Needle-free vaccine delivery has become a global priority, both to eliminate the risk of improper and unsafe needle use and to simplify vaccination procedures. In pursuit of greater ease of vaccination, a number of needle-free delivery routes have been explored, with mucosal routes being perhaps the most prominent. Since the vaccine administration route significantly affects immune responses, numerous researchers are attempting to develop alternative vaccine delivery methods including a mucosal route. My group's recent studies demonstrate the potential of the sublingual (s.l.) route for delivering vaccines capable of inducing mucosal as well as systemic immune responses. Sublingual administration conferred effective protection against a lethal challenge with influenza virus (H1N1) or genital papillomavirus. Moreover, CCR7-CCL19/CCL21-regulated dendritic cells are responsible for activation of T and B cells following s.l. administration. This review highlights current knowledge about the safety and effectiveness of s.l. vaccination and describes how s.l. vaccination can induce both systemic and mucosal immunity.

Multiple injections of low-dose streptozotocin (MLDS) induce lymphocytic insulitis and diabetes in rodents. To test whether the influx of inflammatory cells was associated with changes in the expression of chemokines, we measured the expression of all known chemokine ligands by real-time quantitative PCR in isolated islets. With the exception of CCL20 and CCL19, chemokines were not significantly expressed in islets from wild-type mice before MLDS treatment. Ten days after treatment, the expression of several chemokines, including CXCL9, CCL1, CXCL10, and CCL21, was dramatically up-regulated. The expression of CCL1, CXCL9, and CCL21 protein was confirmed by immunohistochemistry and was mostly associated with the infiltrating cells. The mouse herpesvirus 68-encoded chemokine decoy receptor M3 can broadly engage these chemokines with high affinity. To test whether a blockade of chemokine function would alter the onset or magnitude of insulitis and diabetes, we used transgenic mice expressing M3 in beta cells (rat insulin promoter (RIP)-M3 mice). RIP-M3 mice were normoglycemic and responded normally to glucose challenge but were remarkably resistant to diabetes induced by MLDS. Islets from MLDS-treated RIP-M3 mice had fewer inflammatory cells and expressed lower levels of chemokines than those from MLDS-treated controls. The role of M3 in chemokine blockade during insulitis was further supported by in vitro experiments demonstrating that multiple chemokines up-regulated during islet inflammation are high-affinity M3 ligands that can be simultaneously sequestered. These results implicate chemokines as key mediators of insulitis and suggest that their blockade may represent a novel strategy to prevent insulitis and islet destruction.

Stromal cells generate a complex cellular scaffold that provides specialized microenvironments for lymphocyte activation in secondary lymphoid organs. Here, we assessed whether local activation of stromal cells in the central nervous system (CNS) is mandatory to transfer immune recognition from secondary lymphoid organs into the infected tissue. We report that neurotropic virus infection in mice triggered the establishment of such stromal cell niches in the CNS. CNS stromal cell activation was dominated by a rapid and vigorous production of CC-motif chemokine receptor (CCR) 7 ligands CCL19 and CCL21 by vascular endothelial cells and adjacent fibroblastic reticular cell (FRC)-like cells in the perivascular space. Moreover, CCR7 ligands produced by CNS stromal cells were crucial to support recruitment and local re-activation of antiviral CD8(+) T cells and to protect the host from lethal neuroinflammatory disease, indicating that CNS stromal cells generate confined microenvironments that control protective T cell immunity.

