Decoding the dynamics of T cell–dendritic cell interactions in vivo

Summary: T lymphocytes receive activation signals during their encounters with antigen-bearing dendritic cells (DCs) in secondary lymphoid organs. With the recent application of two-photon imaging to visualize immune responses as they happen, the dynamics of T cell–DC interactions have been dissected in several mouse models. As we are integrating the results of these new studies, we are learning that the dynamics of T cell–DC interactions are regulated by multiple immunological parameters and, most importantly, that the spatiotemporal characteristics of these cell–cell contacts encode part of the T-cell fate.     

Human hepatic lymph nodes contain normal numbers of mature myeloid dendritic cells but few plasmacytoid dendritic cells

We investigated whether the hyporesponsiveness of the adaptive arm of the liver immune system is related to the composition of the dendritic cell (DC) population in hepatic lymph nodes. Noninflamed human hepatic lymph nodes (LN) were obtained from multiorgan donors, inflamed hepatic LN from liver transplant recipients with autoimmune cholestatic liver diseases, and inguinal LN from kidney transplant recipients. Quantitative immunohistochemistry showed that all three types of LN contained comparable numbers of mature and immature myeloid DC, but that noninflamed and inflamed hepatic LN contained significantly fewer plasmacytoid DC as compared to inguinal LN. Likewise, DC-enriched cell preparations from hepatic LN contained relatively few plasmacytoid DC. The difference in numbers of plasmacytoid DC was confirmed in comparisons of hepatic LN and ileacal LN from the same organ-donors. Myeloid DC from hepatic LN showed similar expressions of HLA-DR, CD83, and CD86, and higher expression of CD80 compared to myeloid DC from inguinal LN. In conclusion, hepatic LN contain similar numbers of myeloid DC as muscle/skin lymph-draining LN, with no signs of immaturity, but relatively few plasmacytoid DC.     

Subsets of migrating intestinal dendritic cells

Dendritic cells (DCs) in the intestine are heterogeneous. Phenotypically different populations of conventional DCs have been identified in the intestinal lamina propria, Peyer’s patches, and in the draining mesenteric lymph nodes, to which these DCs constitutively migrate. Markers used to identify these populations include major histocompatibility complex class II, CD11c, CD8α, CD11b, and CD103. Extensive studies in rats, summarized here, which involved collection of migrating DCs by thoracic duct cannulation after mesenteric lymphadenectomy, have clearly demonstrated that the subsets of migrating intestinal lymph DCs have different functional properties. The subsets might play different roles in the induction of oral tolerance and in driving systemic immune responses after vaccination or intestinal stimulation with Toll-like receptor ligands. The use of these surgical techniques allows investigation of the functions of purified subsets of migrating DCs. However, in the rat, these studies are limited by the range of available reagents and are difficult to compare with data from other species in this fast-moving field. Recent refinements have enabled the collection of migrating intestinal DCs from mice; our initial results are described here. We believe that these studies will generate exciting data and have the potential to resolve important questions about the functions of migrating intestinal DC subsets.     

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.     

自体树突状细胞体外增强肿瘤患者引流淋巴结细胞抗肿瘤活性的作用

目的：探讨自体树突状细胞(DC)体外对胃腺癌患者肿瘤引流区)细胞抗肿瘤活性的增强作用。方法：分离肿瘤患者的外周血单个核细胞(PBMC)经IL-4、GM-CSF和TNF-α诱导其成熟，并以自体肿瘤冻融抗原预致敏，诱导其中的DC。另外，分离胃癌患者的TDLN细胞，在含有IL-2的培养体系中培养，并将TDLN细胞分为3组：(1)第1组为肿瘤抗原致敏的DC加TDLN细胞(D组)；(2)第2组为冻融的肿瘤抗原加TDLN细胞(Ag组)；(3)第3组不加DC及肿瘤抗原作为对照组(L组)。比较3组TDLN细胞对胃腺癌细胞系KATO3和黑色素瘤细胞系A375的杀伤作用。结果：D组对KATO3细胞系的杀伤作用明显优于Ag组与L组；而对A375细胞，各组细胞的杀伤作用没有明显差异。结论：自体DC可明显增强胃癌患者TDLN细胞的杀伤作用。     

Expression of multidrug resistance 1 gene in B‐cell lymphomas: association with follicular dendritic cells

