体外诱导成熟树突状细胞的研究

目的：探讨在体外从干细胞中诱导出成熟DC的适宜环境。方法：分别将人胎肝、骨髓和脾细胞以及小鼠骨髓和脾细胞在体外用GM－CSF和TNF－α诱导,观察了第3、5、7天DC的收获情况。进一步用S－P免疫细胞化学染色检测了mBmDC和fLDC有关分子表达。结果：在体外通过GM－CSF和TNF－α的作用,小鼠骨髓、人胎肝、骨髓细胞呈典型的毛刺状胞浆突起,第5天的收获率分别为：39.5％、67.2％、12.9％,而基本不能从脾细胞中诱导出成熟DC,获得的DC能与MAbCD80、MAbCD40呈强阳性反应。结论：在GM－CSF和TNF－α的共同作用下,能在体外从人胎肝、 骨髓和小鼠骨髓干细胞中获得成熟DC,其DC能表达高高水平的B7－1和CD40分子,这为大量获得DC提供了一种简便可行的手段,为研究DC的生物学特征及其抗原提呈功能提供了丰富的材料,为开展以DC为基础的各种免疫治疗研究奠了良好的基础。     

人急性髓系白血病HL-60来源的树突样细胞诱导的CTL体内外作用研究

目的 观察人急性髓系白血病HL－60来源的树突样细胞（HL－60DC）对细胞毒T淋巴细胞（CTL）的免疫调节作用。方法 采用PKC激活剂佛波酯（PMA）及细胞因子GM－CSF、IL－4、TNF－α在体外诱导培养HL－60细胞，获得具有树突状细胞形态、表型（CD1a、CD80、CD86、RelB阳性表达）的HL60DC，观察HL－60DC诱导的CTL体外抗瘤效应，并建立人白血病HL－60／SCID（严重联合免疫缺陷）小鼠异种移植模型进行过继治疗。结果 联合GM－CSF、TNF－α及PMA处理7－11d，获得的HL－60DC体外激活的CTL在体外对HL－60细胞有显著的细胞毒活性。CTL以5：1效靶比多次腹腔回输，能够明显延长荷瘤鼠存活时间，降低瘤块重量，减轻肝、脾、骨髓受肿瘤浸润程度，但流式细胞检测发现部分受治疗的存活小鼠有微小病灶的存在。结论 人急性髓系白血病HL－60来源的树突样细胞诱导的CTL在体内外有一定的抗瘤效应。     

小鼠骨髓间充质干细胞对同种异体骨髓来源的树突状细胞分化和功能的影响

目的:研究小鼠骨髓间充质干细胞(BMSCs)对同种异体骨髓来源的树突状细胞(DCs)分化、成熟及功能的影响,探讨MSCs发挥免疫调节作用的机制.方法:体外分离培养BALB/c小鼠BMSCs,与C57BL/6小鼠骨髓有核细胞(BMCs)在体外按不同的细胞比例混合培养,诱导DC分化,流式细胞术(FCM)分析DC细胞表型及FITC-Dextran内吞能力,ELISA检测分泌的细胞因子IL-12的水平.结果:当MSC/BMC达到较高的比例(1:10)时,细胞表面的CD11c、CD14、CD83、CD86、I-A~b的表达均显著下降(P<0.05),FITC-Dextran内吞能力及IL-12分泌水平亦显著降低(P<0.05).结论:小鼠MSCs在体外能抑制同种异体骨髓来源的DCs的分化、成熟. SCs,与C57BL/6小鼠骨髓有核细胞(BMCs)在体外按不同的细胞比例混合培养,诱导DC分化,流式细胞术(FCM)分析DC细胞表型及FITC-Dextran内吞能力,ELISA检测分泌的细胞因子IL-12的水平.结果:当MSC/BMC达到较高的比例(1:10)时,细胞表面的CD11c、CD14、CD83、CD86、I-Ab的 达均显著下降(P<0.05),FITC     

