Final Report Summary - INTEGRATE (Integrative biology of T cells and dendritic cells in vivo.)
T cells probe the surface of dendritic cells (DCs) in search of cues reflecting the antigenic and inflammatory status of body tissues. We used innovative genetic and proteomic tools to describe under physiological conditions and at the systemic levels the molecular signals that result from the encounter of T cells and DCs and that are at the basis of adaptive immunity. Using a unique resource of 20 knock-in mice, we demonstrated that it is possible to generate - via affinity purification followed by mass spectrometry (AP-MS) - a high-density set of qualitative and quantitative data describing in a time-resolved manner the TCR signaling network of primary T cells. Considering that during physiological T cell responses, the TCR requires costimulatory signals originating from the CD28 receptor, we pursued the study of the mechanisms of action of RLTPR, a novel cytosolic protein that we discovered and that is essential to couple CD28 to key effectors of the NF-KκB signaling pathway. We specifically demonstrated that the scaffolding function of RLTPR explains its essential role in mouse and human T cells, a finding that paved the way to the demonstration that humans with RLTPR mutations show T cell- and B cell-intrinsic deficiency. One caveat that faces systemic approaches is that they often remain descriptive and that they do not permit the underlying biochemical mechanisms to be understood. Using Crispr-Cas9-based genome editing, we developed a fast track approach permitting to prove the functional relevance of the novel components and novel protein-protein interactions that we identified on the basis of our AP-MS-based systemic approach.
The coincident study of DCs is thus mandatory if we are ever to make sense of the complexity of T cell activation under physiological conditions. DCs constitute a network of subsets that differ in terms of origin and function. A substantial part of our efforts have been devoted to disentangle the function of DC subsets in vivo using genetic tools that minimally disturb the physiological conditions. We validated a universal toolbox for the automated identification of DC, through high-dimensional unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse and human tissues and provided the first meta-analysis of the process of DC maturation. A lack of consensus regarding how to identify the immune myeloid cell types present in the skin has hampered the precise identification of the cells that capture the ink particles found in tattoo paste and retain then in situ for extended period. Benefiting of our advanced toolbox, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture-release-recapture without any tattoo vanishing. Therefore, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity. This study reached a high societal visibility with an Altmetric score of 954, that is in the top 5% of all research outputs scored by Altmetric. Finally, using the fundamental knowledge we acquired on a subset of skin DC, we showed that a single laser-assisted intradermal deliveryof a tumor-specific vaccine protected mice against melanoma tumor growth in prophylactic and therapeutic settings, in the absence of exogenous adjuvant. Our original objectives have been obtained on time and without rectification and gave rise to 17 publications, the majority of which was published in the leading journals of the discipline.
The coincident study of DCs is thus mandatory if we are ever to make sense of the complexity of T cell activation under physiological conditions. DCs constitute a network of subsets that differ in terms of origin and function. A substantial part of our efforts have been devoted to disentangle the function of DC subsets in vivo using genetic tools that minimally disturb the physiological conditions. We validated a universal toolbox for the automated identification of DC, through high-dimensional unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse and human tissues and provided the first meta-analysis of the process of DC maturation. A lack of consensus regarding how to identify the immune myeloid cell types present in the skin has hampered the precise identification of the cells that capture the ink particles found in tattoo paste and retain then in situ for extended period. Benefiting of our advanced toolbox, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture-release-recapture without any tattoo vanishing. Therefore, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity. This study reached a high societal visibility with an Altmetric score of 954, that is in the top 5% of all research outputs scored by Altmetric. Finally, using the fundamental knowledge we acquired on a subset of skin DC, we showed that a single laser-assisted intradermal deliveryof a tumor-specific vaccine protected mice against melanoma tumor growth in prophylactic and therapeutic settings, in the absence of exogenous adjuvant. Our original objectives have been obtained on time and without rectification and gave rise to 17 publications, the majority of which was published in the leading journals of the discipline.