Our previous in vitro studies led to proposals that the atypical chemokine receptor CCX-CKR is a scavenger of CCR7 ligand homeostatic chemokines. In the present study, we generated CCX-CKR(-/-) mice and confirm this scavenger function in vivo. Compared with wild-type mice, CCX-CKR(-/-) have a 5-fold increase in the level of CCL21 protein in blood, and 2- to 3-fold increases in CCL19 and CCL21 in peripheral lymph nodes. The effect of these protein increases on immunity was investigated after immunization with MOG(35-55) peptide emulsified in complete Freund adjuvant (CFA). The subsequent characteristic paralysis develops with enhanced kinetics and severity in CCX-CKR(-/-) versus wild-type mice. Despite this effect, antigen-specific immune responses in the draining lymph nodes are diminished in CCX-CKR(-/-) mice. Instead, the earlier onset of disease is associated with enhanced T-cell priming in the CCX-CKR(-/-) spleen and a skewing of CD4(+) T-cell responses toward Th17 rather than Th1. This observation correlates with increased expression of IL-23 in the CCX-CKR(-/-) spleen and increased CCL21 levels in the central nervous system postimmunization. The early onset of disease in CCX-CKR(-/-) mice is reversed by systemic administration of neutralizing anti-CCL21 antibodies. Thus, by regulating homeostatic chemokine bioavailability, CCX-CKR influences the localization, kinetics, and nature of adaptive immune responses in vivo.

c-Jun N-terminal kinase (JNK) is generally thought to be involved in inflammation, proliferation and apoptosis. However, functional role(s) of this molecule in dendritic cells (DCs) has not been well understood. CCR7 ligands, CCL19 and CCL21, induce not only chemotaxis but also endocytosis in mature DCs. In the present study, we examined the role of JNK for inducing chemotaxis and endocytosis in murine mature DCs. CCL19 rapidly enhanced endocytosis of mature DCs within a few minutes, whereas significant migration of mature DCs to this chemokine was detected 30 min or more after incubation. CCL19 significantly activated JNK in mature DCs at 15 min. CCL19 also increased interaction between phospho-JNK and phospho-mitogen-activated protein kinase kinase (MKK) 4 but not phospho-MKK7 in mature DCs, suggesting that the JNK activation is mediated via MKK4. Blocking of this JNK activation significantly inhibited the CCL19-induced migration of mature DCs. Blocking of Rho-associated kinase also inhibited the CCL19-induced migration without affecting the JNK activation. On the other hand, the inhibition of either JNK or Rho-associated kinase showed no significant effects on CCL19-induced endocytosis by mature DCs. These findings suggest that CCL19 activates JNK via a Rho-independent pathway, thereby inducing migration of mature DCs, whereas the JNK activation is dispensable for the CCL19-induced endocytosis. It seems that at least two different pathways, JNK pathway and Rho-associated kinase pathway, are involved in the CCR7-mediated migration of mature DCs. Thus, we demonstrate herein a novel role of JNK for regulating chemokine-induced DC migration.

In hepatitis C virus (HCV) infection the immune response is ineffective, leading to chronic hepatitis and liver damage. Primed CD8 T cells are critical for antiviral immunity and subsets of circulating CD8 T cells have been defined in blood but these do not necessarily reflect the clonality or differentiation of cells within tissue. Current models divide primed CD8 T cells into effector and memory cells, further subdivided into central memory (CCR7+, L-selectin+), recirculating through lymphoid tissues and effector memory (CCR7-, L-selectin-) mediating immune response in peripheral organs. We characterized CD8 T cells derived from organ donors and patients with end-stage HCV infection to show that: 1) all liver-infiltrating CD8 T cells express high levels of CD11a, indicating the effective absence of naive CD8 T cells in the liver. 2) The liver contains distinct subsets of primed CD8+ T cells including a population of CCR7+ L-selectin- cells, which does not reflect current paradigms. The expression of CCR7 by these cells may be induced by the hepatic microenvironment to facilitate recirculation. 3) The CCR7 ligands CCL19 and CCL21 are present on lymphatic, vascular, and sinusoidal endothelium in normal liver and in patients with HCV infection. We suggest that the recirculation of CCR7+/L-selectin- intrahepatic CD8 T cells to regional lymphoid tissue will be facilitated by CCL19 and CCL21 on hepatic sinusoids and lymphatics. This centripetal pathway of migration would allow restimulation in lymph nodes, thereby promoting immune surveillance in normal liver and renewal of effector responses in chronic viral infection.

