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INTEGRATE Report Summary

Project ID: 322465
Funded under: FP7-IDEAS-ERC
Country: France

Mid-Term Report Summary - INTEGRATE (Integrative biology of T cells and dendritic cells in vivo.)

T cells carry out the formidable task of identifying small numbers of foreign antigenic peptides rapidly and specifically. Early steps in T cell activation have thus fascinated biologists and are among the best-studied models of cell stimulation. Our INTEGRATE project aims at describing under physiological conditions and at the systems level the molecular signals that results from the encounter of T cells and dendritic cells (DCs) using novel genetic and proteomic tools. A major methodological achievement has been reached during the first two years of the INTEGRATE project. It consisted in the development of knock-in mice that bear a genetic tag that permits quantitative mass spectrometric analysis of protein complexes in primary mouse T cells stimulated under physiological conditions. The development and validation of this approach revolved around three lines of gene-targeted mice expressing a One-STrEP-tag (OST) at the carboxyl-terminus of endogenous ZAP70, LAT and SLP-76 proteins. We identified a ZAP70-LAT-SLP-76 signalling network consisting of 90 signaling proteins linked via 112 interactions. 70% of the high-confidence proteins identified have never before been observed in the context of TCR signalling. This analysis also highlighted that early TCR signals bifurcate immediately after their onset into a LAT-dependent and a CD6-dependent branch. The description of this novel methodology has been published in Nature Immunology (15, 384-392), and put into a larger context in papers in Nature Immunology (15, 790-797) and in Annual Review of Immunology (33: 539-561). It has been suggested that upon recognition of strong agonist ligands by the TCR, LCK undergoes Ser phosphorylation at position 59, preventing recruitment of SHP1 and subsequent TCR desensitization. Such a positive feedback loop is thought to be inefficiently induced by weak agonist ligands and thus to increase TCR ligand discrimination. To address the importance of LCK-Ser59 in vivo, we generated a line of knock-in mice, in which Ser59 was replaced by Ala. Its comprehensive analysis, showed that Ser59 of LCK has a modest role in the SHP1-based negative feedback pathway thought to implement TCR ligand discrimination, suggesting that the mechanistic model proposed by Germain (Nature Immunology 4: 284-254) to explain T-cell ligand discrimination needs to be revisited. These results have been published in The EMBO Journal (34 : 393-409) as a collaboration with Oreste Acuto.
Our attempts to disentangle the functional complexity of the macrophages and DC subsets that exist in the mouse has led to a rather “simple” picture that we summarized in Nature Review Immunology (Malissen et al., 14: 417-428). Importantly, such knowledge can be used to develop vaccines capable of inducing efficient CD8+ T cell responses. By using a P.L.E.A.S.E. portable laser developed by Pantec Biosolutions, we created laser-generated micropores through the epidermis and targeted a model protein antigen (Ovalbumin : OVA) fused to XCL1, the ligand of XCR1, to dermal XCR1+ DCs. These OVA-XCL1 « vaccibodies » induced antigen-specific CD8+ and CD4+ T cell responses. Moreover, a single intradermal immunization reduced melanoma tumor growth in prophylactic and therapeutic settings, in the absence of exogenous adjuvant. These results have been published in The Journal of Immunology (195, 4953-4961 and 195, 5895-5902). As indicated by its title our project aims at inter- and cross-disciplinary developments. Accordingly we have performed a meta-analysis to resolve at the systems level how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs. Such analysis provides a systems level perspective on the common and divergent features associated with homeostatic and TLR-induced maturation of XCR1+ DCs, a preview of those findings has been published in a review in The EMBO Journal (33, 1104-1116; co-senior authorship with M. Dalod and T. Lawrence).

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