lewis rat.     

IL-10 gene modified dendritic cells induced antigen-specific tolerance in experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) in rats is a T-cell-mediated disorder, and the involvement of Th1/Th2 unbalance has been demonstrated. The induction of antigen-specific tolerance is critical for the treatment of EAM and maintenance of immune tolerance. IL-10 is a pleiotrophic immunomodulatory cytokine that functions at different levels of the immune response, so it has emerged as a promising therapeutic factor for the treatment of autoimmune/inflammatory diseases. This study was designed to explore the effects of IL-10 gene modified bone-marrow-derived immature dendritic cells (iDCs) on the in vitro and in vivo immune response to cardiac myosin in EAM. EAM was induced using the classic methods of cardiac myosin immunization on day 0 and day 7. 2 x 10(6)/per rat mature DC (mDC), immature DC (iDC), pcDNA3 transfected iDC, pcDNA3-IL-10 transfected iDC or PBS were injected intravenously for treatment 5 days after the first immunization. On day 21, transthoracic echocardiogram and HE staining were performed to detect the cardiac function and myocardial inflammation. Th1/Th2 cytokines were detected by ELISA and MHC-II molecules, costimulatory molecules were identified by flow cytometry. In vitro T lymphocyte proliferation assay and adoptive transfer of DCs were performed to determine the antigen-specific tolerance induced by IL-10 gene modified iDCs. IL-10 gene modified iDC-treated EAM rats showed improved cardiac function and reduced infiltration of inflammatory cell into myocardium. Serum cytokines data indicated lower Th1 while higher Th2-type responses were induced in the pcDNA3-IL-10-iDC-treated group, suggesting a Th2 polarization. Moreover, IL-10 gene modified iDCs down-regulated MHC-II and costimulatory molecules on the surface of splenocytes and inhibited the antigen-specific immunological responses towards cardiac myosin. Adoptive transfer of IL-10 producing DCs prevented EAM induction. IL-10 gene modified iDCs ameliorates EAM histopathologically and functionally. The underlying mechanisms may be related to the IL-10 induced Th2 polarization and down-regulation of MHC-II molecules and costimulatory molecules expression.     

IL-10-conditioned dendritic cells prevent autoimmune diabetes in NOD and humanized HLA-DQ8/RIP-B7.1 mice

This study was to determine whether BMDCs cultured in the presence of IL-10 (G/10-DCs) could promote T cell tolerance and prevent autoimmune diabetes in two different animal models of T1D. Our results showed that G/10-DCs suppressed both insulitis and spontaneous diabetes in NOD and HLA-DQ8/RIP-B7.1 mice. The suppression was likely to be mediated by T cells, as we found that regulatory CD4(+)CD25(+)Foxp3(+) cells were significantly increased in G/10-DC treated animals. In vivo, the G/10-DCs inhibited diabetogenic T cell proliferation; in vitro, they had reduced expression of costimulatory molecules and produced little IL-12/23 p40 or IL-6 but a large amount of IL-10 when compared with DCs matured in the presence of IL-4 (G/4-DC). We conclude that IL-10-treated DCs are tolerogenic and induce islet-directed immune tolerance, which was likely to be mediated by T regulatory cells. This non-antigen-specific DC-based approach offers potential for a new therapeutic intervention in T1D.     

Induction of IL-10 producing CD4+ T cells with regulatory activities by stimulation with IL-10 gene-modified bone marrow derived dendritic cells

Dendritic cells (DCs) can induce both tolergenic as well as effective immune responses in the lung. Pulmonary DCs producing interleukin (IL)-10 mediated tolerance induced by respiratory exposure to antigen. IL-10 is an important immunosuppressive cytokine, which inhibits maturation and function of DC. To assess whether IL-10 producing DCs can exert the tolergenic effect through the differentiation of regulatory T cells, bone marrow derived DCs were genetically modified by IL-10 expressing adenovirus. IL-10 gene modified DCs (Ad-IL-10-DC) displayed a characteristic phenotype of immature DCs. Here we showed that in vitro repetitive stimulation of naïve DO11·10 CD4⁺ T cells with Ad-IL-10-DCs resulted in a development of IL-10 producing T-cell regulatory cells. These T cells could not proliferate well but also lost their ability to produce interferon-γ upon restimulation with irradiated splenocytes and ovalbumin peptide. Furthermore, in co-culture experiments these T cells inhibited the antigen-driven proliferation of naïve CD4+ T cells in a dose-dependent manner. Our findings demonstrated that IL-10 producing DCs had the potential to induce the differentiation of Tr1-like cells and suggested their therapeutic use.     

