Dendritic-like leukemia cells (DLLC) originating from leukemic cells could potentially induce a T cell-mediated anti-leukemia immune response. It has been demonstrated that B7-H1, a newly identified homologue of CD80/CD86, is abundant in human carcinomas and dendritic cells (DC), can exert co-stimulatory and immune regulatory functions. We demonstrated that B7-H1 was significantly expressed on AML cells and was strongly enhanced after differentiation to DLLC. Blockade of B7-H1 expressed on DLLC results in increased T cell proliferation and Th1 cytokine production, and decreased Th2 cytokine production. Importantly, autologous CTLs induced by DLLC treated with B7-H1 mAb showed significantly increased specific cytotoxcity against AML blasts. We further demonstrated that a significant decrease in IL-12 production, increase in IL-10 production by DLLC, and an increased CD4(+)CD25(+)Foxp3(+) T regulatory population lead to the defective T cell immune response that is induced by B7-H1 up-regulation on DLLC. Our data suggest that up-regulated B7-H1 on DLLC acts as a strong inhibitor of anti-leukemia T cell response, and that blockade of B7-H1 can improve DLLC-mediated anti-leukemia immunity.

从造血干细胞(HSC)诱导树突细胞(DC)传统方法是在GM-CSF+TNF-α的基础上联合早期细胞因子一步诱导获得[1].但如果采用先扩增、后诱导的两步法则可以在保证DC功能的基础上显著提高DC数量.我们已经成功建立了一个先用SCF+FL+TPO+IL-3扩增10 d再加入GM-CSF、IL-4、TNF-α诱导8 d的两步法从脐血(CB)CD34+细胞大量获得DC的体系[2],但与文献报道[3]类似,所得DC以CD1a+细胞为主,代表DC成熟状态的CD83表达不充分.因此,我们在前期工作的基础上探讨扩增阶段的细胞因子及诱导阶段的细胞因子对两步法所得DC的产量及功能的影响,以期进一步提高两步法所得DC的功能.

To compare the expansion efficiency and function of dendritic cells derived from CB-CD34+ cells and MPB-CD34+ cells by using two-step culture method, enriched CB-CD34+ cells or MPB-CD34+ cells with immunoadsorption were primarily cultured in the presence of FL, SCF, TPO, GM-CSF for 10 days, and then further cultured with a combination of GM-CSF, IL-4, TNF-alpha, CD40Ab and PGE2 to induce DC. The DC phenotypes were detected by flow cytometry, the expansion efficiency and cell function were evaluated by mix-lymphocyte reaction (MLR), IL-12 level was detected by using ELISA and the chemotactic function mediated by secondary lymphoid tissue chemokine (SLC) was determined with Transwell plate. The results indicated that after 10 days of expansion, there were no significant difference in the percentage of CD14+CD1a- cells between CB and MPB [(40.48 +/- 16.85)% vs (28.07 +/- 23.19)%, P > 0.05], but the expansion of total cells in CB was higher than that in MPB (388.88 +/- 84.63-fold vs 79.67 +/- 10.32-fold, P < 0.01), so the yield of CD14+CD1a- cells from CB was significantly higher than that from MPB too (189.42 +/- 25.02-fold vs 28.74 +/- 23.27-fold, P < 0.01). The percentage of CD83+ DCs cultured with CD40Ab/PGE2 derived from CB were higher than those cultured with TNF-alpha derived from MPB respectively [(34.52 +/- 11.22)% vs (3.70 +/- 2.27)% and (36.69 +/- 13.36)% vs (7.34 +/- 3.364)% respectively, P < 0.01]. In the same circumstance, the yield of CD83+ DCs derived from CB was much more than that from MPB (198.72 +/- 117.53 times vs 33.95 +/- 6.19 times, P < 0.01). There were no difference in stimulating capacity, IL-12 secretion and migration capacity between DCs derived from CB and MPB. It is concluded that DCs induced from CB-CD34+ cells by two-step culture possess similar functions with that from MPB-CD34+ cells, but the yield of DCs from CB CD34+ cells is much more than that from MPB CD34+ cells.