Aims: Multidrug resistance (MDR) in B‐cell lymphomas still constitutes a major obstacle to the effectiveness of chemotherapy even in the anti‐CD20 antibody therapy era. The aim of this study was to investigate the expression of MDR‐associated molecules in reactive lymphadenopathy (RL), follicular lymphoma (FL), and diffuse large B‐cell lymphoma (DLBCL). Methods and results: The expression of mRNA for ABC‐transporter family genes was determined by real‐time RT‐PCR in lymph nodes from RL, FL, and DLBCL cases. MDR1 exhibited significantly stronger expression in RL, FL, and DLBCL than Raji B‐cell lymphoma cells. RL and FL showed significantly higher expression than DLBCL. Immunohistochemically, MDR1 positive cells were localized in the germinal centers of RL and center of the nodular lesions of FL showing associations with CD21 positive follicular dendritic cells (FDCs). Raji cells were co‐cultured with FDC sarcoma‐derived cells and the expression of MDR1 and drug resistance were analyzed. The co‐culture of Raji cells with FDCs induced strong expression of MDR1 and introduced resistance to doxorubicin‐induced apoptosis. Conclusions: These results suggest that FDCs induce MDR1 expression in reactive as well as neoplastic B‐cells. Inhibition of the interaction of FDCs with B‐cells may provide a novel strategy for treating the chemotherapy resistant fraction.     

Dendritic reticulum cell sarcoma? Four cases of a lymphoma probably derived from dendritic reticulum cells of the follicular compartment

This paper describes the histological picture of four tumours of the follicular compartment of the lymph node, in which the proliferating cell appeared to be the dendritic reticulum cell (DRC). This assumption was based on the results of light microscopical, ultrastructural, immunological, and enzymehistochemical investigations. The tumour cells resembled DRC's closely in (1) the striking pattern of interdigitations and occasional tight junction-like contacts between the neoplastic cells on electron microscopical analysis; (2) presence of receptors for the activated third component of complement on the membrane of the cells; (3) absence of monoclonal immunoglobulins and T-cell antigen on the surface and of lysozyme, alpha 1-antitrypsin or alpha 1-antichymotrypsin in the cytoplasm of the neoplastic cells. Moreover, (4) the tumour cells showed moderate alpha-naphtyl acetate esterase, weak to absent acid phosphatase and (with one exception) strong 5-nucleotidase activity. Furthermore, (5) the neoplastic cells expressed Ia-like antigens on the surface in all four cases. The relation with follicle centre cell lymphomas, the differential diagnosis and clinical data are discussed.     

Tracking dendritic cells: use of an in situ method to label all blood leukocytes

Here we describe an in situ procedure with a labeling index (percent of labeled blood leukocytes) >98%, which is high enough to permit the direct tracking of dendritic cell (DC) precursors from blood into lymphoid tissues, while circumventing the pitfalls associated with in vitro labeling. DC and lymphocytes have similar blood to afferent lymph migratory capabilities. This method has additional applications in tracking other rare cell populations in both normal and pathological states.     

Mucin-1 is expressed on dendritic cells, both in vitro and in vivo

Dendritic cells (DCs) are the best professional antigen-presenting cells to stimulate cytotoxic as well as T helper cells and are therefore appropriate candidates for establishing immunotherapy. The concept of our vaccination program is to introduce the tumor-associated antigen mucin-1 (MUC1) into DCs. Analysis of immature and mature DCs--before transducing the antigen MUC1--already demonstrated expression of MUC1 on in vitro monocyte-derived DCs upon maturation. Different culture methods as well as maturation cocktails showed similar results concerning the upregulation of MUC1 expression. Furthermore, we studied the expression of MUC1 on DCs in vivo. No MUC1 expression was found on blood DCs, or on thymic or tonsil DCs. On the other hand, synovial fluid from patients with arthritis contained DCs that were found to express MUC1. This study shows for the first time that the tumor-associated antigen MUC1 is expressed on in vivo DCs. We further show that MUC1 is also expressed on in vitro cultured bone marrow-derived DCs of human MUC1 transgenic mice, supporting the relevance of this mouse model to the human situation. The observation that MUC1 is present on in vivo DCs suggests a functional role, but this physiological function remains to be elucidated.     

Follicular dendritic cell tumor with histiocytic characteristics and fibroblastic antigen

A report is presented of a follicular dendritlc cell (FDC) tumor arising In the lymph nodes and Inguen of a 55-year-old Japanese female, who had suffered from schizophrenla for 25 years. The left submandibular lymph nodes had completely lost their normal architecture, except for the capsule, due to tumor cell infiltration. Occaslonal nodular structures resembling epltheliold granulomas, attributable, at least In part, to follicular Involvement of tumor cells, were observed. These nodules were composed of epithellold- or fibroblast-like tumor cells forming interwoven fascicles, to which small lymphocytes were attached. Tumor cells were also scattered in the internodular areas. For more atyplcal tumor cells, arranged in a sheet-like structure, were present In the inguinal specimen, the tumor cells of which expressed Ki-M4p, CD21, CD35 and other antigens known to be expressed on FDC. Furthermore, they also expressed the monocytdmacrophage antlgens, α1-antltrypsin, α-antlchymotrypsin, lysozyme, CD14, CD33, CD68 and Mac387 and fibroblastic antigen. Ultrastructural studies demonstrated lysosomal granules as well as a few desmosomes, Indicating the tumor cells possessed fibrohistiocytic and FDC characteristics.     