BTLA-HVEM通路阻断对树突状细胞功能影响的体内外研究

目的 探讨阻断BTLA-HVEM(B/T淋巴细胞弱化因子疱疹病毒进入介质)通路对树突状细胞功能的影响和相关免疫学机制.方法 构建小鼠BTLA胞外功能区的真核表达载体psBTLA,转染CHO细胞;HSP70-TC-1肿瘤抗原肽刺激小鼠骨髓来源DCs,流式细胞仪检测处理后DCs表面BTLA、HVEM的表达,同时给予转染了psBTLA质粒的CHO细胞的培养上清处理后,检测DCs表面B7-1的表达,ELISA检测上清中IL-12的分泌;处理后的DCs刺激脾细胞,检测淋巴细胞增殖和细胞因子分泌;检测psBTLA体内转染对宫颈癌细胞系TC-1成瘤小鼠DCs表达B7-1和肿瘤生长的影响.结果 成功构建小鼠BTLA胞外段的真核表达载体psBTLA,获得了稳定转染psBTLA的CHO细胞,在其培养上清检测到BTLA胞外段(sBTLA)的表达.DCs经抗原肽刺激后BTLA、HVEM表达均上调,加入含sBTLA的上清处理后上调B7-1,上清中分泌的IL-12增加,与脾细胞共培养时促进细胞增殖和IL-2、IFN-γ的分泌;体内基因转染psBTLA促进DCs表达B7-1以及抑制肿瘤生长.结论 通过sBTLA阻断BTLA-HVEM共抑制通路,可以进一步促进DCs的功能,更好地激活淋巴细胞,促进抗肿瘤免疫应答.     

IL-10 gene modified dendritic cells inhibit T helper type 1-mediated alloimmune responses and promote immunological tolerance in diabetes

Dendritic cells （DCs） have the potency to regulate the outcome of autoimmunity through the modulation of immune responses. The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In the present Study, we transfected IL-10 gene into DCs and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on islet allograft in mice. An IDDM C57BL/6 mouse model was induced by streptozotocin. The islet cells isolated from the BALB/c mice were transplanted into the kidney capules of the model mice followed by injection of IL-10 modified DCs （mDCs）. The results showed that mDCs could significantly inhibit T lymphocyte proliferation mediated by allotype cells and induce its apoptosis, whereas, unmodified DCs （umDCs） could promote the murine lymphocyte proliferation markedly. The injection of mDCs could prolong the survival of allotype islet transplanted IDDM mice. The average plasma glucose （PG） level in mDCs treated mice returned to normal within 3 days and lasted for about 2 weeks. The rejection response in control mice occurred for 5 days after transplantation. The level of IFN-γ was lower while IL-4 was higher in mDCs treated mice than that in umDCs treated mice, which indicated that Thl/Th2 deviation occurred. Our studies suggest that IL-10 gene modified DCs can induce the immune tolerance to islet graft and prolong survival of the recipients by the inhibiting of T cell proliferation in allotype mice. Cellular ＆ Molecular Immunology.     

Response of human dendritic cells to different immunomodulatory polysaccharides derived from mushroom and barley

Polysaccharides derived from fungi and plants have been increasingly used as dietary supplement with therapeutic intention for cancer. However, whether these polysaccharides from different sources and structures can elicit similar immunological effects remain unknown. This study aims to investigate and compare the effects of selected groups of purified and crude polysaccharides on human dendritic cells (DCs), the most potent antigen-presenting cells. The selected polysaccharides were from Ganoderma lucidum [(GL) Lingzhi, Reishi], a medicinal mushroom commonly used by oriental; and barley glucan, a purified polysaccharide with known in vivo immunomodulating effect. We found that purified polysaccharides from GL mycelium could induce human PBMC proliferation and phenotypic and functional maturation of DCs with significant IL-12 and IL-10 production. Polysaccharides of GL spore and barley were both rather weak immunostimulator in vitro. In general, all these polysaccharides did not polarize T cells into either T(h)1 or T(h)2 or regulatory T cells, except for crude spore polysaccharides-treated DCs which could suppress T cell proliferation with IL-10 production. This study revealed the polysaccharides of different sources have different immune potency and effect on human immune cells including DCs. Our study also provides a reproducible biological platform for comparing the potential therapeutic effects of different herbal-derived polysaccharides in the future.     

