Tissue drains fluid and macromolecules through lymphatic vessels (LVs), which are lined by a specialized endothelium that expresses peculiar differentiation proteins, not found in blood vessels (i.e., LYVE-1, Podoplanin, PROX-1, and VEGFR-3). Lymphatic capillaries are characteristically devoid of a continuous basal membrane and are anchored to the ECM by elastic fibers that act as pulling ropes which open the vessel to avoid edema if tissue volume increases, as it occurs upon inflammation. LVs are also crucial for the transit of T lymphocytes and antigen presenting cells from tissue to draining lymph nodes (LN). Importantly, cell traffic control across lymphatic endothelium is differently regulated under resting and inflammatory conditions. Under steady-state non-inflammatory conditions, leukocytes enter into the lymphatic capillaries through basal membrane gaps (portals). This entrance is integrin-independent and seems to be mainly guided by CCL21 chemokine gradients acting on leukocytes expressing CCR7. In contrast, inflammatory processes in lymphatic capillaries involve a plethora of cytokines, chemokines, leukocyte integrins, and other adhesion molecules. Importantly, under inflammation a role for integrins and their ligands becomes apparent and, as a consequence, the number of leukocytes entering the lymphatic capillaries multiplies several-fold. Enhancing transmigration of dendritic cells en route to LN is conceivably useful for vaccination and cancer immunotherapy, whereas interference with such key mechanisms may ameliorate autoimmunity or excessive inflammation. Recent findings illustrate how, transient cell-to-cell interactions between lymphatic endothelial cells and leukocytes contribute to shape the subsequent behavior of leukocytes and condition the LV for subsequent trans-migratory events.

OBJECTIVE

To investigate the effects of CCL21/CCR7 on the proliferation, migration, and invasion of T24 cells and the possible associated mechanisms: expression of MMP-2 and MMP-9, and regulation of BCL-2 and BAX proteins.

METHODS

T24 cells received corresponding treatments including vehicle control, antibody (20 ng/mL CCR7 antibody and 50 ng/ml CCL21), and 50, 100, and 200 ng/ml CCL21. Proliferation was evaluated by MTT assay; cell migration and invasion were assayed using a transwell chamber. Cell apoptosis was induced by Adriamycin (ADM). The rate of cell apoptosis was examined by flow cytometry using annexin V-FITC/PI staining. Western-blot was used to analyze MMP-2 and MMP-9 and BCL-2 and BAX proteins.

RESULTS

CCL21 promoted T24 cell proliferation in concentration-dependent manner with that 200 ng/mL induced the largest amount of proliferation. Significant differences of cell migration were found between CCL21treatment groups and the control group in both the migration and invasion studies (P < 0.001 for all). The expressions of MMP-2 and MMP-9 proteins were significantly increased after CCL21 treatment (p < 0.05 for all). Protein expression of Bcl-21 follows an ascending trend while the expression of Bax follows a descending trend as the concentration of CCL21 increases. No difference was found between the control group and antibody group for all assessments.

CONCLUSIONS

CCL21/CCR7 promoted T24 cell proliferation and enhanced its migration and invasion via the increased expression of MMP-2 and MMP-9. CCL21/CCR7 had antiapoptotic activities on T24 cells via regulation of Bcl-2 and Bax proteins. CCL21/CCR7 may promote bladder cancer development and metastasis.

Neuropilin-2 (NRP2) is well known as a co-receptor for class 3 semaphorins and vascular endothelial growth factors, involved in axon guidance and angiogenesis. Moreover, NRP2 was shown to promote chemotactic migration of human monocyte-derived dendritic cells (DCs) toward the chemokine CCL21, a function that relies on the presence of polysialic acid (polySia). In vertebrates, this posttranslational modification is predominantly found on the neural cell adhesion molecule (NCAM), where it is synthesized on N-glycans by either of the two polysialyltransferases, ST8SiaII or ST8SiaIV. In contrast to NCAM, little is known on the biosynthesis of polySia on NRP2. Here we identified the polySia attachment sites and demonstrate that NRP2 is recognized only by ST8SiaIV. Although polySia-NRP2 was found on bone marrow-derived DCs from wild-type and St8sia2(-/-) mice, polySia was completely lost in DCs from St8sia4(-/-) mice despite normal NRP2 expression. In COS-7 cells, co-expression of NRP2 with ST8SiaIV but not ST8SiaII resulted in the formation of polySia-NRP2, highlighting distinct acceptor specificities of the two polysialyltransferases. Notably, ST8SiaIV synthesized polySia selectively on a NRP2 glycoform that was characterized by the presence of sialylated core 1 and core 2 O-glycans. Based on a comprehensive site-directed mutagenesis study, we localized the polySia attachment sites to an O-glycan cluster located in the linker region between b2 and c domain. Combined alanine exchange of Thr-607, -613, -614, -615, -619, and -624 efficiently blocked polysialylation. Restoration of single sites only partially rescued polysialylation, suggesting that within this cluster, polySia is attached to more than one site.

