Nat. Immunol. 5, 1124–1133 (2004); published online 10 October 2004; corrected online 24 February 2014 In the version of this article initially published, the top row of Figure 2c incorrectly presented identical plots for diffDC and diffDC-LPS. As the source of the original data was unusable, the plots have been replaced with data obtained in new experiments.

目的观察白血病来源的树突状细胞(DC)刺激T细胞对自体急性早幼粒细胞白血病(APL)细胞的毒性作用.方法应用GM-CSF、IL-4和TNF-α诱导APL细胞向DC分化,获得成熟DC.另取患者外周血T细胞,加入经射线灭活的DC或IL-2,培养10d后,收集非贴壁细胞,为效应细胞.采用51Cr4h释放法进行细胞毒性实验,用流式细胞技术检测免疫表型.结果 DC诱导的效应细胞比IL-2活化的效应细胞对靶细胞(自体白血病细胞)具有更强的细胞毒性作用.在效靶比为25∶1时,分别为76.1%±15.3%和18.2%±6.6%,两者之间存在显著性差异(P＜0.01).该效应细胞对MHC匹配的异体正常人细胞反应性很低.流式细胞仪检测结果表明该效应细胞主要是以CD8+细胞为主的杀伤细胞.结论白血病DC刺激的T细胞对APL细胞有明显的细胞毒性作用,DC的诱导作用强于IL-2,这种效应细胞的细胞毒性作用具有明显的特异性,有望用于APL的过继性免疫治疗.

To better understand the immunobiology of dendritic cells (DCs), we took the expressed sequence tag (EST) approach to describe their transcript profile and discovered novel genes. ESTs (n = 25,668) were generated from monocyte-derived DCs, and 15,863 ESTs (61.8%) represented unique genes in GenBank. Integration of ESTs allowed for the generation of a profile of 4,367 known genes and identification of > 100 novel genes. HLA-DR invariant chain p33, cathepsin D, HLA-DR alpha chain, beta2-microglobulin, HLA-DP beta chain, CD11a, and mannose receptor were in the top 30 transcripts, and 451 known genes were potentially associated with the immunobiology of DCs. This transcript profile was consistent with the unique antigen-presenting capacity of DCs and provided invaluable information to better understand the immunobiology of DCs. On the basis of the EST database, a full-length novel gene was identified that exhibited close homology with CD84; it was designated CD84-H1. The full-length cDNA of CD84-H1 contained an open reading frame of 870 bp encoding a type I transmembrane protein of 289 amino acids. Consistent with the structural feature of the CD2 family, the predicted 270-amino acid mature protein of CD84-H1 contained two extracellular immunoglobulin-like domains that shared homology with CD2 family members, e.g., CD84, Ly-9, CD48, and signaling lymphocyte activation molecule. Its intracellular domain was short and contained no putative signaling structure. Northern blot analysis revealed that CD84-H1 expression was predominantly restricted in hematopoietic tissues. Reverse transcription-PCR analysis showed that it was widely expressed in the immune cells, including monocytes, DCs, B cells, and T cells. These data indicate that CD84-H1 may be relevant to immune responses.

Objective To develop a genetically modified fetal liver cells (FLC) based transplantation system that can release therapeutic levels of hematopoietic growth factors into the system circulation which can facilitate treatment of patient receiving cytokine therapy following chemotherapy. Method Examine adeno virus mediated gene transfer to isolated murine FLC and evaluate the biocharacterization of intrasplenic transplantation of gene modified murine FLC. Results Substantial transfection rate of 80%～85% were achieved at a ratio of 50 for 2 hr of exposure. Gene modified FLC (FLC GM) labeled with 111 In were injected into the allogenic mice, spleen, the %ID/g of liver was 20%～25% at 24 hr and 50%～55% at 48 hr after transplantation. In addition, serum concentration of GM CSF in mice with intrasplenic transplantation reached its maximum at 48 hr [(356 ±58 ) pg/ml]. Conclusion Intrasplenic transplantation of FLC GM can be predominantly localized in liver and spleen, and engraft rapidly and maintain normal function, which represent a critical step toward successfully accomplishing liver directed gene therapy.

Objective: To investigate the effects of macrophages transfected with IL 2 gene. Methods: IL 2 gene was transfected into murine peritoneal macrophages by adenovirus. Levels of TNF, IL 1 and NO in the supernatant of macrophages as well as cytotoxicity of macrophages to target cells L1210 and Renca cells were assayed. Results: The productions of TNF,IL 1 and NO in the supernatant of IL 2 gene modified macrophages increased, cytotoxicity of the macrophages to L1210 cells and Renca cells also increased significantly. Conclusion: Transfection of IL 2 gene to macrophages can enhance their cytotoxicity, thus provide experimental basis for immunotherapy of tumor with IL 2 gene modified macrophages.