抗CD40抗体联合诱导人脐血CD34+造血干细胞向DC2分化的作用

目的 探讨从脐血CD34^＋造血细胞诱导DC2的方法及CD40分子在DC2分化诱导中的作用。方法 运用磁珠从脐血中分离出CD34^＋造血干细胞,以rhIL-3（10ng／mL）、rhFlt-3L（100ng／mL）和rhGM-CSF（100ng／mL）诱导其向DC2分化,采用流式细胞仪分析DC2表型,并观察抗人CD40单克隆抗体诱导DC2分化成熟的作用。结果 人脐血CD34^＋造血细胞在rhIL-3、rhFlt-3L和rhGM-CSF联合诱导培养12d,获得具有DC2样（淋巴样DC）形态的细胞,然后分别加入rhIL-3＋抗人CD40 mAb（Ⅰ组）或rhIL-3＋TNF-α（Ⅱ组）诱导DC2的分化成熟,流式细胞仪分析细胞表型,发现Ⅰ组和Ⅱ组Lineage^-（CD3、CD14、CD16、CD19、CD56）CD123^＋细胞的HLA-DR、CD83、CD86、CD80表达率分别为88．78％、37．38％、32．83％和99．08％;78．87％、32．29％、29．57％和98．86％。结论 体外联合多种细胞因子从CD34^＋造血细胞成功地诱导出富集DC2表型的细胞,抗人CD40 mAb可以促进DC2的分化成熟。     

FBL3细胞在小鼠体内外诱导CD4+CD25+调节性T细胞增殖的实验研究

目的：通过小鼠体内外实验观察肿瘤细胞是否可以诱导CD4^＋CD25^＋Treg的分化增殖。方法：将小鼠的红白血病瘤细胞系FBL3接种于C57BL/6小鼠腹壁皮下,流式细胞仪检测小鼠外周血、脾脏及瘤组织CD4^＋CD25^＋Treg细胞含量,RT-PCR检测小鼠脾脏及瘤组织Foxp3 mRNA的表达;体外实验检测FBL3细胞培养上清液对小鼠脾脏CD4^＋CD25^＋Treg细胞的作用。结果：荷瘤鼠外周血CD4^＋CD25^＋Treg比例与正常鼠比较无统计学差异（P〉0.05）,但荷瘤鼠脾脏CD4^＋CD25^＋Treg比例显著高于正常对照（P〈0.01）,荷瘤小鼠脾脏组织Foxp3 mRNA的表达量明显增加;体外实验表明FBL3培养上清液促使CD4^＋CD25^＋Treg细胞比例增高,并诱导Foxp3 mRNA表达增加。结论：FBL3细胞及其分泌的可溶性物质能诱导CD4^＋CD25^＋Treg细胞的增殖,说明是肿瘤的发生促进了CD4^＋CD25^＋Treg增高。     

雷帕霉素对同种移植耐受模型CD4+CD25+ T细胞活性的影响

目的：研究雷帕霉素（Rapa）对同种移植耐受个体CD4^＋CD25^＋ T细胞体内负免疫调节作用的影响.方法：建立同种皮肤移植模型, 受体鼠术前预输注供体鼠脾细胞, 术后给予环孢素A（CsA）进行耐受诱导.移植后第14天提取耐受诱导模型鼠的T细胞, 经不同浓度Rapa和/或IL-2体外处理后, 混合淋巴细胞反应（MLR）确定T细胞特异增殖水平;流式细胞术（FCM）检测CD4^＋CD25^＋ T细胞比例变化;RT-PCR检测Foxp3 mRNA表达情况;ELISA检测细胞培养不同时间后上清中IL-10的变化.然后将Rapa和/或IL-2处理的T细胞过继转移给同种移植后的BALB/c-SCID鼠, 观察移植物存活状态.结果：CsA加供体脾细胞预先注射可明显延长小鼠移植皮片的存活期（P＜0.05）;移植耐受状态的T细胞经Rapa和/或IL-2体外处理后CD4^＋CD25^＋ T细胞比例升高、增殖水平明显降低、 Foxp3表达量明显增加;过继转输给同种移植SCID鼠后, 其移植皮片存活时间显著延长（P＜0.05）.结论：Rapa可体外扩增耐受诱导模型中CD4^＋CD25^＋ T细胞, 使CD4^＋CD25^＋ T细胞相关的Foxp3和IL-10明显升高, 过继免疫后, 小鼠同种移植物存活时间明显延长, 而低浓度IL-2可以协同Rapa的这一作用.     