OBJECTIVE

We have characterized the expression and the function of the chemokine receptor CCR7 on fibroblast-like synoviocytes (FLS) of patients with RA and OA and on dermal fibroblasts.

METHODS

FLS were obtained after enzymatic digestion of synovial tissue (ST) of patients with RA and OA undergoing knee replacement surgery and taken into culture for chemokine receptor analysis by RT-PCR, flow cytometry and functional tests. Immunofluorescence for CCR7, fibroblast and T-cell markers was performed on ST of RA and OA patients. To study the response of FLS to CCR7 ligands and other chemokines, migration assays were performed in modified Boyden chambers. After stimulation of FLS with CCR7 ligands, the secretion of VEGF was evaluated by ELISA and Luminex.

RESULTS

CCR7 is expressed on FLS of patients with RA and OA, but not on dermal fibroblasts. FLS migrated in response to the CCR7 ligands, CCL19 and CCL21. Stimulation of FLS with CCL19 resulted in a significantly increased secretion of VEGF of RA- and OA-FLS.

CONCLUSIONS

Apart from the migration of FLS in response to CCL19 and CCL21, it was shown that activation of the CCR7 receptor on FLS results in an enhanced VEGF secretion. A considerable expression of CCR7 ligands in proximity to perivascular infiltrates has previously been described in inflamed synovial tissue of RA patients. Stimulation of FLS via CCR7 could thereby contribute to angiogenesis in the synovial tissue.

BACKGROUND

The earliest endoscopically-evident lesion in Crohn's disease is the aphthous ulcer, which develops over ectopic lymphoid tissues (ie, inducible lymphoid follicles (ILF), tertiary lymphoid tissue (TLT)) in the chronically inflamed intestine. ILF/TLT are induced within effector sites by homeostatic lymphoid chemokines, but their role in the development of intestinal ILF/TLT and in the pathogenesis of Crohn's disease is poorly understood.

METHODS

Using a mouse model of Crohn's-like ileitis (TNFARE) which develops florid induction of ILF/TLT within its terminal ileum, the contribution of the CCR7/CCL19/CCL21 chemokine axis during the development of TLT and its role in disease pathogenesis were assessed.

RESULTS

Both CCL19 and CCL21 were increased within the inflamed ileum of TNFARE mice, which resulted in CCR7 internalisation and impaired T cell chemotaxis. ILF/TLT were a major source of CCL19 and CCL21 and increased local synthesis, augmented recruitment/retention of effector, naïve and central memory T cell subsets within the inflamed ileum. Immunoblockade of CCR7 resulted in further effector T cell retention and exacerbation of ileitis.

CONCLUSIONS

Induction of ILF/TLT in the chronically inflamed intestine alters the homeostatic CCL19-CCL21 lymphoid-chemokine gradient and increases recruitment/retention of effector CCR7+ T cell subsets within the terminal ileum, contributing to the perpetuation of chronic inflammation. Thus, blockade of CCR7 or its ligands might result in deleterious consequences for subjects with chronic inflammatory diseases.

Myeloablative conditioning before bone marrow transplantation (BMT) results in thymic epithelial cell (TEC) injury, T-cell immune deficiency, and susceptibility to opportunistic infections. Conditioning regimen-induced TEC damage directly contributes to slow thymopoietic recovery after BMT. Keratinocyte growth factor (KGF) is a TEC mitogen that stimulates proliferation and, when given before conditioning, reduces TEC injury. Some TEC subsets are refractory to KGF and functional T-cell responses are not fully restored in KGF-treated BM transplant recipients. Therefore, we investigated whether the addition of a pharmacologic inhibitor, PFT-beta, to transiently inhibit p53 during radiotherapy could spare TECs from radiation-induced damage in congenic and allogeneic BMTs. Combined before BMT KGF + PFT-beta administration additively restored numbers of cortical and medullary TECs and improved thymic function after BMT, resulting in higher numbers of donor-derived, naive peripheral CD4(+) and CD8(+) T cells. Radiation conditioning caused a loss of T-cell zone fibroblastic reticular cells (FRCs) and CCL21 expression in lymphoid stroma. KGF + PFT-beta treatment restored both FRC and CCL21 expression, findings that correlated with improved T-cell reconstitution and an enhanced immune response against Listeria monocytogenes infection. Thus, transient p53 inhibition combined with KGF represents a novel and potentially translatable approach to promote rapid and durable thymic and peripheral T-cell recovery after BMT.