Adaptive immunity is involved in the pathogenesis of atherosclerosis, but the recruitment of T and B lymphocytes to atherosclerotic lesions is not as well studied as that of monocytes. In this review, we summarize the current understanding of the role of lymphocyte subsets in the pathogenesis of atherosclerosis and discuss chemokines and chemokine receptors involved in lymphocyte homing to atherosclerotic lesions. We review evidence for involvement of the chemokines CCL5, CCL19, CCL21, CXCL10, and CXCL16 and macrophage migration inhibitory factor in lymphocyte homing in atherosclerosis. Also, we review the role of their receptors CCR5, CCR6, CCR7, CXCR3, CXCR6, and CXCR2/CXCR4 and the role of the L-selectin in mouse models of atherosclerosis.

The thymus plays a primary role in early-onset Myasthenia Gravis (MG) mediated by anti-acetylcholine receptor (AChR) antibodies. As we recently showed an inflammatory and anti-viral signature in MG thymuses, we investigated in detail the contribution of interferon (IFN)-I and IFN-III subtypes in thymic changes associated with MG. We showed that IFN-I and IFN-III subtypes, but especially IFN-β, induced specifically α-AChR expression in thymic epithelial cells (TECs). We also demonstrated that IFN-β increased TEC death and the uptake of TEC proteins by dendritic cells. In parallel, we showed that IFN-β increased the expression of the chemokines CXCL13 and CCL21 by TECs and lymphatic endothelial cells, respectively. These two chemokines are involved in germinal center (GC) development and overexpressed in MG thymus with follicular hyperplasia. We also demonstrated that the B-cell activating factor (BAFF), which favors autoreactive B-cells, was overexpressed by TECs in MG thymus and was also induced by IFN-β in TEC cultures. Some of IFN-β effects were down-regulated when cell cultures were treated with glucocorticoids, a treatment widely used in MG patients that decreases the number of thymic GCs. Similar changes were observed in vivo. The injections of Poly(I:C) to C57BL/6 mice triggered a thymic overexpression of IFN-β and IFN-α2 associated with increased expressions of CXCL13, CCL21, BAFF, and favored the recruitment of B cells. These changes were not observed in the thymus of IFN-I receptor KO mice injected with Poly(I:C), even if IFN-β and IFN-α2 were overexpressed. Altogether, these results demonstrate that IFN-β could play a central role in thymic events leading to MG by triggering the overexpression of α-AChR probably leading to thymic DC autosensitization, the abnormal recruitment of peripheral cells and GC formation.

Microenvironmental interactions are crucial for the survival and proliferation of chronic lymphocytic leukemia (CLL) cells. CD4+ T cells that express CD40 ligand (CD40L), along with other accessory immune and stromal cells within CLL lymph nodes, provide signals needed for activation and outgrowth of the tumor clone. Furthermore, correct positioning of CLL cells within lymphoid subcompartments is essential for the transmission of these supportive signals. Thereby, interstitial cell migration and adhesion events, influenced by activational stimuli, determine CLL cell localization. CD44 has been implicated in cell activation, migration, and tissue retention via binding to its extracellular matrix ligand hyaluronan (HA). In this study, we investigated the role of CD44-HA interactions for CLL positioning and interaction with supportive microenvironments in peripheral lymph nodes, focusing on its regulation via CD40L-dependent, T-cell-mediated activation of CLL cells. We found that hyaluronan triggered a robust CCL21-induced motility of resting CLL cells. However, CD40L stimulation promoted the firm, CD44-mediated adhesion of CLL cells to hyaluronan, antagonizing their motile behavior. N-linked glycosylations of CD44, particularly associated with the variant isoform CD44v6 after CD40L activation, seemed to facilitate hyaluronan recognition by CD44. We propose that the CD40L-CD40 signaling axis provides a stop signal to motile CLL cells within lymph node compartments by inducing high avidity CD44-HA adhesion. This might retain CLL cells close to T-cell stimuli and facilitate essential interactions with hyaluronan-bearing stromal cells, collectively promoting CLL cell proliferation and survival.

