Restricting Glutamine or Glutamine-Dependent Purine and Pyrimidine Syntheses Promotes Human T Cells with High FOXP3 Expression and Regulatory Properties

Abstract

T cell subsets differ in their metabolic requirements, and further insight into such differences might be harnessed to selectively promote regulatory T cells (Tregs) for therapies in autoimmunity and transplantation. We found that Gln restriction during human T cell activation favored CD4 T cells with high expression of the Treg transcription factor FOXP3. This resulted from shrinking numbers and reduced proliferation of activated FOXP3lo/−CD4 T cells while FOXP3hiCD4 T cell numbers increased. This gain was abolished by blocking Gln synthetase, an enzyme that responds to Gln and purine/pyrimidine deficiencies. The shift toward FOXP3hiCD4 T cells under Gln restriction was recapitulated with inhibitors of Gln-dependent pyrimidine and purine syntheses that together closely mimicked declining cell numbers and cell cycles, and by small interfering RNA knockdown of the respective rate-limiting Gln-consuming enzymes CAD and PPAT. FOXP3hi-enriched CD25hiCD4 T cells from these cultures inhibited proliferation, but they also produced effector cytokines, including IL-17A. The latter was largely confined to CTLA-4hi-expressing FOXP3hi-enriched CD25hiCD4 T cells that suppressed proliferation more weakly than did CTLA-4lo/−CD25hiFOXP3hi–enriched T cells. A causal link between high IL-17A production and impaired suppression of proliferation could not be demonstrated, however. Collectively, these results reveal a Gln synthetase–dependent increase and resilience of FOXP3hi cells under Gln restriction, and they demonstrate that impaired Gln-dependent nucleotide synthesis promotes FOXP3hi cells with regulator properties. It remains to be investigated to what extent the concomitant retention of IL-17A–producing CD4 T cells may limit the therapeutic potential of Tregs enriched through targeting these pathways in vivo.