Regulatory T cells and dendritic cells in transplantation tolerance: molecular markers and mechanisms

Summary: Transplantation tolerance can be induced in adult rodents using monoclonal antibodies against coreceptor or costimulation molecules on the surface of T cells. There are currently two well‐characterized populations of T cells, demonstrating regulatory capacity: the ‘natural’ CD4⁺CD25⁺ T cells and the interleukin (IL)‐10‐producing Tr1 cells. Although both types of regulatory T cells can induce transplantation tolerance under appropriate conditions, it is not clear whether either one plays any role in drug‐induced dominant tolerance, primarily due to a lack of clear‐cut molecular or functional markers. Similarly, although dendritic cells (DCs) can be pharmacologically manipulated to promote tolerance, the phenotype of such populations remains poorly defined. We have used serial analysis of gene expression (SAGE) with 29 different T‐cell and antigen‐presenting cell libraries to identify gene‐expression signatures associated with immune regulation. We found that independently derived, regulatory Tr1‐like clones were highly concordant in their patterns of gene expression but were quite distinct from CD4⁺CD25⁺ regulatory T cells from the spleen. DCs that were treated with the tolerance‐enhancing agents IL‐10 or vitamin D3 expressed a gene signature reflecting a functional specification in common with the most immature DCs derived from embryonic stem cells.     

Activation of invariant NKT cells by toll-like receptor 9-stimulated dendritic cells requires type I interferon and charged glycosphingolipids

Invariant natural killer T (iNKT) cells are a subset of innate lymphocytes that recognize lipid antigens in the context of CD1d and mediate potent immune regulatory functions via the rapid production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). We investigated whether diverse Toll-like receptor (TLR) signals in myeloid dendritic cells (DCs) could differentially stimulate iNKT cells. Together with the lipopolysaccharide-detecting receptor TLR4, activation of the nucleic acid sensors TLR7 and TLR9 in DCs were particularly potent in stimulating iNKT cells to produce IFN-gamma, but not IL-4. iNKT cell activation in response to TLR9 stimulation required combined synthesis of type I interferon and de novo production of charged beta-linked glycosphingolipid(s) by DCs. In addition, DCs stimulated via TLR9 activated both iNKT cells and NK cells in vivo and protected mice against B16F10-induced melanoma metastases. These data underline the role of TLR9 in iNKT cell activation and might have relevance to infectious diseases and cancer.     

Dendritic cells partially abrogate the regulatory activity of CD4+CD25+ T cells present in the human peripheral blood

The factors that influence the functionality of human CD4(+)CD25(+) regulatory T cells are not well understood. We sought to characterize the effects of dendritic cells (DCs) on the in vitro regulatory activity of CD4(+)CD25(+) T cells obtained from peripheral blood of healthy human donors. Flow cytometry showed that a higher proportion of CD4(+)CD25(+(High)) T cells expressed surface glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and CTL-associated antigen 4 than CD4(+)CD25(-) or CD4(+)CD25(+(Med-low)) T cells. Intracellular Foxp3 was equivalently expressed on CD4(+)CD25(+(All)), CD4(+)CD25(+(High)), CD4(+)CD25(+(Med-low)) and CD4(+)CD25(-) T cell populations, irrespective of GITR and CTL-associated antigen 4 expression. CD4(+)CD25(+) T cells were isolated and then cultured in vitro with CD4(+)CD25(-) responder T cells and stimulated with anti-CD3 antibodies, and immature dendritic cells (iDCs), mature dendritic cells (mDCs), PBMCs or PBMCs plus anti-CD28 antibodies to provide co-stimulation. In addition, secretion of the T(h)1 cytokine IFN-gamma, IL-2 and the immunoregulatory cytokines, IL-10 and transforming growth factor (TGF)-beta, were also assessed in these cultures. We found that iDCs and mDCs were capable of reversing the suppression of proliferation mediated by CD4(+)CD25(+) regulatory T cells. However, the reversal of suppression by DCs was not dependent upon the increase of IFN-gamma and IL-2 production or inhibition of IL-10 and/or TGF-beta production. Therefore, DCs are able to reverse the suppressive effect of regulatory T cells independent of cytokine production. These results suggest for the first time that human DCs possess unique abilities which allow them to influence the functions of regulatory T cells in order to provide fine-tuning in the regulation of T cell responses.