Immune adaptors SLP-76, ADAP and SKAP1 (SKAP-55) play central roles in anti-CD3 induced 'inside-out' signalling for LFA-1 activation and ICAM-1 adhesion. However, it has been unclear whether SKAP1 is also required for chemokine-induced T-cell motility. In this study, we found that SDF-1 and CCL21 induced similar motility in SKAP1 deficient (SKAP1-/-) and wild type (SKAP1+/+) resting, primary T-cells. In addition, the speed (i.e. 13 microM/min), tracking distance (i.e. length) and displacement values (i.e. direct distance between the start and the end positions of cell movement) in response to SDF1 were similar for SKAP1-/- and SKAP1+/+ primary, activated T-cells. Relatively high strength anti-CD3 ligation also arrested the migration (i.e. stop-signal) of resting SKAP1+/+ and SKAP1-/- T-cells in the presence of SDF-1 and CCL21. These data demonstrate that contrary to its central role in anti-CD3 induced LFA-1 adhesion, the response of primary T-cells to SDF-1 and CCL21 is not profoundly dependent on SKAP1 expression.

Using cDNA microarray technology, the expression of chemokine genes in the elicitation site of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity (CHS) was examined in mice. Of the 33 genes analyzed, levels of 11 gene expressions changed, and these can be assigned to four groups based on their kinetic patterns; (1) LARC/CCL20 whose mRNA level increased rapidly at 3 h post-challenge and then gradually decreased, (2) JE/CCL2, MARC/CCL7, MIP-1gamma/CCL9, monocyte chemoattractant protein (MCP)-5/CCL12, ELC/CCL19 and BRAK/CXCL14 whose mRNA levels increased with time and reached the maximum at 6-9 h post-challenge, (3) LIX/CXCL5, Mig/CXCL9 and IP-10/CXCL10 whose mRNA levels increased gradually at least up to 12 h post challenge, and (4) SLC/CCL21 whose mRNA level decreased gradually with time after challenge. The findings suggest that sequential expression of chemokine genes is essential for orientating non-specific skin response to hapten-specific CHS response through the recruitment of inflammatory cells such as neutrophils, monocytes/macrophages and T-cells from the circulation into the tissue site.

BACKGROUND

CCL19 and CCL21, acting through CCR7, are termed homeostatic chemokines. Based on their role in concerting immunological responses and their proposed involvement in tissue remodeling, we hypothesized that these chemokines could play a pathogenic role in heart failure (HF).

RESULTS

Our main findings were: (i) Serum levels of CCL19 and particularly CCL21 were markedly raised in patients with chronic HF (n = 150) as compared with healthy controls (n = 20). A CCL21 level above median was independently associated with all-cause mortality. (ii) In patients with HF following acute myocardial infarction (MI; n = 232), high versus low CCL21 levels 1 month post-MI were associated with cardiovascular mortality, even after adjustment for established risk factors. (iii). Explanted failing human LV tissue (n = 29) had markedly increased expression of CCL21 as compared with non-failing myocardium (n = 5). (iv) Our studies in CCR7(-/-) mice showed improved survival and attenuated increase in markers of myocardial dysfunction and wall stress in post-MI HF after 1 week, accompanied by increased myocardial expression of markers of regulatory T cells. (v) Six weeks post-MI, there was an increase in markers of myocardial dysfunction and wall stress in CCR7 deficient mice.

CONCLUSIONS

High serum levels of CCL21 are independently associated with mortality in chronic and acute post-MI HF. Our findings in CCR7 deficient mice may suggest that CCL21 is not only a marker, but also a mediator of myocardial failure. However, while short term inhibition of CCR7 may be beneficial following MI, a total lack of CCR7 during long-term follow-up could be harmful.

Recent studies have revealed new populations of T/B cells, including central/effector memory, follicular T cells and CXCR3+ or CXCR4+ B cells. In the present study, changes in these populations of CD4+ T cells were examined on the basis of the expression of CD62L, CCR7 and CXCR5 in patients with systemic lupus erythematosus (SLE) in relation to CCL21 and CXCL10. Changes in CXCR3+, CXCR4+ and CXCR5+ B cells were also examined. CD62L and various chemokine receptors were examined by flow cytometry analysis using monoclonal antibodies, and CCL21 and CXCL10 were examined by sandwich enzyme-linked immunosorbent assay. In patients with SLE, a decrease of naive T cells and an increase in the ratio of activated effector memory T cells were associated with an increase of CCL21 and CXCL10 in serum, although the correlation was not significant. An increase in the ratio of CXCR3+ B cells was also recognized. These results suggest that naive T cells are transferred to lymphoid tissue by CCL21, and that effector memory T cells are activated by CXCL10. It is also suggested that B cells responsive to follicular helper T cells tend to migrate to inflammatory tissue.

