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.

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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.

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Immune checkpoint (ICP) co-receptors play a key role in the fine modulation of the immune response. While inhibitory ICP co-receptors are mainly characterized in cancer by the exhaustion of the immune response, stimulatory ICP co-receptors are clearly involved in an anarchic activation of the immune system leading to autoimmune diseases. But in another hand, this subtype of co-receptor shows a great asset in cancer therapy by increasing anti-tumoral response. All together, these co-receptors show a great interest for the development of innovative therapies in immunology or immuno-oncology. Nowadays, treatments targeting these co-receptors are currently tested in clinical trials or approved for use in man, especially antibodies targeting both inhibitory and stimulatory ICP. Small molecule-based strategy is tested in early drug discovery and 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, using our powerful proprietary BRET technology: bioSens-AllTM, we successfully develop and validate a cell-based assay platform to assess immune checkpoint pharmacology. The activation or blockade of the signaling of checkpoint co-receptors are monitored in real time and in living cells. BRET assays dedicated to stimulatory ICPs: 4-1BB/4-1BB Ligand and OX-40/OX-40 Ligand were designed. BRET assay development is inspired by the natural recruitment and proximity of specific cytoplasmic partners very close to their co-receptors in an activated pathway. Indeed, the first assay design is based on the co-receptor fused with rGFP and its cytoplasmic partner fused with a rLucII. In the second assay design, the co-receptor is no longer fused, but is co-transfected with a membrane anchor fused with the rGFP and co-transfected with the cytoplasmic partner fused with rLucII. So, in an activated system (soluble ligand, antibody or in co-culture system) a modulation of BRET can be observed. For ICP assays dedicated to 4-1BB, BRET signal is triggered using soluble ligand, agonist antibody, or co-culture where TRAF1 is the biosensor fused with rLucII. Further validation was obtained for OX-40/OX-40 Ligand. Those BRET assays complement once previously developed for inhibitory ICPs axis: PD-1/PD-Ligand 1 or PD-Ligand 2 and CTLA-4/CD80-CD86. The ICP Platform set up here shows a good accuracy and robustness and represents a strong and reliable technology for drug discovery dedicated to ICPs. Our spatio-temporal cell-based functional assays can support broad drug programs, including: High Throughput Functional Screening, Lead Optimization and Bioanalytical QC lot Release. Citation Format: Alice GENTIL DIT MAURIN, Christel FRANCHET, Stephan SCHANN, Xavier LEROY. A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB123.

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 propose 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. At Domain Therapeutics, a diverse library of several dozens of mouse mAbs directed against the hCCR8 was discovered and characterized, providing a unique source for the development of a best-in-class and well differentiated anti-hCCR8 depleting antibody for the treatment of cancers.Special attention was paid on two important characteristics for the selection of our depleting mAb candidates: (1) their ability to detect all CCR8 positive immunosuppressive cells and (2) the combination of different killing modes of action (ADCC, ADCP, CDC) to trigger an optimal depletion.Our selected mAb candidates were nominated based on their capacity to bind to immunosuppressive cells present in different tumor tissues and optimal depleting activity. The impact of our mAbs on tumor growth and on the immune tumor microenvironment (TME) were also studied in mouse hCCR8-KI mice. Finally, following a comprehensive in vitro benchmarking mAb analysis, we identified key differentiating points versus competitor’s mAbs. Nominated candidates wil be ready to enter cell line generation efforts in early 2023. Citation Format: Iseulys Richert, Malaury Schappler, Alice Gentil Dit Maurin, Megane Jeannelle, Solene Rose, Pauline Urquia, Dounia Chraa, Luc Baron, Maria Dolores Garcia Fernandez, Quentin Ruet, Camille Dietsch, Orphee Blanchard, Safia Ayachi, Christel Franchet, Alexandre Fontayne, Claudine Vermot-Desroches, Stephan Schann, Nathalie Lenne. Depleting hCCR8 mAb Therapy #2: Selection of candidates for the development of innovative depleting anti CCR8 therapeutic antibodies to control the immunosuppressive tumor microenvironment [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 2945.

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