树突状细胞向淋巴结归巢的研究进展

树突状细胞（DC）作为专职抗原递呈细胞在诱导机体产生抗肿瘤免疫应答过程中发挥重要作用。以此为基础制备的DC肿瘤疫苗在抗肿瘤免疫治疗中显示出一定的疗效。体外回输DC迁移归巢至局部引流淋巴结是激发特异性免疫应答的关键步骤,归巢DC的数目直接影响免疫应答的强度。了解DC体内归巢至淋巴结的机制,促进DC向淋巴结迁移对于提高DC疫苗的抗肿瘤效果具有重要意义。趋化因子和相应受体、黏附分子、基质金属蛋白酶和脂类介质等多种因素共同调控DC向淋巴结归巢,其中CCR7及其配体CCL19和CCL21是一组最受关注的因子。     

肥大细胞脱颗粒促进外源树突状细胞向引流淋巴结归巢

目的:探讨通过诱导局部肥大细胞脱颗粒促进外源骨髓源性树突状细胞(BM-DC)向淋巴结迁移归巢的方法。方法:采用肥大细胞脱颗粒刺激剂compound 48/80(c48/80)进行C57BL/6小鼠局部皮肤注射,诱导局部组织肥大细胞脱颗粒。将体外培养的荧光微球标记的同系小鼠BM-DC注射入c48/80或生理盐水处理的局部皮肤,48h收集注射部位引流淋巴结细胞进行CD11c荧光抗体染色,流式细胞术测定外源回输的BM-DC的迁移效果。结果:c48/80注射小鼠皮肤后可有效地促进局部组织肥大细胞脱颗粒,c48/80处理侧局部引流淋巴结中外源BM-DC迁移细胞总数和淋巴结细胞总数较对照侧均明显增多,增多比例分别为67%±43%和55%±43%(P0.05)。结论:通过诱导局部皮肤肥大细胞脱颗粒,可促进外源BM-DC向淋巴结归巢。     

淋巴结内树突状细胞研究进展

ＤＣ是具有最强的抗原提呈和启动机体免疫应答能力的一类抗原提呈细胞，居留于淋巴结内的ＤＣ主要为并指状ＤＣ和滤泡状ＤＣ，在功能和特征上均有各自的特性，在外周ＤＣ向淋巴结的迁移中，多种细胞因子发挥着重要的调控作用，有关ＤＣ在肿瘤等疾病中的作用是当前研究的热点之一。     

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。     

树突状细胞和滤泡树突状细胞的研究进展

树突状细胞作为一种专职性抗原递呈细胞，在免疫应答起着很重要的作用。近年来人们对两类ＣＤ，即与Ｔ细胞相关的指突太ＤＣ和与Ｂ细胞相关的滤泡ＤＣ有了较深入的了解。本文介绍各种ＤＣ的内在联系，在组织间的移行，功能上的成熟过程以及ＦＤＣ对Ｂ细胞介导的体液免疫作用的等研究进展作一介绍。     

Compound 48 / 80 活化的肥大细胞对树突状细胞归巢的影响

目的:检测compound 48/80(C48/80)活化的肥大细胞对树突状细胞(DC)向局部引流淋巴结迁移的影响,初步探讨C48/80 增强适应性免疫应答的机制。方法:体外培养C57BL/6 小鼠骨髓来源的DC(BM-DC)和MC/9 肥大细胞,Transwell 趋化小室实验检测C48/80 处理的MC/9 细胞对BM-DC 向趋化因子CCL21 迁移的影响。甲苯胺蓝染色法检测小鼠肩背部皮肤注射C48/80 后肥大细胞脱颗粒状态;流式细胞术检测肩上和腋窝淋巴结CD11c+ DC 的数量。小鼠肩背部两侧各注射荧光微球标记的BM-DC,制备局部引流淋巴结冷冻切片,荧光显微镜观察C48/80 处理对外源性DC 归巢的影响。采用负载卵白蛋白的BM-DC(OVA-DC)免疫C48/80 处理及对照小鼠,WST-8 试剂检测局部引流淋巴结淋巴细胞的增殖活性。结果:采用小鼠骨髓细胞成功培养并获得大量典型的BM-DC。MC/9 肥大细胞活化上清可促进BM-DC 向CCL21 迁移(P<0.01)。皮内注射C48/80 后30 min 可诱导局部皮肤明显肥大细胞脱颗粒。C48/80 注射部位24 h 出现明显炎细胞浸润,局部引流淋巴结细胞总数和DC 细胞数量增多。注射荧光微球标记的BM-DC 后48 h 淋巴结冷冻切片显示C48/80 处理侧荧光阳性的DC细胞数量明显多于C48/80 未处理侧。OVA-DC 免疫小鼠显示C48/80 处理组抗原特异性淋巴细胞增殖能力较对照组明显增高(P<0.05)。结论:C48/80 诱导的肥大细胞活化能够促进DC 向局部引流淋巴结归巢,增强特异性淋巴细胞增殖能力,参与适应性免疫应答。     

