Ricerc@Sapienza

Introduction CD28 is a crucial costimulatory receptor necessary for full T cell activation. One important contribution of CD28 to T cell activation relies on its ability to regulate T cell metabolism by enhancing nutrient uptake, aerobic glycolysis and anabolic pathways. Indeed, CD28 binds class 1A PI3K that in turn recruits and activates the PDK1/Akt/mTOR pathway. We have recently found that CD28 stimulation strongly up-regulates the expression of cytokines related to the Th17 cell phenotype in relapsing-remitting multiple sclerosis (RRMS) patients. Objectives The aim of this work was to characterize the role of CD28 and associated class 1A PI3K in the modulation of the metabolic programs regulating pro-inflammatory Th17 cell responses in RRMS. Patients & methods. 30 patients with a clinically defined MS according to the McDonald criteria and a clinically RRMS were enrolled from S. Camillo Hospital, (Rome, Italy). 30 age-/gender-matched healthy donor (HD) buffy coats from the blood bank of Sapienza University (Rome, Italy) with no previous history of neurological or autoimmune diseases were used as controls. CD4+ T cells isolated from the peripheral blood of HDs or RRMS patients were stimulated with agonistic anti-CD28 antibodies and changes in cellular metabolism (Seahorse technology), surface expression of activation markers and the glucose transporter 1 (Glut -1) (Flow cytometry) as well as the expression of pro-inflammatory cytokines and metabolic enzymes (Real-time PCR, ELISA, western blotting) were analysed. Results: CD28 stimulation up-regulated glycolysis without significantly affecting mitochondrial oxidative phosphorylation in CD4+ cells from RRMS patients. The analysis of the major enzymes regulating the glycolytic pathway revealed that CD28 stimulation induced the increase of c-myc and the glucose transporter Glut1 in CD4+ T cells from RRMS. CD28-induced increase of glycolysis was also associated with the up-regulation of pro-inflammatory cytokines, most of which were related to the Th17 cell phenotype, as demonstrated by the strong inhibition exerted by the glycolysis inhibitor 2-deoxy-D-glucose. Finally, treatment of CD4+ T cells from RRMS patients with a class 1A PI3K inhibitor strongly impaired CD28-induced glycolysis, c-myc and Glut1 expressions, as well as the up-regulation of pro-inflammatory cytokines. Conclusion: Altogether these data strongly suggest a role of CD28 and associated class 1A PI3K in reprogramming the metabolic process that maintain/amplify the inflammatory phenotype of peripheral T cells in RRMS patients.