人骨髓来源的树突状细胞的诱导扩增及鉴定

目的:以人骨髓细胞为来源,建立体外诱导扩增树突状细胞(DC)的方法并进行形态学和免疫表型鉴定。方法:取正常人骨髓,以淋巴细胞分离液分离骨髓单核细胞后,用rhGM-CSF和rhIL-4诱导DC产生,再用rhTNF促进其成熟。收集细胞,用扫描电镜观察细胞的形态特征,用流式细胞术分析细胞的表型。结果:人骨髓细胞经rhGM-CSF、rhIL-4和rhTNF诱导可得到大量成熟的DC,电镜观察具有典型的DC形态。流式细胞术分析细胞的表型表明,诱导后第5天,CD1a 细胞的百分率为70%~75%,CD83 细胞为3%~3.2%;诱导后第7天,CD1a 细胞为84%~86%,CD83 细胞为30%~32%。结论:由人骨髓细胞可成功地诱导出具有典型形态特征的DC,为DC的深入研究和临床应用提供又一细胞来源。     

抗P选择素功能域单抗对人树突状细胞表型及IL-12分泌的影响

观察抗P选择素Lectin EGF功能域单抗 (PsL EGFmAb )对体外培养人树突状细胞 (DC )表型以及促炎细胞因子IL 1 2分泌的影响 ,探讨PsL EGFmAb对DC炎性成熟过程中的调节作用。通过SCF、GM CSF、TGF β1 、Flt 3和TNF α体外培养体系 ,从脐血CD34+ 造血干细胞中诱导扩增获得DC ,并于成熟中用PsL EGFmAb进行干预。采用流式细胞仪分析细胞表型CD1a、CD1 1c、CD83、CD80、CD86和HLA DR ;采用RT PCR检测IL 1 2p35、p4 0mRNA表达 ;以及ELISA法测定IL 1 2p70分泌的含量。结果显示 ,PsL EGFmAb可下调成熟中DC表面CD1 1c、CD83、CD80、CD86和HLA DR的表达 ,同时能抑制DC内IL 1 2p35、p4 0mRNA的转录和IL 1 2p70的分泌。本研究提示 ,PsL EGFmAb对DC黏附共刺激分子表达和促炎细胞因子合成具有抑制作用 ,并可能影响和调抑DC成熟及其提呈抗原功能     

用含有特定细胞因子的无血清培养基培养慢性乙型肝炎病人的树突状细胞

目的　体外扩增慢性乙肝病人的树突状细胞 (dendriticcell,DC) ,从细胞表型和功能上鉴定和研究。方法　用含GM CSF和IL 4的无血清培养基AIM V体外培养慢性乙肝病人外周血单个核细胞 ,获得树突状细胞。流式细胞仪检测细胞表型 ,IL 1 2ELISA试剂盒检测DC分泌IL 1 2的水平 ,并观察加入细胞因子TNF α后对DC培养的影响。结果　慢性乙肝病人的外周血单个核细胞用AIM V培养及细胞因子诱导后 ,经贴壁法纯化 1 0 0mL可获得 0 .5× 1 0 7～ 1 .5× 1 0 7成熟的具有典型形态的DC ,加入TNF α后 ,CD83阳性占 60 .80 % ,明显高于未加组 (P <0 .0 5) ,IL 1 2分泌较未加TNF α组增高近 1 0倍。结论　①慢性乙肝病人的DC可用AIM V无血清培养基及特定的细胞因子诱导在体外大量获得 ,TNF α是诱导DC成熟的重要的细胞因子。②典型的细胞形态和CD1 4 -、HLA DRhigh+、CD86high+的细胞表面分子特征可作为临床上快速鉴定培养DC的标志。     

基因修饰树突状细胞诱导前列腺癌患者外周血T细胞亚群重排的研究

目的探讨以腺相关病毒（AAV）为载体,前列腺特异性抗原（PSA）基因转染树突状细胞（DC）诱导前列腺癌患者外周血T细胞亚群变化特点及临床意义。方法抽取30例前列腺癌患者外周血,采用密度梯度离心法分离外周血单个核细胞,以rAAV/PSA感染DC前体细胞,采用系列细胞因子诱导DC前体细胞成熟。第6天收集成熟DC并与T细胞按比例混合培养,诱导细胞毒性T淋巴细胞（CTL）。分别于DC与T细胞混合培养前后应用流式细胞术分析外周血T细胞亚群及调节性T细胞（CD4^＋CD25^＋FoxP3^＋Treg）的表达水平。结果 PSA基因转染DC刺激T淋巴细胞爆发增殖,与培养前比较,混合培养6d后CD8^＋、CD8^＋CD69^＋、CD8^＋CD28^＋T细胞的比例和CD8^＋/CD4^＋比值均明显增高,差异有统计学意义（P〈0.01）;而CD8＋CD28-T细胞和Treg细胞的比例均显著降低,差异有统计学意义（P〈0.01）。CD4^＋T细胞比例较前略有升高,但差异无统计学意义（P〉0.05）。结论 PSA基因转染DC能够有效地激活CD8＋抗原特异性CTL,下调免疫抑制性T细胞,提高患者的细胞免疫功能,为前列腺癌的免疫治疗提供新的有效策略。     

