SUMMARY Regulatory T (Treg) cells suppress immune responses to a broad range of non-microbial and microbial antigens and indirectly limit immune inflammation-in-flicted tissue damage by employing multiple mechanisms of suppression. Here, we demonstrate that selective Treg cell deficiency in amphiregulin leads to severe acute lung damage and decreased blood oxygen concentration during influenza virus infection without any measureable alterations in Treg cell suppressor function, antiviral immune responses, or viral load. This tissue repair modality is mobilized in Treg cells in response to inflammatory mediator IL-18 or alarmin IL-33, but not by TCR signaling that is required for suppressor function. These results suggest that, during infectious lung injury, Treg cells have a major direct and non-redundant role in tissue repair and maintenance—distinct from their role in suppression of immune responses and inflammation—and that these two essential Treg cell functions are invoked by separable cues.

Summary Regulatory T (Treg) cells, whose differentiation and function are controlled by X-chromosome-encoded transcription factor Foxp3, are generated in the thymus (tTreg) and extrathymically (peripheral, pTreg) and their deficiency results in fatal autoimmunity. Here we demonstrate that a Foxp3 enhancer, conserved non-coding sequence 1 (CNS1), essential for pTreg, but dispensable for tTreg cell generation, is present only in placental mammals. CNS1 is largely composed of mammalian-wide interspersed repeats (MIR) that have undergone retrotransposition during early mammalian radiation. During pregnancy pTreg cells specific to a model paternal alloantigen were generated in a CNS1-dependent manner and accumulated in the placenta. Furthermore, when mated with allogeneic, but not syngeneic males, CNS1-deficient females showed increased fetal resorption accompanied by increased immune cell infiltration and defective remodeling of spiral arteries. Our results suggest that during evolution a CNS1-dependent mechanism of extrathymic differentiation of Treg cells emerged in placental animals to enforce maternal-fetal tolerance.

Foxp3+ regulatory T cells (Treg cells) maintain immunological tolerance and their deficiency results in fatal multi-organ autoimmunity. Although heightened T cell receptor (TCR) signaling is critical for the differentiation of Treg cells, the role of TCR signaling in Treg cell function remains largely unknown. Here we demonstrate inducible ablation of the TCR results in Treg cell dysfunction which cannot be attributed to impaired Foxp3 expression, decreased expression of Treg cell signature genes or altered ability to sense and consume interleukin 2. Rather, TCR signaling was required for maintaining the expression of a limited subset of genes comprising 25% of the activated Treg cell transcriptional signature. Our results reveal a critical role for the TCR in Treg cell suppressor capacity.

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.

SUMMARY In multicellular organisms, specialized functions are delegated to distinct cell types whose identity and functional integrity is maintained upon challenge. However, little is known about the mechanisms enabling lineage inheritance and their biological implications. Regulatory T (Treg) cells, which express the transcription factor Foxp3, suppress fatal autoimmunity throughout the lifespan of animals. Here, we show that a dedicated Foxp3 intronic element CNS2 maintains Treg cell lineage identity by acting as a sensor of the essential Treg cell growth factor IL-2 and its downstream target STAT5. CNS2 sustains Foxp3 expression during division of mature Treg cells when IL-2 is limiting and counteracts pro-inflammatory cytokine signaling that leads to the loss of Foxp3. CNS2 mediated stable inheritance of Foxp3 expression is critical for adequate suppression of diverse types of chronic inflammation by Treg cells and prevents their differentiation into inflammatory effector cells. The described mechanism may represent a general principle of the inheritance of differentiated cell states.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (Treg cells). To gain insights into the molecular mechanisms of Foxp-mediated gene expression we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multi-protein complexes of 400–800 kDa or larger and identified 361 associated proteins, ~30% of which are transcription-related. Foxp3 directly regulated expression of a large proportion of the genes encoding its co-factors. Reciprocally, some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of Foxp3 cooperation with one such partner, GATA-3, provided further evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of Treg cell biology.

Post-transcriptional regulation by microRNAs and siRNAs depends not only on characteristics of individual binding sites in target mRNA molecules, but also on system-level properties such as overall molecular concentrations. We hypothesize that an intracellular pool of microRNAs/siRNAs faced with a larger number of available predicted target transcripts will downregulate each individual target gene to a lesser extent. To test this hypothesis, we analyzed mRNA expression change from 178 microRNA and siRNA transfection experiments in two cell lines. We find that downregulation of particular genes mediated by microRNAs and siRNAs indeed varies with the total concentration of available target transcripts. We conclude that to interpret and design experiments involving gene regulation by small RNAs, global properties, such as target mRNA abundance, need to be considered in addition to local determinants. We propose that analysis of microRNA/siRNA targeting would benefit from a more quantitative definition, rather than simple categorization of genes as ‘target' or ‘not a target.' Our results are important for understanding microRNA regulation and may also have implications for siRNA design and small RNA therapeutics.