Transient receptor potential vanilloid 1 expression and function in splenic dendritic cells: a potential role in immune homeostasis

Neuro‐immune interactions, particularly those driven by neuropeptides, are increasingly implicated in immune responses. For instance, triggering calcium‐channel transient receptor potential vanilloid 1 (TRPV1) on sensory nerves induces the release of calcitonin‐gene‐related peptide (CGRP), a neuropeptide known to moderate dendritic cell activation and T helper cell type 1 polarization. Despite observations that CGRP is not confined to the nervous system, few studies have addressed the possibility that immune cells can respond to well‐documented ‘neural’ ligands independently of peripheral nerves. Here we have identified functionally relevant TRPV1 on primary antigen‐presenting cells of the spleen and have demonstrated both calcium influx and CGRP release in three separate strains of mice using natural agonists. Furthermore, we have shown down‐regulation of activation markers CD80/86 on dendritic cells, and up‐regulation of interleukin‐6 and interleukin‐10 in response to CGRP treatment. We suggest that dendritic cell responses to neural ligands can amplify neuropeptide release, but more importantly that variability in CGRP release across individuals may have important implications for immune cell homeostasis.     

Follicular dendritic cell sarcoma of the colon mimicking stromal tumour

AIMS: Follicular dendritic cell tumours are very rare neoplasms that often occur in lymph nodes. We report here a case in the colon, a hitherto unreported site, in a 37-year-old female. The differentiation from gastrointestinal stromal tumour is emphasized. METHODS and RESULTS: The tumour was tan, elastic and solid with surface ulceration. Microscopically, it was composed of oval to spindle tumour cells with syncytial cytoplasm arranged in fascicular and whorled patterns. There were many infiltrating lymphocytes. The histological appearance resembled gastrointestinal stromal tumour, thymoma or meningioma. Distinct from the stromal tumour, the lymph node was also involved by the tumour. Immunohistochemically, the tumour cells were positive for CD21, CD35 and CD68, but negative for cytokeratin, CD34, smooth muscle actin, desmin, S100 protein, epithelial membrane antigen, leukocyte common antigen, HMB-45 and c-kit. In-situ hybridization study was negative for Epstein-Barr virus RNA sequences. Ultrastructurally, the tumour cells possessed cytoplasmic processes joined by desmosomes. CONCLUSIONS: This entity should be considered in the list of differential diagnoses for gastrointestinal stromal tumour. The lymph node metastasis and immunohistochemical features are of value for identification of this rare neoplasm.     

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。     

Folliculocentric B-cell-rich follicular dendritic cells sarcoma: a hitherto unreported morphological variant mimicking lymphoproliferative disorders

We report three cases of follicular dendritic cell sarcoma (FDCS) showing a hitherto undescribed histological pattern consisting of nodular tumor growth associated with small B lymphocytes. FDCS tumor cells consistently showed large epithelioid features and were intermingled with small lymphocytes in the nodules in two cases, whereas they formed cohesive aggregates surrounded by lymphocyte mantle in the other. These features were easily confused with lymphomatous proliferations and, in particular, subtypes of Hodgkin lymphoma, high-grade follicular lymphoma, and germinotropic large B-cell lymphomas. The diagnosis was established by the use of a broad panel of antibodies that showed a variable expression of the FDC markers CD21, CD23, CD35, clusterin, podoplanin, claudin 4, epidermal growth factor receptor, and CXCL13. The associated B lymphocytes revealed a mantle zone B phenotype, with expression of CD20 and PAX5, together with TCL1 and IgD. Of notice, in all cases, morphological features suggesting hyaline-vascular Castleman disease were recognized in the interfollicular areas, containing scattered epithelioid cells similar to those found in the nodules, thus providing a useful clue for FDCS diagnosis. Of the 3 cases, 1 presented multiple recurrences unresponsive to chemotherapy and radiotherapy and finally died of disease 14 years after diagnosis. This study further emphasizes the extreme variability of morphological presentation of FDCS and expands the spectrum of lesions showing a nodular growth pattern occurring in human lymph nodes.     

The effects of in vivo cyclosporin A administration on rat thymic dendritic cells.