γδ T cells continuously survey extralymphoid tissues, providing key effector functions during infection and inflammation. Despite their importance, the function and the molecules that drive migration of skin-recirculating γδ T cells are poorly described. Here we found that γδ T cells traveling in the skin-draining afferent lymph of sheep are effectors that produce IFN-γ or IL-17 and express high levels of the skin- and inflammation-seeking molecule E-selectin ligand. Consistent with a role for chemokine receptor CCR7 in mediating T cell exit from extralymphoid tissues, conventional CD4 and CD8T cells in skin-draining lymph were enriched in their expression of CCR7 compared to their skin-residing counterparts. In contrast, co-isolated γδ T cells in skin or lymph lacked expression of CCR7, indicating that they use alternative receptors for egress. Skin-draining γδ T cells were unresponsive to many cutaneous and inflammatory chemokines, including ligands for CCR2, CCR4, CCR5, CCR8, CCR10, and CXCR3, but showed selective chemotaxis toward the cutaneously expressed CCR6 ligand CCL20. Moreover, IL-17(+) γδ T cells were the most CCL20-responsive subset of γδ T cells. The data suggest that γδ T cells survey the skin and sites of inflammation and infection, entering via CCR6 and E-selectin ligand and leaving independent of the CCR7-CCL21 axis.

BACKGROUND

Although dendritic cells (DCs) play significant roles in intestinal immune responses, little is known regarding the direct effects of luminal foods on DC functions in the intestinal mucosa. In this study, we examined the effects of fatty acids (FAs) with various chain length on the phagocytic function, antigen presentation, and chemotaxis of intestinal DCs.

METHODS

DCs obtained from the thoracic duct lymph of mesenteric lymphadenectomized rats were cultured with long [arachidonic acid (AA) or oleic acid] or medium (octanoic acid) chain FAs with interleukin-4 and granulocyte macrophage-colony stimulating factor. Tumor necrosis factor-alpha was added in the maturation group. Phagocytic function was examined by the intake of fluorescent microbeads. The expression of cell surface molecules was determined by immunocytochemistry or fluorescence-activated cell sorting (FACS). Antigen presentation ability was evaluated by coincubating keyhole limpet hemocyanin (KLH)-sensitized spleen lymphocytes and KLH-pulsed DCs. Migratory ability of DCs toward the chemokines CC chemokine ligand (CCL) 20 and CCL21 was also assessed.

RESULTS

There was a maturation-induced decrease in phagocytic function, and an increased expression of major histocompatibility complex (MHC) class II molecules. Exposure of DCs to both long- and medium-chain FAs maintained phagocytic ability. The expression of MHC class II molecules was significantly suppressed only by long-chain FAs. The expression of costimulatory factors was suppressed only by AA. Long- but not medium-chain FAs suppressed the antigen presentation ability of DCs induced by maturation. Chemotactic ability of mature DCs toward CCL21 was abrogated only by long-chain FAs.

CONCLUSIONS

The data suggest that intraluminal exposure to long- and medium-chain FAs may differentially modulate the immune functions of intestinal DCs.

It is well documented that inflammatory chemokines play a significant role in the development of multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE). Recently, the involvement of homeostatic (or lymphoid) chemokines in the pathogenesis of autoimmune diseases has become an object of intensive study. In this work, quantitative analysis of CCL19, CCL21 and CCR7 expression in the central nervous system (CNS), as well as in inflammatory mononuclear cells isolated from several organs during the first attack, remission and the second attack of chronic-relapsing EAE (ChREAE), was performed. Using real-time PCR, RNAse Protection Assay and immunohistochemistry, the expression of both chemokines, as well as of their common receptor CCR7, was analyzed in the brain, spleen, lymph nodes and peripheral blood mononuclear cells. Increased expression of CCL19 and CCL21 was observed mostly in mononuclear inflammatory cells isolated from the CNS during active ChREAE. At the same time the expression of CCR7 in blood mononuclear leukocytes was reduced. This observation extends our current knowledge about the possible role of chemokines CCL19, CCL21 and their receptor CCR7 in the pathogenesis of ChREAE and, by extension, MS.