Although dendritic cell (DC)-based immunotherapy is considered a promising approach for cancer treatment, a large quantity of DC vaccine is required for effective sensitization/activation of immune cells because of the poor migratory ability of administered DCs into regional lymphoid tissue. In this study, we created a DC vaccine sufficiently transduced with CC chemokine receptor-7 gene (CCR7/DCs) by applying RGD fiber-mutant adenovirus vector (AdRGD), and investigated its immunological characteristics and therapeutic efficacy. CCR7/DCs acquired strong chemotactic activity for CC chemokine ligand-21 (CCL21) and exhibited an immunophenotype similar to mature DCs but not immature DCs with regard to major histocompatibility complex/costimulatory molecule-expression levels and allogenic T cell proliferation-stimulating ability, while maintaining inherent endocytotic activity. Importantly, CCR7/DCs injected intradermally into mice could accumulate in draining lymph nodes about 5.5-fold more efficiently than control AdRGD-applied DCs. Reflecting these properties of CCR7/DCs, DC vaccine genetically engineered to simultaneously express endogenous antigen and CCR7 could elicit more effective antigen-specific immune response in vivo using a lower dosage than DC vaccine transduced with antigen alone. Therefore, the application of CCR7/DCs having positive migratory ability to lymphoid tissues may contribute to reduction of efforts and costs associated with DC vaccine preparation by considerably reducing the DC vaccine dosage needed to achieve effective treatment by DC-based immunotherapy.

Adoptive T-cell transfer has achieved significant clinical success in advanced melanoma. However, therapeutic efficacy is limited by poor T-cell survival after adoptive transfer and by inefficient trafficking to tumor sites. Here, we report that intratumoral expression of the chemokine CCL21 enhances the efficacy of adoptive T-cell therapy in a mouse model of melanoma. Based on our novel observation that CCL21 is highly chemotactic for activated OT-1 T cells in vitro and down-regulates expression of CD62L, we hypothesized that tumor cell-mediated expression of this chemokine might recruit, and retain, adoptively transferred T cells to the sites of tumor growth. Mice bearing metastatic tumors stably transduced with CCL21 survived significantly longer following adoptive T-cell transfer than mice bearing non-CCL21-expressing tumors. However, although we could not detect increased trafficking of the adoptively transferred T cells to tumors, tumor-expressed CCL21 promoted the survival and cytotoxic activity of the adoptively transferred T cells and led to the priming of antitumor immunity following T-cell transfer. To translate these observations into a protocol of real clinical usefulness, we showed that adsorption of a retrovirus encoding CCL21 to OT-1 T cells before adoptive transfer increased the therapeutic efficacy of a subsequently administered dose of OT-1 T cells, resulting in cure of metastatic disease and the generation of immunologic memory in the majority of treated mice. These studies indicate a promising role for CCL21 in enhancing the therapeutic efficacy of adoptive T-cell therapy.