L-selectin functions as an important adhesion molecule that mediates tethering and rolling of lymphocytes by binding to high endothelial venule (HEV)-expressed ligands during recirculation. Subsequent lymphocyte arrest and transmigration require activation through binding of HEV-decorated homeostatic chemokines such as secondary lymphoid tissue chemokine (SLC; CCL21) to its counterreceptor, CCR7. Importantly, L-selectin also functions as a signaling molecule. In this study, signaling induced by ligation of L-selectin using mAb or endothelial cell-expressed ligand significantly enhanced the chemotaxis of murine T cells and B cells to SLC but not to other homeostatic chemokines. Consistent with the expression levels of L-selectin in different lymphocyte subsets, L-selectin-mediated enhancement of chemotaxis to SLC was observed for all naive lymphocytes and effector/memory CD8(+) T cells, whereas only a subpopulation of effector/memory CD4(+) T cells responded. During in vivo mesenteric lymph node migration assays, the absence of L-selectin on lymphocytes significantly attenuated both their ability to migrate out of the HEV and their chemotaxis away from the vessel wall. Notably, ligation of L-selectin and/or CCR7 did not result in increased CCR7 expression levels, internalization, or re-expression. Pharmacologic inhibitor studies showed that L-selectin-mediated enhanced chemotaxis to SLC required intact intracellular kinase function. Furthermore, treatment of lymphocytes with the spleen tyrosine kinase family inhibitor piceatannol reduced their ability to migrate across the HEV in peripheral lymph nodes. Therefore, these results suggest that "cross-talk" in the signaling pathways initiated by L-selectin and CCR7 provides a novel mechanism for functional synergy between these two molecules during lymphocyte migration.

BACKGROUND

IFN-γ is presently the only soluble immunological marker used to help diagnose latent Mycobacterium tuberculosis (M.tb) infection. However, IFN-γ is not available to distinguish latent from active TB infection. Moreover, extrapulmonary tuberculosis, such as tuberculous pleurisy, cannot be properly diagnosed by IFN-γ release assay. As a result, other disease- or infection-related immunological biomarkers that would be more effective need to be screened and identified.

METHODS

A panel of 41 soluble immunological molecules (17 cytokines and 24 chemokines) was tested using Luminex liquid array-based multiplexed immunoassays. Samples, including plasma and pleural effusions, from healthy donors (HD, n = 12) or patients with latent tuberculosis infection (LTBI, n = 20), pulmonary tuberculosis (TB, n = 12), tuberculous pleurisy (TP, n = 15) or lung cancer (LC, n = 15) were collected and screened for soluble markers. Peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) were also isolated to investigate antigen-specific immune factors.

RESULTS

For the 41 examined factors, our results indicated that three patterns were closely associated with infection and disease. (1) Significantly elevated plasma levels of IL-2, IP-10, CXCL11 and CXCL12 were present in both patients with tuberculosis and in a sub-group participant with latent tuberculosis infection who showed a higher level of IFN-γ producing cells by ELISPOT assay compared with other latently infected individuals. (2) IL-6 and IL-9 were only significantly increased in plasma from active TB patients, and the two factors were consistently highly secreted after M.tb antigen stimulation. (3) When patients developed tuberculous pleurisy, CCL1, CCL21 and IL-6 were specifically increased in the pleural effusions. In particular, these three factors were consistently highly secreted by pleural fluid mononuclear cells following M.tb-specific antigen stimulation. In conclusion, our data imply that the specific secretion of soluble immunological factors, in addition to IFN-γ, may be used to evaluate M.tb infection and tuberculosis disease.

T-cell infiltration into tumors represents a critical bottleneck for immune-mediated control of cancer. We previously showed that this bottleneck can be overcome by depleting immunosuppressive Foxp3+ regulatory T cells (Tregs), a process that can increase frequencies of tumor-infiltrating lymphocytes through promoting the development of specialized portals for lymphocyte entry, namely high endothelial venules (HEVs). In this paper, we used a carcinogen-induced tumor model that allows for coevolution of the tumor microenvironment and the immune response to demonstrate that Treg depletion not only results in widespread disruption to HEV networks in lymph nodes (LNs) but also activates CD8+ T cells, which then drive intratumoral HEV development. Formation of these vessels contrasts with ontogenic HEV development in LNs in that the process is dependent on the TNF receptor and independent of lymphotoxin β receptor-mediated signaling. These intratumoral HEVs do not express the chemokine CCL21, revealing a previously undescribed intratumoral blood vessel phenotype. We propose a model where Treg depletion enables a self-amplifying loop of T-cell activation, which promotes HEV development, T-cell infiltration, and ultimately, tumor destruction. The findings point to a need to test for HEV development as part of ongoing clinical studies in patients with cancer. Cancer Immunol Res; 5(11); 1005-15. ©2017 AACR.