This study was aimed to investigate the specific anti-L615 leukemia cell immunity induced by L615/DC fused cell vaccine in vivo and in vitro. BM-derived DCs were generated from bone marrow of 615 mice by culturing for 9 - 10 days in culture medium supplemented with GM-CSF and IL-4. Irradiated L615 tumor cells were fused with DC by using PEG to form fused cell vaccine, with which 615 mice were immunized. After immunization, the specific proliferation ability and cytotoxicity against L615 leukemia cells in vitro were examined by MTT and LDH methods. Anti-leukemia effect of fused cell vaccine in vivo was studied by observing the immunotherapy effects on L615 tumor-bearing mice. The results showed that fully mature and functional bone marrow-derived DC were obtained. L615/DC fused cell vaccine could elicit potent specific proliferation response of spleen T cells from immunized mice when contacting with the same antigen at the second time, and could also elicit the effective cytotoxic activity against L615 leukemia cells in vitro, which were significantly different from other groups. In vivo the average survival time of the tumor-bearing mice received immunotherapy with L615/DC fused cell vaccine was 25.7 +/- 1 days, and one fourth of treated tumor-bearing mice survived for long time, but the mice of control group died all, their average of survival time was 17.5 +/- 1 days. The immunized mice survived with no evidence of recurrence when exposed to the second attack of lethal dose of living L615 cells 2 months later. It is concluded that L615/DC fused cell vaccine can improve the immunogenecity of L615 and induce effectively the specific anti-leukemia immunity against L615 leukemia cells to eliminate the residual leukemia cells, prolong the survival time and induce the immune memory to avoid the relapse. Thus, the fused cell vaccine may be an attractive strategy for malignance immunotherapy.

OBJECTIVE:To assess the feasibility and efficiency of eliciting leukemia-specific T cell responses in acute myeloid leukemia patients in complete remission (AML-CR) in vitro by dendritic cells (DC) pulsed with the leukemia cells total RNA.METHODS:The immature DCs were generated from the adherent bone marrow mononuclear cell in vitro in the presence of combined cytokines (GM-CSF 100 ng/ml, IL-4 500 U/ml), and pulsed with total RNA isolated from autologous leukemic cells by cationic lipid 1,2-dioleoyloxy-3-trimethyl ammonium propane (DOTAP) at day 5 of culture. Then the cells were incubated for another 24 h in a medium containing 10 ng/ml of TNF-alpha for maturation of DC. After the total 7 days culture, the cells were harvested as the mRNA-DC and the expression of mature DC markers were determined by FACS. The proliferative capacity of T cell activated by mRNA-DC was determined by MTT assay. Meanwhile, the mRNA-DC was co-cultured with T lymphocytes at a ratio of 1:3 for 7 days. The activated T lymphocytes were harvested, the secretion of IFN-gamma was determined by ELISPOT assay, and the cytotoxicity was analyzed in vitro by LDH release assay.RESULTS:After culture, the BMMNC from 14 AML-CR patients developed morphologic and phenotypic characteristics of mature DC. At a stimulator/reactor ratio of 1:16, auto-T lymphocytes primed with mRNA-DC exhibited significant proliferative activity compared with T lymphocyte primed with non-pulsed DC [(36.84 +/- 5.68)% vs (12.20 +/- 3.16)%, (P < 0.05)]. An expansion of mRNA reacted T cell secreting IFN-gamma could be observed on ELISPOT assay. At an effector/target ratio of 20:1, the auto-T lymphocytes primed with mRNA-DC exhibited significant killing activity to auto-AML cells (45.46 +/- 6.34 )% as compared with that stimulated by IL-2 alone (13.26 +/- 2.28)% or primed by non-pulsed DC (12.32 +/- 1.32)% (P < 0.05).CONCLUSION:Immunization with DC-leukemia cell RNA vaccines may be a simple, rapid and potent approach to elicitation of T cell-mediated anti-leukemia immunity.

