Achilles is a circadian clock-controlled gene that regulates immune function in Drosophila.

The circadian clock is a transcriptional/translational feedback loop that drives the rhythmic expression of downstream mRNAs. Termed "clock-controlled genes," these molecular outputs of the circadian clock orchestrate cellular, metabolic, and behavioral rhythms. As part of our on-going work to characterize key upstream regulators of circadian mRNA expression, we have identified a novel clock-controlled gene in Drosophila melanogaster, Achilles (Achl), which is rhythmic at the mRNA level in the brain and which represses expression of antimicrobial peptides in the immune system. Achilles knock-down in neurons dramatically elevates expression of crucial immune response genes, including IM1 (Immune induced molecule 1), Mtk (Metchnikowin), and Drs (Drosomysin). As a result, flies with knocked-down Achilles expression are resistant to bacterial challenges. Meanwhile, no significant change in core clock gene expression and locomotor activity is observed, suggesting that Achilles influences rhythmic mRNA outputs rather than directly regulating the core timekeeping mechanism. Notably, Achilles knock-down in the absence of immune challenge significantly diminishes the fly's overall lifespan, indicating a behavioral or metabolic cost of constitutively activating this pathway. Together, our data demonstrate that (1) Achilles is a novel clock-controlled gene that (2) regulates the immune system, and (3) participates in signaling from neurons to immunological tissues.

Recombinant CD95-Fc (APG101) prevents graft-versus-host disease in mice without disabling antitumor cytotoxicity and T-cell functions.

Graft-versus-host disease (GVHD) induced by transplant-derived T cells represents a major complication after allogeneic bone marrow transplantation (BMT). However, these T cells support engraftment, early T-cell immunity, and mediate the graft-versus-tumor (GVT) effect. Cytotoxic effector functions by transplanted T cells are predominantly mediated by the perforin/granzyme and the CD95/CD95L system. APG101, a novel recombinant human fusion protein consisting of the extracellular domain of CD95 and the Fc domain of an IgG1 antibody inhibited CD95L-induced apoptosis without interfering with T-cell function in vitro and was therefore tested for its ability to prevent GVHD in murine BMT models across minor or major histocompatibility barriers. Starting APG101 treatment either 1 day before or 6 days after transplantation effectively reduced clinical GVHD and rescued survival between 60% and 100% if GVHD was CD95L mediated. APG101 did not interfere with the GVT effect, because P815 mastocytoma and most importantly primary Bcr-Abl-transformed B-cell leukemias were completely eradicated by the alloantigen-specific T cells. Phenotype and homing of alloantigen-specific T cells or their perforin/granzyme-mediated cytotoxicity and proliferative capacity were not affected by APG101 treatment suggesting that APG101 therapy might be useful in GVHD prophylaxis without impairing T-cell function and most importantly preserving GVT activity.

Endothelial plasticity governs the site-specific leukocyte recruitment in hepatocellular cancer.

The correct programming of the endothelial cell phenotype is crucial for efficient leukocyte recruitment to tumor tissue. It has been previously described that T cells infiltrated hepatocellular cancer (HCC) tissue mainly in peritumoral, stromal and tumor border areas. In the current study, phenotype features of tumor endothelial cells and their potential impact on leukocyte recruitment were analyzed in murine tissue of HCC. In the murine model, proinflammatory stimulation with IL-1ß induced leukocyte recruitment in the blood vessels of peripheral tumor areas and in nonmalignant liver tissue, but not in deeper tumor blood vessels. Furthermore, peripheral tumor endothelium, but not deeper tumor blood vessels exhibited a "normalized" hepatic sinusoidal endothelial cell (HSEC)-like phenotype with regard to the expression of adhesion molecules and liver sinusoidal endothelial markers. When tumor endothelial cells were isolated and incubated in vitro, their phenotype rapidly changed and became almost identical to normal hepatic endothelial cells. Interestingly, cytokine production in HCC was strongly dysregulated as compared to normal liver, with IL-1RN exhibiting the most prominent elevation. Experiments with isolated hepatic endothelial cells showed that IL-1RN effectively antagonized the activating action of IL-1ß on the expression of adhesion molecules and T cell attachment. These novel insights indicate that tumor endothelium of HCC represents a plastic system that is susceptible to microenvironmental changes. The peritumoral and tumor border areas have distinct endothelial cell phenotype, which promotes leukocyte recruitment to HCC tissue.

