Due to their unstable phenotype, iTregs are not currently considered for use in adoptive T cell therapy despite their potential advantages, such as high scalability [12]. Exploring agents that induce Foxp3 and those that stabilize its expression is necessary to enhance the stability of iTregs.

Previously, we reported that STAT6 deficiency in iTregs preserved a stable phenotype and caused the cells to express high levels of Foxp3 and CD25 during long expansion periods, even in the presence of proinflammatory cytokines such as IL-6 [4], which is known to trigger Treg instability and Th17-like phenotype [13]. Furthermore, we observed that iTregs generated under STAT6 deficient conditions exhibited an elevated demethylation status for the FOXP3 TSDR, accompanied by reduced mRNA expression of DNA methyltransferase 1 (DNMT1) [4]. This observation implies that STAT6 might contribute to the silencing of the FOXP3 gene via DNMT1 recruitment [14]. Our previous results showed that STAT6 deficiency in naïve CD4+ T cells promoted the permanence of Foxp3 expression and suppressive function after iTreg differentiation for up to 15 days of expansion. In this study, we searched for the optimal way to generate stable iTregs using STAT6 deficiency with targeted demethylation of the TSDR with a CRISPR-TET1 tool. Epigenetic editing of the TSDR in STAT6-/- iTregs resulted in a significant increase in the expression of Foxp3. In addition, the mRNA expression of suppressive markers such as CTLA-4, PD-1, IL-10, and TGF-β increased.

Previous reports using the CRISPR-Cas9-based method for targeted demethylation of the FOXP3-TSDR demonstrated that epigenetic editing resulted in Foxp3 expression. However, epigenetic TSDR modification could not induce a functional Treg phenotype [7]. This is the first report that demonstrates a protocol where the combination of epigenetic editing with STAT6 deficiency improves the expression of Foxp3 and the classical markers for Treg identification, indicating that the RNA expression profile is switched to a Treg signature. While TSDR demethylation alone may not be adequate to induce functional Tregs, simultaneous STAT6 deficiency can enhance functional alterations, as evidenced by the increased capacity to suppress CD4 and CD8 T lymphocytes observed in transfected STAT6-/- iTregs. STAT6 deficiency could also have other Treg-stabilizing effects such as inhibition of the IL-4 mediated conversion of iTregs to Th2-like effector T cells that typically occurs in allergy settings [15]. However, in this study, we did not analyze whether STAT6 deficiency, in conjunction with targeted demethylation of the TSDR, could be detrimental to cells in the long term. Previously, we demonstrated that in an in vivo model of colitis-associated cancer (CAC), characterized by inflammatory conditions, significant increases in the number of CD4+CD25+Foxp3+ cells were observed in the colon, circulation, and spleen when STAT6 was absent. These increases were accompanied by overexpression of TGF-β, IL-10, and Foxp3 compared to WT mice [4]. Therefore, at least in vivo, the loss of STAT6 improves the stability of Tregs. It would be intriguing to analyze the in vitro culture over longer periods of time to verify the viability of STAT6-/- iTregs within pSgTSDR-transfected cells, as well as to confirm their stability after being transferred in vivo.

IL-4 signaling through the IL-4R in naive CD4+ T cells leads to the phosphorylation and activation of STAT6. Subsequently, STAT6 translocates into the nucleus, initiating the expression of IL-4-responsive genes driving Th2 differentiation [16]. Several investigations have suggested the close relationship between Tregs and Th2 cells [17]. In the scurfy (Sf) mutant mouse, characterized by the absence of functional Foxp3, Tregs lose their in vitro suppressive capacity and exhibit elevated production of Th2-type cytokines. This is further indicated by an increased expression of GATA-3, implying a degree of plasticity between Th2 cells and iTregs [18]. Th2 cytokines IL-4/13 that use STAT6 signaling inhibit the immunosuppression and tolerance induced by Foxp3+ Tregs [19]. Indeed, IL-4 and GATA-3 hamper the in vitro differentiation of naive CD4+ T cells into Foxp3+ Tregs in the presence of TGF-β, diminishing their capacity to suppress T cell proliferation. Conversely, Foxp3 can bind to GATA-3, inhibiting the expression of IL-5 and impeding Th2 differentiation [20]. Previous reports have highlighted the antagonistic interaction between STAT6 and Foxp3. Th2 development plays a significant role in the decline in iTreg viability during prolonged culture, primarily due to the direct binding of STAT6 to the FOXP3 promoter [21]. In accordance with previous results [22], this study showed that STAT6 deficiency significantly decreased GATA-3 expression in iTregs, impairing the Th2 response development which could enhance Tregs` differentiation. Previously, we demonstrated that during in vitro Treg expansion (day 15), the secretion of IL-4 increased in cultures from WT cells, contributing to iTreg fragility [4]. In a mouse model, Tregs expressing reduced Foxp3 levels resulted in the onset of a severe autoimmune syndrome, where these cells tend to adopt a Th2-type effector phenotype, even in a Th1-polarizing environment [23]. In addition, in vitro iTreg induction diminished the expression of Foxp3 and caused Tregs to revert to effector T cells (especially Th2-like cells), which produce cytokines such as IL-2 and IL-4 [24]. Therefore, modifying the STAT6 signaling axis could be an excellent strategy for improving iTreg cell generation.