Cyclosporin A (CsA) induces a graft-versus-host-like disease (GVHD) in lethally irradiated Lewis rats reconstituted with syngeneic bone marrow. The role of the thymus in the generation of disease has been unequivocally established. It has been suggested that the CsA-induced disappearance of thymic dendritic cells (DC) is responsible for the generation of the autoaggressive cells. In this study we quantify the loss of DC upon in vivo CsA administration in normal and bone marrow-reconstituted rats using an isolation technique. The phenotype of the DC is determined using MoAbs recognizing antigens which are expressed on thymic medullary DC. Furthermore, the functional aspects are assessed by determining the antigen presentation capacity. Short-term CsA exposure clearly affects the number of DC isolated from the thymus in a concentration-dependent manner. However, in all instances a substantial number of DC can be isolated from CsA-treated animals. These isolated DC exhibit an identical phenotype and function as DC isolated from control animals. Therefore, the partial deficiency of DC can not be held as essential for loss of tolerance.     

Presence and maturity of dendritic cells in melanoma lymph node metastases

Immune avoidance mechanisms play a key role in the successful dissemination of melanoma. One mechanism whereby this could be achieved is by interfering with dendritic cell (DC) presentation of tumour‐associated antigens to naïve T cells. In particular, immature DCs characterized by the absence of accessory molecules are known to be immunosuppressive and to be involved in the induction of tolerance. The present study has investigated the presence and activation status of DCs within melanoma metastases in the regional lymph nodes. Using image analysis techniques, the expression of Factor XIIIa (FXIIIa), CD40, CD83 and HLA‐DR and the morphological features of DCs were examined in paraffin sections from 26 lymph nodes containing melanoma metastases. DCs expressing FXIIIa were found in 70% of the lymph nodes. The number of DCs identified was generally small but there were more concentrated areas of DCs designated as hotspots. In these areas of high FXIIIa staining, the percentage area occupied by DCs varied between 0.1% and 10%. The majority of FXIIIa‐positive cells did not express the DC maturation markers CD83 or CD40 and morphologically were rounded with few dendrites, indicating that they were immature. The cells did, however, express high levels of HLA‐DR, suggesting that they have the ability to present antigen but lack the accessory molecules required to initiate an immune response. Immature DCs, characterized by phenotypic and morphological features, are therefore present within the tumour deposits in lymph nodes infiltrated by melanoma and may specifically modulate the anti‐melanoma immune response. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Identification of three distinct subsets of migrating dendritic cells from oral mucosa within the regional lymph nodes

To investigate the phenotypic and migrational properties of oral mucosal dendritic cells (OMDCs), fluorescein isothiocyanate (FITC) was painted onto mouse buccal mucosa and the expression patterns of functional molecules in FITC-bearing migrating DCs within the regional lymph nodes (RLNs) were analysed. We found three distinct subpopulations of migrating OMDCs within the RLNs: CD11c^hi CD207⁻ (F1), CD11c^int/lo CD207⁻ (F2) and CD11c^int/lo CD207⁺ (F3). The F1 DCs reached the RLNs earlier (after 24 hr) but diminished immediately. Additionally, F1 DCs expressed high levels of CD11b. The F2 DCs migrated continuously to the RLNs and maintained the highest ratio of all three fractions. The F3 DCs migrated slowly to the RLNs and demonstrated a late peak at 96 hr. In addition, F3 DCs showed the highest CD205 expression levels of all three subsets. All fractions of migrating OMDCs expressed CD80, CD86 and major histocompatibility complex class II at high levels, suggesting that all OMDCs are in a mature stage and have the potential for antigen presentation. All migrating OMDCs lacked CD8α expression. Taken together, our results indicate that the lack of CD207 is one factor that identifies submucosal DCs. Both F1 and F2 DCs lack CD207; F1 DCs are resident and F2 DCs are newly recruited following FITC application. The F3 DCs, which express CD207, are mucosal Langerhans cells that migrate later. The identification of OMDC subsets should facilitate further studies investigating the functional roles of each fraction.     

Distinct antigen trafficking from skin in the steady and active states

In antigen trafficking from the skin, it has been postulated that Langerhans cells/dendritic cells are activated after capturing exogenous antigens, up-regulate the expression of the chemokine receptor, CCR7, and migrate into lymphoid organs in response to the signaling of a chemokine, CCL21, which is expressed in lymphatic vessels and T cell zone stromal cells. Here we demonstrate that there is a distinct pathway of antigen trafficking from skin in the steady state that is independent of CCL21-CCR7 signaling. Employing melanin granules as an endogenous traceable antigen, we developed a system for visualizing antigen trafficking using mice with melanocytosis in the skin. We found the abrogation of antigen trafficking into regional lymph nodes (LN) in CCL21-Ser-deficient paucity of lymph node T cells (plt) mice in the active state induced by lipopolysaccharide injection, corresponding with previous reports, but normal accumulation of antigen in regional LN under steady-state conditions. These findings suggest that self-antigen is trafficking constitutively using pathway(s) other than that of the active state and the constitutive trafficking might regulate self-reactivity of the immune system.