Das Immunsystem unter besonderer Berücksichtigung der Erkennungsstrukturen auf B- und T-Lymphozyten

The essential function of the immune system is to defend against infection. Individuals with defects in central parts of this system continously suffer from infections and without medical care often die. The immune system consists of lymphocytes, macrophages and antibodies which are synthesized by the lymphocytes. Specific immune reactions are induced by antigens. Any substance can serve as antigen and induce immune responses in the host. Antigenic determinants are called epitopes. In case of proteins, an epitope is formed by about 10 aminoacids. Epitopes are recognized by antibodies or immunoglobulins. These are built up of 4 polypeptide chains, namely two identical heavy and two identical light chains. Within each chain one can distinguish a constant and a variable part. The variable parts of heavy and light chains form the antigenbinding site or paratope. Each paratope fits to an epitope as a key fits to its lock. As there exist millions of epitopes there must exist millions of paratopes formed by the variable regions of heavy and light chains. It is not yet fully understood how nature creates this enormous variability.The immune response is initiated by the recognition of epitopes by receptors located on the surface of lymphocytes. Once the antigen is recognized, the lymphocytes get activated and start to produce antibodies which have the same specificity that means the same variable regions as the lymphocyte receptors. The production of specific antibodies is only one branch, namely the humoral response of the immune answer. The other branch includes the production of specific lymphocytes which represents the cellular immune response.The lymphocytes can be divided into two major classes, namely B and T lymphocytes. Both have their stemcells in the bone marrow and B lymphocytes mature under the influence of a not yet fully characterized central lymphoid organ. In birds this organ is the Bursa of Fabricii. T lymphocytes mature in the thymus. B lymphocytes are responsible for the production of antibodies. Once activated, B cells differentiate either into plasmacells which secrete the immunoglobulins or into memory cells which wait within the body for a further contact with the same antigen against which the memory cells can very rapidly response in a so-called secondary immune response. In order to produce antibodies, B lymphocytes need the help of T lymphocytes. These T cells are called helper cells which represent one of the three subpopulation of T cells. The other two are killer cells and suppressor cells. The killer T cells can eliminate other cells and are e. g. activated in the course of a transplant rejection and responsible for it. Suppressor cells, once activated can specifically suppress an immune response.A central problem of modern immunology is the question how lymphocytes can recognize epitopes. B cells do that by the use of immunoglobulin receptors. At the B cell level surface receptors and the effector molecules, the antibodies, are more or less the same. The nature of the T cell receptor is not yet known. There is ample evidence that T cells use in part the same variable regions as B cells in order to create the antigenbinding regions

Analysis of adenovirus trans-complementation-mediated gene expression controlled by melanoma-specific TETP promoter in vitro

BACKGROUND: Human adenoviruses (Ads) have substantial potential for clinical applications in cancer patients. Conditionally replicating adenoviruses (CRAds) include oncolytic adenoviruses in which expression of the immediate early viral transactivator protein E1A is controlled by a cancer cell-selective promoter. To enhance efficacy, CRAds are further armed to contain therapeutic genes. Due to size constraints of the capsid geometry, the capacity for packaging transgenes into Ads is, however, limited. To overcome this limitation, the employment of E1A-deleted replication-deficient viruses carrying therapeutic genes in combination with replication-competent CRAd vectors expressing E1A in trans has been proposed. Most trans-complementing studies involved transgene expressions from strong ubiquitous promoters, and thereby relied entirely on the cancer cell specificity of the CRAd vector. RESULTS: Here we tested the trans-complementation of a CRAd and a replication-deficient transgene vector containing the same cancer cell-selective promoter. Hereto, we generated two new vectors expressing IL-2 and CD40L from a bicistronic expression cassette under the control of the melanoma/melanocyte-specific tyrosinase enhancer tyrosinase promoter (TETP), which we previously described for the melanoma-specific CRAd vector AdDeltaEP-TETP. These vectors gave rise to tightly controlled melanoma-specific transgene expression levels, which were only 5 to 40-fold lower than those from vectors controlled by the nonselective CMV promoter. Reporter analyses using Ad-CMV-eGFP in combination with AdDeltaEP-TETP revealed a high level of trans-complementation in melanoma cells (up to about 30-fold), but not in non-melanoma cells, unlike the AdCMV-eGFP/wtAd5 binary vector system, which was equally efficient in melanoma and non-melanoma cells. Similar findings were obtained when replacing the transgene vector AdCMV-eGFP with AdCMV-IL-2 or AdCMV-CD40L. However, the combination of the novel AdTETP-CD40L/IL-2 vector with AdDeltaEP-TETP or wtAd5 gave reproducible moderate 3-fold enhancements of IL-2 by trans-complementation only. CONCLUSIONS: The cancer cell-selective TETP tested here did not give the expected enforceable transgene expression typically achieved in the Ad trans-complementing system. Reasons for this could include virus-mediated down regulation of limiting transcription factors, and/or competition for such factors by different promoters. Whether this finding is unique to the particular promoter system tested here, or also occurs with other promoters warrants further investigations

