*Purpose: TCR-induced calcineurin phosphatase activation and subsequent Nfat dephosphorylation, leading to Nfat translocation to the nucleus, is a key event in conventional T cell activation. Such translocation in Treg cells leads to Foxp3/Nfat interactions that repress expression of IL-2, induce CD25 and CTLA4, and promote Treg suppressive functions. Subsequent phosphorylation of nuclear Nfat by Dyrk1a leads to its nuclear export, switching of these events. Hence, we hypothesized that Dyrk1a deletion in Tregs would promote nuclear residence and enhance Treg functions.

*Methods: We undertook biochemical, cellular and in vivo studies using WT or C57BL/6 mice with conditional deletion of Dyrk1a in their Tregs as a result of mating floxed Dyrk1a and Foxp3cre mice, as well as testing the effects of potent Dyrk1a inhibitors (Harmine, INDY).

*Results: RNAseq and/or qPCR studies showed that conditional deletion of Dyrk1a in Foxp3+ Tregs led to increased expression of many Kmt2 genes (whose products promote H3K4me3 and gene expression), as well as Tbet, multiple Irf genes, Ifng and Il2. At the cellular level, Dyrk1a deletion led to increased phospho-Nfat1c and phospho-Foxo1, and Dyrk1a gene deletion or pharmacologic targeting led to impaired Treg suppressive function. Likewise, whereas peritransplant CD154 mAb/donor specific transfusion (DST, 5×10⁶cells) induced permanent survival of BALB/c cardiac allografts in WT B6 recipients, mice with conditional deletion of Dyrk1a in their Tregs were unable to maintain long-term allograft survival despite CD154/DST therapy (p<0.01). Conditionally deleted mice also showed enhanced antitumor immunity in syngeneic models.

*Conclusions: These studies indicate nuclear translocation of Nfat is required for optimal Treg functions but that sustained nuclear residence of Nfat in Tregs is detrimental. The balance of cytoplasmic/nuclear Nfat needs to be keep within certain limits and sustained Nfat phosphorylation and translocation needs to be counter-regulated through the actions of Dyrk1a. Without this enzyme’s activity, excess nuclear Nfat overcome and disrupts the normal repressive mechanisms of Foxp3. Hence, unexpectedly, Dyrk1a targeting is not a useful strategy to enhance Treg functions in allograft recipients but rather can be used to degrade Treg function and promote host antitumor immunity.

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