The level of TGF-#beta#1 was evaluated by enzyme linked immunosorbant assay (ELISA); after the TGF-#beta#1 was neutralized by its correspondig mAb, the ability of T lymphocyte-proliferation was measured by ~(3)H-Thydimine incorporation; and the level of interferon-gamma (IFN-#gamma#) was measured by Eli-spot kit. MSCs reduced the level of IFN-#gamma# secreted by activated T lymphocytes; inhibited the ability of T-lymphocyte proliferation and such an effect could be reversed partly by anti-TGF-#beta#1 antibody. MSCs inhibit T-lymphocyte proliferation through secreting TGF-#beta#1.

随着免疫学和移植学的发展, 人类对自身免疫性疾病认识的不断提高和对器官移植的日益接受, 免疫抑制剂在临床医学中的应用愈来愈广泛.

OBJECTIVE To explore whether coculture of dendritic cells (DC) with cytokine-induced killer (CIK) lead to an increase of cytotoxicity against tumor cells in vitro and in vivo. METHODS DC and CIK were prepared from human peripheral blood mononuclear cells (PBMC) by conventional methods, the DC pulsed with or without NB4 leukemia cell lyses (LCL) was cocultured with the CIK (LCL-DC + CIK and DC + CIK), CIK was used as control. Cells phenotypes were analyzed by flow cytometry, secretion of IFN-gamma was determined by ELISPOT assay, and cytotoxicity was assayed in vitro with (51)Cr-release assay. A human leukemia cell NB4-bearing nude mice model was established to test in vivo antitumor efficacy and cell homing. RESULTS Compared with CIK, LCL-DC + CIK got a significant increasing of proliferation rate [(18.2 +/- 2.1) times vs (11.6 +/- 2.3) times, P < 0.05] and CD(3)(+)CD(56)(+) expression rate [(51.05 +/- 2.63)% vs (30.18 +/- 1.45)%, P < 0.05], and the number of IFN-gamma secreting cells was increased significantly [(13.86 +/- 3.28)/10(4) cells vs (8.74 +/- 2.53)/10(4) cells, n = 12, P < 0.05]. Meanwhile, LCL-DC + CIK led to an increase of cytotoxic activity to NB4, K562, and KG1a cells, and showed significant inhibition of the growth of transplanted tumor cells and increased tumor free survival rate of nude mice (100% vs 66.7%, P < 0.05), DiI labeled LCL-DC + CIK were detected in spleen, lymph node and tumor within a week after injection. There was no significant different in antitumor activity between LCL-DC + CIK cell and DC + CIK cell. CONCLUSION Coculture of CIK with DCs can promote the effect of CIK against tumor in vitro and in vivo. DC-CIK is promising as an immuno-therapeutic strategy for patients with leukemia.

OBJECTIVE To Explore a two-step culture system to generate a large number of dendritic cells (DC) differentiated from cord blood (CB) CD(34)(+) cells. METHODS Enriched CB CD(34)(+) cells with immunoadsorption were primarily cultured in the presence of stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (Tpo) and interleukin-3 (IL-3) for 7 (group I), 10 (group II) or 14 days (group III) respectively, and then further cultured with GM-CSF, IL-4 and TNF-alpha for 5 - 8 days to induce DC. The expansion and cell function were evaluated by flow cytometry (FCM) and mix-lymphocyte reaction (MLR), and detection of IL-12 in the supernatant by using ELISA. RESULTS The total nucleated cells with 53.39 +/- 20.59-, 307.17 +/- 119.59- and 1117.25 +/- 335.49-folds expansion could be respectively obtained after 7 - 14 days of expansion culture. After DC induction, CD(1a)(+) cells were 21.40 +/- 16.70-, 143.2 +/- 60.35- and 150.8 +/- 42.16-fold increase as compared to the initial nucleated cells. Comparing with that in group I, the CD(1a)(+) cells were much more in groups II and III; but there was no difference between the latter two groups (P > 0.05). The cultured cells in the three groups showed almost the same allo-stimulatory capability and IL-12 excretion when the second culture duration maintained 8 days, while the capability and excretion were greatly decreased when the duration shortened to 5 days (P < 0.05). CONCLUSION A plenty of functionally mature DC could be obtained from the CD(34)(+) cells in the two-step culture system of 7 - 10 days HSC expansion followed by 8 days DC induction.

