Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells

Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro–differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.Bronchus-associated lymphoid tissue (BALT) is part of the mucosal immune system of the lung and is characterized by the aggregation of lymphoid cells at the bifurcations of the upper bronchioles (Bienenstock and Befus, 1984). Like other lymphoid follicles, BALT is composed of B cells surrounded by a parafollicular region of T cells (Sminia et al., 1989). Recirculating lymphocytes are believed to enter BALT via high endothelial venules and leave these structures by efferent lymphatics (Lührmann et al., 2002/2003; Xu et al., 2003). Although BALT is largely absent in normal mice, it spontaneously forms in mice deficient for the chemokine receptor CCR7 (Kocks et al., 2007). In humans, it is neither found at birth nor in healthy adults but transiently arises during childhood and adolescence (Tschernig and Pabst, 2000). In both humans and mice, pulmonary infection and inflammation can induce BALT (Moyron-Quiroz et al., 2004). Data derived from splenectomized lymphotoxin-α–deficient mice, which lack all secondary lymphoid organs but do develop BALT, suggest that BALT can serve as induction sites for adaptive immune responses to pathogens with lung tropism (Moyron-Quiroz et al., 2004). However, mechanisms that control the development and maintenance of BALT are largely unknown.Modified vaccinia virus Ankara (MVA) is a highly attenuated orthopoxvirus that lost its capacity to replicate in mammalian cells (Meyer et al., 1991). Recently, MVA was proposed to represent a useful agent for mucosal vaccination via the respiratory route in a nonhuman primate model (Corbett et al., 2008). In mice, MVA delivered via the intranasal (i.n.) route has been shown to induce long-lasting and protective antibody and T cell immune responses (Gherardi and Esteban, 2005; Kastenmuller et al., 2009). However, little is known about the immunological events after respiratory MVA infection.The present report demonstrates that a single i.n. application of the replication-deficient MVA is sufficient to induce the long-lasting presence of BALT and that the lung-specific depletion of DCs interferes with BALT maintenance. Ex vivo imaging of antigen-specific T cell–DC interactions within BALT by two-photon microscopy indicates that, independent of the specific antigenic challenge inducing its formation, BALT can function as a general priming site for T cell responses directed against antigens that reach the lower respiratory tract.     

Constitutively active phosphatase inhibitor-1 improves cardiac contractility in young mice but is deleterious after catecholaminergic stress and with aging

Phosphatase inhibitor-1 (I-1) is a distal amplifier element of β-adrenergic signaling that functions by preventing dephosphorylation of downstream targets. I-1 is downregulated in human failing hearts, while overexpression of a constitutively active mutant form (I-1c) reverses contractile dysfunction in mouse failing hearts, suggesting that I-1c may be a candidate for gene therapy. We generated mice with conditional cardiomyocyte-restricted expression of I-1c (referred to herein as dTG^I-1c mice) on an I-1–deficient background. Young adult dTG^I-1c mice exhibited enhanced cardiac contractility but exaggerated contractile dysfunction and ventricular dilation upon catecholamine infusion. Telemetric ECG recordings revealed typical catecholamine-induced ventricular tachycardia and sudden death. Doxycycline feeding switched off expression of cardiomyocyte-restricted I-1c and reversed all abnormalities. Hearts from dTG^I-1c mice showed hyperphosphorylation of phospholamban and the ryanodine receptor, and this was associated with an increased number of catecholamine-induced Ca²⁺ sparks in isolated myocytes. Aged dTG^I-1c mice spontaneously developed a cardiomyopathic phenotype. These data were confirmed in a second independent transgenic mouse line, expressing a full-length I-1 mutant that could not be phosphorylated and thereby inactivated by PKC-α (I-1^S67A). In conclusion, conditional expression of I-1c or I-1^S67A enhanced steady-state phosphorylation of 2 key Ca²⁺-regulating sarcoplasmic reticulum enzymes. This was associated with increased contractile function in young animals but also with arrhythmias and cardiomyopathy after adrenergic stress and with aging. These data should be considered in the development of novel therapies for heart failure.     

Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice

Injury and loss of podocytes are leading factors of glomerular disease and renal failure. The postmitotic podocyte is the primary glomerular target for toxic, immune, metabolic, and oxidant stress, but little is known about how this cell type copes with stress. Recently, autophagy has been identified as a major pathway that delivers damaged proteins and organelles to lysosomes in order to maintain cellular homeostasis. Here we report that podocytes exhibit an unusually high level of constitutive autophagy. Podocyte-specific deletion of autophagy-related 5 (Atg5) led to a glomerulopathy in aging mice that was accompanied by an accumulation of oxidized and ubiquitinated proteins, ER stress, and proteinuria. These changes resulted ultimately in podocyte loss and late-onset glomerulosclerosis. Analysis of pathophysiological conditions indicated that autophagy was substantially increased in glomeruli from mice with induced proteinuria and in glomeruli from patients with acquired proteinuric diseases. Further, mice lacking Atg5 in podocytes exhibited strongly increased susceptibility to models of glomerular disease. These findings highlight the importance of induced autophagy as a key homeostatic mechanism to maintain podocyte integrity. We postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomerular disease and aging-related loss of renal function.     

Tumor-specific crosslinking of GITR as costimulation for immunotherapy

Activation of murine glucocorticoid-induced tumor necrosis factor-related receptor (mGITR) by its natural ligand (GITRL) or antiGITR agonist mAb enhances T-cell responses, inhibits regulatory T-cell (Treg)-mediated suppression and induces tumor immunity in a variety of murine tumor models. However, systemic administration of these costimulatory agents can lead to global T-cell activation and autoimmunity. To specifically manipulate the T-cell compartment in the tumor microenvironment we propose to target the tumor infiltrating T cells with a bispecific mGITRL fusion protein. For that purpose, mGITRL is linked to a single-chain antibody targeting fibroblast activation protein (FAP) as FAP expression is restricted to cancer-associated fibroblasts (CAFs) found in the stroma of epithelial cancers. AntiFAP-mGITRL fusion protein forms dimers and binds to murine GITR with 1.2?μM affinity and to murine FAP with 4.5?nM. The construct is able to costimulate CD8+ and CD4+ effector T cells resulting in increased proliferation, IFN-γ and IL-2 production. This costimulatory effect is enhanced when the fusion protein is bound to a FAP-positive cell line mimicking FAP CAFs. In suppression assays, membrane-bound antiFAP-mGITRL is 100-fold more effective in overcoming Treg-mediated suppression than unbound fusion protein. These studies suggest that targeted tumor therapy with antiFAP-mGITRL fusion protein could induce tumor rejection while minimizing autoimmune side effects.     

Therapiemonitoring mittels 2-[18F]-FDG-Positronenemissionstomographie nach neoadjuvanter Strahlenbehandlung des Mundhöhlenkarzinoms

BACKGROUND: Combined protocols of radiation therapy and surgical resection, as applied in advanced oral cancer, rely on objective and early assessment of treatment response to radiation therapy. Non-responders require immediate radical salvage surgery even in spite of substantial operative risks, while complete or subtotal response may give reasons for continuing the conservative approach. Therefore, we investigated radiation response by FDG-PET for early monitoring of oral cancer. PATIENTS AND METHODS: In 30 patients with advanced stages of oral cancer (Table 1), FDG-PET (Siemens, ECAT EXACT 922) was performed within 4 weeks after completion of preoperative radiation therapy (36 Gy). SUV of tumor regions were compared to the histologic degree of tumor regression in complete resection specimens. Statistic evaluation included correlation analysis of SUV vs tumor regression and ROC analysis for SUV cut-off values. RESULTS: While low FDG accumulation was found in tumors with histological complete remission (2.3 +/- 0.4) as well as in cases of residual tumor (3.4 +/- 1.8), high FDG uptake was a rather specific indicator of vital tumor tissue (Figure 2). Significant correlation (p = 0.045) between postradiotherapeutic FDG-uptake and histological tumor regression was recognized. A SUV > 2.75 as a clinically practicable threshold value for the identification of residual vital tumor resulted in a specificity of 88%, sensitivity of 68%, a positive predictive value of 94% and a negative predictive value of 50% (Figure 3). Based on our actual follow-up data we could not confirm a significant correlation between postradiotherapeutic SUV and patients' survival. CONCLUSION: Within a standardized protocol, FDG-PET recognize treatment response to radiation therapy in oral squamous cell carcinoma with a reasonable specificity and thus provides a basis for further therapeutic decisions. An increased SUV (> 2.75) may be the rational to justify an aggressive surgical approach even when patients face substantial surgical or anesthesiological risk. However, the posttherapeutic pattern of glucose uptake varies with the applied treatment modalities and has to be explored for the protocol applied.