This paper presents a series of 10 hypotheses on the etiology of type 1 diabetes. We begin with the hypothesis that wheat gluten is one of the elusive environmental triggers in type 1 diabetes. Habitual consumption of wheat gluten increases the intestinal synthesis of dipeptidyl peptidase IV. This enzyme helps to shape the repertoire of peptides released into the small intestine following the ingestion of wheat gluten by catalyzing the release of X-Pro dipeptides from the N-terminus of the proline-rich glutenins and gliadins in wheat gluten. The release of gluten-derived peptides causes the tight junctions of the small intestine to open through a zonulin-dependent mechanism, which allows these peptides to enter the lamina propria where they get presented as antigens by HLA-DQ, -DR and CD1d molecules. Binding of one or more gluten peptides by CD1d leads to abrogation of oral tolerance, and a marked increase in peripheral immune responses to wheat proteins. Furthermore, it is our contention, that in response to beta cell apoptosis during normal remodeling of the pancreas and CCL19/CCL21 expression within the pancreatic lymph nodes (PLNs), gluten-loaded dendritic cells migrate from the small intestine to the PLNs. These dendritic cells present gluten-derived antigens on the surface of the PLNs, which leads to migration of CD4(-)CD8(-) gammadelta and CD4(-)CD8(+) alphabeta T cells to the pancreas where they mediate Fas and perforin dependent cytotoxicity. We also hypothesize that at least one of the type 1 diabetes associated HLA-DR molecules that bind and present wheat-derived peptide(s) also bind and present an islet cell antigen(s), activating plasma cell synthesis of islet cell autoantibodies and irrevocable, complement-dependent destruction of islet cells. Our final two hypotheses state that type 1 diabetes morbidity is reduced in those areas of globe where genetically susceptible individuals get adequate amounts of vitamin D, in the diet and/or through exposure to sunlight, and in areas where people are exposed to bacterial, viral, or parasitic infections in early childhood.

The lack of immune cells in mid-gestational fetal skin is often mentioned as a key factor underlying scarless healing. However, the scarless healing ability is conserved until long after the immune system in the fetus is fully developed. Therefore, we studied human second-trimester fetal skin and compared the numbers of immune cells and chemokine levels from fetal skin with adult skin. By using immunohistochemistry, we show that healthy fetal skin contains significant lower numbers of CD68(+) -macrophages, Tryptase(+) -mast cells, Langerin(+) -Langerhans cells, CD1a(+) -dendritic cells, and CD3(+) -T cells compared to adult skin. Staining with an early lineage leukocyte marker, i.e., CD45, verified that the number of CD45(+) -immune cells was indeed significantly lower in fetal skin but that sufficient numbers of immune cells were present in the fetal lymph node. No differences in the vascular network were observed between fetal and adult skin. Moreover, significant lower levels of lymphocyte chemokines CCL17, CCL21, and CCL27 were observed in fetal skin. However, levels of inflammatory interleukins such as IL-6, IL-8, and IL-10 were undetectable and levels of CCL2 were similar in healthy fetal and adult skin. In conclusion, this study shows that second-trimester fetal skin contains low levels of immune cells and leukocyte chemokines compared to adult skin. This immune cell deficiency includes CD45(+) leukocytes, despite the abundant presence of these cells in the lymph node. The immune deficiency in healthy second-trimester fetal skin may result in reduced inflammation during wound healing, and could underlie the scarless healing capacities of the fetal skin.