Objective to explore whether AML patients have imbalanced cytokine levels and abnormal ratio of T lymphocyte subsets and their clinical value. Methods Peripheral blood and bone marrow were collected from AML patients during different periods. PBMNCs were detected by flowcytometry. The purified T lymphocytes were cultured and their supernatant was collected on different days.ELISA and RIA were used to measure the IL、4、IFN、γ and IL、2 level,respectively. Results The IL、4 levels in serum of pretreated AML cases were higher than those of the controls,while the IFN、γ levels on the contrary. IL、4 levels of high risk cases were found to be significantly higher than that of other cases, while IFN、γ levels much lower than the latter. The ratios of CD4+/CD8+ T lymphocyte subsets in high risk cases were much lower than that of the other cases. The duration of CR closely related to the ratio of CD4+/CD8+ T subsets(r=0.835、3,linear regression equation:y= 1.068+0.017x). Conclusion The imbalanced cytokine levels of Th1/Th2 subsets and reversed ratios of CD4+/CD8+ T lymphocyte subsets occurred in AML patients,which is of prognostic value.

The aim of this study was to explore effect of CD8+CD28-T-lymphocyte in the inhibition of mesenchymal stem cells (MSC) on T-lymphocyte proliferation. T cells were harvested by using nylon column and CD8+T cells were sorted by magnetic beads; the T-lymphoc yte proliferation in the presence of PHA was evaluated by MTT; the proportion of CD8+CD28-T cells was assayed by fluorescence-activated cell sorter (FACS). The results showed that MSC inhibited T-lymphocyte proliferation and the inhib itory eff ect depended on the amount of MSC; the data of FACS indicated that in the CD8 +T cells co-cultured with MSC, CD8+CD28-T cells were up-regulated signific antly, com pared with the non-treated CD8+T cells. In conclusion, MSC perform their imm unosuppressive function by up-regulation of CD8+CD28-T cells.

BMSCs not only can enter the immunoorgans of allogeneic mouse, but also can exist there for at last 30 days. Ex vivo tests show: (1)BALB/C BMSCs can suppress the T cells proliferation of BALB/C, B6 and BXSB mice stimulated by ConA. (2)BALB/C BMSCs can promote the B cells proliferation and Ig secretion of both BALB/C and B6 mice stimulated by LPS. But they decreased the B cells proliferation and Ig secretion of BXSB mice. (3)BALB/C BMSCs can reduce the IFN#gamma#-producing cell count of BALB/C and B6 mice stimulated by ConA. But showed no affect on their IL-4-producing cell count. For BXSB mice, BALB/C BMSC can cut down the IL-4-producing cell count, but can augment the IFN#gamma#-producing cell count. And all above effects appear as a dose-dependant fashion. Allogeneic BMSC can enter the immunoorgans of recipient, and can exist there for relatively long time (at last 30 days). In addition, BMSC shows some immunoregulatory effects on normal syngeneic, normal allogeneic and allogeneic mice with autoimmune disease.

免疫耐受与自身免疫性疾病、超敏反应性疾病、骨髓移植及器官移植等密切相关.对免疫耐受机制及人工诱导免疫耐受方法的研究进展近年来不断有报道.本文就近年来在人工诱导免疫耐受方面的进展作一综述.