小肠系膜淋巴结外滤泡树突状细胞肉瘤

目的 探讨淋巴结外滤泡树突状细胞肉瘤的临床病理学特征和免疫学表型。方法 对1例发生于小肠系膜的淋巴结外滤泡树突状细胞肉瘤进行光镜观察和免疫组化标记。结果 镜下显示特征性的双相性细胞结构，由旋涡状排列的胖梭形、卵圆形或多边性瘤细胞和大量混杂的小淋巴细胞组成。瘤细胞胞界不清，呈合体状。胞质淡嗜伊红色，核圆形或卯圆形，有清晰的核膜，染色质呈空泡状或点彩状，可见小核仁，灶性区域可见分叶状或多核性瘤细胞。瘤细胞核显示轻到中度的异型性，可见核分裂象。肿瘤内可见凝固性坏死灶。除与瘤细胞相混杂外，部分区域内可见淋巴细胞聚集于血管周围，形成袖套结构。免疫表型：瘤细胞表达CD21，弱阳性表达EMA，而CD35、S100蛋白、desmin和AE1／AE3等标记均为阴性，大多数小淋巴细胞表达CD45RO(UCHL-1)和CD3。结论 淋巴结外滤泡树突状细胞肉瘤是一种罕见的免疫辅助细胞中度恶性肿瘤，应予以完整切除，必要时辅以化疗和(或)放疗。     

树突状细胞和滤泡树突状细胞的研究进展

树突状细胞（ＤＣ）作为一种专职性抗原递呈细胞，在免疫应答起着很重要的作用。近年来人们对两类ＣＤ，即与Ｔ细胞相关的指突状ＤＣ（ＩＤＣ）和与Ｂ细胞相关的滤泡ＤＣ（ＦＤＣ）有了较深入的了解。本文介绍各种ＤＣ的内在联系，在组织间的移行、功能上的成熟过程以及ＦＤＣ对Ｂ细胞介导的体液免疫的作用等研究进展作一介绍。     

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。     

P-gp对树突状细胞从皮肤迁移至引流淋巴结的影响

细胞膜P-糖蛋白(P-gp，P-glycoprotein)是多药耐药(mdr)基因表达产物，广泛分布在各种排泄器官包括肠道、肝、肾等上皮细胞，脑及胎盘血管内皮细胞、造血干细胞及外周血细胞的细胞膜上。人类mdrl基因，小鼠mdrla基因表达产物与多药耐药有关。目前，有关P-gp的研究多集中在肿瘤化学治疗的药物抵抗方面，对其在免疫调节中的作用还不清楚。     

Taking the lymphatic route: dendritic cell migration to draining lymph nodes

In contrast to leukocyte migration through blood vessels, trafficking via lymphatic vessels (LVs) is much less well characterized. An important cell type migrating via this route is antigen-presenting dendritic cells (DCs), which are key for the induction of protective immunity as well as for the maintenance of immunological tolerance. In this review, we will summarize and discuss current knowledge of the cellular and molecular events that control DC migration from the skin towards, into, and within LVs, followed by DC arrival and migration in draining lymph nodes. Finally, we will discuss potential strategies to therapeutically target this migratory step to modulate immune responses.     