Development of dendritic cells from GM-CSF-/- mice in vitro : GM-CSF enhances production and survival of cells

The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF-/- mice. Multiple cultures from GM-CSF-/- and wild type mice were established and compared for cell production. GM-CSF-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.     

Development of Dendritic Cells from GM-CSF-/- Mice in vitro : GM-CSF Enhances Production and Survival of Cells

The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF^-/- mice. Multiple cultures from GM-CSF^-/- and wild type mice were established and compared for cell production. GM-CSF^-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF^-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF^-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF^-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.     

A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells

We established a novel culture method for generating dendritic cells (DC) from mouse bone marrow (BM) cells. Unfractionated bulk BM cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5-7 days and a DC population was isolated by gradient centrifugation with 14.5% (w/v) metrizamide. Through this method, 30-40 x 10(6)/mouse DC with 85-95% purity was obtained on day 7; this yield was higher than those of conventional DC generated by Inaba's method either with GM-CSF alone (conventional-GM DC) or GM-CSF and IL-4 (conventional-GM/4 DC). Bulk-cultured DC have a more matured phenotype than both conventional-GM and -GM/4 DC as shown by higher expression of CD86, MHC class II and CD40. Functional analyses reveal that (1) bulk-DC show less ability in endocytosis than conventional-GM DC and are comparable in IL-12 p70 production with conventional-GM and -GM/4 DC. (2) Bulk-DC exhibit stronger stimulatory capacity in allogeneic T-cell proliferation than conventional DC. (3) By using ovalbumin (OVA) and OVA-specific T-cell receptor (TCR) transgenic mice (DO11.10) system, OVA protein-loaded bulk-DC stimulated CD4 T cells of DO11.10 mice more than conventional-GM DC and comparable with conventional-GM/4 DC. (4) Furthermore, OVA peptide-pulsed bulk-DC stimulated CD4 T cells more than conventional-GM and -GM/4 DC. These data indicate that bulk-DC are functionally more mature than conventional DC. Taken together, bulk-culture method is a simple technique for generating functionally mature BM-DC in large quantities and high purity.     

Interleukin-3 and granulocyte-macrophage colony-stimulating factor enhance the generation and function of dendritic cells.

Dendritic cells, well-known for their potent antigen-presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allo-stimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin-peptide specific class I- and class II-restricted hybridomas containing the lacZ reporter gene, we found that these cytokine-generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)-presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine-generated DC. These findings indicate that exposure in vivo to the cytokines IL-3 and GM-CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of cross-priming and the subsequent generation of tumour-reactive cytotoxic T lymphocytes (CTL).     

The influence of granulocyte/macrophage colony-stimulating factor on dendritic cell levels in mouse lymphoid organs

To ascertain whether the development of dendritic cells (DC) in mouse lymphoid organs is dependent on granulocyte/macrophage colony-stimulating factor (GM-CSF), we determined the number of DC in the thymus, spleen and lymph nodes of normal mice, of mice with the genes coding for GM-CSF or its receptor inactivated, and of transgenic mice with excessive levels of GM-CSF. DC were extracted from the tissues and enriched prior to flow cytometric analysis. The total DC level and the incidence of DC expressing lymphoid-related markers (CD8^hiCD11b^lo) and myeloid-related markers (CD8^loCD11b^hi) were monitored. Both in GM-CSF null mice, and GM-CSF receptor null mice, DC of all surface phenotypes were present in all lymphoid organs; only small decreases in DC levels were recorded, except for the lymph nodes of GM-CSF receptor null mice which showed a more pronounced (threefold) decrease in DC numbers. Since the GM-CSF receptor null mice lacked the β chain common to the GM-CSF, interleukin (IL)-3 and IL-5 receptors, the development of DC in the absence of GM-CSF was not due to common β chain mediated developmental signals elicited by IL-3 or IL-5. In GM-CSF transgenic mice, there was only a 50 % increase in DC numbers in thymus and spleen, paralleling an increase in overall cellularity, but a more pronounced (threefold) increase in DC numbers in lymph nodes. There was no evidence that GM-CSF had a selective effect on any particular DC subpopulation defined by CD8 or CD11b expression. We conclude that the development of most lymphoid tissue DC can proceed in the absence of GM-CSF, although this cytokine can produce some elevation of DC levels. It is not clear whether the enhancing effect of GM-CSF is direct or an indirect effect mediated by other cytokines.     