树突状细胞(DC)是免疫调节的关键细胞,为唯一能提呈抗原给初始型T细胞并诱导其活化和功能分化的细胞,可作为治疗恶性肿瘤或感染等疾病的细胞疫苗.随着基于DC的免疫治疗进入临床研究,稳定地产生足量有效的DC是技术关键.获得DC的方法除直接分离纯化外,还可用细胞因子(CK)体外培养.前一方法产量很低,后一方法也有其不尽如人意之处,如培养时间较长以及有的急性髓系白血病(AML)细胞不能被CK诱导分化为DC.虽然有报道乙酸豆蔻佛波酯(PMA)也能诱导CD34+细胞分化为DC,但由于PMA的促癌作用,不能用于临床.因此有必要寻找其他获得DC的方法.最近发现钙动员(calcium mobilization)可诱导髓系细胞变成DC,以下就钙动员和CK诱导正常和恶性髓系细胞分化为DC的比较研究以及钙动员诱导DC分化的信号转导机制的研究等方面的内容作一综述.

Objective To investigate the possibility of immune tole rance induced by bone marrow-derived mesenchymal stem cells in allogeneic organ transplantation. Methods Allogeneic bone marrow-derived mesenchymal stem cells and syngeneic bone marrow cells were cotransplanted to lethally irradiated female C57BL/6 recipient mice. FACS was used to analyze the chimerism 150 days later. Mixed lymphocyte reaction and ConA induced proliferation test were performed to evaluate proliferative activity of mice spleen cells in cell-transplanted group. Skin transplantation test was done to observe immune response of cell-transplanted group mice against organ graft from donor mice. Results About 5 97% donor T cells were detected in splenocytes of cell-transplanted group mice. MLR showed that mean SI of cell-transplanted group mice was 1.79, and that of untreated group mice was 7 28. ConA induced proliferation test showed that mean SI of cell-transplanted group mice was 31 92; and that of untreated group mice was 34 99. Mean survival time of donor-derived skin graft in cell-transplanted group mice was more than 90 days; and that in untreated group mice was 8 days. Conclusion Our results showed for the first time that induction of stable mixed hematopoietic chimerism after allogeneic bone marrow derived mesenchymal stem cells transplantation lead to stable donor-specific tolerance in allogeneic host and skin graft survival from donor mice.

OBJECTIVES:To explore the feasibility of DC being in vitro induced from AML cells with cytokine cocktails and their biological properties.METHODS:AML cells were cultured in either presence or absence of cytokine cocktails. DC were studied for morphology, and cytochemical and immunofluorescent staining. Functions of DC were examined by MLC, FITC-conjugated dextran uptake test, and LDH release assay. RT-PCR and FISH were used to analyze the specific fusion genes of culture-derived DC.RESULTS:Classical DC morphological changes occurred in all 15 cultured AML cells. DC-associated surface molecules such as CD(1a), CD(80), CD(86), CD(106), CD(83) and HLA-DR were upregulated (P < 0.05). The allostimulatory abilities of culture-derived DC were significantly higher than those of AML cells uncultured or cultured in the absence of cytokines (P < 0.05). Culture-derived DC only in the presence of GM-CSF + IL-4 have phagocytotic activities. CTL assay was performed in 5 of the 15 samples. At effector/target ratio of 20:1, auto-T lymphocytes primed with the culture-derived DC exhibited no more killing activity to auto-AML cells than those stimulated by IL-2 or uncultured AML cells. Culture-derived DC presenced the native AML-specific aberrant karyotype and related fusion gene.CONCLUSIONS:Cytokine cocktails could in vitro induce AML cells into DC with classical morphology, immunophenotype and function. DC maturity induced by different cytokine cocktails could be variable. Culture-derived DC were originated from the native AML cells. AML cells could make the auto-T lymphocyte anergy.

Recently stem cell plasticity has arouse great interests in stem cell research for its potential therapeutic application in degenerative or inherited diseases. Transplantation of bone marrow-derived stem cells in vivo has been shown to give rise to cells of muscle,liver,nerve,endothelium,epithelium etc. But there are still disputes as to stem cell plasticity, as is in the case of contribution of bone marrow-derived cell to skin cells. Here we show that cells co-purified from bone marrow mesenchymal stem cells can evidently give rise to skin tissue in vivo. In the present study, we first isolated and expanded bone marrow-derived multipotentianl mesenchymal stem cells from BALB/C mice(H-2K~(d), white hair), then co-transplanted them with certain bone marrow cells from C57BL/6 mice via tail vein into lethally-irradiated 12 weeks old C57BL/6 mice(H-2K~(b),black hair).The recipient mice grew out white hair on the back about forty days later.

