Molecular Mechanism of Generation of LTreg Applied to Inflammatory Processes

Objectives: To identify the molecular mechanism for generation of regulatory T cells, through the association of CD5 with RasGAP. Understanding the molecular mechanism involved in the generation of nTreg and the regulation of the inflammatory response through the association of CD5 with RasGAP; may serve as a therapeutic strategy to control inflammatory diseases, including periodontitis or progression control of oral lymphomas.
Methods: EL-4 cells were cultured and transfected with human CD5 (hCD5) WT or hCD5 cytoplasmic tail punctual mutant molecule in functional tyrosines as Y429, Y441, Y463; and were stimulated with biotinylated antibodies specific for murine CD3 (mCD3), murine CD5 (mCD5) and hCD5 molecules; pervanadate was used as a positive control. Following stimulation, cells were lysed and immunoprecipitated with anti-RasGAP, and separated by electrophoresis (SDS-PAGE). The phosphorylated and co-associated proteins, including CD5 and RasGAP, were detected by Western Blot using antibodies anti-phosphotyrosine and anti-hCD5. Analyse information was performed by evaluating the presence or absence of protein bands corresponding with the molecular weight 120kDa RasGAP and 65 kDa CD5 with the molecular weight standard.
Results: The association of RasGAP to hCD5 may be observed in all stimuli even in unstimulated cells, which could suggest a constitutive association between them; however, after stimuli the phosphorylated hCD5 is not seen after the crosslinking of mCD3hCD5, which suggests the role of RasGAP in the regulation of CD5 function.
Conclusions: RasGAP was constitutively associated to CD5. In the absence of the tyrosines studied (Y429, Y441, Y463) RasGAP-CD5 association was found when cells were stimulated with mCD3mCD5 or mCD3hCD5, which suggests the presence of other potential binding sites such as serines. Y429 is not the only determinant site in the association of RasGAP with hCD5. The association of RasGAP to CD5 constitutive may be directly by Y441 or indirectly by Y429 and Y463 due to its association with PI3k.