Plasmacytoid dendritic cells from human lung cancer draining lymph nodes induce Tc1 responses

Dendritic cells (DC) resident in draining lymph nodes (LN) of patients with lung cancer are proposed to have a critical role in stimulating anti-tumor immunity. CpG oligodeoxynucleotides are undergoing clinical trials in patients with lung cancer and are likely to target plasmacytoid-DC. The present study, therefore, investigated the capacity of plasmacytoid-DC from human lung cancer draining LN to respond to CpG for activation of T cell responses relevant to anti-tumor immunity. The phenotype of DC was examined by flow cytometry, and cytokine production by cytometric bead array (CBA) and ELISA. Plasmacytoid-DC, purified by cell sorting, were immature but expressed the toll-like receptor, TLR9. Plasmacytoid-DC responded to the CpG oligodeoxynucleotide, CpG 2216, by production of the proinflammatory cytokines, IFN-alpha and IL-6. DC were cocultured with normal, allogeneic T cells, and cytokine production determined by CBA and immunophenotyping. CpG 2216 enhanced IFN-gamma production and induced intracellular production of IFN-gamma by CD8(+) and CD4(+), granzyme B by CD8(+), and IL-2 by CD4(+) T cells, respectively. Ligation of CD40 on plasmacytoid-DC combined with exposure to CpG 2216 also strongly enhanced IFN-gamma production. There was no significant difference between the responses of plasmacytoid-DC from patients with lung cancer and patients with benign carcinoid tumors with no pathologic LN involvement. These results indicate that plasmacytoid DC from the draining LN of patients with lung cancer effectively induce Tc1 immunity and could, therefore, represent a novel and attractive target for immunotherapeutic intervention.     

Antigen-bearing dendritic cells in the draining lymph nodes of contact sensitized mice: cluster formation with lymphocytes.

Following topical exposure to skin-sensitizing chemicals, Langerhans' cells, a significant proportion of which bear antigen, are induced to migrate from the epidermis to the regional lymph node. There is evidence that the antigen-bearing cells which arrive in the draining lymph nodes have the functional characteristics of mature dendritic cells (DC) and efficiently induce T-lymphocyte activation in vitro and contact sensitization in vivo. In contrast, freshly isolated Langerhans' cells are known to be relatively inefficient antigen-presenting cells. Evidence exists that during culture in the presence of granulocyte/macrophage colony-stimulating factor, Langerhans' cells undergo a functional maturation and assume the characteristics of dendritic cells. We have speculated that, in response to chemical exposure and the stimulus to migrate. Langerhans' cells undergo a similar maturation in vivo. To investigate this we have examined the capacity of draining lymph node DC to form antigen-independent clusters with T lymphocytes. Previous studies have confirmed that freshly isolated Langerhans' cells are unable to form such clusters. We report, however, that the antigen-bearing DC which arrive in the draining lymph nodes following skin sensitization, and which are recently derived from epidermal Langerhans' cells, efficiently form clusters with lymphocytes. Thus, antigen-bearing DC were found to have formed clusters with lymphocytes in situ in the draining lymph node, and to readily form clusters with lymphocytes in vitro. In both cases a higher proportion of lymphocytes associated with DC in clusters were T cells. An interesting observation was that DC within draining nodes appeared more efficient at cluster formation than DC in resting nodes, and that within draining nodes antigen-bearing DC formed clusters with greater affinity and/or greater stability than DC which lacked antigen. Taken together these data demonstrate that Langerhans' cell-derived antigen-bearing cells which accumulate in the draining lymph nodes following skin sensitization form clusters with lymphocytes in the manner of mature DC. This is compatible with the hypothesis that, while in transit from the skin, Langerhans' cells are subject to a functional maturation comparable to that witnessed in vitro.     

Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells

We have previously reported a novel method for the production of tumour‐antigen‐presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma‐derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell‐based immunotherapy induces T‐cell‐mediated immune responses resulting in improved long‐term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti‐tumour immunization. This mobilization of DCs is mainly driven by the C‐C chemokine receptor type 7 (CCR7), which is up‐regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient‐derived DCs, and also on the monocytic/macrophage cell line THP‐1. Moreover, in vitro assays showed that TRIMEL‐stimulated DCs and THP‐1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC‐based vaccines for treating aggressive melanomas.     