1973年Steinman和Cohn第一次系统地描述了小鼠脾脏树突细胞(dendritic cells,DC).其独特的形态及功能吸引了众多后来的科研人员投入到与之有关的研究中来.目前普遍认为DC是功能最强也是唯一可以激活初始T细胞(native T cell)的抗原递呈细胞(antigen present cell,APC),其在免疫激活、免疫耐受、肿瘤免疫、移植免疫及抗感染免疫中都扮演着重要角色.DC广泛分布于除脑、睾丸、眼以外的人体各脏器,但数量极少.同时DC缺乏特异性抗原标记,难以纯化.多年来人们不断探索有效获得DC的方法,包括最初从含有DC的组织(如皮肤、扁桃体、外周血等)通过密度梯度离心获得DC,以及后来从脐血、骨髓、动员的外周血(MPB)中的单核细胞或CD34+造血干细胞[1](hematopoietic progenitor cell,HPC)甚至从血液系统肿瘤细胞[2, 3]培养获得DC.这中间又由于脐血相对容易获得、脐血CD34+ HPC扩增能力较强而吸引人们广泛研究从脐血中获得DC并用于基础及临床前研究.现将近年来有关文献综述如下.

慢性粒细胞白血病(CML)是人造血干细胞发生的一种恶性肿瘤,临床上慢粒是以异常的、不成熟的恶性造血祖细胞的大量增生为特征.病理特征是因第九与第22位染色体易位{t(9:22)}形成的pH1染色体,该染色体易位产生的BCR/ABL融合基因表达的具有高酪氨酸激酶活性的p210BCR/ABL融合蛋白是慢粒发生的分子基础,它能结合或激活细胞内多种信号相关的分子,为造血细胞的恶性转化必要而充分的条件.该病的p210BCR/ABL是通过什么分子机制引起这些变化仍不清楚,目前临床上除干细胞移植外尚无治愈的方法.

目的 研究热休克对慢性髓系白血病 （CML）患者自身T细胞杀伤 （ATK）肿瘤细胞活性的影响。方法 用5 1 Cr释放法检测自身T细胞对热休克处理的CML靶细胞 （ΔCML）和未热休克处理的CML靶细胞 （δCML）的ATK活性 ;用流式细胞仪检测CML细胞热休克蛋白 70 （HSP70 ）的表达 ;淋巴细胞混合培养 （MLTC）法刺激扩增T细胞 ,并检测其免疫表型和ATK活性。结果 2 1例CML患者中有 4例显示对δCML有ATK活性 ,有 10例对ΔCML有ATK活性。IL 2刺激的自身T细胞对ΔCML的ATK活性为 （2 0 .37± 10 .72 ） % ,不仅高于对δCML的ATK活性 [（7.42± 6 .49） % ,P ＜0 .0 1],也明显高于未刺激的自身T细胞对δCML的ATK活性 [（1.5 1± 3.42 ） % ,P ＜0 .0 0 1]。δCML和ΔCML胞浆HSP70的表达差异无显著性 ,未检测到细胞膜HSP70表达增加。MLTC扩增的自身T细胞表型为TCRγδ CD3,以CD8+ T细胞为主 ,部分细胞表达CD2 5 和HLA DR ,γδT细胞仅占 （0 .33± 0 .2 4） % ;其对ΔCML和δCML的ATK活性分别为 （5 1.2 5± 4.2 6 ） %和 （36 .5 2± 3.83） % ,而对K5 6 2细胞杀伤活性仅为 （2 .92± 1.19） %。结论 热休克可诱导或促进自身T细胞 ,尤其是IL 2刺激的T细胞的ATK活性 ,热休克诱导的ATK活性似与γδT细胞或CML细胞膜HSP70表达无关。