Artery tertiary lymphoid organs (ATLOs) are atherosclerosis-associated lymphoid aggregates with varying degrees of complexity ranging from small T/B-cell clusters to well-structured lymph node-like though unencapsulated lymphoid tissues. ATLOs arise in the connective tissue that surrounds diseased arteries, i.e., the adventitia. ATLOs have been identified in aged atherosclerosis-prone hyperlipidemic apolipoprotein E-deficient (ApoE-/-) mice: they are organized into distinct immune cell compartments, including separate T-cell areas, activated B-cell follicles, and plasma cell niches. Analyses of ATLO immune cell subsets indicate antigen-specific T- and B-cell immune reactions within the atherosclerotic arterial wall adventitia. Moreover, ATLOs harbor innate immune cells, including a large component of inflammatory macrophages, B-1 cells, and an aberrant set of antigen-presenting cells. There is marked neoangiogenesis, irregular lymphangiogenesis, neoformation of high endothelial venules, and de novo synthesis of lymph node-like conduits. Molecular mechanisms of ATLO formation remain to be identified though media vascular smooth muscle cells may adopt features of lymphoid tissue organizer-like cells by expressing lymphorganogenic chemokines, i.e., CXCL13 and CCL21. Although these data are consistent with the view that ATLOs participate in primary T- and B-cell responses against elusive atherosclerosis-specific autoantigens, their specific protective or disease-promoting roles remain to be identified. In this review, we discuss what is currently known about ATLOs and their potential impact on atherosclerosis and make attempts to define challenges ahead.

Dendritic cells (DCs) are the most potent antigen-presenting cells, yet little data are available on the differential characteristics of donor and recipient DCs (dDCs and rDCs, respectively) during the process of islet allograft rejection. DTR-GFP-DC mice provide a novel tool to monitor DC trafficking and characteristics during allograft rejection. We show rapid migration of dDCs to recipient lymphoid tissues as early as 3 h post-islet allotransplantation. Compared with rDCs, dDCs express different patterns of chemokine receptors, display differential proliferative capacity, and exhibit a higher level of maturity; these findings could be attributed to the effects of injury that dDCs undergo during islet cell preparation and engraftment. Intriguingly, we detected dDCs in the spleen of recipients long after rejection of islet allografts. Given that dDCs express high levels of CCR7, islets were cultured before transplant with the ligand for CCR7 (CCL21). This novel method, which enabled us to enhance the efflux of dDCs from islet preparations, resulted in a prolongation of islet allograft survival in immunocompetent recipients. This study introduces dDCs and rDCs as two distinct types of DCs and provides novel data with clinical implications to use chemokine-based DC-depleting strategies to prolong islet allograft survival.

CD3(-)CD4(+)CD45(+) inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer's patches (PP) in the aerodigestive tract. CXCL13(-/-) mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3(-)CD4(+)CD45(+) cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13(-/-) and plt/plt mice, the formation of germinal centers in CXCL13(-/-) mice was affected, and their population of B cells was much lower than in the NALT of CXCL13(+/-) mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.

We analyzed the modulation of human B cell chemotaxis by the gp120 proteins of various HIV-1 strains. X4 and X4/R5 gp120 inhibited B cell chemotaxis toward CXCL12, CCL20, and CCL21 by 40-50%, whereas R5 gp120 decreased inhibition by 20%. This gp120-induced inhibition was strictly dependent on CXCR4 or CCR5 and lipid rafts but not on CD4 or V(H)3-expressing BCR. Inhibition did not impair the expression or ligand-induced internalization of CCR6 and CCR7. Our data suggest that gp120/CXCR4 and gp120/CCR5 interactions lead to the cross-desensitization of CCR6 and CCR7 because gp120 does not bind CCR6 and CCR7. Unlike CXCL12, gp120 did not induce the activation of phospholipase Cbeta3 and PI3K downstream from CXCR4, whereas p38 MAPK activation was observed. Similar results were obtained if gp120-treated cells were triggered by CCL21 and CCL20. Our results are consistent with a blockade restricted to signaling pathways using phosphatidylinositol-4,5-bisphosphate as a substrate. X4 and X4/R5 gp120 induced the cleavage of CD62 ligand by a mechanism dependent on matrix metalloproteinase 1 and 3, CD4, CXCR4, Galpha(i), and p38 MAPK, whereas R5 gp120 did not. X4 and X4/R5 gp120 also induced the relocalization of cytoplasmic CD95 to the membrane and a 23% increase in CD95-mediated apoptosis. No such effects were observed with R5 gp120. The gp120-induced decrease in B cell chemotaxis and CD62 ligand expression, and increase in CD95-mediated B cell apoptosis probably have major deleterious effects on B cell responsiveness during HIV infection and in vaccination trials.