Objective To observe the activation effect on cytotoxic T lymphocytes (CTL) by murine dendritic cells (DCs) pulsed with mild acid eluted tumor antigen peptides presented on erythroleukemia cell surface. Methods FBL 3 erythroleukemia cells were treated with citrate phosphate buffer (pH3.3) at room temperature for 5 min. Flow cytometry was used to analyze the elution effect. The acid eluted peptides were subsequently purified through SepPark C18 column. DCs pulsed with these peptides were used to stimulate the proliferation of the specific CTL line in vitro and to induce specific anti tumor response in vivo. Results Mild acid could elute tumor antigen peptides from tumor cells effectively. DCs pulsed with these peptides could stimulate the proliferation of the specific CD8 + CTL line which could recognize Friend murine leukemia viral nonapeptides CCLCLTVFL presented on the surface of FLB 3 cells. Vaccination with DCs pulsed with the peptides could also induce high level of specific CTL activity in vivo and make mice resistant to the subsequent challenge of parental tumor cells. The in vivo effects induced by DCs pulsed with thawed tumor antigens were also achieved, but were less effective than that induced by DCs pulsed with acid eluted antigen peptides. Conclusions Mild acid could elute class Ⅰ restricted peptides from tumor cells. DCs pulsed with acid eluted tumor antigen peptides could activate CTL and induce specific anti tumor immunity effectively.

利用减法杂交技术筛选人树突状细胞(DC)经抗原刺激后特异表达的基因, 成功地获得了十多个特异表达基因, 其中包括一种核不均一核糖核蛋白(heterogeneous nuclear ribonucleoprotein, hnRNP)M4第159～197位氨基酸缺失的新基因. 对部分肿瘤细胞系及原代培养细胞进行RT-PCR检测发现, 它的表达与hnRNP M4基因是一致的, 同时证实了该基因表达于经KLH刺激后的DC而非正常的DC. 组织分布分析显示, 该基因高表达于脾脏、外周血淋巴细胞、肺和肝脏. 另外, 通过多种细胞因子刺激的骨髓基质细胞(bone marrow-derived stromal cells, BMSC)也表达这两种分子, 但TNF-a 处理后mRNA的表达消失. 这一发现增加了对DC在抗原提呈过程中基因表达变化的认识, 为hnRNP家族增添了新成员.

目的:观察体外肿瘤抗原冲击致敏的白细胞介素2(IL-2)基因修饰的巨噬细胞对肾癌小鼠的治疗效果并探讨其相关的免疫机理.方法:通过重组腺病毒的介导,将IL-2基因转入新鲜分离的小鼠腹腔巨噬细胞,经肿瘤抗原冲击致敏后回输治疗原位肾癌小鼠,采用4 h 51 Cr释放法检测脾脏NK和CTL活性.结果:IL-2基因修饰的巨噬细胞经肿瘤抗原冲击后体内回输可使肾癌小鼠肺转移结节明显减少,存活期明显延长,40%肾癌小鼠达到长期存活.治疗后荷瘤小鼠脾脏NK和CTL活性显著提高.结论:IL-2基因修饰的巨噬细胞经肿瘤抗原冲击后自体回输是治疗肾癌的有效方法.

To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-α. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

目的:建立人树突状细胞(DC)的cDNA文库,通过大规模随机测序克隆免疫新分子.方法:来源于正常人外周血的单核细胞,体外经GM-CSF和IL-4培养,扩增出高纯度的DC,抽提纯化mRNA,转录成cDNA,定向插入pSPORT2.0载体,建立人DC的cDNA质粒文库;用ABI377全自动测序仪对该文库进行随机测序,将得到的EST在Sun-E450服务器中与EMBL及Swissprot数据库进行同源性比较,筛选出代表未知基因EST,然后在GCG软件中进行分析,对重要的未知EST克隆其全长cDNA.结果:所扩增的DC经流式细胞仪分析高表达MHC-Ⅰ、MHC-Ⅱ、B7、CDla和CD83等表面标志,能强烈刺激同种异体T淋巴细胞的体外增殖反应;建立的DC cDNA文库92%以上克隆有平均1.6 kb大小的插入片段;从所测的12 000余条EST中筛选出代表未知基因的3 000余条,克隆到109条全长新基因,包括属于细胞因子、细胞因子受体、趋化因子和粘附分子等家族的免疫新分子,部分全长基因已在Genebank中登录.结论:成功建立了人DC的cDNA基因文库及其大规模随机测序体系,并克隆到免疫分子.

目的：观察白细胞介素２ （ＩＬ２） 基因修饰的巨噬细胞ＩＬ２ 动态分泌水平及对其表面分子的表达和抗原提呈能力变化的影响。方法：通过重组腺病毒介导，将ＩＬ２ 基因转染至腹腔巨噬细胞，ＭＴＴ法检测其ＩＬ２ 动态分泌水平，ＦＡＣＳ检测巨噬细胞表型，混合淋巴细胞反应法（ＭＬＲ） 测定巨噬细胞抗原提呈能力。结果：ＩＬ２ 基因修饰４ ｈ 后巨噬细胞即可表达较高水平的ＩＬ２ ，１８ ～４８ ｈ 之间的ＩＬ２ 分泌处于高峰。ＩＬ２ 基因修饰的巨噬细胞ＭＨＣＩＩ、Ｂ７１、Ｂ７２ 和ＣＤ４０ 表达增加，抗原提呈能力提高。结论：ＩＬ２ 基因修饰的巨噬细胞能有效表达ＩＬ２ ，与其功能密切相关的ＭＨＣＩＩ、Ｂ７ 、ＣＤ４０ 表达增加，其抗原提呈能力增强，本实验为巨噬细胞免疫治疗肿瘤提供了实验依据。

