Graded Foxo1 Activity in Tregs Differentiates Tumor Immunity from Spontaenous Autoimmunity

Regulatory T cells (Tregs) expressing the transcription factor Foxp3 have a pivotal role in maintaining immunological self-tolerance1-5; yet, excessive Treg activities suppress anti-tumor immune responses6-8. Compared to resting phenotype Tregs (rTregs) in the secondary lymphoid organs, Tregs in non-lymphoid tissues including solid tumors exhibit an activated Treg (aTreg) phenotype9-11. However, aTreg function and whether its generation can be manipulated to promote tumor immunity without evoking autoimmunity are largely unexplored. Here we show that the transcription factor Foxo1, previously demonstrated to promote Treg suppression of lymphoproliferative diseases12,13, has an unexpected function in inhibiting aTreg-mediated immune tolerance. We found that aTregs turned over at a slower rate than rTregs, but were not locally maintained in tissues. Transcriptome analysis revealed that aTreg differentiation was associated with repression of Foxo1-dependent gene transcription, concomitant with reduced Foxo1 expression, cytoplasmic Foxo1 localization, and enhanced Foxo1 phosphorylation at sites of the Akt kinase. Treg-specific expression of an Akt-insensitive Foxo1 mutant prevented downregulation of lymphoid organ homing molecules, and impeded Treg homing to non-lymphoid organs, causing CD8+ T cell-mediated autoimmune diseases. Compared to Tregs from healthy tissues, tumor-infiltrating Tregs downregulated Foxo1 target genes more substantially. Expression of the Foxo1 mutant at a lower dose was sufficient to deplete tumor-associated Tregs, activate effector CD8+ T cells, and inhibit tumor growth without inflicting autoimmunity. Thus, Foxo1 inactivation is essential for the migration of aTregs that have a crucial function in suppressing CD8+ T cell responses; and the Foxo signaling pathway in Tregs can be titrated to preferentially break tumor immune tolerance. Overall design: Transcriptome of splenic rTreg (CD4+Foxp3+CD62LhiCD44lo) and aTreg (CD4+Foxp3+CD62LhiCD44lo) were compared. Duplicates from biologically independent animials were used.

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Luo CT, Liao W, Dadi S, Toure A, Li MO