Human cytomegalovirus encodes the G protein-coupled chemokine receptor homologue US28 that binds several CC chemokines and sequesters extracellular chemokines from the environment of infected cells. Mechanistically, it has been shown that US28 undergoes rapid constitutive receptor endocytosis and recycling. Monoclonal antibodies were raised that allowed the characterization of a ligand-independent phosphorylation and low surface expression of the US28 receptor in transiently transfected HEK293A cells. Phosphoamino acid analysis defined C-terminal serine and threonine residues as phospho-acceptor sites for constitutive receptor phosphorylation. Coexpression of G protein-coupled receptor kinase-2 and US28 enhanced ligand-independent receptor phosphorylation. C-terminal serine to alanine mutagenesis of US28 resulted in a decreased phosphorylation rate that correlated with enhanced surface expression. Maximal surface expression was detected when all C-terminal serines were substituted. Exchange of all C-terminal serines also significantly reduced receptor endocytosis. Thus, constitutive US28 phosphorylation regulates receptor endocytosis and receptor surface display and may thereby provide a pathogenic mechanism for a potential decoy function of the virally encoded receptor.

The chemokine receptor CCR7 and its ligands regulate migration and colocalization of T cells and mature dendritic cells to and within secondary lymphoid organs. The requirement of CCR7 in efficient priming of allospecific cytotoxic CD8 + T cells is poorly characterized. Here, we demonstrate a role for CCR7 in the initiation of an alloimmune response and in the development of transplant rejection. Remarkably, in a model of acute allogeneic tumor rejection, CCR7 -/-mice completely failed to reject subcutaneously injected MHC class I mismatched tumor cells and cytotoxic activity of allospecific T cells was severely compromised. When solid tumors derived from wild-type mice were transplanted, recipient CCR7 -/-mice were capable of rejecting the allografts. In contrast, tumor allografts transplanted from CCR7 -/-donors onto CCR7 -/-recipients showed allograft survival up to 28 days, suggesting a critical function of CCR7 on donor-type passenger leukocytes in the initiation of cytotoxic CD8 + T cell responses. In a heterotopic heart transplantation model CCR7 deficiency resulted in significantly prolonged but not indefinite allograft survival. Additional prolongation of graft survival was observed when hearts from CCR7 -/-mice were used as donor organs. Our results define a key role for CCR7 in allogeneic T cell priming within the context of draining lymph nodes.

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Dendritic cells (DC) either induce T cell tolerance or contribute to the initiation and modulation of T and B cell responses. Since many of the variables determining the thresholds of naive T cell priming were defined in vitro using a homogeneously matured DC population, we here focused on partially mature DC which might reflect the occurrence of tumor-infiltrating and thymic DC. To predict how those DC regulate the induction of antigen-specific T cell proliferation and T cell tolerance, we co-cultured ovalbumin-pulsed murine DC at different ratios with antigen-specific DO11.10 transgenic T cells. Whereas partially mature DC at a DC/T cell ratio of 1 : 10 supported proliferation, a DC/T cell ratio of 1 : 2 induced proliferation arrest in naive CD4 + T cells. The acquisition of the NK cell inhibitory markers NK1.1 and KLRG on T cells exposed to high numbers of DC suggests a role for these molecules in the protection of antigen-responsive T cells from exhaustion by overstimulation. Mechanistically, abortive T cell proliferation upon encounter of high numbers of partially mature DC is caused by an apoptosis-related pathway, suggesting that excessive antigen density without sufficient costimulation results in activation-induced cell death.

Background: Sensory neuron opioid receptors are targets for spinal, epidural, and peripheral opioid application. Although local nerve growth factor (NGF) has been identified as a mediator of sensory neuron -opioid receptor (MOR) up-regulation, the signaling pathways involved have not been yet identified. Methods: Wistar rats were treated with intraplantar vehicle, Freund's complete adjuvant, NGF, NGF plus intrathecal p38 mitogen-activated protein kinase (MAPK) inhibitors, or NGF plus extracellular signal-regulated kinase-1/2 MAPK inhibitors. After 4 days of treatment, paw pressure thresholds of an intraplantar full (fentanyl) or partial (buprenorphine) opioid agonist were determined by algesiometry. Tissue samples from rat dorsal root ganglia were subjected to radiolabeled ligand binding, Western blot analysis, and confocal immunofluorescence. Results: Exogenous and endogenous NGF resulting from Freund's complete adjuvant inflammation produced significant potentiation and enhanced efficacy in fentanyl-and buprenorphine-induced dose-dependent antinociception, respectively. Furthermore, in the ipsilateral dorsal root ganglia, NGF produced a significant increase in MOR binding sites, proteins, and immunoreactive neurons. In parallel,

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