Stability of Foxp3 mRNA is controlled by SAMHD1 in human T regulatory cells

Clinical application of antigen-specific T regulatory cells (Tregs) offers promise for the treatment of undesirable immune diseases. To achieve this goal, long-term expansion of Tregs is required to obtain sufficient numbers of cells. However, human Tregs are not stable ex vivo. Therefore, we previously developed an innovative Treg expansion protocol using phosphorothioated random oligonucleotides (ODNps25). The addition of ODNps25 successfully resulted in the stabilization of engineered antigen-specific Tregs, however, the mechanism is not fully characterized. We first identified SAMHD1 (sterile alpha motif and HD domain 1) as an ODNps25 binding protein using a UV-crosslinking pull-down strategy. SAMHD1 physically interacted with the 3′ untranslated region (UTR) of Foxp3 mRNA and was translocated from nucleus to cytoplasm after ODNps25 treatment. Importantly, addition of ODNps25 enhanced the interaction of SAMHD1 and Foxp3 mRNA significantly, and this interaction was increased by TCR stimulation. Since ODNps25 binds to the nuclease (HD) domain of SAMHD1, we then established that overexpression of a nuclease-deficient mutant (D137N) in Tregs significantly stabilized the expression level of Foxp3 protein. This mutant also enhanced stability of a luciferase reporter, followed by the 3′ UTR of Foxp3 mRNA. Additionally, we also found that phosphorylation of the threonine residue (Thr592), which is a regulatory site to control SAMHD1 activity, was clearly downregulated in Tregs. Taken together, we suggest that ODNs interact with the HD of SAMHD1 to interfere with its nuclease activity, thereby stabilizing 3′ UTR of FoxP3 mRNA in long-term culture.