T-bet is a transcription factor critical for Th1 differentiation and directly regulates the expression of IFNℽ [25]. T-bet blocks the differentiation of other CD4+ Th cell subsets. Mainly, T-bet binds to the evolutionarily conserved region (ECR) upstream of the FOXP3 promoter site, blocking its expression [26]. In iTregs with epigenetic editing, we observed a decrease in T-bet and IFNℽ mRNA expression, which correlated with higher Foxp3 expression, compared to control cells. Neuropilin-1 (Nrp-1)+ iTregs have enhanced suppressive function and stability compared to their Nrp-1- counterpart both in vivo and in vitro [27]. Nrp-1-/- Tregs exhibit elevated T-bet expression and IFN-γ production, resulting in increased Treg fragility [28]. While, in this study, we did not specifically analyze the expression of Nrp1, the reduction in T-bet and IFNℽ expression suggests a potentially more stable phenotype for Tregs.

Multiple redundancies exist in the Treg switch. Some studies have reported the ability of transcription factors (TFs) such as Eos, Irf4, Satb1, Lef1, and Gata1 to flip the switch [11]. Co-immunoprecipitation experiments in transduced Tregs showed interactions between Foxp3 and Gata1, Satb1, and Lef1, suggesting that combining these TFs with Foxp3 could induce the Treg signature [11]. Our experiments detected a significant increase in EOS, IRF4, and SATB1 expression, particularly in pSgTSDR-transfected STAT6-/- iTregs. The Treg signature, along with its regulatory elements, is structured with regulatory feedback loops, both positive and negative. This organization allows for the potential to self-assemble and stabilize once the expression of Foxp3 and specific cofactors surpasses that of Tconv cells. Interestingly, in our system, the differentiation of Tregs triggered, directly or indirectly, the transient expression of Foxp3 and one of its cofactors. In the future, it would be interesting to analyze how these cofactors affect the localization of Foxp3 throughout the genome.

Retroviral expression of Foxp3 has been reported to increase CTLA-4 expression, conferring a suppressive function to conventional T cells [29]. However, previous reports using dCas9-TET1 constructs targeted to FOXP3-TSDR failed to induce CTLA-4 expression despite successful TSDR demethylation [7]. In a prior study [6], dCas9-TET1 mediated demethylation of the human TSDR in Jurkat cells. The results indicated augmented Foxp3 expression; however, no substantial alterations were noted in other master regulators associated with various T-helper lineages, such as RORγT, GATA-3, and Tbet. This lack of significant changes suggests that there was no complete commitment to the Treg lineage. In this study, we demonstrated that the synergy between TSDR-mediated Foxp3 induction and STAT6 deficiency not only significantly enhanced Foxp3 expression but also induced the expression of CTLA-4 and PD-1. However, CTLA-4 proteins in Tregs are not stably expressed on the cell surface but are rapidly cycling between the cell surface and the cell interior [30]. One limitation of this study, stemming from the small number of cells obtained post-transfection, was the challenge in assessing extended culture times and confirming the stability of the transfected iTregs and the CTLA-4 expression. Therefore, it would be valuable to explore the tool described here in conjunction with other iTreg-induction protocols that have been previously reported, such as in vitro retinoic acid treatment with TGF-β1 that reduces STAT6 binding to the Foxp3 promoter and enhances histone acetylation [21]. In addition, utilizing antagonistic agents to neutralize IL-4 or diminishing STAT6 binding to the Foxp3 promoter could represent novel strategies to enhance the generation of inducible Treg cells and promote tolerance. The efficacy of Ruxolitinib (RUX), a JAK 1/2 inhibitor, in promoting the expansion of Tregs has been studied [31]. Although RUX expands Tregs and impedes the differentiation of CD4+ T cells into IFN‐γ‐ and IL‐17A‐producing cells, its effects are immune-context dependent [32]. In addition, RUX decreases the phosphorylation of STAT5, a transcription factor essential for Foxp3 transcriptional regulation. Thus, the use of specific inhibitors for STAT6 could be a more appropriate strategy.

The utilization of alloantigen-specific regulatory T cells (alloTregs) through adoptive transfer has emerged as a potential immunotherapeutic strategy for kidney transplantation. These expanded alloTregs have demonstrated effective suppression of T cell proliferation. Nevertheless, prolonged expansion has been associated with heightened methylation of the TSDR [33]. To address this challenge, a novel approach involving epigenetic editing through CRISPR-dCas9-TET1 and the inhibition of STAT6 signaling could offer a promising avenue to prevent the loss of Foxp3 expression during extensive in vitro expansion of Tregs.