De novo lymphangiogenesis influences different pathological courses via modulating tissue fluid homeostasis, macromolecule absorption, and leukocyte transmigration. During the past decade, improved understanding of lymphatic biology, especially VEGF-C/-D/VEGFR-3-mediating lymphangiogenesis has substantially promoted clinical research in lymphatic insufficiency. The role of lymphangiogenesis in the setting of the inflammatory processes observed in transplants, inflamed cornea, wound healing, acquired lymphedema and tumor invasion, however, remains to be elucidated. The chemokine family of peptide chemoattractants and other mediators, e.g., CCL21-CCR7, D6, NF-kappaB and TNF-alpha may be important contributors to pathophysiological changes of lymphatic endothelial cells (LECs) and inflammatory lymphangiogenesis. Dendritic cells and macrophages may also involve in LEC proliferation and differentiation. Increased knowledge of LEC-specific modulators in inflammatory microenvironment is vital for prevention and treatment of lymphatic-associated diseases.

The physiologic function of the secondary lymphoid organs to recruit large numbers of naïve lymphocytes increases the probability that antigens encounter their rare, sometimes unique, specific T lymphocytes and initiate a specific immune response. In peripheral lymph nodes (LNs), this recruitment is a multistep process, initiated predominantly within the high endothelial venules (HEVs), beginning with rolling and chemokine-dependent firm adhesion of the lymphocytes on the venular endothelium surface. We report here that, in C57BL/6 mice, the recruitment of naïve lymphocytes is impaired in LNs draining a B16 melanoma tumor. Intravital microscopy analysis of the tumor-draining LNs revealed that this effect is associated with an important defect in lymphocyte adhesion in the HEVs and a progressive decrease in the expression of the LN chemokine CCL21. In parallel with these effects, the tumor up-regulated, essentially through a P-selectin-dependent mechanism, the rolling and sticking of circulating polymorphonuclear cells within the LN low-order venules where few rolling and sticking events are usually observed. These effects of the tumor were independent of the presence of metastasis into the LN and occurred as long as the tumor developed. Together, these results indicate that the tumor proximity disturbs the LN physiology by modifying the molecular, spatial, and cellular rules that usually control leukocyte-endothelium interactions into the peripheral LNs. In addition, they emphasize a new role for the low-order venules of the peripheral LNs, which compared with the HEVs, seem to be the preferential port of entry for cells linked to inflammatory processes.

Little is known about the dynamics of the interactions between thymocytes and other cell types, as well as the spatiotemporal distribution of thymocytes during positive selection in the microenvironment of the cortex. We used two-photon laser scanning microscopy of the mouse thymus to visualize thymocytes and dendritic cells (DCs) and to characterize their interactions in the cortex. We show that thymocytes make frequent contacts with DCs in the thymic cortex and that these associations increase when thymocytes express T cell receptors that mediate positive selection. We also show that cortical DCs and the chemokine CCL21 expression are closely associated with capillaries throughout the cortex. The overexpression of the chemokine receptor CCR7 in thymocytes results in an increase in DC-thymocyte interactions, while the loss of CCR7 in the background of a positive-selecting TCR reduces the extent of DC-thymocyte interactions. These observations identify a vasculature-associated microenvironment within the thymic cortex that promotes interactions between DCs and thymocytes that are receiving positive selection signals.

