TLR4在HSP70_(EC-TCV)冲激的DC成熟过程中的作用

观察TLR4在Hca-F榄香烯复合瘤苗来源的HSP70(HSP70EC-TCV)冲激处理的小鼠骨髓来源的DC成熟过程中的作用。用rmGM-CSF和rmIL-4诱导小鼠骨髓来源的DC,以HSP70EC-TCV或加入抗TLR4抗体30 min后再用HSP70EC-TCV冲激处理DC,流式细胞仪检测DC的CD40和CD86表达,ELISA法检测DC上清中IL-12和T细胞上清中IL-2浓度,MTT法检测T细胞对Hca-F细胞的杀伤率。结果表明,抗TLR4抗体对DC的CD40和CD86表达无明显影响,但对HSP70EC-TCV诱导DC分泌IL-12和进而致敏的T细胞分泌IL-2及产生杀瘤功能有明显的抑制作用。TLR4信号通路参与HSP70EC-TCV诱导DC成熟过程。     

约氏疟MIF对小鼠BM-DC细胞作用的探索

 摘要：目的 探索约氏疟原虫来源巨噬细胞迁移抑制因子（PyMIF）对小鼠髓系树突状细胞（BM-DC）表型和功能的影响。方法分离小鼠骨髓细胞并经GM-CSF和IL-4诱导培养得到BM-DC：经PyMIF刺激后,通过流式细胞术检测其TLR2、TLR4、CD80、CD86、CD40、MHCII分子表达,通过ELISA方法检测IL-12、IL-10分泌;经PyMIF刺激的BM-DC与CD4+T或CD8+T细胞共培养,同样方法检测T细胞CD69表达、IL-2分泌情况,并检测CD8+T细胞对靶细胞杀伤能力。结果PyMIF可以下调小鼠骨髓来源树突状细胞TLR-4的表达;但不能影响BM-DC细胞表面分子TLR2、CD80、CD86、CD40、MHCII的表达,也不改变BM-DC分泌IL-12、IL-10。PyMIF可通过BM-DC下调CD8+T的CD69表达,但不能通过BM-DC改变CD4+T细胞CD69表达、IL-2分泌,及CD8+T细胞IL-2分泌。结论PyMIF可能是通过下调BM-DC细胞TLR4表达来抑制免疫细胞对疟原虫的识别,使之逃逸机体的攻击而存活。     

体外DCs在抗CD45RB抗体诱导免疫耐受中的作用机制

目的:探讨树突状细胞(dendritic cells,DCs)在抗CD45RB抗体诱导的免疫耐受中所发挥的作用,从而阐明抗CD45RB抗体诱导免疫耐受的机制。方法:采用DCs的常规诱导方法(rmGM-CSF、IL-4和LPS),在诱导过程中加入不同剂量的抗CD45RB抗体,成熟后利用流式细胞仪检测细胞表型、周期和吞噬能力,ELISA法检测IL-12分泌量,混合淋巴细胞培养检测DCs对T细胞增殖能力的影响。结果:DCs经抗CD45RB抗体处理后,CD11C和CD83表达升高,CD86表达下降,自身增殖和吞噬能力增强,但分泌IL-12和刺激T细胞增殖的能力明显下降。结论:耐受性树突状细胞(tolerogenic dendritic cells,tDCs)能显著抑制T细胞的增殖,它的产生是抗CD45RB抗体诱导免疫耐受的主要机制之一。     

吞噬自身抗原对骨髓来源树突状细胞体外分化发育及功能的影响

目的:观察小鼠骨髓来源树突状细胞吞噬自身抗原后体外分化发育及功能的变化,研究其对免疫状态的影响.方法:小鼠骨髓细胞经GM-CSF+IL-4诱导分化为DCs,采用荧光显微镜和流式细胞仪检测DCs的纯度;运用免疫组化法及透射电镜鉴定DCs能否吞噬自身抗原;流式细胞仪检测吞噬自身抗原后DCs的CD86、MHCⅡ的变化; MTT法检测吞噬自身抗原后DCs刺激同基因背景小鼠脾淋巴细胞增殖能力的改变;ELISA法检测脾淋巴细胞经DCs刺激后,分泌IL-4、IFN-γ量的改变.结果:所培养DCs的纯度为90.6%,DCs能够吞噬自身抗原,吞噬自身抗原后的DCs体外分化发育异常,抑制同基因背景小鼠脾淋巴细胞的增殖,刺激同基因背景小鼠脾淋巴细胞分泌IL-4增加,分泌IFN-γ减少.结论:DCs吞噬自身抗原后能够抑制自身成熟,促进淋巴细胞免疫耐受,促进体液免疫.     

