In vivo differentiated cytokine-producing CD4(+) T cells express functional CCR7

Chemokines and their receptors fulfill specialized roles in inflammation and under homeostatic conditions. CCR7 and its ligands, CCL19 and CCL21, are involved in lymphocyte recirculation through secondary lymphoid organs and additionally navigate lymphocytes into distinct tissue compartments. The role of CCR7 in the migration of polarized T effector/memory cell subsets in vivo is still poorly understood. We therefore analyzed murine and human CD4(+) cytokine-producing cells developed in vivo for their chemotactic reactivity to CCR7 ligands. The responses of cells producing cytokines, such as IFN-gamma, IL-4, and IL-10, as well as of subsets defined by memory or activation markers were comparable to that of naive CD4(+) cells, with slightly lower reactivity in cells expressing IL-10 or CD69. This indicates that CCR7 ligands are able to attract naive as well as the vast majority of activated and effector/memory T cell stages. Chemotactic reactivity of these cells toward CCL21 was absent in CCR7-deficient cells, proving that effector cells do not use alternative receptors for this chemokine. Th1 cells generated from CCR7(-/-) mice failed to enter lymph nodes and Peyer's patches, but did enter a site of inflammation. These findings indicate that CD4(+) cells producing effector cytokines upon stimulation retain the capacity to recirculate through lymphoid tissues via CCR7.     

Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis

The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-gamma by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-gamma+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.     

The HIV-1 Gp120/CXCR4 Axis Promotes CCR7 Ligand-Dependent CD4 T Cell Migration: CCR7 Homo- and CCR7/CXCR4 Hetero-Oligomer Formation as a Possible Mechanism for Up-Regulation of Functional CCR7

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.     

Cross-talk between TCR and CCR7 signaling sets a temporal threshold for enhanced T lymphocyte migration

Lymphocyte homing to, and motility within, lymph nodes is regulated by the chemokine receptor CCR7 and its two ligands CCL19 and CCL21. There, lymphocytes are exposed to a number of extracellular stimuli that influence cellular functions and determine the cell fate. In this study, we assessed the effect of TCR engagement on CCR7-mediated cell migration. We found that long-term TCR triggering of freshly isolated human T cells through CD3/CD28 attenuated CCR7-driven chemotaxis, whereas short-term activation significantly enhanced CCR7-mediated, but not CXCR4-mediated, migration efficiency. Short-term activation most prominently enhanced the migratory response of naive T cells of both CD4 and CD8 subsets. We identified distinct roles for Src family kinases in modulating CCR7-mediated T cell migration. We provide evidence that Fyn, together with Ca(2+)-independent protein kinase C isoforms, kept the migratory response of naive T cells toward CCL21 at a low level. In nonactivated T cells, CCR7 triggering induced a Fyn-dependent phosphorylation of the inhibitory Tyr505 of Lck. Inhibiting Fyn in these nonactivated T cells prevented the negative regulation of Lck and facilitated high CCR7-driven T cell chemotaxis. Moreover, we found that the enhanced migration of short-term activated T cells was accompanied by a synergistic, Src-dependent activation of the adaptor molecule linker for activation of T cells. Collectively, we characterize a cross-talk between the TCR and CCR7 and provide mechanistic evidence that the activation status of T cells controls lymphocyte motility and sets a threshold for their migratory response.     

Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 +/- 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 +/- 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2(-/-)/arrestin 3(-/-) murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.     

CCR7 signaling inhibits T cell proliferation

CCR7 and its ligands, CCL19 and CCL21, are responsible for directing the migration of T cells and dendritic cells into lymph nodes, where these cells play an important role in the initiation of the immune response. Recently, we have shown that systemic application of CCL19-IgG is able to inhibit the colocalization of T cells and dendritic cells within secondary lymphoid organs, resulting in pronounced immunosuppression with reduced allograft rejection after organ transplantation. In this study, we demonstrate that the application of sustained high concentrations of either soluble or immobilized CCL19 and CCL21 elicits an inhibitory program in T cells. We show that these ligands specifically interfere with cell proliferation and IL-2 secretion of CCR7(+) cells. This could be demonstrated for human and murine T cells and was valid for both CD4(+) and CD8(+) T cells. In contrast, CCL19 had no inhibitory effect on T cells from CCR7 knockout mice, but CCR7(-/-) T cells showed a proliferative response upon TCR-stimulation similar to that of CCL19-treated wild-type cells. Furthermore, the inhibition of proliferation is associated with delayed degradation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) and the down-regulation of CDK1. This shows that CCR7 signaling is linked to cell cycle control and that sustained engagement of CCR7, either by high concentrations of soluble ligands or by high density of immobilized ligands, is capable of inducing cell cycle arrest in TCR-stimulated cells. Thus, CCR7, a chemokine receptor that has been demonstrated to play an essential role during activation of the immune response, is also competent to directly inhibit T cell proliferation.     

