C-C chemokine receptor 7 (CCR7) controls lymphocyte migration to secondary lymphoid organs. Although CCR7 has been implicated in targeting the metastasis of cancers to lymph nodes, the role of CCR7 in the metastasis of breast cancer, along with the molecular mechanisms that are controlled by CCR7 that target breast cancer metastasis to the lymph nodes, has yet to be defined. To explore the cellular and molecular mechanisms of breast cancer cell migration to the lymph nodes, we used the mouse MMTV-PyVmT mammary tumor cells (PyVmT) transfected with CCR7 and the human CCR7-expressing MCF10A and MCF7 mammary cell lines. We found that the CCR7 ligands CCL19 and CCL21, controlled cell migration using the β(1)-integrin heterodimeric adhesion molecules. To define a physiological significance for CCR7 regulation of migration, we used the FVB syngeneic mouse model of metastatic breast cancer. When CCR7-negative PyVmT cells transfected with control vector were orthotopically transferred to the mammary fat pad of FVB mice, tumors metastasized to the lungs (10/10 mice) but not to the lymph nodes (0/10). In contrast, CCR7-expressing PyVmT (CCR7-PyVmT) cells metastasized to the lymph nodes (6/10 mice) and had a reduced rate of metastasis to the lungs (4/10 mice). CCR7-PyVmT tumors grew significantly faster than PyVmT tumors, which mirrored the growth in vitro, of CCR7-PyVmT, MCF7, and MCF10A mammospheres. This model provides tools for studying lymph node metastasis, CCR7 regulation of tumor cell growth, and targeting of breast cancer cells to the lymph nodes.

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 ␤1 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 C␥1 (PLC␥1) can mediate phosphorylation of ERK1/2, which is required for ␤1 integrin activation. Paradoxically, we found that inhibition of PLC␥1 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 PLC␥1, we reduced PLC␥1 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 PLC␥1, suggesting a feedback loop between ERK1/2 and PLC␥1. Overall, these results suggest that the CCR7 signaling pathway leading to T lymphocyte migration on fibronectin is a ␤1 integrin-dependent pathway involving transient ERK1/2 phosphorylation, which is modulated by PLC␥1. G protein-coupled receptors (GPCRs)2 are responsive to many types of stimuli, such as hormones, neurotransmitters, and chemoattractants. Ligand activation of a GPCR leads to a conformational change in the receptor, resulting in an exchange of GDP for GTP in the G␣ subunit and dissociation of G␣ from the G␤␥ subunits. The subunits then initiate downstream signaling events to regulate the activation state of adenylyl cyclase or phospholipase C (PLC) (1). CCR7 (C-C chemokine receptor 7), a G protein-coupled receptor, is expressed on naive T lymphocytes and is required for migration into and within lymph nodes. CCR7 binds to two ligands, CCL19 and CCL21. CCL21 is expressed in high endothelial venules, the entry route into lymph nodes (2). However, it is currently unknown how T lymphocytes signal through CCR7 to mediate migration via ␤1 integrins in response to CCL21.Integrins, which are key mediators of lymphocyte adhesion and migration, are heterodimeric adhesion proteins consisting of an ␣ and a ␤ subunit, for which they are named. ␤1 integrins, which are expressed by T lymphocytes, bind to the vascular cell adhesion molecule, collagen, and fibronectin (3). Fibronectin is a major component of the lymph node and is highly expressed in the cortex in the presence of fibroblastic reticular cells, which express CCL21 (4, 5). This network provides an environment that allows CCR7-expressing T lymphocytes to migrate throughout the lymph node.PLC␤ is a...

Dendritic cells (DCs) are potent professional antigen presenting cells (APC) that activate naïve T cells. Interaction of ICAM-1 and LFA-1 molecules on each cell is required for T cell conjugation to DCs which leads to naïve CD4+ T cell activation and proliferation. Nanoparticles capable of blocking LFA-1/ICAM-1 interaction were studied as inhibitors of T cell conjugation to DCs. Primary DCs were primed with ovalbumin, then treated with a peptide that binds ICAM-1 (LABL), a peptide that binds LFA-1 (cIBR) or the same peptides covalently linked to the surface of poly(dl-lactic-co-glycolic acid) nanoparticles (NPs). LABL-NPs and cIBR-NPs rapidly bound to DCs and inhibited T cell conjugation to DCs to a greater extent than the free peptides, unconjugated nanoparticles (NPs), anti-ICAM-1 antibodies and anti-LFA-1 antibodies. In addition, DCs treated with NPs or with cIBR-NPs stimulated the proliferation of T cells, but DCs treated with LABL-NPs did not stimulate T cell proliferation. Nanoparticles targeting ICAM-1 or LFA-1 also altered cytokine production by DC cocultured with T cells when compared to free ligands suggesting these NPs may offer a unique tool for shaping T cell response.

T lymphocytes circulate between the blood, tissues, and lymph. These T cells carry out immune functions, using the C-C chemokine receptor 7 (CCR7) and its cognate ligands, CCL19 and CCL21, to enter and travel through the lymph nodes. Distinct roles for each ligand in regulating T lymphocyte trafficking have remained elusive. We report that in the human T cell line HuT78 and in primary murine T lymphocytes, signaling from CCR7/CCL19 leads to increased expression and phosphorylation of extracellular signal-regulated kinase 5 (ERK5) within eight hours of stimulation. Within 48–72 h we observed peak levels of endothelial differentiation gene 1 (EDG-1), which mediates the egress of T lymphocytes from lymph nodes. The increased expression of EDG-1 was preceded by up-regulation of its transcription factor, Krüppel-like factor 2 (KLF-2). To determine the cellular effect of disrupting ERK5 signaling from CCR7, we examined the migration of ERK5flox/flox/Lck-Cre murine T cells to EDG-1 ligands. While CCL19-stimulated ERK5flox/flox naïve T cells showed increased migration to EDG-1 ligands at 48 h, the migration of ERK5flox/flox/Lck-Cre T cells remained at a basal level. Accordingly, we define a novel signaling pathway that controls EDG-1 up-regulation following stimulation of T cells by CCR7/CCL19. This is the first report to link the two signaling events that control migration through the lymph nodes: CCR7 mediates entry into the lymph nodes and EDG-1 signaling controls their subsequent exit.