不同刺激诱导肺癌患者引流淋巴结细胞抗自体肿瘤的体外研究

目的：探索适合于临床大规模应用的有效刺激、活化肿瘤引流淋巴结（Tumor-draining lymph node，TDLN）细胞的方法。方法：对3种刺激剂（IL-2、IL-2＋自身肺癌细胞抗原和IL-2＋GM-CSF＋IL-4＋自身肺癌细胞抗原）诱导TDLN细胞体外抗自体肿瘤作用，通过乳酸脱氢酶法测定了CTL杀伤活性，观察了细胞形态学变化，通过流式细胞技术测定了细胞CD83的阳性表达率。结果：经MTT法检测，IL-2＋GM-CSF＋IL-4＋自身肺癌细胞抗原诱导的TDLN细胞的增殖程度明显高于IL-2＋自身肺癌细胞抗原刺激组和IL-2刺激组（P〈0．01）。IL-2＋GM-CSF＋IL-4＋自身肺癌细胞抗原诱导的肿瘤特异性CTL活性明显高于IL-2＋自身肺癌细胞抗原刺激组和IL-2刺激组，CD83阳性表达细胞率也明显高于IL-2＋自身肺癌细胞抗原刺激组和IL-2刺激组。结论：用IL-2＋GM-CSF＋IL-4＋自身肺癌细胞抗原刺激和活化TDLN，优于单独用IL-2刺激或IL-2＋自身肺癌细胞抗原刺激，该作用可能与其诱导TDLN细胞产生DC数量的增加有关。     

肿瘤引流淋巴结中免疫活性细胞分布的原位分析

目的:观察人乳腺癌和胃癌局部引流淋巴结(LDLN)从无转移、微转移到晚期转移过程中,免疫组织学变化及免疫活性细胞(ICC)的分布特征。方法:采用传统的病理学方法,对22例乳腺癌LDLN(71个)和7例进展期胃癌LDLN(28个)进行组织形态学分类,并以抗穿孔素、抗颗粒酶B、抗CD8、抗CD56、抗CD68、抗S-100、抗CD134及抗CD25单克隆抗体(mAb)进行催化信号放大(Catalyzedsignalamplification,CSA)免疫组化染色,检测肿瘤LNDN中ICC的分布。结果:肿瘤LDLN中以副皮质区增生和窦组织细胞增生为主,细胞毒性T淋巴细胞(CTL)及树突状细胞(DC)的数量,从无转移、微转移到晚期转移过程中有逐渐减少的趋势。在无和微转移的淋巴结内,穿孔素 、颗粒酶B 及S100 DC的数量高于晚期转移淋巴结(P<0.05);而S100 DC不仅数量减少,而且其形态也有变化,呈多角形、星形,并有胞质突起,与周围淋巴细胞接触呈活化状态的DC变为椭圆形,少有胞质突起或呈短突起的静止状态的DC。CD134 细胞及CD25 细胞的数量在晚期转移淋巴结中明显高于无和微转移淋巴结(P<0.01)。ICC在无和微转移的前哨和非前哨淋巴结中的分布无统计学意义(P>0.05)。结论:肿瘤LDLN中ICC分布的变化,提示随着肿瘤的进展,其免疫微环境向抑制机体抗肿瘤免疫的方向偏移。     

自体树突状细胞诱导TDLN细胞对胃癌细胞系KATO3的体外杀伤作用

目的：研究用冻融的自体胃癌抗原冲击树突状细胞(DC)来诱导肿瘤引流区淋巴结(TDLN)细胞对胃癌细胞系的体外杀伤作用。方法：采用胃癌患者外周血粘附细胞(PBAC)，经GM-CSF、IL-4、TNF-α诱导和自体肿瘤冻融抗原(Ag)刺激诱生所获得的DC与TDLN细胞1：50比例共培养3天，获得DC激活的TDLN，即DC-TDLN作效应细胞；分别以Ag和培液代替DC同样培养TDLN，即Ag-TDLN和TDLN作对照，对胃癌细胞系KATO3和黑色素瘤细胞系A375进行杀伤。结果：DC-TDLN、Ag-TDLN、TDLN各组细胞对KATO3，均有显著杀伤活性，其中DC-TDLN组的杀伤作用明显优于后两组，且效靶比20：1的杀伤率优于10：1，显示可能有量效关系；而TDLN各组不同效靶比对A375杀伤率则无显著差异。结论：自PBAC获得的DC，经自身肿瘤Ag冲击并与自身TDLN共培养，可使后者对胃癌细胞系细胞的杀伤作用明显增强。     

