*Purpose: The use of in vitro-differentiated induced Tregs (iTregs) in transplantation and various diseases is a promising therapeutic that is limited by challenges to iTreg stability and function in vivo. A greater understanding of the transcription factors and epigenetics that contribute to iTreg differentiation and stability is needed to advance the therapeutic. We have previously identified Batf3 as a potent inhibitor of FoxP3 expression in iTregs. However, Batf3 is known to interact with the potent transcription factor IRF4. We sought to further understand how Batf3 interacts with IRF4 to control the FoxP3 locus and contribute to iTreg stability and function.

*Methods: We took naïve CD4 T cells from WT, IRF4KO, Batf3Tg, and IRF4KO-Batf3Tg mice and differentiated them under iTreg-inducing conditions in vitro. We also overexpressed these transcription factors and a mutant Batf3 designed to prevent IRF4/Batf3 interactions using a retroviral vector. We utilized the respective genetically modified iTregs in an in vitro suppression assay and in an in vivo disease model to test the impact of these transcription factors on iTregs.

*Results: Our results indicate that Batf3/IRF4 interactions are necessary for FoxP3 inhibition by Batf3. IRF4 by itself is necessary, but not sufficient, for FoxP3 inhibition as IRF4 overexpression was minimally impactful on FoxP3 expression but its absence allowed for near complete conversion of naïve CD4 T cells to iTregs. Batf3 overexpression not only inhibited iTreg differentiation, but destabilized differentiated iTregs. Alternatively, IRF4KO iTregs maintained stable FoxP3 expression in in vitro suppression assays. In contrast to the effect of these transcription factors on FoxP3 expression and stability, Batf3Tg iTregs were more effective at inhibiting effector T cells in vitro and preventing inflammatory disease in vivo. Similarly, IRF4KO iTregs were functionally inferior in vitro and in vivo despite increased FoxP3 expression and stability. This is due at least in part to the impact of these transcription factors on iTreg survival.

*Conclusions: Batf3 and IRF4 are antagonistic toward FoxP3 expression but play other crucial roles in iTreg biology, such as contributing to their survival and function. Although FoxP3 is the hallmark transcription factor of Tregs and necessary for their suppressive function, our results highlight the importance of considering more than FoxP3 expression when determining the impact of various interventions on Treg populations.

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