To examine the effects of high-fat diet (HFD) on melanoma progression, HFD-fed C57BL/6N mice were subcutaneously injected with syngeneic B16F10 melanoma cells. At 3 weeks post-injection, the tumors were resected; the mice were then sacrificed at 2 weeks post-resection. HFD stimulated melanoma growth and lymph node (LN) metastasis as well as tumor and LN lymphangiogenesis. Lipid vacuoles in the tumor and M2-macrophage (MΦ)s in the adipose and tumor tissues were increased in HFD-fed mice. CCL19 and CCL21 contents were higher in LNs than in tumors. HFD increased both CCL19 and CCL21 levels in LNs and CCR7 in tumors. Adipose tissue-conditioned media (CM) from HFD-fed mice enhanced lymphangiogenesis, and mature adipocyte (MA)/M2-MΦ co-culture CM markedly stimulated the tube formation of lymphatic endothelial cell (LEC)s and B16F10 migration. Monocyte migration was moderately stimulated by B16F10 or MA CM, but tremendously stimulated by B16F10/M2-MΦ co-culture CM, which was enhanced by MA/B16F10/M2-MΦ co-culture CM. The co-culture results revealed that MAs increased CCL2, M-CSF and CCR7 mRNAs in B16F10s; vascular endothelial growth factor (VEGF)-D mRNA in M2-MΦs; and CCL19, CCL21 and VEGF receptor (VEGFR)3 mRNA in LECs. M2-MΦs increased CCL2, M-CSF and VEGF-A mRNAs in B16F10s, whereas B16F10s increased VEGF-C mRNAs in M2-MΦs and VEGFR3 mRNA in LECs. These results indicate that in HFD-fed mice, MA-induced CCL2 and M-CSF in tumor cells increase M2-MΦs in tumor; the crosstalk between tumor cells and M2-MΦs further increases cytokines and angiogenic and lymphangiogenic factors. Additionally, MA-stimulated CCL19, CCL21/CCR7 axis contributes to increased LN metastasis in HFD-fed mice.

CD4(+)CD3(-) lymphoid tissue inducer (LTi) cells are crucial for the development and organisation of lymph nodes and gut associated lymphoid tissues. In this report, we characterise their appearance in the developing spleen and highlight their importance in relation to the development of splenic T cell zones. LTi cells were detected in embryonic spleen from embryonic day 13, although their progenitors were present at embryonic day 12. These cells clustered initially around splenic blood vessels in a lymphotoxin (LT)-independent manner, but up-regulation of VCAM-1 expression on stromal cells associated with the blood vessels was LT dependent. After birth, T cell colonisation of these clusters to form nascent white pulp areas was also LT dependent. Transfer experiments reconstituting RAG(-/-) mice with either WT or LTalpha(-/-) splenocytes demonstrated that lymphocyte expression of LT was not essential for the organisation of a discrete CD3(+) T cell zone with localised podoplanin and CCL21 expression. Our studies indicate that a combination of LT signals from LTi cells and LT-independent signals from lymphocytes is sufficient for expression of podoplanin and CCL21 on splenic T cell zone stroma and subsequent T cell organisation.

Human papillomavirus (HPV) type 16 infects the epithelial layer of cervical mucosa and is causally associated with the generation of cervical cancer. Langerhans cells (LC) are the resident APCs at the site of infection and therefore are responsible for initiating an immune response against HPV16. On the contrary, LC exposed to HPV16 do not induce a specific T cell immune response, which leads to the immune evasion of HPV16. Demonstrating that TLR7 and TLR8 are expressed on human LC, we hypothesized that imidazoquinolines would activate LC exposed to HPV16, leading to the induction of an HPV16-specific cell-mediated immune response. Surprisingly, both phenotypic and functional hallmarks of activation are not observed when LC are exposed to HPV16 virus-like particles and treated with imiquimod (TLR7 agonist). However, we found that LC are activated by 3M-002 (TLR8 agonist) and resiquimod (TLR8/7 agonist). LC exposed to HPV16 virus-like particles and subsequently treated with 3M-002 or resiquimod highly up-regulate surface activation markers, secrete proinflammatory cytokines and chemokines, induce CCL21-directed migration, and initiate an HPV16-specific CD8(+) T cell response. These data strongly indicate that 3M-002 and resiquimod are promising therapeutics for treatment of HPV infections and HPV-induced cervical lesions.

