CD28 signaling in T regulatory precursors requires p56^lck and rafts integrity to stabilize the Foxp3 message

Naturally occurring CD4⁺25^highFoxp3⁺ T regulatory (T-reg) cells are critical for maintaining tolerance to self and non-self Ags. The Foxp3 master-regulatory gene and CD28 costimulation are both required for thymic development and suppressogenic function of CD4 ⁺25^highFoxp3⁺ T-regs. Herein, we show that the sole CD28 stimulation of T-reg thymic precursors augments Foxp3 expression through the increase in Foxp3 mRNA span life by a mechanism involving p56 ^lck and its binding motif on CD28 cytosolic tail, as well as the lipid rafts. We found that 1) the glycosphingolipids and cholesterol components of lipid rafts were highly expressed and unusually partitioned in T-reg thymic precursors as compared with the conventional T cell precursors, 2) the CD28 receptor density on cell membrane is proportional with the content of cholesterol in lipid rafts and with the level of Foxp3 mRNA expression in T-reg precursors, and 3) the CD28-mediated increase of Foxp3 mRNA span life was paralleled by an increased proliferative and suppressogenic capacity of terminally differentiated CD4⁺25^highFoxp3⁺ T-reg precursors. Thus, the functional integrity of CD28 receptor p56 ^lck and plasma membrane lipid rafts are all prerequisites for up-regulation and long-term expression of Foxp3 mRNA transcripts in CD4 ⁺25^highFoxp3⁺ T-reg precursors.