Anti-CD44 induces apoptosis in T lymphoma via mitochondrial depolarization.

A blockade of CD44 can interfere with haematopoietic and leukemic stem cell homing, the latter being considered as a therapeutic option in haematological malignancies. We here aimed to explore the molecular mechanism underlying the therapeutic efficacy of anti-CD44. We noted that in irradiated mice reconstituted with a bone marrow cell transplant, anti-CD44 exerts a stronger effect on haematopoietic reconstitution than on T lymphoma (EL4) growth. Nonetheless, in the non-reconstituted mouse anti-CD44 suffices for a prolonged survival of EL4-bearing mice, where anti-CD44-prohibited homing actively drives EL4 cells into apoptosis. In vitro, a CD44 occupancy results in a 2-4-fold increase in apoptotic EL4 cells. Death receptor expression (CD95, TRAIL, TNFRI) remains unaltered and CD95 cross-linking-mediated apoptosis is not affected. Instead, CD44 ligation promotes mitochondrial depolarization that is accompanied by caspase-9 cleavage and is inhibited in the presence of a caspase-9 inhibitor. Apoptosis becomes initiated by activation of CD44-associated phosphatase 2A (PP2A) and proceeds via ERK1/2 dephosphorylation without ERK1/2 degradation. Accordingly, CD44-induced apoptosis could be mimicked by ERK1/2 inhibition, that also promotes EL4 cell apoptosis through the mitochondrial pathway. Thus, during haematopoietic stem cell reconstitution care should be taken not to interfere by a blockade of CD44 with haematopoiesis, which could be circumvented by selectively targeting leukemic CD44 isoforms. Beyond homing/settlement in the bone marrow niche, anti-CD44 drives leukemic T cells into apoptosis via the mitochondrial death pathway by CD44 associating with PP2A. Uncovering this new pathway of CD44-induced leukemic cell death provides new options of therapeutic interference.

Prevailing role of contact guidance in intrastromal T-cell trapping in human pancreatic cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive collagen-rich stroma. T cells that infiltrate pancreatic cancers frequently become trapped in the stroma and do not contact tumor cells. Here, we aimed to analyze how chemokines and extracellular matrix (ECM) collagen interact in mediating T-cell infiltration in PDAC. EXPERIMENTAL DESIGN: T-cell distribution and ECM structure within tumors were analyzed. Chemokine concentrations in human PDAC were compared with the levels of immune cell infiltration. We assessed the influences of selected chemokines and collagen on directed and random T-cell movement using in vitro migration systems. RESULTS: PDAC overproduced several T-cell-active chemokines, but their levels were not correlated with intratumoral T-cell infiltration. In the absence of collagen, directed migration of activated T cells was induced by chemokines. Interestingly, collagen itself promoted high migratory activity of T cells, but completely abolished chemokine-guided movement. This effect was not altered by a ß1-integrin blocking antibody. Activated T cells actively migrated in low-density collagen matrices, but migration was inhibited in dense collagen. Accordingly, T cells were heterogeneously distributed in the pancreatic cancer stroma, with the majority residing in areas of low-density collagen far from tumor clusters. CONCLUSION: The excessive desmoplasia in PDAC promotes T-cell migration by contact guidance, which abrogates tumor cell-directed movement. Furthermore, dense collagen networks represent a physical barrier, additionally rearranging T-cell distribution to favor tumor stroma. These mechanisms are mainly responsible for intrastromal T-cell trapping in pancreatic cancer and may hinder the development of T-cell-based immunotherapies.