Cariogenic and periodontal pathogens are thought to be etiological factors in the development of cardiovascular disease. We assessed the involvement of the periodontal pathogen Aggregatibacter actinomycetemcomitans and cariogenic pathogen Streptococcus mutans in the development of atherosclerosis in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice. The mice were treated intravenously with A. actinomycetemcomitans HK1651, S. mutans GS-5, or phosphate-buffered saline three times a week for 3 weeks and killed at 15 weeks of age. The areas of the aortic sinus that were covered with atherosclerotic plaque were significantly larger in Apoe(shl) mice challenged with A. actinomycetemcomitans compared with S. mutans- or vehicle-challenged mice. Aggregatibacter actinomycetemcomitans challenge increased serum high-sensitive C-reactive protein and lipopolysaccharide levels. Bacterial DNA was detected in the blood, heart, and spleen, but not in the liver. Furthermore, serum interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, and MCP-1 levels and Toll-like receptor (TLR)2, TLR4, ICAM-1, E-selectin, P-selectin, LOX-1, HSP60, CCL19, CCL21, CCR7, and MCP-1 expressions in the aorta were significantly increased in mice challenged with A. actinomycetemcomitans. These results suggest that systemic infection with A. actinomycetemcomitans accelerates atherosclerosis in Apoe(shl) mice by exposing the whole microorganisms or their products, followed by initiating inflammation. Increases in proatherogenic factors may explain the aggravation of atherosclerosis by A. actinomycetemcomitans infection.

It has long been demonstrated that a subset of patients with Sjogren's syndrome (SS) develop ectopic lymphoid structures (ELS) in the salivary glands (SG). These structures are characterised by periductal clusters of T and B lymphocytes, development of high endothelial venules and differentiation of follicular dendritic cells (FDC) networks. Evidence in patients with and animal models of SS demonstrated that the formation and maintenance of ELS in the SG is critically dependent on the ectopic expression of lymphotoxins (LT) and lymphoid chemokines CXCL13, CCL19, CCL21 and CXCL12. Several cell types, including resident epithelial, stromal and endothelial cells as well as different subsets of infiltrating immune cells, have been shown to be capable of producing some of these factors during chronic inflammation in SS. In this review we focus on the cellular and molecular mechanisms regulating the formation of ELS in SS SG, with particular emphasis on the role of lymphoid chemokines. In addition, we summarise accumulating data in support of the notion that ELS in SS represent functional niches whereby autoreactive B cells undergo affinity maturation, clonal selection and differentiation into autoantibody producing cells, thus contributing to autoimmunity over and above secondary lymphoid organs. Furthermore, we review the emerging role of ELS and lymphoid chemokines in driving extranodal B cell lymphomagenesis in SS and we focus on recent evidence suggesting that ELS identify subsets of SS patients at increased risk of developing systemic manifestations and lymphoma.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). Immature DCs (iDCs) are situated in the periphery where they capture pathogen. Subsequently, they migrate as mature DCs (mDCs) to draining lymph nodes to activate T cells. CCR7 and CCL21 contribute to the migratory capacity of the DC, but it is not completely understood what molecular requirements are involved. Here we demonstrate that monocyte-derived DCs dramatically change ST8Sia IV expression during maturation, leading to the generation of polysialic acid (polySia). PolySia expression is highly upregulated after 2 days Toll-like receptor-4 (TLR4) triggering. Surprisingly, only immunogenic and not tolerogenic mDCs upregulated polySia expression. Furthermore, we show that polySia expression on DCs is required for CCL21-directed migration, whereby polySia directly captures CCL21. Corresponding to polySia, the expression level of CCR7 is maximal two days after TLR4 triggering. In contrast, although TLR agonists other than LPS induce upregulation of CCR7, they achieve only a moderate polySia expression. In situ we could detect polySia-expressing APCs in the T cell zone of the lymph node and in the deep dermis. Together our results indicate that prolonged TLR4 engagement is required for the generation of polySia-expressing DCs that facilitate CCL21 capture and subsequent CCL21-directed migration.