Statins inhibit lymphocyte homing to peripheral lymph nodes

Lymphocyte homing to peripheral lymph nodes is governed by adhesion molecules, including lymphocyte function-associated antigen 1 (LFA-1). Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and exert anti-inflammatory effects, e.g. inhibition of LFA-1. It is still not known whether statin compounds are capable of inhibiting lymphocyte homing in vivo. We used a cervical lymph node preparation to study the effects of simvastatin on lymphocyte adhesion to high endothelial venules (HEVs) by means of intravital fluorescence microscopy (IVM). IVM revealed that firm adhesion of lymphocytes to HEV endothelium critically depends on the adhesive function of LFA-1. The number of firmly adherent lymphocytes was reduced by 58% in LFA-1-deficient mice (P < 0.05 versus wild-type controls). As in mutant mice, acute treatment with simvastatin (i.p. injection at 2 hr prior to IVM) inhibited the firm adhesion of lymphocytes to HEV endothelium of wild-type animals by 63% (P < 0.05 versus vehicle-treated wild-type controls). In addition, acute treatment with the synthetic statin-derivate LFA878 also reduced firm lymphocyte adhesion in HEVs by 63% (P > 0.05 versus placebo-treated controls). Histological analysis after a 10-day treatment with simvastatin showed reduced cellularity of cervical lymph nodes, as indicated by a reduction of the relative area of haematoxylin-stained cell nuclei in cervical lymph node cross-sections from 94 +/- 0% in vehicle-treated controls to 77 +/- 3% in simvastatin-treated mice (P < 0.05). We conclude that statin compounds are capable of inhibiting lymphocyte homing to murine peripheral lymph nodes in vivo. This may have novel implications for the treatment of adaptive immune responses, e.g. transplant rejection.     

The appearance of non-specific antibody-forming cells in the efferent lymph draining antigen-stimulated single lymph nodes.

Immunization of single lymph nodes with various antigens led to the appearance of cells in the efferent lymph that secreted antibody specific for the antigen which induced their formation and for a number of unrelated, non-crossreacting antigens. Immunization of single lymph nodes with mitogens led to the appearance of cells secreting antibodies specific for an even greater number of antigens, including one (TNP) that in all probability is not present in the animals' natural environment. When the node was primed with one antigen, a subsequent challenge with an unrelated antigen 12 weeks later led to the appearance of greater numbers of cells containing and secreting antibody against the previously experienced antigen, than was the case in unprimed lymph nodes. These findings indicate that the immune response to antigen provokes the maturation of lymphocytes of specificities unrelated to that of the injected immunogen. Such a mechanism may be important in maintaining immunological memory. Mitogens may directly activate lymphocytes into maturation and expression as antibody-secreting cells, whereas antigens appear to act indirectly.     

IL-2 production by dendritic cells is not critical for the activation of cognate and innate effectors in draining lymph nodes

Dendritic cells (DC) are unique antigen-presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL-2 production by mouse DC. Previous reports demonstrated that IL-2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC-derived IL-2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone marrow-derived DC results in early IL-2 production unless IL-4 is introduced in DC cultures. Here we show that IL-2 produced by LPS-activated DC is dispensable for cognate T cell responses since IL-2 loss of function DC elicit OVA-specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL-2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL-2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.     

In vivo magnetic resonance imaging of dendritic cell migration into the draining lymph nodes of mice

Dendritic cell (DC) migration into the draining lymph nodes is critical for T cell priming. Here, we show that magnetic resonance imaging (MRI) can be used to visualize DC migration in vivo. We combined clinically approved small particles of iron oxide (SPIO) with protamine sulfate to achieve efficient uptake by murine bone marrow-derived DC. SPIO-DC were largely unaltered and after injection into the footpads of mice, they migrated into the T cell areas of the draining lymph nodes, which could be visualized by MRI. Distinct MRI signal reduction patterns correlated with the detection of SPIO-DC mainly within Thy-1.2+ B220- T cell areas, as confirmed by iron staining and immunohistology. Clear signal reduction patterns could still be observed with 1x10(6) injected SPIO-DC at high resolution, resulting in the detection of about 2000 DC. Control injections of homing-incompetent SPIO-DC derived from CCR7-/- mice or SPIO alone did not reach the T cell areas. Taken together, the results demonstrate that clinically approved contrast agents allow the non-invasive visualization of DC migration into the draining lymph node by MRI in vivo at high resolution. This protocol therefore also allows dynamic imaging of immune responses and MRI-based tracking of human DC in patients.