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 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. These complex processes determine the fate of T cells whether they 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.
Sensory adaptation of T cell signaling threshold
(Maria Elena Marquez et Vincenzo Di Bartolo)
Besides clonal selection, the modulation of the T cell signalling threshold in response to self antigens has been proposed as another mechanism to control T lymphocyte self-reactivity. To address the importance of such a mechanism, we generated a transgenic mouse with increased TCR signalling sensitivity by expressing a ZAP-70 gain-of-function mutant (ZAP-YEEI) in post-selection CD8+thymocytes and T cells. Despite an increased basal phosphorylation of the ZAP-70-specific substrate LAT, ZAP-YEEI-expressing CD8 T cells showed no sign of activation and/or auto-reactivity in vivo but appeared rather hypo-responsive in vitro, since their secretion of cytokines was reduced. This correlated with a small decrease in TCR (T cell antigen receptor) and CD5 levels and partial modifications of signalling pathways (e.g., MAP kinase activation kinetics). The effect of avidity for self ligand(s) on adaptation was monitored by crossing ZAPYEEI mice onto P14 and HY TCR transgenic mice. In these animals, ZAP-YEEI expression began in double-positive thymocytes, and tuning of TCR and CD5 expression suggested some adaptation during thymic maturation. Moreover, while P14xZAP-YEEI mature CD8 T cells showed reduced antigen-induced cytokine production and homeostatic proliferation, peripheral survival/expansion and antigen responsiveness of HYxZAP-YEEI cells were enhanced. Our studies suggest that a TCR avidity-dependent sensory adaptation to self-induced stimuli takes place during and/or after T cell thymic development. We have proposed that such a process may prevent dangerous auto-reactivity, as an alternative to T cell deletion and consequent repertoire waste.
Activation of transmethylation in T cells by CD28 co-stimulatory signal
S-Adenosyl-L-Methionine (AdoMet)-dependent methyltransferases (MTases) regulate many biological processes, including signal transduction and gene expression. Every cellular methylation generates S-adenosyl-L-homocysteine (AdoHcy), a potent feedback inhibitor of MTases, normally removed by the enzyme AdoHcyase. T cell proliferation and differentiation are severely affected by blocking AdoHcyase activity and the resulting inhibition of methylation. We have found that the co-stimulatory receptor CD28 interacts with AdoHcyase, stimulates its activity and induces AdoMet-dependent arginine methylation of several proteins, including Vav1, a key effector of CD28 signalling. Methylated Vav1 is found in the nucleus. All these events require intact CD28 cytoplasmic region and are weakly induced by TCR ligation. Our findings uncover a novel role of CD28 in activating a pathway critical for T cell activation. Our future research in this field 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.
Role of the adapter protein Dok-2 as a modulator of TCR signalling and T cell differentiation
The family of protein adapters, Dok (Dok1-5), appears to negatively regulate cellular proliferation, interfere with cell transformation induced by oncogenes and also control cellular differentiation. Dok phosphorylation on tyrosine is associated to its recruitment to the cell membrane and the "build-up" of a negative signaling pathway targeting Ras and Map kinases Erk1/2. The interest of these studies resides in the characterisation of signalling circuits capable of finely modulating the TCR-mediated activation signal and understanding their role in the polarisation of T helper cells towards Th1/2-cell types. We have shown that in CD4 T cells from human blood, Dok-2 (expressed particularly in T cells) is tyrosine phosphorylated after TCR triggering. We have partially characterised the composition of the multimolecular complex including Dok-2. To determine the role of Dok-2 in T cell activation, we are setting up the technique of siRNA in primary CD4 human T cells and utilising loss-of-function mutants of Dok-2. Our preliminary data indicate that Dok-2 recruitment at the membrane of the activated T cell is long-lasting and that it would play a role in the down-modulation of the activation signal.
Keywords: T cells, kinases, phosphatases, co-stimulation, methyltransferases