We previously reported that mac25/angiomodulin (AGM), a 30-kDa secretory protein, is abundantly expressed in high endothelial venules (HEVs), which play a crucial role in lymphocyte trafficking to the lymph nodes and Peyer's patches. We report that mac25/AGM interacts preferentially with certain molecules that are expressed in or around HEVs. In particular, mac25/AGM interacted with not only the extracellular matrix proteins and glycosaminoglycans that are expressed in most blood vessels including HEVs, but also with some chemokines that are implicated in the regulation of lymphocyte trafficking, such as the secondary lymphoid-tissue chemokine (SLC; CCL21), IFN-gamma-inducible protein 10 (IP-10; CXCL10), and RANTES (CCL5). The binding of mac25/AGM to SLC and IP-10 was dose-dependent and saturable. The binding to IP-10 could be inhibited by SLC but not by a non-mac25/AGM-binding chemokine, EBI1-ligand chemokine (ELC; CCL19). Interestingly, mac25/AGM failed to interact with 18 other chemokines, suggesting that it binds to certain chemokines preferentially. Immunohistochemical analysis indicated that mac25/AGM colocalizes at least partially with SLC and IP-10 at the basal lamina of HEVs. Upon binding with mac25/AGM, SLC and IP-10 retained all their Ca(2+)-signaling activity in vitro, suggesting that mac25/AGM can hold and present chemokines in the basal lamina of HEVs. These results imply that mac25/AGM plays a multifunctional role, serving not only as an adhesion protein to interact with glycosaminoglycans and extracellular matrix proteins but also as a molecule to present chemokines so that lymphocytes extravasating through HEVs receive further directional cues subsequent to the luminal encounter with lymphoid chemokines.

Triptolide (TPT) is a chemically defined, potent immunosuppressive compound isolated from an anti-inflammatory Chinese herbal medicine. TPT has been reported to inhibit autoimmunity, allograft rejection, and graft-versus-host disease (GVHD), and its efficacy was previously attributed to the suppression of T cells. Since dendritic cells (DCs) play a major role in the initiation of T-cell-mediated immunity, we studied the effects of TPT on the phenotype, function, and migration of human monocyte-derived DCs. TPT treatment, over a pharmacologic concentration range, inhibited the lipopolysaccharide (LPS)-induced phenotypic changes, characteristic of mature DCs and the production of interleukin-12p70 (IL-12p70). Consequently, the allostimulatory functions of DCs were impaired by TPT treatment. Furthermore, the calcium mobilization and chemotactic responses of LPS-stimulated DCs to secondary lymphoid tissue chemokine (SLC)/CC chemokine ligand 21 (CCL21) were significantly lower in TPT-treated than untreated DCs, in association with lower chemokine receptor 7 (CCR7) and higher CCR5 expression. Egress of Langerhans cells (LCs) from explanted mouse skin in response to macrophage inflammatory protein-3beta (MIP-3beta)/CCL19 was arrested by TPT. In vivo administration of TPT markedly inhibited hapten (fluorescein isothiocyanate [FITC])-stimulated migration of mouse skin LCs to the draining lymph nodes. These data provide new insight into the mechanism of action of TPT and indicate that the inhibition of maturation and trafficking of DCs by TPT contributes to its immunosuppressive effects.

OBJECTIVE

Idiopathic interstitial pneumonias (IIPs) are a diverse grouping of chronic pulmonary diseases characterised by varying degrees of pulmonary fibrosis. The triggers of the fibroproliferative process in IIP remain enigmatic but recent attention has been directed towards chemokine involvement in this process.

METHODS

The expression of two chemokine receptors, CCR7 and CXCR4, and their respective ligands, CCL19, CCL21, and CXCL12, were examined in surgical lung biopsies (SLBs) from patients with IIP. Transcript and protein expression of these receptors and their ligands was compared with that detected in histologically normal margin SLBs.