HTA-HSP70BCG冲激的小鼠骨髓树突状细胞的生物学活性变化

利用HTA、HSP70BCG、HTA-HSP70BCG体外冲激小鼠骨髓树突状细胞,观察树突状细胞的抗原捕获能力及抗原提呈能力的变化。用流式细胞仪检测不同冲激组树突状细胞对FITC-Dextran的摄取率;用MTT法检测不同冲激组树突状细胞对HCa-F的增殖抑制作用、对脾不黏附细胞的刺激增殖活性及活化后的脾不黏附细胞对HCa-F的杀伤活性;分别用ELISA法和一氧化氮硝酸还原酶法检测树突状细胞培养上清中IL-12和NO的浓度。结果显示,冲激后的树突状细胞对FITC-Dextran的摄取率降低,对HCa-F的增殖抑制率、脾不黏附细胞的刺激增殖指数、活化后的脾不黏附细胞对HCa-F的杀伤率及树突状细胞培养上清中IL-12和NO的浓度均较对照组树突状细胞增高(P<0.05)。结果表明HTA-HSP70BCG及单独HTA、HSP70BCG均能促进小鼠骨髓树突状细胞的成熟,成熟后的树突状细胞抗原捕获能力下降,而抗原提呈能力增强。     

约氏疟MIF影响小鼠脾CD8~+DC分泌细胞因子

目的研究约氏疟原虫巨噬细胞迁移抑制因子(PyMIF)对宿主小鼠树突状细胞(DC)表型和功能的影响,为阐明PyMIF对寄主免疫反应的作用机理提供理论依据。方法制备及纯化小鼠MIF及PyMIF重组蛋白,磁珠分选法得到小鼠脾DCs两种主要亚群CD8+DC及CD4+DC,在体外培养的情况下,流式细胞仪检测CD8+DC及CD4+DC TLR4、CD40、CD80、CD86、MHCⅠ、MHCⅡ等细胞表面分子表达,用ELISA方法检测细胞因子IL-12、TGF-β1的分泌。结果 PyMIF未改变脾DCs细胞表面CD40、CD80、CD86、MHCⅠ、MHCⅡ等分子水平,但可以下调脾DCs表面TLR4水平;此外,在CD8+DC,PyMIF能够拮抗LPS刺激所引起IL-12分泌的上升,由(433±48)pg/mL降至(373±30)pg/mL(P<0.05),并促进未成熟DCs分泌TGF-β1,由(136±4)pg/mL升至(182±7)pg/mL(P<0.05),而对CD4+DC分泌细胞因子并无影响。结论 PyMIF不参与DCs成熟过程,但有助于感染后宿主体内免疫抑制环境的产生,以逃避宿主免疫攻击有利于自身的存活。     

骨髓来源间充质干细胞通过JAK-STAT信号通路抑制树突状细胞功能的体外研究

目的探讨白介素-10(interleukin-10,IL-10)及其下游的JAK/STAT信号转导通路在骨髓来源的间充质干细胞(mesenchymal stem cells,MSCs)对树突状细胞(dendritic cells,DCs)的免疫抑制机制中的作用。方法 SD大鼠骨髓来源的MSCs和DCs按1∶10比例共培养,在共培养体系中分别加入IL-10、IL-10中和抗体(neutralizing antibody,NA)和JAK抑制剂(AG490)干预。ELISA法检测培养细胞上清液IL-10和IL-12水平的变化;流式检测DCs表面分子标志物CD80、CD86、OX62及MHC-II表达情况;Western blot检测(P-)JAK1,(P-)STAT3蛋白的表达情况。结果成功在体外分离和培养MSCs和DCs并得到流式鉴定证实。MSCs与DCs培养后上清液分泌IL-10水平明显升高。MSCs处理后DCs没有典型的成熟DCs表面标志,CD80、CD86、MHC-II及OX62的表达下降,IL-12分泌的水平也降低。实验中还发现外源性IL-10加到共培养板IL-10水平增加而IL-12水平减少,DCs的表面标志物表达下降,而加入IL-10NA与IL-10恰恰表现了相反的作用效果。加入JAK抑制剂(AG490)到共培养板后,IL-10分泌和(P-)JAK1和(P-)STAT3表达明显下降。结论大鼠骨髓来源的MSCs能抑制DCs成熟和功能,可能是通过IL-10和JAK1/STAT3信号转导通路来发挥作用的。     

