During this reporting period, the research has focused on different targets. A thorough analysis of the repertoires of HLADP4 and HLADR-restricted FVIII peptides presented by antigen presenting cells such as dendritic cells and endothelial cells has been performed. Further, the protocol to derive endothelial cells (liver sinusoidal endothelial-like) from healthy donors and haemophilia A patients with different haemophilia-causing gene abnormalities has been refined and validated. Work is now focusing on confirming the presence of endogenous of FVIII, its localization into different cellular compartment, the expression of HLADR molecules, as a pre-requisite to perform peptide repertoire analyses. Likewise, the protocols to study HLADP4-restrcited FVIII peptides have been validated and the results are summarized in an article under submission for publication. The goal here was to deliver a list of 5 most immunogenic HALDP4-restricted FVIII peptides. Another aspect has been to delineate the repertoire of FVIII-specific CD4+ T cells. More than 800 FVIII-specific T cells lines were successfully generated from more than 22 healthy donors altogether. The specific T cell lines have been identified and tested for the recognition of 63 predicted HLADR-restricted T cell epitopes. The analysis of the repertoires of the the Vbeta CDR3 regions of the T cell receptors was performed which provided a full picture of the heterogeneity of this peculiar cell population. In parallel, several recombinant proteins have been cloned and produced including Fc-fused FVIII domains, immunogenic FVIII A2 or C2 domains linked to tolerogenic molecules. Immunodominant FVIII peptides were selected and synthesized in collaboration with other Beneficiaries and the protocols were delineated for the conjugation of synthetic immuno-dominant FVIII peptides to DC4U’s GlycoDCTM platform dedicated to the induction of T cell mediated immune tolerance in constant interaction with the partner organization DC4U. Likewise, second and third generation FVIII-specific CAR constructs with different intracellular domain combinations have been cloned into lentiviral vectors and used to transduce T cells for further in vitro characterization. In addition, several in vitro culture assays have been validated including the characterization of liver sinusoidal endothelial cells from different from commercial sources, or the preliminary cell culture to study the response to FVIII of transduced CAR CD4+ T cells from healthy donors. Likewise, the experimental protocols to study transcytosis in vitro by human placental cells have been performed; the results are now summarized in an article under submission for publication. In conclusion, most of the tools (peptides, recombinant proteins, primary cell preparation, establishment of cell lines, in vivo experimentation) have been validated and the fundamental experimental protocols for our research have been established. For most of the ESRs, sufficiently original and numerous results have been accumulated to allow the redaction of several research articles, some have been published and some are under submission.
