The pool of innate immune effector cells is wired to rapidly respond to pathogens, whereas only few specificities within the naïve adaptive repertoire expand clonally, undergo epigenetic remodelling and differentiate into effector and memory cells. However, innate cells can differentiate upon pathogen encounter and remember past experiences as well, thereby challenging this strict dichotomy. In particular, us and others have shown that human memory Natural Killer (NK) cells with global epigenetic remodelling can be generated in response to specific signals during cytomegalovirus (CMV) infection. We have recently identified two major types of open chromatin domains in human memory NK (mNK) cells ex vivo: first, a shared signature featured by all mNK cells across CMV-seropositive donors (“public memory”); second, a diverse set of unique open chromatin regions associated with the drastic expansions of individual and stable NK cell clones (“private memory”). Based on this unexpected finding, we hypothesise that the shared and the unique clonal memory might provide mNK cells with increased fitness and high effector potential, but also enhance the risk of oncogenic mutations. The ultimate goal of this project is to identify the signals and molecular mechanisms driving acquisition, selection and maintenance of human NK cell public and private clonal memory. To this end, we will combine multiomic single cell assays and lineage tracing of human NK cells from healthy donors and patients ex vivo, or under various stimuli in vitro, with genome-wide CRISPR perturbation studies to directly link ex vivo features with functional read outs. Success of this project will not only lead to new insights into the key networks promoting persistence and effector functions of mNK cells, but also reveal promising novel targets for cellular anti-tumour therapies.

3TR is a transdisciplinary consortium made of experts in all areas of medicine, basic sciences and bioinformatics from academic institutions, SMEs, and 8 major pharmaceutical companies, teamed to study a fundamental issue in medicine: the mechanisms of response and non-response to therapies, the major aim of 3TR, both within single disease entities and across diseases, where molecular stratification may identify shared disease taxonomies. The molecular identification of groups of patients to whom a drug will benefit, will allow focusing on those who are drug orphan. Harmonization of data from existing academy or industry-sponsored studies will identify biomarkers to inform a new collection. Specimens of diseased tissues, blood, stools, and other fluids will be obtained in a de novo observational prospective trial with standard of care medication prior, during and after first or second line of treatment. Because the studies will be at different phases of progression, a carrousel model of work was designed for input and output of data to be continuously analysed, and interpreted, to inform those measurements to be undertaken and allow cross-validation of results. The 3TR team will elucidate the role of the microbiome, genetics and regulatory genomic features in disease progression. The working aims of 3TR are: 1) establish a centralized data management platform; 2) perform comprehensive molecular and clinical characterisation of a prospective patient cohort; 3) establish integrated analysis of all data using advanced bioinformatics/statistical and modelling methods; 4) identify sets of predictive biomarkers of response/non-response to therapies; 5) improve the competitiveness of European industry and support development of novel solutions. 3TR will sustain beyond the project end the samples and its knowledge base. 3TR will challenge and revolutionize the conventional single-disease based approach with important implications in future disease treatment.

B cells can act both as negative regulators and as drivers of immunity through the production of cytokines. Through secretion of interleukin (IL)-10 B cells inhibited immunity in autoimmune and infectious diseases. For instance, IL-10 from B cells drove complete recovery from disease in experimental autoimmune encephalomyelitis (EAE), the primary animal model for multiple sclerosis (MS), while a lack of IL-10 production by B cells resulted in a severe chronic EAE. B cells can also suppress immunity via IL-35. Human B cells might similarly play inhibitory roles. In few patients with immune-mediated diseases B cell depletion therapy with Rituximab was associated with exacerbation of symptoms, or onset of new pathologies. Conversely, an opposite role of B cells as drivers of immunity was highlighted by the beneficial effect of Rituximab in some patients with rheumatoid arthritis or MS. Clinical improvement often precedes reduction in autoantibody levels in Rituximab treated patients, indicating that B cell-mediated pathogenesis is largely antibody-independent. A candidate factor for the deleterious effects of B cells in MS is IL-6. IL-6 secretion is a major mechanism of B cell-mediated pathogenesis in EAE, and B cells from MS patients produced more IL-6 than cells from healthy individuals. There is now an urgent need for the characterization of the phenotypes of the B cells producing IL-6, IL-10, and IL-35 in vivo at single cell and molecular levels. Markers for these cells might allow understanding the paradoxical effects of B cell-depletion therapy, and guide the development of novel agents depleting distinctively pro-inflammatory B cells, while sparing the remaining of the B cell compartment. Using advanced genetic models to identify and track cytokine-expressing cells, our project aims at characterizing B cells with pro- and anti-inflammatory functions in mice in vivo, to subsequently guide the identification of comparable markers in human.