Lymphadenopathy in autoimmune and other lymphoproliferative diseases is in part characterized by immunoblasts and vascular proliferation. The lymph node vasculature, along with the nonvascular stromal compartment, supports lymphocyte function, and targeting vascular-stromal expansion in inflamed nodes may modulate lymphocyte function in disease. CD11c(+) cells are essential for vascular-stromal proliferation and the upregulation of vascular endothelial growth factor (VEGF) needed for vascular proliferation. However, targetable CD11c(+) cell-derived molecular mediators, the identity of relevant CD11c(+) cells, and whether CD11c(+) cells directly stimulate VEGF-expressing stromal cells are poorly understood. In this study we show that CD11c(+) CD11b(+) CCR2-dependent monocytes and CCR7-dependent dendritic cells express IL-1β. IL-1β blockade, IL-1β deficiency in radiosensitive cells, and CCR2/CCR7 double deficiency but not single deficiency all attenuate immunization-induced vascular-stromal proliferation. gp38(+) stromal fibroblastic reticular cells (FRCs) that express VEGF are enriched for Thy1(+) cells and partially overlap with CCL21-expressing FRCs, and FRC VEGF is attenuated with IL-1β deficiency or blockade. IL-1β localizes to the outer borders of the T zone, where VEGF-expressing cells are also enriched. Ex vivo, CD11b(+) cells enriched for IL-1β(+) cells can directly induce cultured gp38(+)Thy1(+) FRCs to upregulate VEGF. Taken together, these results suggest a mechanism whereby multiple recruited CD11c(+) populations express IL-1β and directly modulate FRC function to help promote the initiation of vascular-stromal growth in stimulated lymph nodes. These data provide new insight into how CD11c(+) cells regulate the lymph node vascular-stromal compartment, add to the evolving understanding of functional stromal subsets, and suggest a possible utility for IL-1β blockade in preventing inflammatory lymph node growth.

We have learned various data on the role of purinoceptors (P2X4, P2X7, P2Y6 and P2Y12 receptors) expressed in spinal microglia and several factors that presumably activate microglia in neuropathic pain after peripheral nerve injury. Especially P2X4 receptors (P2X4Rs) make a critical contribution to the pain processing. P2X4Rs of microglia might be promising targets for treating neuropathic pain. A predicted therapeutic benefit of interfering with microglial P2X4Rs may be that normal pain sensitivity would be unaffected since expression or activity of most of these receptors are upregulated or enhanced predominantly in activated microglia in the spinal cord where damaged sensory fibers project. Recently, we found that CCL21 regulates the expression of P2X4Rs in different manners, respectively. These new findings also provide novel targets for developing anti-neuropathic pain medicines.

Lenalidomide, an analog of thalidomide, modified responses of stimulated T cells from healthy young (ages 21-40 years) and old (≥ age 65 years) subjects. At 0.03 μM to 1 μM, lenalidomide enhanced generation of IL-2 and IFN-γ by T cell receptor-stimulated T cells of young subjects up to respective maximum increases of 17-fold and three-fold, but at 0.3 μM and 1 μM suppressed IL-17 generation. The same concentrations of lenalidomide enhanced IL-2 and IFN-γ generation by stimulated T cells of old subjects more, with greater respective maximal increases of up to 120-fold and six-fold, without suppressing IL-17 generation. Lenalidomide enhanced proliferation and suppressed apoptosis of stimulated T cells from old subjects, by IL-2-dependent mechanisms, and restored diminished T cell chemotactic responses to CCL21 and sphingosine 1-phosphate. The reversal of T cell abnormalities of immunosenescence by low concentrations of lenalidomide suggest a potential for improvement of immunity in the elderly.

BACKGROUND

Vitiligo is an autoimmune depigmentation disease, and defects in regulatory T cells (Tregs) have been proposed in the pathogenesis of generalized vitiligo (GV). However, the role of programmed cell death (PD)1(+) Tregs has not been studied.

OBJECTIVE

To investigate the status of Tregs, PD1(+) Tregs and associated parameters in active GV (aGV) during the first episode of disease attack and to establish the clinical correlation.

METHODS

The percentages of circulating Tregs, PD1(+) Tregs and CD3(+) CD4(+) PD1(+) T cells were evaluated in 50 patients with aGV and 51 controls. Expression levels of FOXP3, TGFB1, CTLA4 and genes for chemokine receptors (CCR4, CCR7) and their ligands (CCL21, CCL22) were quantified in peripheral blood and in lesional, perilesional, nonlesional and normal skin sections. The corresponding proteins were immunolocalized in tissue of aGV.

RESULTS

The percentage of Tregs was decreased (P = 0·001) and that of PD1(+) Tregs increased (P = 0·001) in peripheral blood of patients with aGV compared with controls. The abundance of TGFB1 and CCL21 mRNA was significantly decreased in the peripheral blood of patients with aGV. Significant differences in forkhead box P3, transforming growth factor-β and CCL21 protein expression were found in skin sections.

