Mechanisms of action of the protein RLTPR member of the Carmil family. Rltpr is essential for the CD28 costimulatory pathway. RLTPR is at the interface between CD28 at the membrane, the cytoskeleton, CARMA1 and NF-kB activation pathway.
The CD28 Coreceptor is essential for T cell activation and development and function of regulatory T cells. CD28 has been studied extensively but remains poorly characterized. We propose a study of the protein interactome between three partners in this activation pathway: CD28-RLTPR-CARMA1. This study is conducted in primary T cells in mice.
Mouse models produce for this study are of two types :
- Tagged protein in vivo for mass spectrometry studies
- Structure function studies in knock out or point mutation.
To date the project is in its launch phase with the production of all animal models.
T cell activation regulation by coactivator and co-inhibitor receptors is a topic of great interest in clinical research in areas as diverse as auto-immunity, cancer or vaccination.
Integrative Biology of T cell activation. Nature Immunol, 2014, 15 : 790-797
Review summarizes recent results using the strategy of analysis that will be developed in the BASILIC project
A wealth of molecular signals results from the encounter of T cells and dendritic cells (DCs) and is at the basis of adaptive immunity. Early studies have led to a "two-signal model" of T cell activation in which the first signal is delivered via the T cell antigen receptor (TCR) following recognition of antigenic peptides bound to molecules of the major histocompatibility complex. This signal provides antigen specificity to T cell responses. The second signal is known as the costimulatory signal and is delivered by the CD28 molecule upon recognition of the CD80 and CD86 ligands that are expressed on DCs. By synergizing with the TCR, CD28 activates the NF-kB signaling pathway and leads to the transcription of genes involved in T cell survival and proliferation. The importance of CD28 is demonstrated by the failure of CD28-deficient mice to produce T cell-dependent antibody responses and to clear infections. By using a N-ethyl-N-nitrosurea mutagenesis screen, we identified a mutation in a lymphoid-specific gene that is called Basilic and the product of which turned out to be a novel and essential component of the CD28 signaling pathway. These findings have led us to amend the existing model of CD28 costimulation into a new more complete model in which Basilic plays an essential role owing to its capacity to couples CD28 to PKCq and Carma1, two essential inducers of the NF-kB pathway. The present proposal aims at defining the mechanisms of action of this novel protein essential for T cell physiology.
The BASILIC project is based on a large constellation of pilot experiments that we have recently performed. The corresponding results that are under submission are extensively presented to support the feasibility of the whole BASILIC proposal and to allow reviewing the likelihood of its success. Due to its multidisciplinary nature, the BASILIC project will involve the collaboration of Ruedi Aebersold (Institute of Molecular System Biology, ETH, Zurich), of Takashi Saito (Riken Center for Allergy and Immunology, Yokohama) and of Christoph Wulfing (University of Bristol, Bristol). The BASILIC project is organized into the following complementary tasks: (1) The first task is based on a robust approach that we have recently developed and that is based on knockin mice that permits to generate primary (untransformed) T cells amenable to Mass Spectrometry (MS) analysis. Task 1 will allow us to determine in an unbiased and comprehensive manner the composition and dynamics of the signaling complexes that assemble around Basilic in primary CD4+ T cells following engagement of the TCR and CD28 signaling patwhays; (2) We recently demonstrated that it is possible to use transcription activator like effector nucleases (TALENs) for genome editing in a mouse T cell line. Therefore, such approach offers the long sought possibility of editing the genome of a T cell line in a fast track manner and thereby of performing structure-function analysis of the novel components of the CD28 pathway to be identified during the BASILIC project; (3) We have developed two lines of knockin mice to perform MS analysis of the CD28 and Carma1 interactomes. On that basis, we expect to precisely define the position of Basilic within the CD28-Carma1 signaling axis. (4) the outcome of adaptive immune responses results in large part from the quality of the interactions between T cells and DCs. This last task constitutes a « grand challenge » since it aims at stimulating primary T cells with CD80-positive or –negative DCs and at defining via MS and for the first time the CD28 costimulatory interactome in primary T cells stimulated under physiological conditions.
Madame Marie MALISSEN (Institut National de la Santé et de la Recherche Médicale)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
INSERM-CIML Institut National de la Santé et de la Recherche Médicale
Help of the ANR 400,000 euros
Beginning and duration of the scientific project: December 2013 - 42 Months