Dendritic cells in germ-free and specific pathogen-free mice have similar phenotypes and in vitro antigen presenting function

Dendritic cells (DC) can direct downstream T-cell responses. Although bacterial adjuvants are strong activators of DC in vitro, the effects of normal enteric bacteria on DC in vivo are not well defined. We used germ-free (GF) mice to determine whether enteric bacteria alter DC phenotype and ability to stimulate na?ve T cells. Surface expression of CD11c, CD86, and MHCII was measured on splenic and mesenteric lymph node (MLN) DC. In addition, we tested the ability of T-cell depleted splenocytes from mice injected with LPS to stimulate allogeneic T cells, as determined by cell proliferation. The absolute numbers of CD11c+ DC were decreased in the MLN and spleen of GF mice. Freshly isolated CD11c+ DC from spleens or MLN of SPF and GF mice expressed similar levels of CD86 and MHCII by FACS analysis. Proportions of splenic DC expressing CD4 or CD8 were not different in GF versus SPF mice, although the percentage of CD8alpha-/CD11b+ DC was higher in GF MLN. Intraperitoneal injection of LPS upregulated MHCII and CD86 to a similar extent on splenic DC from GF or SPF mice. Splenic antigen-presenting cells, as well as unseparated spleen or MLN cells, from GF or SPF mice also induced similar levels of T-cell proliferation in vitro. We conclude that commensal bacterial flora do not affect co-stimulatory molecule expression of DC in the spleen or MLN, which exhibit a predominantly immature phenotype. In addition, splenic APC from GF mice are fully competent to stimulate na?ve T-cell proliferation in vitro.     

IL-4 supports the generation of a dendritic cell subset from murine bone marrow with altered endocytosis capacity

Dendritic cells (DC) of myeloid origin can be generated from mouse bone marrow (BM) using granulocyte macrophage-colony stimulating factor (GM-CSF). Immature major histocompatibility complex (MHC) II(low) DC are known to bear a high endocytosis capacity, in contrast to DC precursors and mature DC. Now we found that a subset of MHC II(low) DC in BM-DC cultures is unable to exert mannose receptor-mediated endocytosis of fluorescein isothiocyanate (FITC)-dextran (DX) and resembles immature Langerhans cells (LC). The FITC-DX endocytosis activity of LC-like cells occurs at an earlier stage of development, where the surface MHC II expression is absent or very weak. This LC-like subset expresses higher levels of E-cadherin but lower amounts of the markers Gr-1, scavenger receptor 2F8, and CD11b, when compared with the highly endocytic DC subset. The latter myeloid DC resemble monocyte-derived DC (MoDC). The sorted LC-like population develops completely and exclusively into mature MHC IIhigh DC, and the MoDC-like cells remain immature MHC II(low) DC or develop into adherent MHC IIneg macrophages or mature into MHC IIhigh DC. The development of LC-like cells is promoted by interleukin-4. Thus, we show here that the simultaneous development of LC-like and MoDC-like DC subsets occurs in standard bulk cultures with GM-CSF, suggesting the existence of two different precursors for LC and MoDC in BM.     