CONCLUSIONS

Deficiency in Treg frequency and decreased expression of Treg-associated parameters (TGFB and CCL21) suggested a possible defect in Tregs that may alter their suppression function and skin homing in aGV. The increased PD1(+) Tregs suggests that the PD1/PD ligand pathway may be involved in aGV and may have a role in Treg exhaustion. Further study is required to delineate the effect of PD1 in regulating Treg function in aGV.

Navigation of cells along gradients of guidance cues is a determining step in many developmental and immunological processes. Gradients can either be soluble or immobilized to tissues as demonstrated for the haptotactic migration of dendritic cells (DCs) toward higher concentrations of immobilized chemokine CCL21. To elucidate how gradient characteristics govern cellular response patterns, we here introduce an in vitro system allowing to track migratory responses of DCs to precisely controlled immobilized gradients of CCL21. We find that haptotactic sensing depends on the absolute CCL21 concentration and local steepness of the gradient, consistent with a scenario where DC directionality is governed by the signal-to-noise ratio of CCL21 binding to the receptor CCR7. We find that the conditions for optimal DC guidance are perfectly provided by the CCL21 gradients we measure in vivo. Furthermore, we find that CCR7 signal termination by the G-protein-coupled receptor kinase 6 (GRK6) is crucial for haptotactic but dispensable for chemotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. These findings suggest that stable, tissue-bound CCL21 gradients as sustainable "roads" ensure optimal guidance in vivo.

During human immunodeficiency virus (HIV) infection, enhanced migration of infected cells to lymph nodes leads to efficient propagation of HIV-1. The selective chemokine receptors, including CXCR4 and CCR7, may play a role in this process, yet the viral factors regulating chemokine-dependent T cell migration remain relatively unclear. The functional cooperation between the CXCR4 ligand chemokine CXCL12 and the CCR7 ligand chemokines CCL19 and CCL21 enhances CCR7-dependent T cell motility in vitro as well as cell trafficking into the lymph nodes in vivo. In this study, we report that a recombinant form of a viral CXCR4 ligand, X4-tropic HIV-1 gp120, enhanced the CD4 T cell response to CCR7 ligands in a manner dependent on CXCR4 and CD4, and that this effect was recapitulated by HIV-1 virions. HIV-1 gp120 significantly enhanced CCR7-dependent CD4 T cell migration from the footpad of mice to the draining lymph nodes in in vivo transfer experiments. We also demonstrated that CXCR4 expression is required for stable CCR7 expression on the CD4 T cell surface, whereas CXCR4 signaling facilitated CCR7 ligand binding to the cell surface and increased the level of CCR7 homo- as well as CXCR4/CCR7 hetero-oligomers without affecting CCR7 expression levels. Our findings indicate that HIV-evoked CXCR4 signaling promotes CCR7-dependent CD4 T cell migration by up-regulating CCR7 function, which is likely to be induced by increased formation of CCR7 homo- and CXCR4/CCR7 hetero-oligomers on the surface of CD4 T cells.

The close cooperation of both innate and acquired immunity is essential for the induction of truly effective antitumor immunity. We tested a strategy to enhance the cross-talk between NKT cells and conventional antigen-specific T cells with the use of alpha GalCer-loaded dendritic cells genetically engineered to express antigen plus chemokine, attracting both conventional T cells and NKT cells. DC genetically engineered to express a model antigen, OVA, along with SLC/CCL21 or monokine induced by IFN-gamma/CXCL9, had been generated using a method based on in vitro differentiation of DC from mouse ES cells. The ES-DC were loaded with alpha-GalCer and transferred to mice bearing MO4, an OVA-expressing melanoma, and their capacity to evoke antitumor immunity was evaluated. In vivo transfer of either OVA-expressing ES-DC, stimulating OVA-reactive T cells, or alpha-GalCer-loaded non-transfectant ES-DC, stimulating NKT cells, elicited a significant but limited degree of protection against the i.p. disseminated MO4. A more potent antitumor effect was observed when alpha-GalCer was loaded to ES-DC expressing OVA before in vivo transfer, and the effect was abrogated by the administration of anti-CD8, anti-NK1.1 or anti-asialo GM1 antibody. alpha-GalCer-loaded double transfectant ES-DC expressing SLC along with OVA induced the most potent antitumor immunity. Thus, alpha-GalCer-loaded ES-DC expressing tumor-associated antigen along with SLC can stimulate multiple subsets of effector cells to induce a potent therapeutic effect against peritoneally disseminated tumor cells. The present study suggests a novel way to use alpha-GalCer in immunotherapy for peritoneally