RESULTS

CCR7 and CXCR4 were detected by gene array and real time polymerase chain reaction analysis and CCR7, but not CXCR4, expression was significantly raised in usual interstitial pneumonia (UIP) relative to biopsies from patients diagnosed with non-specific interstitial pneumonia (NSIP) or respiratory bronchiolitis/interstitial lung disease (RBILD). CCR7 protein was expressed in interstitial areas of all upper and lower lobe UIP SLBs analysed. CCR7 expression was present in 50% of NSIP SLBs, and CCR7 was restricted to blood vessels and mononuclear cells in 75% of RBILD SLBs. Immune cell specific CXCR4 expression was seen in IIP and normal margin biopsies. CCR7 positive areas in UIP biopsies were concomitantly positive for CD45 (the leucocyte common antigen) but CCR7 positive areas in all IIP SLBs lacked the haemopoietic stem cell antigen CD34, collagen 1, and alpha smooth muscle actin.

CONCLUSIONS

This molecular and immunohistochemical analysis showed that IIPs are associated with abnormal CCR7 transcript and protein expression.

T cells exhibit high-speed migration within the paracortical T zone of lymph nodes (LNs) as they scan cognate Ags displayed by dendritic cells in the tissue microenvironment supported by the network of stromal cells. Although intranodal T cell migration is controlled in part by chemokines and LFA-1/ICAM-1, the mechanisms underlying their migratory activity independent of these factors remain to be elucidated. In this study, we show that LN stromal cells constitutively express autotaxin (ATX), an ectoenzyme that is important for the generation of lysophosphatidic acid (LPA). Importantly, CCL21(+) stromal cells in the T zone produced and immobilized ATX on their cell surface. Two-photon imaging using LN tissue slices revealed that pharmacological inhibition of ATX or LPA receptors significantly reduced T cell migration, and this was further exacerbated by blockage of Gαi signaling or LFA-1. Therefore, T cell motility mediated by the ATX-LPA axis was independent of Gαi and LFA-1. LPA induced slow intermittent movement of T cells in vitro in a LFA-1-independent manner and enhanced CCL21-induced migration. Moreover, LPA and CCL21 cooperatively augmented RhoA activity in T cells, which was necessary for efficient intranodal T cell migration via the downstream ROCK-myosin II pathway. Taken together, T zone stromal cells control optimal migratory behavior of T cells via multiple signaling cues mediated by chemokines and ATX/LPA.

Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. The present study was designed to examine the expression of chemokine receptor CCR7 in oral squamous cell carcinoma (OSCC), and to investigate the possible role of CCR7/CCL21 interaction in neck lymph node metastasis of OSCC. By using immunohistochemistry, RT-PCR and Western Blot, expression of CCR7 was examined in 85 cases of oral squamous cell carcinoma, and Tca8113 and ACC cell lines. CCL21-mediated cell migration was assayed in Matrigel-coated chemotaxis chamber. In vitro adhesion assay was shown for banding of tumor cell lines to submandibular lymph nodes with or without anti-CCR7 antibody treatment. Immunohistochemical staining showed 65.9% (56/85) of positive CCR7 expression in OSCC tissues. CCR7 expression was significantly higher in patients with lymph node metastasis compared with those without lymph node metastasis (P=0.015) and was also associated with tumor size (P=0.014), and clinical stage (P=0.009). RT-PCR and Western Blot also confirmed positive CCR7 expression in oral squamous cell carcinoma and Tca8113 cell line, and negative CCR7 expression in normal oral mucosa and ACC cell line. CCL21 stimulation increased the ability of CCR7-positive Tca8113 cells passing through the Matrigel membrane. CCR7-positive Tca8113 cells also showed stronger adhesion to lymph nodes, which could be partly blocked by anti-CCR7 antibody incubation. These results indicated that the chemotactic CCR7/CCL21 interaction may be a possible mechanism for induction of directional lymph node metastasis by oral squamous cell carcinoma.

