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“Natural” regulatory T cells (nTregs) that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. “Induced” regulatory T cells (iTregs) are nonredundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg-produced IL-10 and iTreg fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1−/− mice by the adoptive transfer of naive CD4+ T cells carrying a nonfunctional Foxp3 allele. At the onset of weight loss, mice were treated with both iTregs and nTregs where one marked subset was selectively IL-10 deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTregs were present but IL-10 deficient, iTreg-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ∼85% of the transferred iTregs lost Foxp3 expression (ex-iTregs) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTregs and ex-iTregs, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10–dependent functional reciprocity between regulatory T cell subsets that maintains mucosal tolerance. The niche supporting stable iTregs is limited and readily saturated, which promotes a large population of ex-iTregs with pathogenic potential during immunotherapy.

Individuals with atopic dermatitis immunized with the small pox vaccine, vaccinia virus (VV), are susceptible to eczema vaccinatum (EV), a potentially fatal disseminated infection. Dysfunction of Forkhead box P3 (FoxP3)-positive regulatory T cells (Treg) has been implicated in the pathogenesis of atopic dermatitis. To test whether Treg deficiency predisposes to EV, we percutaneously VV infected FoxP3-deficient (FoxP3KO) mice, which completely lack FoxP3+ Treg. These animals generated both fewer VV-specific CD8+ effector T cells and IFN-γ–producing CD8+ T cells than controls, had higher viral loads, and exhibited abnormal Th2-polarized responses to the virus. To focus on the consequences of Treg deficiency confined to the skin, we generated mixed CCR4KO FoxP3KO bone marrow (CCR4/FoxP3) chimeras in which skin, but not other tissues or central lymphoid organs, lack Treg. Like FoxP3KO mice, the chimeras had impaired VV-specific effector T cell responses and higher viral loads. Skin cytokine expression was significantly altered in infected chimeras compared with controls. Levels of the antiviral cytokines, type I and II IFNs and IL-12, were reduced, whereas expression of the proinflammatory cytokines, IL-6, IL-10, TGF-β, and IL-23, was increased. Importantly, infection of CCR4/FoxP3 chimeras by a noncutaneous route (i.p.) induced immune responses comparable to controls. Our findings implicate allergic skin inflammation resulting from local Treg deficiency in the pathogenesis of EV.

Foxp3-deficient regulatory T (Treg) cells lack suppressor function and manifest a T effector (Teff) cell-like phenotype. We demonstrate that Foxp3 deficiency dysregulates mTORC2 signaling and gives rise to augmented aerobic glycolysis and oxidative phosphorylation. Mutant Treg cell-specific deletion of the mTORC2 adaptor gene Rictor or expression of a Foxo1 transgene improved regulatory function and ameliorated disease. Rictor deficiency reestablished a subset of Treg cell genetic circuits and suppressed the Teff cell-like metabolic program. Treatment of mutant Treg cells of patients with FOXP3 deficiency with mTOR inhibitors similarly antagonized their Teff cell-like program and restored suppressive function. Thus, regulatory function can be reestablished in Foxp3-deficient Treg cells by targeting their metabolic pathways, providing opportunities to restore tolerance in Treg cell disorders.

In addition to thymus-derived or natural T regulatory (nTreg) cells, a second subset of induced T regulatory (iTreg) cells arises de novo from conventional CD4+ T cells in the periphery. The function of iTreg cells in tolerance was examined in a CD45RBhighCD4+ T cell transfer model of colitis. In situ-generated iTreg cells were similar to nTreg cells in their capacity to suppress T cell proliferation in vitro and their absence in vivo accelerated bowel disease. Treatment with nTreg cells resolved the colitis, but only when iTreg cells were also present. Although iTreg cells required Foxp3 for suppressive activity and phenotypic stability, their gene expression profile was distinct from the established nTreg “genetic signature,” indicative of developmental and possibly mechanistic differences. These results identified a functional role for iTreg cells in vivo and demonstrated that both iTreg and nTreg cells can act in concert to maintain tolerance.

CD40 ligand (L), FasL, and TNF-α are members of the TNF family of cytokines. All are expressed by T lymphocytes shortly after activation but have distinct effector functions. Transcription of these genes can be induced by stimulation of T cells by calcium ionophore alone and requires the calcineurin-dependent transcription factor NF of activated T cells. We have examined a second calcium-dependent signaling pathway, mediated by calcium/calmodulin-dependent kinase IV (CaMKIV) in transcriptional activation of TNF family genes. In reporter gene assays using constructs driven by the promoters of human CD40L, FasL, or TNF-α along with vectors expressing constitutively active CaMKIV and calcineurin, we have demonstrated that each promoter is activated by calcineurin and CaMKIV in a synergistic fashion. Furthermore, specific inhibition of CaMKIV by chemical means and by a dominant negative mutant of CaMKIV impairs the ionomycin-induced activity of all three promoters as well as protein expression of CD40L and TNF-α. Our results indicate that activation of gene expression by calcineurin and CaMKIV is common to members of the TNF cytokine family.

We have found that FOXP3 is an oligomeric component of a large supramolecular complex. The Zinc-Leuzip region of FOXP3 is sufficient to mediate its homotetramerization. Wild type FOXP3 directly binds to the human IL-2 promoter, but the E251 deletion in FOXP3 in XLAAD/IPEX patient’s T cells disrupts its homo-oligomerization, heteromerization with FOXP1, its association with the IL-2 promoter, and limits repression of IL-2 transcription after T cell activation. Additionally we report that transcriptional repression by FOXP3 involves a histone acetylase-deacetylase complex that includes TIP60 and HDAC7 and HDAC9. FOXP3 can be acetylated, and TIP60 promotes FOXP3 acetylation in vivo. Knockdown of endogenous TIP60 relieved FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation, and can be restored by the Trichostatin A indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a novel complex based mechanism by which FOXP3 actively mediates transcriptional repression.