Función del diacilglicerol en el desarrollo, activación y homeostasis de las células T

Resumen

Discrimination between self and non-self antigens is essential for the immune system to control its own response. T cells achieve this self-tolerance acquisition during development, where pre-TCR (T cell receptor) and TCR functions promote thymocytes to survive, mature and choose a cell fate. The mechanisms controlling these events are based on signal intensity after receptor stimulation; thus positive and negative regulators of TCR signaling are important in generating gradual intensities that dictate the cell output. One of the factors implied in this control downstream of the TCR is the extent and duration of Ras-Erk activation, as addressed by numerous studies. Diacylglycerol is a lipid second messenger generated after TCR stimulation by Phospholipase C ¿ (PLC¿) and consumed by Diacylglycerol Kinases (DGK). DAG binds to C1 domain containing proteins to activate them. RasGRP1, one of these DAG-regulated proteins, is responsible of Ras-Erk activation during T cell development. It is known that RasGRP1 activity depends, in vitro, on DAG binding to its C1 domain, but the relevance of this fact in vivo has not been shown. Furthermore, little is known about DAG accumulation during receptor stimulation, which could be a key event in controlling Ras-Erk signal intensity or duration. Thus, we decided to generate tools to study these aspects of T cell biology. Firstly, we developed a method to sense DAG production and localization, in both thymocytes and peripheral T cells. We generated GFP or GST tagged C1 domains to monitor DAG dynamics during antigen recognition. The use of these bio-probes in transgenic TCR primary cells allowed us to manipulate the strength of the stimulus given. Our data show that different pools of DAG exist in T cells: reservoir DAG remains at internal membranes and receptor-induced DAG is produced at the immune synapse. In this last situation, lipid accumulation is strictly controlled by the affinity of the peptide-MHCTCR interaction. Finally, we have analyzed the impact that DAG metabolism plays in T cell tolerance in vivo. To that aim we generated a constitutive active DGK located at the plasma membrane (caDGK), thus diminishing the lipid levels in this specific location after TCR stimulation. Our results have shown a general attenuation of TCR signaling when caDGK is overexpressed in cell lines. The consequences of this fact in T cell selection and homeostasis were evaluated in transgenic mice expressing caDGK in T cell lineage. T cell subsets in thymus were altered at the ISP (immature simple positive cells) level and defects in selection of CD4 and CD8 SP (simple positives cells) were evident. Analysis of peripheral T cells showed general lymphopenia, unbalanced ratio of CD4 and CD8 populations and hyperactivation phenotype of both lineages. Regarding these results, DAG concentration at the plasma membrane is a critical parameter in the development.