Objective:It has been considered that activation-induced apoptosis is a down-regulation mechanism in immune response. The antigen presenting function of the BMSCs has been demonstrated in our laboratory. Whether activation-induced apoptosis would be involved in the antigen presenting cells is still puzzling.Methods:Activation-induced apoptosis of antigen presenting cells were studied BMSCs as a model in this experiment. C57BL/6 murine bone marrow stromal cells were cultured with recombinant murine GM-CSF to maturation and then were stimulated by the FBL-3 tumor antigen. The immuno-associated molecules on the BMSCs were assayed by FACS.Results:It was found that MHC-Ⅰ, MHC-Ⅱ, VCAM-1 and B7-2 molecules essential to antigen presenting cells were expressed abundantly. F4/80 and NLDC145, the maker molecules of monocyte-macrophage and dendritic cell were also highly expressed on BMSCs. The GM-CSF induced BMSCs could strongly stimulate the proliferation of allogeneic T lymphocytes before the stimulation of the tumor antigen. BMSCs changed into dendritic-like cells morphologically after stimulation by the antigen and then exfoliated. Fas and Fas ligand expressed on the antigen-pulsed BMSCs has been determined, and the specific ultrastructural changes of apoptosis were found in this cells with electromicroscope.Conclusions:The results indicate that an activation-induced apoptosis occurs in the BMSCs after antigen stimulation.

Macrophages, with potent cytotoxic and antigen-presenting activities, can be used in cancer treatment. The biological characteristics and antitumor effect of GM-CSF gene-modified and tumor antigen-pulsed macrophages were investigated. The high levels of GM-CSF could be detected in the supernatants of macrophages after gene transfer. The cytotoxicity and the expression of MHC class II molecules of the gene-modified macrophages increased significantly and the antigen-presenting ability was enhanced. The gene-modified macrophages were then pulsed with tumor antigen and used to treat the experimental pulmonary metastastic mice. The number of pulmonary metastases was reduced significantly and the cytotoxicity of the CTL induced from the splenocytes of the tumor-bearing mice also increased. The results demonstrated that adenovirus-mediated GM-CSF gene transfer can activate macrophages to some extent and GM-CSF gene-modified, antigen-pulsed macrophages may be a new type of effective effector cells in the immunogene therapy of cancer.

Vascular cell adhesion molecule 1 (VCAM-1) is a member of immunoglobulin superfamily. The principal ligand for VCAM-1 is integrin α4β/VLA-4 (very late antigen 4). It was reported that VCAM-1 was expressed on macrophages and dendritic cells, but little is known about its function on these professional antigen presenting cells (APC). The present study was performed to investigate the expression of VCAM-1 on macrophages and the role of VCAM-l/VLA-4 in the activation of allogenic T cells by murine macrophages. We analyzed VCAM-1 expression on peritoneal macrophages and macrophage cell line J774A.1 by fluorescence-activated cell sorting (FACS). Using neutralizing antibodies, we further analyzed the role of VCAM-l/VLA-4 interaction in macrophage and allogenic T cell mixed lymphocyte reaction (MLR). We found that VCAM-1 was constitutively expressed on macrophages and its expression level was upregulated by soluble tumor associated antigen (freeze-thaw lysates of FBL-3 leukemia cells) and TNF-a. In MLR assays, we observed that blocking VCAM-l/VLA-4 interaction with anti-VCAM-1 or anti-VLA-4 mAbs caused significant inhibition of the proliferative response and IL-2 production. These results suggest that VCAM-lon macrophages not only facilitates the cell-to-cell contact through adhesive interaction but also plays a role in the costimulation of T cells via its interaction with VLA-4 on the T cells.

研究将对巨噬细胞双重功能均具有激活作用的细胞因子GM-CSF的基因转染小鼠腹腔巨噬细胞,再经肿瘤抗原致敏后通过静脉注射用于实验性CT26结肠癌肺转移小鼠的治疗.结果表明,腺病毒介导的GM-CSF基因转染小鼠腹腔巨噬细胞在转染后4h即可分泌较高水平的GM-CSF.转染后10d仍可有效表达;转染后16h左右小鼠腹腔巨噬细胞MHCⅡ类分子的表达明显增强,其抗原提呈能力也达最高水平;对肿瘤细胞的杀伤活性显著增高;荷瘤3d的实验性CT26结肠癌肺转移小鼠经肿瘤抗原致敏的GM-CSF基因转染巨噬细胞治疗后第16天肺部转移结节数明显减少;经治疗小鼠脾细胞经诱导的CTL杀伤活性也明显升高.以上结果提示,GM-CSF基因转染及表达能有效增强小鼠腹腔巨噬细胞的抗原提呈能力及效应功能;经肿瘤抗原刺激后其对转移性肿瘤也有明显的治疗作用.