The chemokine CCL21 is a potent chemoattractant for T cells and dendritic cells. IL-15 elicits powerful antitumor immune responses through the stimulation of natural killer cells. We constructed a CCL21/IL-15-expressing adenovirus (Ad-CCL21-IL-15) and evaluated its antitumor effects in vitro and in vivo. We found that the intratumoral injection of Ad-CCL21-IL-15 into murine colon carcinomas significantly inhibited tumor growth. Splenocytes from mice treated with Ad-CCL21-IL-15 developed tumor-specific cytotoxic T cells and were protected from subsequent challenges with tumor cells. This study indicates that providing cancer therapy by combining CCL21 and IL-15 can induce antitumor immune responses and is an effective strategy for cancer immunotherapy.

Two approaches have been pursued to elicit antitumour immunity: (i) induce recruitment of immature dendritic cells or their precursors at a site of antigen delivery, and (ii) induce activation of tumour-infiltrating dendritic cells (DCs). The recruitment of selected DC subtype conditions the class of the immune response. Each immature DC population displays a unique spectrum of chemokine responsiveness. For examples, Langerhans cells (LCs) migrate selectively in response to CCL20/MIP-3alpha (through CCR6), blood CD11c+ DC to MCP chemokines (through CCR2). All these chemokines are inducible in response to inflammatory stimuli. CCL20/MIP-3alpha in particular is only detected within inflamed epithelium, at the site of antigen entry, which is infiltrated by immature DCs. Furthermore, to reach the site of injury, sequential responsiveness might operate, blood DC precursors are recruited by a set of chemokines (MIP, MCP) while within the tissue other chemokines will direct their navigation (CCL20/MIP-3alpha). Of interest, when injected in vivo together with antigen, MCP-4/CCL13, but not CCL20/MIP-3alpha, recruits blood monocytes or blood DC precursors that promptly differentiate into typical DCs and that improve antitumour immune responses. After antigen uptake, DCs acquire, upon maturation, responsiveness to CCR7 ligands (CCL21/SLC/6Ckine, CCL19/ELC/MIP-3beta) due to receptor up-regulation. In particular, in the periphery, CCL21/SLC/6Ckine expressed by lymphatic vessels may direct into the lymph stream, antigen-loaded maturing DCs leaving the site of infection; while within lymph-node, CCL21/SLC/6Ckine plays a critical role in the entry of naïve T cells from the blood through HEV. In regard to its central role, we decided to investigate whether the expression of CCL21/SLC/6Ckine in tumour may lead to antitumour immune responses. C26 colon carcinoma tumour cell line transduced with CCL21/SLC/6Ckine showed reduced tumorigenicity when injected in vivo into immunocompetent mice. The protection was CD8 dependent and associated with an important intratumoral infiltration of DCs. Most tumour infiltrating DCs (TIDCs) had an immature phenotype, were able to present TAA in the context of MHC class I, but were refractory to stimulation with the combination of LPS, IFNgamma and anti-CD40 antibody. TIDC paralysis could be reverted, however, by in vitro or in vivo stimulation with the combination of a CpG immunostimulatory sequence and an anti-interleukin 10 receptor (IL10R) antibody. CpG or anti-IL10R alone were inactive in TIDC, while CpG triggered activation in normal DC. In particular, CpG plus anti-IL10R enhanced the TAA-specific immune response and triggered de novo IL-12 production. Subsequently, CpG plus anti-IL10R treatment showed robust antitumour therapeutic activity exceeding by far that of CpG alone, and elicited antitumour immune memory.