肿瘤患者淋巴细胞的抗肿瘤活性

本文用乳酸脱氢酶(LDH)释放法检测了肿瘤患者肿瘤浸润淋巴细胞(TIL)、肿瘤引流区域淋巴结淋巴细胞(RNL)、外周血淋巴细胞(PBL)和脾细胞(SPC)抗K562、Daudi、同种和自身新鲜肿瘤靶细胞的细胞毒活性、ADCC、及其经rIL-2培养不同时期的变化。结果表明肿瘤患者淋巴细胞的NK活性ADCC均明显低于正常人,IL-2可以使之逆转并能诱导LAK活性的产生,以TIL和RNL抗自身肿瘤活性提高最明显。     

不同刺激剂诱导肺癌患者引流淋巴结细胞分泌细胞因子的研究

目的:探讨多种刺激剂对肿瘤引流淋巴结(TDLN)细胞分泌细胞因子的影响及其抗肿瘤效应和机制。方法:采用ELISA和Griess法测定不同刺激组(分别为IL2、IL2 自身肺癌细胞抗原、IL2 GMCSF IL4 自身肺癌细胞抗原和IL2 GMCSF IL4 LPS)刺激TDLN后第7、14、21天,分泌IL12p70、IFNγ、TNFα和NO的水平。结果:IL2 GMCSF IL4 自身肺癌细胞抗原和IL2 GMCSF IL4 LPS组刺激的TDLN细胞中,CD83 细胞的比率明显增多,分泌IL12p70、IFNγ及TNFα的水平也明显高于IL2和IL2 自身肺癌细胞抗原刺激组,IL2 GMCSF IL4 LPS组刺激的TDLN细胞分泌NO的水平明显高于其他3组。各种刺激剂刺激TDLN细胞后,分泌IL12p70、IFNγ和NO的水平在第14天时达到高峰。结论:不同刺激剂诱导TDLN细胞中的CD83 细胞数量不同,故具有不同的抗肿瘤活性。     

肿瘤引流淋巴结的免疫学性质及其抗肿瘤作用的研究进展

肿瘤引流淋巴结（ＴＤＬＮ）主要由抗原提呈细胞（ＡＰＣ）和免疫活性细胞组成，前者主要执行提呈抗原的功能，后者介导免疫应答。ＴＤＬＮ既是暂时阻止瘤细胞转移的天然屏障，也是肿瘤细胞远处转移的中介者。免疫制剂或某些化疗药物可以通过解除ＴＤＬＮ局部的免疫受抑状态，增强局部的抗肿瘤作用，也可以通过离体激活和扩增ＴＤＬＮ细胞用于过继免疫治疗（ＡＩＴ）。肿瘤基因治疗的发展使基因治疗专家和肿瘤治疗专家注意到ＴＤＬＮ的潜在用途，ＴＤＬＮ细胞疗法治疗肿瘤是一条很有前途的肿瘤治疗途径。     

慢性肝炎患者树突状细胞体外负载抗原后的抗病毒作用

近来在抗肿瘤免疫研究中发现，树突状细胞（DC）在抗原的提呈及激活淋巴细胞使其成为肿瘤特异性细胞毒T淋巴细胞（CTL）方面有重要作用，因而有广泛的应用价值。本研究对慢性肝炎外周血树突状细胞进行体外诱导扩增，DC在未成熟阶段摄取纯的HBsAg后发育为成熟的DC，再与自身T淋巴细胞共培养，观察特异性CTL对2．2．15细胞培养液中HBeAg和HBsAg的表达抑制以及产生IFN-γ和IL-12的情况。     