骨髓间充质干细胞联合胰岛移植对受体鼠树突状细胞成熟和功能的影响

目的:研究骨髓间充质干细胞(MSCs)对骨髓细胞(BMC)分化为成熟树突状细胞(DCs),以及MSCs联合胰岛移植对受体鼠BMC来源DCs成熟和功能的影响,以探讨MSCs如何通过DCs发挥免疫抑制作用的机制。方法:将BALB/c小鼠分离纯化骨髓MSCs按比例加入C57BL/6小鼠BMC,在含重组鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和重组鼠白介素4(rmIL-4)的培养条件下制备DCs,加脂多糖(LPS)促DCs成熟,检测各组DCs免疫表型的变化,抗原摄取及其分泌白细胞介素-12的能力。观察MSCs联合胰岛移植的同种异基因糖尿病模型小鼠的血糖和组织学变化,并取受体鼠BMC体外诱导为DCs,检测成熟DCs免疫表型、抗原摄取和分泌IL-12能力。结果:体内外实验证明MSCs可使BMC来源的DCs表型CD11c,成熟表面标志CD83、协同刺激分子CD86和I-Ab表达明显降低(P0.05),抗原摄取和分泌IL-12能力显著下降(P0.01)。MSCs联合胰岛移植,可抑制同种异基因受体鼠免疫排斥反应的发生。结论:MSCs可通过抑制BMC来源的DCs的成熟和功能,降低DCs抗原摄取和分泌IL-12能力,诱导免疫耐受,减轻对移植胰岛的排斥反应的发生。     

HSP70BCG修饰的DC抗肿瘤作用的机制研究

近年来发现Toll样受体（TLR）是结合病原微生物的受体,参与固有免疫及适应性免疫,在DC上有所表达.结核杆菌中有多种抗原物质可激活细胞免疫功能,并对多种肿瘤细胞株有细胞毒作用.在上述研究的基础上,本文应用卡介苗来源的热休克蛋白70（HSP70BCG）修饰小鼠骨髓DC及经抗-TLR4抗体封闭TLR4后的小鼠骨髓DC,观察其抑瘤效果及TLR4的作用.     

骨髓问充质干细胞联合胰岛移植对受体鼠树突状细胞成熟和功能的影响

目的:研究骨髓问充质干细胞(MSCs)对骨髓细胞(BMC)分化为成熟树突状细胞(DCs),以及MSCs联合胰岛移植对受体鼠BMC来源DCs成熟和功能的影响,以探讨MSCs如何通过DCs发挥免疫抑制作用的机制.方法:将BALB/c小鼠分离纯化骨髓MSCs按比例加入C57BL/6小鼠BMC,在含重组鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和重组鼠白介素4(rmIL-4)的培养条件下制备DCs,加脂多糖(LPS)促DCs成熟,检测各组DCs免疫表型的变化,抗原摄取及其分泌白细胞介素-12的能力.观察MSCs联合胰岛移植的同种异基因糖尿病模型小鼠的血糖和组织学变化,并取受体鼠BMC体外诱导为DCs,检测成熟DCs免疫表型、抗原摄取和分泌IL-12能力.结果:体内外实验证明MSCs可使BMC来源的DCs表型CD11c,成熟表面标志CD83、协同刺激分子CD86和I-A<'b>表达明显降低(P<0.05),抗原摄取和分泌IL-12能力显著下降(P<0.01).MSCs联合胰岛移植,可抑制同种异基因受体鼠免疫排斥反应的发生.结论:MSCs可通过抑制BMC来源的DCs的成熟和功能,降低DCs抗原摄取和分泌IL-12能力,诱导免疫耐受,减轻对移植胰岛的排斥反应的发生.     