CCL21 is known to attract dendritic cells (DCs) and T cells that may reverse tumor-mediated immune suppression. The massive infiltration of tumors by regulatory T cells (Tregs) prevents the development of a successful helper immune response. In this study, we investigated whether elimination of CD4(+) CD25(+) Tregs in the tumor microenvironment using anti-CD25 monoclonal antibodies (mAbs) was capable of enhancing CCL21-mediated antitumor immunity in a mouse hepatocellular carcinoma (HCC) model. We found that CCL21 in combination with anti-CD25 mAbs (PC61) resulted in improved antitumor efficacy and prolonged survival, not only inhibited tumor angiogenesis and cell proliferation, but also led to significant increases in the frequency of CD4(+), CD8(+) T cells and CD11c(+) DCs within the tumor, coincident with marked induction of tumor-specific CD8(+) cytotoxic T lymphocytes (CTLs) at the local tumor site. The intratumoral immune responses were accompanied by the enhanced elaboration of IL-12 and IFN-γ, but reduced release of the immunosuppressive mediators IL-10 and TGF-β1. The results indicated that depletion of Tregs in the tumor microenvironment could enhance CCL21-mediated antitumor immunity, and CCL21 combined with anti-CD25 mAbs may be a more effective immunotherapy to promote tumor rejection.

CCR7 and its ligands CCL19 and CCL21 are a key chemokine system in T cell priming in secondary lymphoid organs, and are rarely expressed in normal muscle tissue. We immunohistochemically investigated the expression of this chemokine system in the muscles of seven patients with inclusion body myositis (IBM). In all cases, CCR7+ mononuclear cells infiltrated in the endomysium, preferentially surrounded and invaded nonnecrotic muscle fibers. Double immunostaining revealed that such CCR7+ mononuclear cells included BDCA-1+ myeloid dendritic cells as well as CD8+ cells, CD4+ cells and CD68+ macrophages. On the other hand, CCL19 was widely expressed on muscle fibers including those invaded by mononuclear cells. CCL19 was also expressed diffusely on endomysial mononuclear cells and endothelium of vessels. Immunoreactivities of CCL21 were detected on some muscle fibers and mononuclear cells. By RT-PCR analyses, mRNA of CCR7 was detected in all the patients and that of CCL19 and CCL21 was detected in six. These findings showed that the CCL19, CCL21/CCR7 chemokine system is expressed in muscles of IBM. The chemokine mediated attraction in dendritic and other immune cells and muscle cells may be crucial in sustained antigen presentation, T cell activation and immune attack to muscles in the pathogenesis of IBM.

Dendritic cells (DCs) are powerful antigen-presenting cells (APCs), that have so far been applied for cancer specific immunotherapy. Recent results suggest that matured DCs derived from human monocytes have a significant impact on the outcome of vaccination. The conventional generation of mature DCs from human monocytes in vitro has been reported to require 5 days for differentiation with granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and 2 days for stimulation. We herein report a new strategy for the functional maturation of monocyte-derived DCs within only 2 days of in vitro culture and the induction of specific cytotoxic T lymphocytes (CTLs) to tumor rejection peptide. The monocytes were incubated for 1 day with GM-CSF and IL-4, followed by activation with a bacterial product, OK-432 and prostaglandin E2 (PGE2) for another 1 day (rapid DC). Rapid DC expressed mature DC surface markers as well as chemokine receptor 7 and secreted Th1-type cytokines. The DCs generated in this study mobilized Ca2+ in response to CCL21/6Ckine and SDF-1, but only marginally did so to Mip-1alpha. Moreover, when rapid DC were compared with mature conventional 7-day DCs, they were equally potent in inducing specific CTLs in vitro. These results indicate that the rapid DC is as effective as the monocyte-derived conventional DCs. The rapid DC would be a potentially useful new cancer-specific immunotherapy.

Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

Metastasis is an important cause of cancer-related mortality. In this study, we investigated the role of CCR7 in the lymph node metastasis of tongue carcinoma. Immunohistochemistry and Western blot revealed the expression of CCR7 in tongue SCC tissues and cell lines. In addition, we examined the expression of CCL21, a ligand of CCR7, in normal and diseased lymph nodes using immunohistochemistry and/or real-time PCR. The CCR7 expression was significantly correlated with cervical lymph node metastasis, tumor staging, and histological grade (P = 0.015, 0.040, and 0.015, respectively). The multivariate analysis showed that regional lymph node metastasis, the expression of CCR7, and LVD were the independent poor prognostic factors. Knockdown of CCR7 gene resulted in a significant inhibition of migration and invasion of SCC4 cells in vitro without affecting the proliferation and apoptosis of tumor cells. Also, CCR7 knockdown obviously inhibited cervical lymph node metastasis in an animal tumor model. Our study indicated that CCR7 may play an important role in progression of tongue SCC and could be a promising target for tongue SCC therapy.