人LAK细胞的体外抗肿瘤作用

用重组IL-2(rIL-2)以及部分纯化的IL-2(PPIL-2)体外激活人外周血单个核细胞.(PBM),使之形成LAK细胞,然后借助于~(51)Cr释放实验,研究了正常人和肿瘤病人的LAk细胞对传代的肿瘤细胞系和新鲜实体瘤细胞的杀伤能力。实验结果表明:1.二种来源的LAK细胞均能明显杀伤传代的肿瘤细胞系,包括NK敏感的K562细胞和NK抵抗的Daudi细胞。2.采用数种新鲜实体瘤细胞作靶,二种来源的LAg细胞同样具有明显的广谱杀伤力,这证实该群杀伤细胞确系LAK细胞。3.不同来源的实体瘤细胞对LAK细胞的杀伤敏感性不同,表现为杀伤程度上的差异,这似乎提示某些肿瘤对LAK细胞杀伤存在抗性。     

脐血树突状细胞的诱导和体外抗肿瘤研究

目的：用细胞因子诱导脐血干细胞分化为树突状细胞(dendritic cells，DC)，并观察脐血来源DC疫苗在体外对人肝癌细胞的杀伤活性。方法：用CD34^ 细胞分选试剂盒和Mini MACS从脐血单个核细胞中分离CD34^ 干细胞。重组人粒细胞-巨噬细胞集落刺激因子100μg／ml、重组人肿瘤坏死因子-α50U／ml诱导CD34^ 干细胞向DC分化，     

肿瘤引流淋巴结的免疫学性质及其抗肿瘤作用的研究进展

肿瘤引流淋巴结（ＴＤＬＮ）主要由抗原提呈细胞（ＡＰＣ）和免疫活性细胞组成，前者主要执行提呈抗原的功能，后者介导免疫应答。ＴＤＬＮ既是暂时阻止瘤细胞转移的天然屏障，也是肿瘤细胞远外转移的中介者。免疫制剂或某些化疗药物可以通过解除ＴＤＬＮ局部的免疫受抑状态，增强局部的抗肿瘤作用，也可以通过离体激活和扩增ＴＤＬＮ细胞用于过继免疫治疗（ＡＩＴ）。肿瘤基因治疗的发展使基因治疗专家和肿瘤治疗专家注意到ＴＤＬＮ     

Ex vivo stimulation of tumor-draining lymph node cells from lung cancer patients: a potential resource for adoptive immunotherapy

To find a feasible method for the stimulation of tumor-draining lymph node （TDLN） cells in preparation for use in the clinic, the CTL activity of TDLN cells induced by different stimuli （IL-2 alone, IL-2 ＋ autologous tumor antigen （atAg）, IL-2 ＋ GM-CSF ＋ IL-4 ＋ atAg） was measured by maximal LDH enzyme release. The mechanisms were explored by the observation of morphology and the detection of CD83^＋ TDLN cells. The expansion of TDLN cells by IL-2 ＋ GM-CSF ＋ IL-4 ＋ atAg was significantly higher than that by IL-2 alone or IL-2 ＋ atAg （p 〈 0.01）. Antitumor CTL activity of TDLN cells induced by IL-2 ＋ GM-CSF ＋ IL-4 ＋ atAg was significantly higher than those of other groups. The number of CD83^＋ cells within the TDLN population treated with IL-2 ＋ GM-CSF ＋ IL-4 ＋ atAg was significantly elevated. The method of stimulating TDLN cells by IL-2 ＋ GM-CSF ＋ IL-4 ＋ atAg was better than the stimulation with IL-2 or IL-2 ＋ atAg. TDLN cells induced by IL-2 ＋ GM-CSF ＋ IL-4 ＋ atAg produced more dendritic cells （DCs）. In our study, we established a system that T cells and DCs were stimulated together ex vivo, which was easy to conduct and produce promising results. It provided a new method for improving TDLN cell antitumor activity which might be used in the clinical cancer therapy. Cellular ＆ Molecular Immunology. 2008;5（4）：307-313.     