CC chemokines, a subfamily of 27 chemotactic cytokines, are a component of intercellular communication, which is crucial for the functioning of the tumor microenvironment. Although many individual chemokines have been well researched, there has been no comprehensive review presenting the role of all known human CC chemokines in the hallmarks of cancer, and this paper aims at filling this gap. The first part of this review discusses the importance of CCL1, CCL3, CCL4, CCL5, CCL18, CCL19, CCL20, CCL21, CCL25, CCL27, and CCL28 in cancer. Here, we discuss the significance of CCL2 (MCP-1), CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL22, CCL23, CCL24, and CCL26. The presentation of each chemokine includes its physiological function and then the role in tumor, including proliferation, drug resistance, migration, invasion, and organ-specific metastasis of tumor cells, as well as the effects on angiogenesis and lymphangiogenesis. We also discuss the effects of each CC chemokine on the recruitment of cancer-associated cells to the tumor niche (eosinophils, myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), regulatory T cells (T_reg)). On the other hand, we also present the anti-cancer properties of CC chemokines, consisting in the recruitment of tumor-infiltrating lymphocytes (TIL).

Keywords: CC chemokine; MCP-1; angiogenesis; anti-cancer therapy; cancer; chemokine; lymphangiogenesis; organ-specific metastasis; tumor; tumor microenvironment.

Here we clarified the morphology and phenotype of interleukin (IL)-17- and interferon (IFN)-gamma-producing cells in both in vitro and in vivo situations. Oligoclonal activation of normal peripheral blood mononuclear cells with the superantigen Staphylococcus aureus enterotoxin B and polyclonal activation with phorbol myristate acetate/phytohemagglutinin were used as in vitro models. This study was extended to various in vivo situations such as rheumatoid arthritis, dermatomyositis, and normal activated lymph nodes. The phenotype of IL-17- and IFN-gamma-producing cells was evaluated by immunohistochemistry using the CD3 and CD4 T-cell markers, the CD20, CD38, kappa and lambda light chain B-cell lineage markers. The expression of two chemokine receptors, CCR6 and CCR7, involved with their associated ligands CCL20 and CCL19/CCL21 in the migration of T lymphocytes, was evaluated in tissue sections. After both polyclonal and oligoclonal activation, IL-17+ and IFN-gamma+ cells acquired a plasma cell-like morphology associated with a high secretory activity, the reduced expression of CD3, and no change of CD4 expression. In rheumatoid arthritis, dermatomyositis, and activated lymph nodes, both IL-17- and IFN-gamma-producing cells had the same morphology. These Th1 cytokine-producing cells were CD4(+)-, CD3-, and B-cell lineage marker-negative. In both in vitro and in vivo situations, expression of CCR6 or CCR7 was not associated with a particular subset. In conclusion, activated T-helper CD4(+) T cells, by their release of cytokines, seem to have functional similarities with plasma cells secreting immunoglobulins.

Inherent immune suppression represents a major challenge in the treatment of human cancer. The extracellular matrix molecule tenascin-C promotes cancer by multiple mechanisms, yet the roles of tenascin-C in tumor immunity are incompletely understood. Using a 4NQO-induced oral squamous cell carcinoma (OSCC) model with abundant and absent tenascin-C, we demonstrated that tenascin-C enforced an immune suppressive lymphoid stroma via CCL21/CCR7 signaling, leading to increased metastatic tumors. Through TLR4, tenascin-C increased expression of CCR7 in CD11c+ myeloid cells. By inducing CCL21 in lymphatic endothelial cells via integrin α9β1 and binding to CCL21, tenascin-C immobilized CD11c+ cells in the stroma. Inversion of the lymph node-to-tumor CCL21 gradient, recruitment of T regulatory cells, high expression of anti-inflammatory cytokines and matrisomal components were hallmarks of the tenascin-C-instructed lymphoid stroma. Ablation of tenascin-C or CCR7 blockade inhibited the lymphoid immune suppressive stromal properties, reducing tumor growth, progression and metastasis. Thus, targeting CCR7 could be relevant in human head and neck tumors as high tenascin-C expression and an immune suppressive stroma correlate to poor patient survival.

OBJECTIVE

Anticancer dendritic cell (DC) vaccines require the DCs to relocate to lymph nodes (LN) to trigger immune responses. However, these migration rates are typically very poor. Improving the targeting of ex vivo generated DCs to LNs might increase vaccine efficacy and reduce costs. We investigated DC migration in vivo in humans under different conditions.

METHODS

HLA-A*02:01 patients with melanoma were vaccinated with mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (NCT00243594). For this study, patients received an additional intradermal vaccination with (111)In-labeled mature DCs. The injection site was pretreated with nonloaded, activated DCs, TNFα, or Imiquimod; granulocyte macrophage colony-stimulating factor was coinjected or smaller numbers of DCs were injected. Migration was measured by scintigraphy and compared with an intrapatient control vaccination. In an ex vivo tissue model, we measured CCL21-directed migration of (19)F-labeled DCs over a period of up to 12 hours using (19)F MRI to supplement our patient data.

