The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan-1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan-1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan-1 or Xylosyltransferase 2, a key enzyme of Syndecan-1 biosynthesis. Simultaneous knockdown of Syndecan-1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan-1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan-1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan-1 and Xylosyltransferase 2 was altered within days post-infection, and the remaining Syndecan-1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.
The transcription factor NF-κB plays a key role in numerous physiological processes such as inflammation, immunity, cell proliferation or control of cell death. Its activation is tightly controlled by a kinase complex, IκB kinase (IKK), composed of three core proteins: IKK1/IKKα, IKK2/IKKβ and NEMO/IKKγ. The first two are structurally related kinases whereas the third one is a regulatory subunit exhibiting affinity for upstream activators modified by polyubiquitin chains. Over the years, several inherited diseases caused by mutations of each of the three subunits of IKK have been identified in humans together with diseases caused by mutations of several of its substrates. They are associated with very specific and complex phenotypes involving a broad range of abnormalities such as impaired innate and acquired immune response, perturbed skin development and defects of the central nervous system. Here, we summarize the diverse clinical, cellular and molecular manifestations of IKK-related genetic diseases and show that studying patient-related mutations affecting the IKK subunits and some of their substrates offers the opportunity to understand the various functions of NF-κB in humans, complementing studies performed with mouse models. This analysis also provides glimpses about putative functions of IKK subunits that may be NF-κB-independent.
OTHER ARTICLES PUBLISHED IN THIS SERIESDying autologous cells as instructors of the immune system. Clinical and Experimental Immunology 2015, 179: 1-4. Anti-dsDNA antibodies as a classification criterion and a diagnostic marker for systemic lupus erythematosus: critical remarks. Clinical and Experimental Immunology 2015, 179: 5-10. The effect of cell death in the initiation of lupus nephritis. Clinical and Experimental Immunology 2015, 179: 11-16 SummaryThe production of T cell receptor αβ + (TCRαβ + ) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-β) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4 + CD8 + double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radiosensitivity was already found to be high in immature CD4 − CD8 − (doublenegative, DN) cKit + CD25 + thymocytes, where TCR-β gene rearrangement is initiated. However, TCR-αβ − CD8 + immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αβ + thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αβ − progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.
Background: Tumor-infiltrating Tregs (TITR) constitute a sub-family of immuno-suppressive cells abundantly found in multiple solid cancers. They play a critical role in tumor progression and their specific depletion has been recently proposed as a novel therapeutic strategy to fight cancers. Based on the transcriptional analysis of tumor infiltrating immune cells, the G-protein coupled receptor CCR8 was found to be expressed on murine and human TITR, but not on proinflammatory effector T cells. The CCR8 receptor therefore represents a unique TITR target for the development of novel depletive antibody-based therapeutics. Methods: We have generated a broad collection of monoclonal antibodies (mAb) directed against the human CCR8 receptor. This collection was widely characterized using different sources of recombinant and native hCCR8 positive cell subsets. Moreover, their binding and activities in several functional assays were assessed with different approaches (BRET, Flow Cytometry, including the inhibition of recruitment of different G-proteins to the CCR8 receptor). A comprehensive molecular and pharmacological characterization of each mAb of our proprietary library was generated. mAb binding against different peptides mimicking different states of post-translational modifications of the N-terminus of CCR8 was also monitored. Finally, using two surrogate antibodies directed against the mouse receptor, we tested the role of CCR8 antagonism and Treg depletion in multiple syngeneic models. Results: A comprehensive characterization of Domain Therapeutics’ mAb library revealed distinct mAb cellular reactivity profiles, different epitope recognition patterns (based on binding to different peptides with different post-translational modifications), subnanomolar antagonist activity of downstream signaling, and insurmountable property with regards to the CCR8 ligand CCL1. Moreover, we demonstrated that treatment with a depleting anti-mCCR8 mAb translated into robust anti-tumor activity in multiple syngeneic mouse models. Conclusion: Due to its high and relatively specific expression on tumor-infiltrating Tregs, CCR8 represents an attractive novel target to derive novel immunotherapies. At Domain Therapeutics, a mAb library of several dozen antibodies with distinct and differentiated binding and pharmacological activity profile was successfully discovered and patent-protected. This collection constitutes a unique source of future clinical candidates for the development of a best-in-class well-differentiated anti-hCCR8 depleting antibody for the treatment of cancers. Citation Format: Claudine Vermot-Desroches, Iseulys Richert, Malaury Schappler, Alice Gentil Dit Maurin, Megane Jeannelle, Solene Rose, Luc Baron, Quentin Ruet, Camille Dietsch, Pauline Urquia, Safia Ayachi, Orphee Blanchard, Maria Dolores Garcia Fernandez, Christel Franchet, Nathalie Lenne, Stephan Schann. Depleting hCCR8 mAb Therapy #1: Characterization of a broad collection of anti-hCCR8 mAbs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2946.