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.     

Spontaneous and homeostatic proliferation of CD4 T cells are regulated by different mechanisms

Transfer of naive CD4 T cells into lymphopenic mice initiates a proliferative response of the transferred cells, often referred to as homeostatic proliferation. Careful analysis reveals that some of the transferred cells proliferate rapidly and undergo robust differentiation to memory cells, a process we have designated spontaneous proliferation, and other cells proliferate relatively slowly and show more limited evidence of differentiation. In this study we report that spontaneous proliferation is IL-7 independent, whereas the slow proliferation (referred to as homeostatic proliferation) is IL-7 dependent. Administration of IL-7 induces homeostatic proliferation of naive CD4 T cells even within wild-type recipients. Moreover, the activation/differentiation pattern of the two responses are clearly distinguishable, indicating that different activation mechanisms may be involved. Our results reveal the complexity and heterogeneity of lymphopenia-driven T cell proliferation and suggest that they may have fundamentally distinct roles in the maintenance of CD4 T cell homeostasis.     

A myriad of functions and complex regulation of the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune system

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 control a diverse array of migratory events in adaptive immune function. Most prominently, CCR7 promotes homing of T cells and DCs to T cell areas of lymphoid tissues where T cell priming occurs. However, CCR7 and its ligands also contribute to a multitude of adaptive immune functions including thymocyte development, secondary lymphoid organogenesis, high affinity antibody responses, regulatory and memory T cell function, and lymphocyte egress from tissues. In this survey, we summarise the role of CCR7 in adaptive immunity and describe recent progress in understanding how this axis is regulated. In particular we highlight CCX-CKR, which scavenges both CCR7 ligands, and discuss its emerging significance in the immune system.     

Recruitment and activation of macrophages by pathogenic CD4 T cells in type 1 diabetes: evidence for involvement of CCR8 and CCL1

Adoptive transfer of diabetogenic CD4 Th1 T cell clones into young NOD or NOD.scid recipients rapidly induces onset of diabetes and also provides a system for analysis of the pancreatic infiltrate. Although many reports have suggested a role for macrophages in the inflammatory response, there has been little direct characterization of macrophage activity in the pancreas. We showed previously that after migration to the pancreas, diabetogenic CD4 T cell clones produce a variety of inflammatory cytokines and chemokines, resulting in the recruitment of macrophages. In this study, we investigated mechanisms by which macrophages are recruited and activated by T cells. Analysis of infiltrating cells after adoptive transfer by the diabetogenic T cell clone BDC-2.5 indicates that large numbers of cells staining for both F4/80 and CD11b are recruited into the pancreas where they are activated to make IL-1beta, TNF-alpha, and NO, and express the chemokine receptors CCR5, CXCR3, and CCR8. Diabetogenic CD4 T cell clones produce several inflammatory chemokines in vitro, but after adoptive transfer we found that the only chemokine that could be detected ex vivo was CCL1. These results provide the first evidence that CCR8/CCL1 interaction may play a role in type 1 diabetes through macrophage recruitment and activation.     

Cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells

To examine the different roles of myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs) in the induction and regulation of immune response, we have studied chemokine secretion by freshly isolated DC subsets in response to bacterial, viral, and T cell-derived stimuli. M-DCs selectively produced very high levels of the homeostatic chemokines CC chemokine ligand (CCL)17 and CCL22, while P-DCs produced very little if any. In contrast, the proinflammatory chemokine CCL3 was secreted mostly by P-DCs, whereas CCL4 and CXC chemokine ligand 8 were produced by both subsets. The selective production of CCL17 and CCL22 by M-DCs but not P-DCs was confirmed in vivo by immunohistology on human reactive lymph node sections. The high production of CCR4 ligands by M-DCs suggests their capacity to selectively recruit at sites of inflammation T cells with regulatory properties or with a Th2 phenotype, whereas P-DCs, by preferentially secreting CCR1/CCR5 ligands, would mostly recruit effector T cells and, in particular, Th1-type cells.     