RESULTS

Pretreatment of the injection site induced local inflammatory reactions but did not improve migration rates. Both in vitro and in vivo, reduction of cell numbers to 5 × 10(6) or less cells per injection improved migration. Furthermore, scintigraphy is insufficient to study migration of such small numbers of (111)In-labeled DCs in vivo.

CONCLUSIONS

Reduction of cell density, not pretreatment of the injection site, is crucial for improved migration of DCs to LNs in vivo.

In epidermal Langerhans cells (LCs), the expression pattern and the functions of TLRs have been poorly characterized. By using mAb, we show that LCs from human skin express TLR1, -2, -5, -6, and -9, the cognate receptors for detection of specific bacteria-derived molecules. As compared with other TLR agonists, LCs acquired a more matured phenotype when activated by specific bacterial or synthetic TLR2 agonists. In addition, monocyte-derived Langerin(+)/CD1c(+)LCs (CD1c(+)MoLCs) secreted higher amounts of IL-6 and TNF-alpha by stimulation via TLR2 than by stimulation via TLR3, -4, -5, -8, and -9. In contrast to MoLCs, dendritic cells, generated from the same donor monocytes, were activated by agonists of TLRs other than TLR2 as well. Lipopeptides triggering TLR2 induced IL-1R-associated kinase-1 phosphorylation and migration toward the chemokines CCL19 and CCL21 in epidermal LCs and CD1c(+)MoLCs. Up-regulation of CD86, CD83, and CCR7, TNF-alpha and IL-6, and NF-kappaB activation and proliferation of CD4(+)T cells could be inhibited TLR2-specific blockage using antibodies prior to TLR2 activation. Application of anti-TLR1, anti-TLR6, and anti-TLR2 indicated an exclusive role of TLR2 in IL-6 induction in human LCs. Collectively, our results show that TLR2 expressed by LCs mediates inflammatory responses to lipopeptides, which implicates a central role in sensing pathogens in human skin.

The lymphoid chemokines CCL19 and CCL21 are known to be crucial both for lymphoid cell trafficking and for the structural organization of lymphoid tissues such as nasopharynx-associated lymphoid tissue (NALT). However, their role in allergic responses remains unclear, and so our current study aims to shed light on the role of CCL19/CCL21 in the development of allergic rhinitis. After nasal challenge with OVA, OVA-sensitized plt (paucity of lymph node T cells) mice, which are deficient in CCL19/CCL21, showed more severe allergic symptoms than did identically treated wild-type mice. OVA-specific IgE production, eosinophil infiltration, and Th2 responses were enhanced in the upper airway of plt mice. Moreover, in plt mice, the number of CD4(+)CD25(+) regulatory T cells declined in the secondary lymphoid tissues, whereas the number of Th2-inducer-type CD8alpha(-)CD11b(+) myeloid dendritic cells (m-DCs) increased in cervical lymph nodes and NALT. Nasal administration of the plasmid-encoding DNA of CCL19 resulted in the reduction of m-DCs in the secondary lymphoid tissues and the suppression of allergic responses in plt mice. These results suggest that CCL19/CCL21 act as regulatory chemokines for the control of airway allergic disease and so may offer a new strategy for the control of allergic disease.

CCR7 binds to its cognate ligand, CCL21, to mediate the migration of circulating naive T lymphocytes to the lymph nodes. T lymphocytes can bind to fibronectin, a constituent of lymph nodes, via their β1 integrins, which is a primary mechanism of T lymphocyte migration; however, the signaling pathways involved are unclear. We report that rapid (within 2 min) and transient phosphorylation of ERK1/2 is required for T cell migration on fibronectin in response to CCL21. Conversely, prevention of ERK1/2 phosphorylation by inhibition of its kinase, MAPK/MEK, prevented T lymphocyte migration. Previous studies have suggested that phospholipase Cγ1 (PLCγ1) can mediate phosphorylation of ERK1/2, which is required for β1 integrin activation. Paradoxically, we found that inhibition of PLCγ1 phosphorylation by the general PLC inhibitor U73122 was associated with a delayed and reduced phosphorylation of ERK1/2 and reduced migration of T lymphocytes on fibronectin. To further characterize the relationship between ERK1/2 and PLCγ1, we reduced PLCγ1 levels by 85% using shRNA and observed a reduced phosphorylation of ERK1/2 and a significant loss of CCR7-mediated migration of T lymphocytes on fibronectin. In addition, we found that inhibition of ERK1/2 phosphorylation by U0126 resulted in a decreased phosphorylation of PLCγ1, suggesting a feedback loop between ERK1/2 and PLCγ1. Overall, these results suggest that the CCR7 signaling pathway leading to T lymphocyte migration on fibronectin is a β1 integrin-dependent pathway involving transient ERK1/2 phosphorylation, which is modulated by PLCγ1.