The tumor-draining lymph node as an immune-privileged site

Summary:  Lymph nodes that lie immediately downstream of tumors [tumor-draining lymph nodes (TDLNs)] undergo profound alterations due to the presence of the upstream tumor. The antigen-presenting cell population in TDLNs becomes modified such that tumor-derived antigens are cross-presented by host cells in a tolerizing fashion. In addition, the number and suppressor activity of regulatory T cells (Tregs) are increased in the TDLN. Emerging evidence suggests that some of these Tregs may be generated de novo against specific tumor-derived antigens, and thus they arise as a direct consequence of antigen presentation in the TDLN. Others may represent Tregs against self-antigens, which undergo preferential activation in the tolerogenic milieu of the TDLN. The TDLN thus becomes an anatomic context in which presentation of new antigens not only fails to elicit a protective immune response but also actively creates systemic tolerance. In this regard, the TDLN displays features analogous to classical immune privilege. Accumulating evidence thus suggests that the TDLNs, although small in size, may exert a profound tolerizing influence on the rest of the immune system. These mechanisms will need to be interrupted in order for clinical anti-tumor immunotherapy to be successful.     

Differential expression of CCL19 by DC-Lamp+ mature dendritic cells in human lymph node versus chronically inflamed skin

De novo formation of lymphoid tissue is one of the characteristic features of chronic inflammation. The formation of T cell–mature dendritic cell (DC) clusters has been previously demonstrated in chronically inflamed skin infected with Candida albicans. A functional similarity was also found between chronic inflammation and the T‐cell zone of lymph nodes (LNs), since a substantial fraction of phenotypically mature DCs in both tissues expressed CCL22 (macrophage‐derived chemokine; MDC) and were closely surrounded by memory‐type T cells expressing its receptor, CCR4. To analyse the nature of T cell–mature DC interactions further in chronically inflamed skin and LNs, the present study focuses on another chemokine system, namely CCL19 (EBI1 ligand chemokine; ELC), CCL21 (secondary lymphoid tissue chemokine; SLC) and their shared receptor, CCR7. RT‐PCR analysis revealed expression of CCL19, CCL21, and CCR7 at high levels in LNs and at low levels in inflamed skin. Using immunohistochemistry, the majority of DC‐Lamp⁺ mature DCs in the T‐cell area of LNs expressed CCL19 and were surrounded by CCR7⁺ naïve‐type lymphocytes, while CCL21 was expressed in reticular stromal cells and vascular endothelial cells. Very few mature DCs in LNs were found to express CCR7. In contrast, the majority of DC‐Lamp⁺ mature DCs in inflamed skin were totally negative for CCL19 and were surrounded by CCR7^? memory‐type T cells. Furthermore, CCL21 expression in the inflamed skin was detected in dermal lymphatic endothelial cells and rare CCR7⁺ mature DCs were mostly seen within the lymphatic vessels. In normal skin, on the other hand, no cells immunoreactive for CCL19, CCL21, or CCR7 were found. The present study thus reveals a striking difference in the function of mature DCs between LNs and chronically inflamed skin. Copyright © 2002 John Wiley & Sons, Ltd.     

Targeting the tumor-draining lymph node with adjuvanted nanoparticles reshapes the anti-tumor immune response

Accumulating evidence implicates the tumor-draining lymph node (TDLN) in tumor-induced immune escape, as it drains regulatory molecules and leukocytes from the tumor microenvironment. We asked whether targeted delivery of adjuvant to the TDLN, presumably already bathed in tumor antigens, could promote anti-tumor immunity and hinder tumor growth. To this end, we used 30 nm polymeric nanoparticles (NPs) that effectively target dendritic cells (DCs, CD11c⁺) within the lymph node (LN) after intradermal administration. These NPs accumulated within the TDLN when administered in the limb ipsilateral (i.l.) to the tumor or in the non-TDLN when administered in the contralateral (c.l.) limb. Incorporating the adjuvants CpG or paclitaxel into the NPs (CpG-NP and PXL-NP) induced DC maturation in vitro. When administered daily i.l. and thus targeting the TDLN of a B16–F10 melanoma, adjuvanted NPs induced DC maturation within the TDLN and reshaped the CD4⁺ T cell distribution within the tumor towards a Th1 (CXCR3⁺) phenotype. Importantly, this also led to an increase in the frequency of antigen-specific CD8⁺ T cells within the tumor. This correlated with slowed tumor growth, in contrast to unhindered tumor growth after c.l. delivery of adjuvanted NPs (targeting a non-TDLN) or i.l. delivery of free adjuvant. CpG-NP treatment in the i.l. limb also was associated with an increase in CD8⁺/CD4⁺ T cell ratios and frequencies of activated (CD25⁺) CD8⁺ T cells within the TDLN whereas PXL-NP treatment reduced the frequency of regulatory T (FoxP3⁺ CD4⁺) cells in the TDLN. Together, these data implicate the TDLN as a delivery target for adjuvant therapy of solid tumors.     