Cell Cycle-Dependent Kinase Cdk9 Is a Postexposure Drug Target against Human Adenoviruses

Human adenoviruses (HAdVs) infect respiratory, gastrointestinal, and urinary tracts and give rise to eye infections and epidemic keratoconjunctivitis (EKC). They persist in lymphoid tissue and cause morbidity and mortality in immunocompromised people. Treatments with significant postexposure efficacy are not available. Here, we report that inhibition of the cell cycle-dependent kinase 9 (Cdk9) by RNA interference, or the compound flavopiridol, blocked infections with HAdV-C2/5, EKC-causing HAdV-D8/37, and progeny formation in human corneal epithelial and cancer cells. Flavopiridol abrogated the production of the immediate early viral transactivating protein E1A without affecting nuclear import of viral DNA. In morphometric plaque assays, the compound exhibited antiviral efficacy in both pre- and postexposure regimens with therapeutic indexes exceeding 10. The study identifies Cdk9 as a postexposure drug target against adenovirus infections in vitro and suggests that the clinically tested anticancer drug flavopiridol is a candidate for treating adenoviral EKC or adenovirus emergence upon immune suppression

Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the αv integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on αv integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn–expressing cells. Surprisingly, 30–50% of the endosomal contents were released into the cytosol of control and also K44A-dyn–expressing cells, and the number of fluid phase–positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection

Pseudotyping serotype 5 adenovirus with the fiber from other serotypes uncovers a key role of the fiber protein in adenovirus 5-induced thrombocytopenia

Adenovirus (Ad) infection in humans is associated with inflammatory responses and thrombocytopenia. Although several studies were conducted in mice models to understand molecular and cellular mechanisms of Ad-induced inflammatory responses, only few of them turned their interest toward the mechanisms of Ad-induced thrombocytopenia. Using different depletion methods, the present study ruled out any significant role of spleen, macrophages, and vitamin K-dependent factor in Ad-induced thrombocytopenia. Interestingly, mice displaying thrombocytopenia expressed high levels of cytokines/chemokines after Ad administration. Most importantly, pseudotyping adenovirus with the fiber protein from other serotypes was associated with reduction of both cytokine/chemokine production and thrombocytopenia. Altogether, our results suggest that capsid fiber protein (and more precisely its shaft) of Ad serotype 5 triggers the cytokine production that leads to Ad-induced thrombocytopenia

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor

<div><p>Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.</p></div

The triggering receptor expressed on myeloid cells (TREM) in inflammatory bowel disease pathogenesis

The Triggering Receptors Expressed on Myeloid cells (TREM) are a family of cell-surface molecules that control inflammation, bone homeostasis, neurological development and blood coagulation. TREM-1 and TREM-2, the best-characterized receptors so far, play divergent roles in several infectious diseases. In the intestine, TREM-1 is highly expressed by macrophages, contributing to inflammatory bowel disease (IBD) pathogenesis. Contrary to current understanding, TREM-2 also promotes inflammation in IBD by fueling dendritic cell functions. This review will focus specifically on recent insights into the role of TREM proteins in IBD development, and discuss opportunities for novel treatment approaches

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Endosomophagy clears disrupted early endosomes but not virus particles during virus entry into cells

Enveloped viruses fuse with host membranes without affecting cell integrity. Non-enveloped viruses and bacteria penetrate by rupturing endosomal membranes, and thereby expose complex-type carbohydrates from the endosome lumen to cytosolic proteins. Here we report on the dynamics and initial marker analyses of Galectin-3 (Gal3)-positive membranes triggered by incoming adenovirus species B/C in HeLa cells. Using mCherry-Gal3 reporter constructs, immuno-labeling, confocal and electron microscopy, we detected robust signals from Gal3-containing, early endosomal antigen 1-positive membranes 1h post-infection (pi). Adenoviruses penetrate from non-acidic endosomes with high efficiency, 15min pi, and largely outnumbered the Gal3-positive membranes, suggesting that Gal3 recruitment to broken membranes is transient, or Gal3-positive membranes are rapidly turned-over. In support of rapid turn-over, Gal3 was found within single membrane vesicles and degradative autophagosomes. The Gal3-membranes contained ubiquitin and the poly-ubiquitin binding protein p62/sequestosome-1, but only low amounts of virus, or membrane-lytic protein VI exposed from virions. Remarkably, the Gal3-positive membranes were cleared 3h pi, slower than protein VI, which was cleared 30 min pi. The data show that broken early endosomes but not virus particles are rapidly removed by a process involving autophagy, which we term ‘endosomophagy’. We speculate that endosomophagy is pro-viral, and attenuates innate immunity