Regulation of immune responses is tightly controlled through a balance of co-stimulatory and inhibitory checkpoint receptors, often exploited by many cancers. Therapeutics that block inhibitory receptors or activate immunostimulatory checkpoint receptors have proven to be powerful agents for the restoration of anti-tumor immune responses. While some antibodies targeting these checkpoint receptors have been tested and approved for use in man, many others are in development. For the same targets, there are also small molecules being tested in early clinical trials. In order to screen and assess the potency and efficiency of these therapies, robust and reliable cell-based assays are urgently needed. At Domain Therapeutics, we have successfully developed and validated immune checkpoint pharmacological assays based on our proprietary BRET technology, bioSensAllTM. In these assays, the activation or blockade of the signaling of checkpoint receptors are assessed. PD-1 is expressed on T-cells, while it's ligands, PD-L1 or PD-L2, are expressed on the surface of tumor cells or antigen presenting cells. Like many other immunoglobulin receptors, PD-1 harbors immunoreceptor tyrosine inhibitory motifs (ITIMs) in its cytoplasmic tail that are important signaling motifs. When its ligand, e.g. PD-L1, binds to PD-1, Src family kinases phosphorylate the ITIM motif, resulting in the recruitment of SH2-domain containing phosphatases, SHP-1 and SHP-2, which are involved in inhibiting the T-cell response. As a translation of this concept, we present PD-1 signaling assays using our proprietary BRET technology. For each inhibitory co-receptor, two types of assays were generated. In the first assay, cells expressing the PD-1 and SHP-2 proteins, fused respectively to rGFP and rLucII, are co-incubated with ligand-presenting cells. This results in PD-1 activation and SHP-2 recruitment to the PD-1 receptor, generating a specific BRET signal. In the second assay design, PD-1 positive cells are transfected with SHP-2 and membrane anchor protein, fused respectively with rLucII and rGFP. Upon target engagement following co-culture with cells expressing PD-L1, translocation of SHP-2 close to the membrane triggers a BRET response. Interestingly, the BRET signal is inhibited with either neutralizing antibodies (against PD-L1 or PD-1) or inhibitory small molecules. Further validation was obtained for PD-L2/PD-1 and CD86/CTLA-4. The Immune Checkpoint Platform shows a good accuracy and robustness. These spatio-temporal cell-based functional assays can support broad drug programs, including: High Throughput Functional Screening, Lead Optimization and Bioanalytical QC lot Release. Based on their complementarity, those assays can be used in tandem for primary and secondary screenings and offer a strong and reliable platform for drug discovery. Citation Format: Alice Gentil Dit Maurin, Christel Franchet, Xavier Leroy, Stephane Schann. Development of a pharmacological platform to study in real time immune checkpoints signaling pathways: validation with therapeutic mAbs and small molecules [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6308.
Unfortunately, the given and family name of one of the coauthors, Dr. Alice Gentil Dit Maurin, was incorrectly published in the original publication. The correct given name and family name should read as 'Alice' and 'Gentil Dit Maurin', respectively.
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