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes

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乳腺癌前哨淋巴结不同转移状态下局部淋巴结成熟树突状细胞的研究

目的：研究乳腺癌前哨淋巴结不同转移状态 下前哨淋巴结与非前哨淋巴结成熟树突状细胞密度的改变。方法:回顾 性分析79例符合研究标准的女性乳腺癌患者。根据前哨淋巴结的转移状况分为3组：A组（2 8例），所有淋巴结转移阴性；B组（25例），仅微小肿瘤出现于前哨淋巴结，包括B1组（16 例），仅游离肿瘤细胞出现于前哨淋巴结；B2组（9例），仅微转移出现于前哨淋巴结；C组 （26例），转移出现于前哨淋巴结，非前哨淋巴结有或无转移。所有前哨淋巴结及非前哨淋 巴结蜡块切片均行与DC-LAMP抗体免疫反应的免疫组织学检查以确认成熟树突状细胞。蔡司 图像分析系统定量分析每个淋巴结DC-LAMP阳性细胞的相对密度（DC-LAMP阳性细胞面积/淋 巴结面积）。Wicoxon检验和Mann-Whitney检验分别用于DC-LAMP阳性细胞的相对密度的组内 和组间比较。结果:DC-LAMP阳性细胞密度的组内比较显示A组和B组前哨 淋巴结DC-LAMP阳性细胞平均密度较非前哨淋巴结高（P<0.05，P<0.01）；C组前哨淋 巴结DC-LAMP阳性细胞平均密度与非前哨淋巴结比较无明显差异（P>0.05）。组间比较 显示各组前哨淋巴结DC-LAMP阳性细胞平均密度无显著差异；而B组(尤其B2组)非前哨淋巴结 DC-LAMP阳性细胞密度较A组和C组显著升高（P<0.05，P<0.01）。结论： 前哨淋巴结和非前哨淋巴结DC-LAMP阳性细胞平均密度在淋巴结肿瘤转移形成过程发生改变,揭示前哨淋巴结在肿瘤与引流淋巴结间相互作用中起重要作用。     

17β-雌二醇调节小鼠淋巴结树突状细胞的表型和吞噬功能

为探索17β-雌二醇(E2)对免疫应答能力的调节是否与树突状细胞(DC)的成熟和功能相关,用Metrizamide密度梯度离心方法分离BALB/c小鼠淋巴结内的淋巴细胞得到DC,不同浓度的E2处理DC 24 h后,使用PE标记的单抗CD11c和FITC标记的单抗MHC I/MHC II/CD40/CD54/CD80/CD86、IL-10/TNF-α以及右旋糖酐(dextran)双标DC,流式细胞仪分别检测其表型和胞内细胞因子的表达及吞噬能力的变化。结果显示,不同浓度的E2处理DC 24 h后,MHC分子、黏附分子CD54和共刺激分子CD86与对照组相比显著提高,而CD40显著降低,但CD80的表达无明显改变。胞内细胞因子IL-10和TNF-α的表达水平随E2的处理而显著增加,其中TNF-α的变化呈剂量依赖性。与对照组相比,DC的吞噬能力随E2的处理浓度增加而显著降低。以上结果表明,E2能够改变DC的成熟和功能,这提示E2对机体的免疫应答的影响可能通过DC的改变而改变。