Long recognized as an evolutionarily ancient cell type involved in tissue homeostasis and immune defense against pathogens, macrophages are being rediscovered as regulators of several diseases including cancer. Here we show that in mice, mammary tumor growth induces the accumulation of tumor-associated macrophages (TAMs) that are phenotypically and functionally distinct from mammary tissue macrophages (MTMs). TAMs express the adhesion molecule Vcam1 and proliferate upon their differentiation from inflammatory monocytes, but do not exhibit an “alternatively activated” phenotype. TAM differentiation depends on the transcriptional regulator of Notch signaling, RBPJ; and TAM, but not MTM, depletion restores tumor-infiltrating cytotoxic T cell responses and suppresses tumor growth. These findings reveal the ontogeny of TAMs and a discrete tumor-elicited inflammatory response, which may provide new opportunities for cancer immunotherapy.

Summary Regulatory T (Treg) cells, whose identity and function are defined by the transcription factor Foxp3, are indispensable for immune homeostasis. It is unclear whether Foxp3 exerts its Treg lineage specification function through active modification of the chromatin landscape and establishment of new enhancers or by exploiting a pre-existing enhancer landscape. Analysis of the chromatin accessibility of Foxp3-bound enhancers in Treg and Foxp3-negative T cells showed that Foxp3 was bound overwhelmingly to pre-accessible enhancers occupied by its cofactors in precursor cells or a structurally related predecessor. Furthermore, the bulk of Foxp3- bound Treg cell enhancers lacking in Foxp3− CD4+ cells became accessible upon T cell receptor activation prior to Foxp3 expression with only a small subset associated with several functionally important genes being exclusively Treg cell-specific. Thus, in a late cellular differentiation process Foxp3 defines Treg cell functionality in an “opportunistic” manner by largely exploiting the preformed enhancer network instead of establishing a new enhancer landscape.

Regulatory T (Treg) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses1–4. Foxp3 operates as a late-acting differentiation factor controlling Treg cell homeostasis and function5, whereas the early Treg-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors6–10. However, whether Foxo proteins act beyond the Treg-cell-commitment stage to control Treg cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulatorof Treg cell function. Treg cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with Treg-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of Treg cells. Genome-wide analysis of Foxo1 binding sites reveals ~300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt–Foxo1 signalling module controls a novel genetic program indispensable for Treg cell function.