激动型CD40单克隆抗体体外抑制结肠癌细胞增殖的实验研究

目的 探讨激动型CD40单克隆抗体在体外对结肠癌细胞增殖的抑制作用.方法 树突状细胞(DCs)经结肠癌冻融抗原致敏后予以不同条件激活,分为激动型CD40单克隆抗体组、阴性对照组及肿瘤坏死因子-α(TNF-α)阳性对照组,诱导培养至第7天,用流式细胞仪检测各组DCs表面分化相关抗原CD80、CD83、CD86和HLA-DR的表达,酶联免疫吸附测定法检测DCs培养液上清中白细胞介素-12(IL-12)的质量浓度,噻唑蓝比色法检测DCs体外刺激T淋巴细胞增殖的能力,进而检测DCs所诱导的肿瘤特异性细胞毒性T淋巴细胞(CTL)对人结肠癌细胞HCT116的杀伤作用.结果 与阴性对照组相比,激动型CD40单克隆抗体组活化的DCs表面抗原CD80、CD83、CD86和HLA-DR的表达率均显著升高(均P＜0.05),DCs上清中IL-12的质量浓度亦显著升高((716.80±53.43) pg/ml比(405.51±12.17) pg/ml,P＜0.05),活化的DCs具有更强的刺激T淋巴细胞增殖的能力(刺激指数2.006 2±0.438 3比1.365 0±0.209 8,P＜0.05),活化的DCs所诱导的CTL对HCT116细胞具有更强的杀伤作用(抑制率(66.08±0.41)％比(46.60±1.10)％,P＜0.05);而与TNF-α阳性对照组相比,其差异均无统计学意义(均P＞0.05).结论 激动型CD40单克隆抗体在体外可促进DCs的活化与成熟,进而诱导肿瘤特异性CTL的产生,从而抑制人结肠癌细胞HCT116的增殖.     

Dendritic cells differentiated in the presence of a single-stranded viral RNA sequence conserve their ability to activate CD4 T lymphocytes but lose their capacity for Th1 polarization

Monocyte-derived dendritic cells (DCs) differentiate in the presence of Toll-like-receptor (TLR) ligands in the course of ongoing infections. A single-stranded RNA (ssRNA) sequence, corresponding to the sequence of the U5 region of human immunodeficiency virus type 1 RNA, was used to mimic viral activation of TLR7 in human DCs. We determined the effector potential of DCs differentiated in the presence of this ssRNA molecule (ssRNA-DCs). ssRNA-DCs phenotypically resembled mature DCs. In contrast, their capacity to allostimulate naive CD4(+) T cells resembled that of conventional immature DCs and could be increased by TLR4 stimulation. Th1 polarization of CD4(+) T cells and production of interleukin 12p70 (IL-12p70) by ssRNA-DCs were selectively abrogated in response to a late TLR4, but not in response to a CD40, maturation signal. Inhibition of p38 mitogen-activated protein kinase partially restored IL-12p70 secretion but did not restore Th1 polarization, whereas addition of exogenous IL-12 led to recovery of Th1 polarization. In contrast to lipopolysaccharide, ssRNA induced IL-12p70 production at the very earliest stages of DC differentiation, indicating a particular role for TLR7 in monocyte-derived DCs recently engaged in differentiation. These data demonstrate generation of phenotypically mature DCs with the ability to expand CD4(+) T lymphocytes lacking Th1/2-polarizing capacity.     

Complement C5a anaphylatoxin is an innate determinant of dendritic cell-induced Th1 immunity to Mycobacterium bovis BCG infection in mice

