Unit: Molecular Immunology
Director: ACUTO, Oreste
We study the molecular mechanisms of T cell activation. The molecular information carried by activated antigen presenting cells (APC) loaded with antigen and/or cytokines (or chemokines) is relayed to the T cell through a complex device formed by membrane receptors and a vast array of intracellular signaling that decodes positive (activators) and negative (tolerising) signals. The outcome of these complex processes determines whether T cells become effectors of the immune response or memory cells, or "anergized" cells. Our research is focused on understanding the role and regulation of some key signaling components on the signaling machinery. Moreover, we search for new signaling pathways by the use of mass spectrometry applied to intracellular protein complexes and explore new findings indicating that CD28 induces activation of a signaling pathway regulating protein arginine methylation in T cells.
Regulation of SLP-76 by serine phosphorylations (Britta Jungwirth, Benjamin Montagne et Vincenzo DI Bartolo)
Spatio-temporal assembly of transient protein complexes is required to integrate signals emanating from the T cell antigen receptor (TCR) and the co-stimulatory receptor CD28. These cues are translated into activation of signaling pathways to coordinate gene expression. We are studying the physical connectivity and biological significance of signaling complexes assembled upon TCR engagement. Towards this goal, we systematically isolate and functional characterize proteins interacting with critical components of the TCR-induced "signalosome". Our approach is based on epitope tagging of known components of the TCR "signalosome" , their isolation by immunoaffinity and identification of their binding partners by mass spectrometry. One initial target has been the adapter protein SLP-76, which plays a pivotal role in both thymocyte development and mature T cell activation. We have discovered two previously unidentified partners of SLP-76, and their role in T cell signaling is currently being investigated. These studies led us to reveal that TCR inducible binding of signaling proteins to SLP-76 is regulated by phosphorylation not only on tyrosine but also on serine residues whose physiological relevance is being addressed.
A novel signaling pathway induced by CD28 co-stimulatory signal : protein arginine methylation (Fabien Blanchet)
S-Adenosyl-L-Methionine (SAM)-dependent methyltransferases (MTases) regulate many biological processes, including signal transduction and gene expression. T cell proliferation and differentiation were previously shown to be particularly sensitive to cellular methylations but the temporal and specific role of MTAases in T cell activation is unknown. We have found that the co-stimulatory receptor CD28 stimulates Protein aRginine MTAse activity arginine methylation of several proteins, including Vav1, a key effector of CD28 signaling. Methylated Vav1 is found in the nucleus. All these events require CD28 cytoplasmic region, protein tyrosine kinase activity but are weakly induced by TCR ligation. Our findings uncover a novel role of CD28 in activating a pathway likely to be critical for T cell activation. Our future research in this area is aimed at dissecting the underlying molecular mechanism of this pathway, at establishing its physiological role in T cell differentiation and its potential exploitation for novel drug therapies in immuno-suppression.
Functional role of signaling complexes inducing negative signaling in T cells (B. Corre, S. Dong et F. Michel)
The adapters Dok-1 and Dok-2 are thought to be negative regulators of myeloid and lymphoid cells proliferation. Their negative function is attributed in part to their interaction with Ras-GAP, which inactivates Ras and consequently Erk1/2. We have shown that Dok-1 and Dok-2 are both tyrosine phosphorylated in primary T cells stimulated via the TCR. These results imply that negative modulator signals are initiated concomitantly with the activator signals and suggest that together they may shape amplitude and duration of the Ras signaling pathway. Indeed, we have observed that Dok-1 and Dok-2 compose a large complex together with the lipid phosphatase SHIP, and another key adapter of TCR signaling. We are now examining the assembly mechanism of this complex and how it integrates positive and negative signals. These studies are pursued by an approach of siRNA for Dok-1 and Dok-2 in normal CD4 T cells to investigate their potential as modulators of the transcriptional response after activation.
Keywords: T cells, kinases, phosphatases, co-stimulation, protein arginine methyltransferases