The migration of dendritic cells (DCs) to lymph nodes (LNs) is pivotal to the establishment of the immune response. DCs have been proved to pass through the afferent lymphatic pathway to enter LNs from the peripheral tissues after they have scanned for self or nonself antigens. In response to danger signals, both myeloid and plasmacytoid DC precursors (mDC and pDC precursors) are rapidly mobilized into the circulation. mDC precursors are recruited to inflamed tissues in response to inflammatory chemokines and then remobilized to regional LNs in response to CCL21. In contrast, pDC precursors directly transmigrate to regional LNs via high endothelial venules in a CXCL9- and E-selectin-dependent manner. Such migration is largely dependent on systemic inflammatory reactions. After accumulating in the LNs through distinct trafficking pathways, DCs interact with lymphocytes temporally and spatially to establish effective immune responses. The inflammation-dependent, chemokine-driven property of DC precursor trafficking is a very sophisticated host defense system.

CCL19 and CCL21 and their receptor CCR7 are expressed constitutively within lymphoid organs, regulating lymphocyte homing. Recent studies suggest that these chemokines may have inflammatory properties. We hypothesized a role of CCL19/CCL21 in human immunodeficiency virus (HIV) infection by promoting inflammation. We examined the expression of CCL19 and CCL21 in mononuclear cells from peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) in HIV-infected patients before and during highly active anti-retroviral therapy (HAART). We also examined the ability of CCL19/CCL21 to promote inflammatory responses in these patients. PBMC from untreated HIV-infected patients (n = 29) released enhanced levels of CCL19 spontaneously compared with cells from controls (n = 20), particularly in those with symptomatic disease (n = 15, P < 0.01 versus controls). During HAART (n = 9), there was a decrease in the spontaneous CCL19 release and an increase in the phytohaemagglutinin-stimulated CCL19 release in both PBMC (P < 0.01) and BMMC (P < 0.05). In patients with enhanced HIV replication there was an increased proportion of inflammatory CD8(+)CCR7(-)CD45RA(-) T cells in peripheral blood [P < 0.01 and P < 0.05 versus controls, untreated (n = 9) and treatment failure (n = 8), respectively]. In vitro, CCL19/CCL21 promoted an inflammatory response in PBMC when accompanied by high viral load, irrespective of HAART. The HIV-tat protein significantly boosted the inflammatory effect of CCL19/CCL21 in PBMC. These findings link a dysregulated CCL19/CCL21/CCR7 system in HIV-infected patients to persistent inflammation and HIV replication, not only in untreated HIV infection, but also in treatment failure during HAART.

BACKGROUND

The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL).

RESULTS

A detailed analysis of inflammatory cells (neutrophils, eosinophils, mast cells, lymphocytes, and macrophages) as well as the expression of chemokines (CCL2, CCL4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8) was carried out in dermis skin samples from 35 dogs that were naturally infected with L. infantum. The analysis was based on real-time polymerase chain reaction (PCR) in the context of skin parasitism and the clinical status of CVL. We demonstrated increased inflammatory infiltrate composed mainly of mononuclear cells in the skin of animals with severe forms of CVL and high parasite density. Analysis of the inflammatory cell profile of the skin revealed an increase in the number of macrophages and reductions in lymphocytes, eosinophils, and mast cells that correlated with clinical progression of the disease. Additionally, enhanced parasite density was correlated with an increase in macrophages and decreases in eosinophils and mast cells. The chemokine mRNA expression demonstrated that enhanced parasite density was positively correlated with the expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. In contrast, there was a negative correlation between parasite density and CCL24 expression.

CONCLUSIONS

These findings represent an advance in the knowledge about skin inflammatory infiltrates in CVL and the systemic consequences. Additionally, the findings may contribute to the design of new and more efficient prophylactic tools and immunological therapies against CVL.