During acquired immunity to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in mice, dendritic cells (DCs) present mycobacterial antigens to naive T cells to prime an immune response. Complement C5a (anaphylatoxin) secreted by mycobacteria-infected macrophages regulates IL-12p70 production. As IL-12p70 regulates Th1 immunity against mycobacteria in mice, we examined the effects of C5a on IL-12p70 secretion by murine DCs and Th1 immunity. DCs cultured from C5-deficient (C5(-/-)) and -sufficient (C5(+/+)) mice were infected with BCG in the presence or absence of the C5a peptide. ELISA showed that C5(-/-) DCs secreted less IL-12p70 (600 pg/mL vs. 100 pg/mL) than C5(+/+) DCs, and they secreted more IL-10. Using immunophenotyping, reduced CD40 expression was found on C5(-/-) DCs after BCG infection. BCG-primed DCs were then cocultured with naive or BCG-immune T cells to differentiate them into IFN-gamma-secreting Th1 T cells. Coincident with increased IL-12p70 levels, BCG-primed C5(+/+) DCs cocultured with naive or immune C5(+/+) T cells showed a larger increase in CD4+ IFN-gamma/CD8+ IFN-gamma+ T cells compared with cocultured DCs and T cells from C5(-/-) mice. Thus, BCG-primed C5(+/+) DCs were better able to drive a Th1 response. Furthermore, BCG aerosol-infected C5(-/-) mice showed reduced CD4 and CD8 IFN-gamma-secreting T cells in the lungs, concurrent with an increased growth of BCG. Thus, C5a, an innate peptide, appears to play an important role in the generation of acquired immune responses in mice by regulating the Th1 response through modulation of IL-12p70 secretion from DCs.     

Antigen-specific and nonspecific determinants of cytokine production during topical sensitization of mice to chemical allergens

BACKGROUND: Topical exposure to chemical allergens such as trimellitic anhydride or 1-chloro-2,4-dinitrochlorobenzene results in the accumulation of dendritic cells (DCs) and subsequent rapid up-regulation of CD4 T-cell proliferation and cytokine secretion within draining lymph nodes. OBJECTIVE: We investigated the contribution of antigen-specific and CD40 ligand (CD40L)-mediated signals to chemical allergen-induced CD4 T-cell growth and cytokine production. METHODS: DCs enriched from lymph nodes of allergen-challenged animals by metrizamide centrifugation were used to stimulate cytokine and proliferative responses by magnetic immunobead-sorted CD4 T cells primed in vivo with the same or unrelated allergen. Cultures of DCs and T cells were supplemented with antibodies that block IL-12 and CD40L activity. RESULTS: Proliferation of CD4 T cells was stimulated by DCs primed with the same but not unrelated antigen, whereas IFN-gamma, IL-12, and IL-10 secretion were provoked equally well by DCs primed with either hapten. Blockade of CD40L engagement abrogated production of IFN-gamma (80%) and IL-12 (95%) under antigen-nonspecific stimulatory conditions. In contrast, IL-10 secretion was enhanced after CD40L blockade under both antigen-specific and nonspecific conditions. Primary CD4 T cells activated by mitogen were also influenced by DCs in the same way. CONCLUSION: These results show that during the development of chemical sensitization emerging CD4 T-cell growth and cytokine production are regulated by independent mechanisms requiring antigen presentation and CD40 signaling, respectively.     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFalpha, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFalpha and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.     

体外LPS刺激及CD40的配基化对sCD40基因修饰DCs TLR4-MD2表达及IL-12分泌的影响

目的： 研究体外LPS刺激及CD40的配基化对可溶性CD40（sCD40）基因修饰树突状细胞TLR4-MD2表达及IL-12分泌的影响,为有效利用树突状细胞诱导特异性移植免疫耐受提供实验依据。方法: 脂质体法将质粒pEGFP-N1/sCD40及空质粒pEGFP-N1转染DC2.4细胞株;应用LPS及抗CD40单抗刺激6 h,流式细胞仪检测DC表面TLR4-MD2的表达,RT-PCR法检测DC 的TLR4 mRNA 表达水平,并用ELISA法检测细胞因子IL-12p70的分泌。结果: LPS刺激下调DC表面TLR4-MD2的表达,同时给予CD40配基化可引起TLR4-MD2的表达显著增高;CD40配基化对DC TLR4mRNA 水平表达无影响，但可部分地增高LPS引起的TLR4mRNA 表达降低;此外,CD40的配基化可显著诱导LPS刺激后IL-12分泌增加。sCD40基因修饰DC可拮抗以上作用。结论: 体外LPS及抗CD40单抗刺激下,sCD40基因修饰树突状细胞可显著下调其表面TLR4-MD2的表达,IL-12p70分泌减少，可能与阻断胞浆内的TLR4-MD2的转运过程有关。     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFα, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFα and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.     

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.     

Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.