A two-step culture method starting with early growth factors permits enhanced production of functional dendritic cells from murine splenocytes

Dendritic cells (DC) are professional antigen presenting cells (APC) able to activate naive T cells and initiate the immune response. They are present in most tissues at very low concentrations and are difficult to isolate. DC can be obtained in larger numbers by their propagation from progenitors present in blood, bone marrow and spleen. However, biochemical studies and biological analysis of DC functions require very large numbers of these cells. In this paper, we described a two-step culture system using unfractionated splenocytes from BALB/c mice as a source of DC progenitors. The proliferative capacity of the progenitors is amplified in the first step of the culture (day 0-6) using different combinations of early acting cytokines combined or not with granulocyte-macrophage CSF (GM-CSF). The second step of the culture starts at day 6 with the removal of early growth factors in order to allow the differentiation and final maturation of DC during 2-3 weeks of culture with flt-3 ligand (flt-3L) and GM-CSF. The addition of Stem Cell Factor (SCF) or IL-6 to the standard combination of flt-3L+/-GM-CSF produces a large increase in the proliferation of GM and DC progenitors (28 times and 11 times respectively) in the first step of the culture. This proliferative wave of DC progenitors is followed by the production of a high percentage of immature and mature DC in flt-3L+GM-CSF stimulated cultures. The best combination of early cytokines in terms of proliferative activity and subsequent level of DC production was flt-3L+IL-6+GM-CSF, which permitted the generation of 1 to 2x10(9) DC from one single spleen. Using this growth factor cocktail, a mixture of immature (2/3) and mature (1/3) DC was produced until day 14 of culture, and levels of MHC class II and costimulatory molecules (CD40, B7.2) increased between 2 and 4 weeks of incubation, or within 2 days when stimulated by IL-4 or LPS. The splenic DC produced after 2 weeks of culture are fully functional, exhibiting a high capacity of endocytosis when immature, a strong stimulatory reactivity in mixed leukocyte reaction and consistently producing high levels of bioactive IL-12 p70 after CD 40 ligation in the presence of LPS between 13 and 43 days of culture.     

Culture of bone marrow cells in GM-CSF plus high doses of lipopolysaccharide generates exclusively immature dendritic cells which induce alloantigen-specific CD4 T cell anergy in vitro

Dendritic cells (DC) can be generated from mouse bone marrow (BM) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Bacterial stimuli such as endotoxin / lipopolysaccharide (LPS) can induce their final maturation. When BM-DC cultures were treated at day 6 or later with LPS, this final maturation was induced in vitro within 24 h. Such mature DC exhibited high levels of surface MHC II molecules and potent T cell sensitizing, but reduced endocytosis capacity. In contrast, immature DC express only few MHC II molecules and are weak T cell stimulators but highly endocytic. When BM-DC cultures in GM-CSF were treated with 1 microg / ml LPS at day 0 of the culture or throughout the culture, only immature DC developed as defined by phenotype (MHC II low) and function (high endocytosis, weak primary mixed lymphocyte reaction). Those early LPS-treated immature DC induced alloantigen-specific anergy of CD4(+) T cells in vitro. These findings might contribute to the understanding of reduced T cell immunity in the course of septic shock and find application in DC-mediated tolerogenic immunotherapy strategies.     

Passive transfer of flt-3L-derived dendritic cells delays diabetes development in NOD mice and associates with early production of interleukin (IL)-4 and IL-10 in the spleen of recipient mice

CD11c+/CD11b+dendritic cells (DC) with high levels of major histocompatibility complex (MHC) class II and co-stimulatory molecules have been derived from spleen cells cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) + flt-3L + interleukin (IL)-6 (flt-3L-DC). Investigating in vivo the function of DC in non-obese diabetic mice (NOD), we showed that a single injection of this in vitro-derived subset of DC prevents the development of diabetes into prediabetic female mice. In contrast, DC derived from bone marrow cells cultured with GM-CSF + IL-4 [bone marrow (BM)-DC] induced no protection. Moreover, protection against diabetes following injection of flt-3L-DC was associated with IL-4 and IL-10 production in the spleen and the pancreatic lymph nodes of recipient mice, indicating that this DC population is able to polarize the immune response towards a Th2 pathway. As we shown previously, NOD BM-DC exhibit an enhanced capacity to produce IL-12p70 in response to lipopolysaccharide (LPS) and anti-CD40 stimulation compared to BM-DC from control mice. In contrast, NOD flt-3L-DC, as their control mouse counterpart, produced no IL-12p70 to these stimuli. Our findings show that a subset of DC, characterized by a mature phenotype and the absence of IL-12p70 production can be derived from NOD mouse spleen favouring IL-4 and IL-10 regulatory responses and protection from diabetes development.