The death receptor Fas (CD95/APO-1) mediates the deletion of T lymphocytes undergoing homeostatic proliferation

Murine T cells adoptively transferred into syngeneic lymphopenic recipients undergo proliferation. Despite continued cell division, this lymphopenia-induced or homeostatic proliferation of a limited number of transferred T cells does not fill the T cell compartment. The continued expansion of the transferred T cells, even after stable T cell numbers have been reached, suggests that active cell death prevents further increase in T cell number. In this study, we show that wild-type T cells undergoing homeostatic proliferation are sensitive to Fas-mediated cell death. In the absence of Fas, T cells accumulate to significantly higher levels after transfer into lymphopenic recipients. Fas is, thus, a principal regulator of the expansion of peripheral T cells in response to self-peptide/MHC during T cell homeostasis. As Fas-deficient lpr mice manifest no significant abnormalities in thymic negative selection or in foreign Ag-induced peripheral T cell deletion, their lymphadenopathy may result from unrestrained homeostatic proliferation.     

Naive T cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.     

Distinct chemokine triggers and in vivo migratory paths of fluorescein dye-labeled T Lymphocytes in acutely simian immunodeficiency virus SIVmac251-infected and uninfected macaques

To define the possible impact of T-lymphocyte trafficking parameters on simian immunodeficiency virus (SIV) pathogenesis, we examined migratory profiles of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T lymphocytes in acutely SIVmac251-infected and uninfected macaques within 48 h after autologous transfer. Despite significant upregulation of homeostatic chemokine CCL19/macrophage inflammatory protein 3beta and proinflammatory chemokine CXCL9/monokine induced by gamma interferon in secondary lymphoid tissue in SIV infection, no differences in CFSE+ T-lymphocyte frequencies or cell compartmentalization in lymph nodes were identified between animal groups. By contrast, a higher frequency of CFSE+ T lymphocytes in the small intestine was detected in acute SIV infection. This result correlated with increased numbers of gut CD4 T lymphocytes expressing chemokine receptors CCR9, CCR7, and CXCR3 and high levels of their respective chemokine ligands in the small intestine. The changes in trafficking parameters in SIV-infected macaques occurred concomitantly with acute gut CD4 T-lymphocyte depletion. Here, we present the first in vivo T-lymphocyte trafficking study in SIV infection and a novel approach to delineate T-lymphocyte recruitment into tissues in the nonhuman primate animal model for AIDS. Such studies are likely to provide unique insights into T-lymphocyte sequestration in distinct tissue compartments and possible mechanisms of CD4 T-lymphocyte depletion and immune dysfunction in simian AIDS.     

CCR5 deficiency aggravates crescentic glomerulonephritis in mice

The chemokine receptor CCR5 is predominantly expressed on monocytes and Th1-polarized T cells, and plays an important role in T cell and monocyte recruitment in inflammatory diseases. To investigate the functional role of CCR5 in renal inflammation, we induced a T cell-dependent model of glomerulonephritis (nephrotoxic serum nephritis) in CCR5(-/-) mice. Induction of nephritis in wild-type mice resulted in up-regulation of renal mRNA expression of the three CCR5 chemokine ligands, CCL5 (15-fold), CCL3 (4.9-fold), and CCL4 (3.4-fold), in the autologous phase of the disease at day 10. The up-regulated chemokine expression was paralleled by infiltration of monocytes and T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Nephritic CCR5(-/-) mice showed a 3- to 4-fold increased renal expression of CCL5 (61.6-fold vs controls) and CCL3 (14.1-fold vs controls), but not of CCL4, in comparison with nephritic wild-type mice, which was accompanied by augmented renal T cell and monocyte recruitment and increased lethality due to uremia. Furthermore, CCR5(-/-) mice showed an increased renal Th1 response, whereas their systemic humoral and cellular immune responses were unaltered. Because the CCR5 ligands CCL5 and CCL3 also act via CCR1, we investigated the effects of the pharmacological CCR1 antagonist BX471. CCR1 blockade in CCR5(-/-) mice significantly reduced renal chemokine expression, T cell infiltration, and glomerular crescent formation, indicating that increased renal leukocyte recruitment and consecutive tissue damage in nephritic CCR5(-/-) mice depended on functional CCR1. In conclusion, this study shows that CCR5 deficiency aggravates glomerulonephritis via enhanced CCL3/CCL5-CCR1-driven renal T cell recruitment.     

Expression and function of T cell homing molecules in Hodgkin’s lymphoma

Circulating T lymphocytes enter a tissue if they express appropriate chemokine receptors and adhesion molecules to engage ligands presented at this site. To aid rational development of T cell-based therapies for Hodgkin's lymphoma (HL), we have assessed the expression and function of homing receptors on tumour-infiltrating T cells in HL and compared them with T cells from unaffected lymph nodes and colorectal cancer tissue. Chemokine receptors CXCR3, CXCR4 and CCR7 were expressed on a large proportion of T cells within HL tissue and mediated chemotaxis to purified chemokine. The corresponding ligands (CXCL10, CXCL12, CCL21) were expressed on the malignant cells and/or vascular endothelium. Adhesion molecules including CD62L were widely expressed on HL-derived T cells and their corresponding ligands were detected on vessels within the tumour. This homing phenotype was distinct from T cells isolated from colorectal cancer, but matched closely the phenotype of T cells from unaffected lymph nodes. Thus, T cell recruitment to HL resembles entry of na?ve/central memory T cells into normal lymph nodes. This has important implications for current approaches to treat HL using T cells activated and expanded in vitro that lack CCR7 and CD62L expression.     

Chemokine receptor CCR4 on CD4+ T cells in juvenile rheumatoid arthritis synovial fluid defines a subset of cells with increased IL-4:IFN-gamma mRNA ratios

To understand the mechanisms that promote recruitment and survival of T cells within the pediatric inflamed joint, we have studied the expression of CCR4 and CCR5 on synovial fluid T cells and matched peripheral blood samples from juvenile rheumatoid arthritis (JRA) patients using three-color flow cytometric analysis. Thymus- and activation-regulated chemokine and macrophage-derived chemokine, ligands for CCR4, were measured by ELISA in JRA synovial fluid, JRA plasma, adult rheumatoid arthritis synovial fluid, and normal plasma. IL-4 and IFN-gamma mRNA production was assessed in CD4+/CCR4+ and CD4+/CCR4(-) cell subsets. We found accumulations of both CCR4+ and CCR5+ T cells in JRA synovial fluids and a correlation for increased numbers of CCR4+ T cells in samples collected early in the disease process. Thymus- and activation-regulated chemokine was detected in JRA synovial fluid and plasma samples, but not in adult rheumatoid arthritis synovial fluid or control plasma. Macrophage-derived chemokine was present in all samples. CD4+/CCR4+ synovial lymphocytes produced more IL-4 and less IFN-gamma than CD4+/CCR4(-) cells. These findings suggest that CCR4+ T cells in the JRA joint may function early in disease in an anti-inflammatory capacity through the production of type 2 cytokines and may play a role in determining disease phenotype.     

Signaling through L-selectin mediates enhanced chemotaxis of lymphocyte subsets to secondary lymphoid tissue chemokine

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.     

Genetic co-transfer of CCR7 ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus

The CC chemokine receptor 7 (CCR7) and cognate CCR7 ligands, CCL19 and CCL21, help establish microenvironments in lymphoid tissue that can facilitate encounters between naive T cells and mature dendritic cells (DCs). This study was conducted to determine if CCR7 ligands can augment the immunogenicity of a DNA vaccine that expresses glycoprotein B (gB) of the pseudorabies virus (PrV). The genetic co-transfer of CCR7 ligands along with a PrV DNA vaccine increased the levels of serum PrV-specific immunoglobulin (Ig) G by 2- to 2.5-fold. In addition, the level of PrV-specific IgG2a isotype was significantly enhanced by co-injection of CCR7 ligand DNA, which indicates that CCR7 ligand biases the humoral immunity toward the Th1-type pattern. The co-injection of CCR7 ligand DNA consistently enhanced the level of Th1-type cytokines (IL-2 and IFN-gamma) produced by stimulated immune cells when compared with a group that was vaccinated with the PrV DNA vaccine. Also, the genetic co-transfer of CCR7 ligand DNAs with PrV DNA vaccine provided prolonged survival against a virulent challenge by PrV. Moreover, the co-administration of CCR7 ligand DNA increased the number of mature DCs into the secondary lymphoid tissues, which appeared to enhance the proliferation of PrV-immune CD4(+) T cells. Taken together, these findings indicate that CCR7 ligands are an attractive adjuvant for a PrV DNA vaccine that can offer protective immunity against the PrV.