Intestinal microbes provide multicellular hosts with nutrients and confer resistance to infection. The delicate balance between pro- and anti-inflammatory mechanisms, essential for gut immune homeostasis, is affected by the composition of the commensal microbial community. Regulatory T (Treg) cells expressing transcription factor Foxp3 play a key role in limiting inflammatory responses in the intestine1. Although specific members of the commensal microbial community have been found to potentiate the generation of anti-inflammatory Treg or pro-inflammatory Th17 cells2-6, the molecular cues driving this process remain elusive. Considering the vital metabolic function afforded by commensal microorganisms, we hypothesized that their metabolic by-products are sensed by cells of the immune system and affect the balance between pro- and anti-inflammatory cells. We found that a short-chain fatty acid (SCFA), butyrate, produced by commensal microorganisms during starch fermentation, facilitated extrathymic generation of Treg cells. A boost in Treg cell numbers upon provision of butyrate was due to potentiation of extrathymic differentiation of Treg cells as the observed phenomenon was dependent upon intronic enhancer CNS1, essential for extrathymic, but dispensable for thymic Treg cell differentiation1, 7. In addition to butyrate, de novo Treg cell generation in the periphery was potentiated by propionate, another SCFA of microbial origin capable of HDAC inhibition, but not acetate, lacking this activity. Our results suggest that bacterial metabolites mediate communication between the commensal microbiota and the immune system, affecting the balance between pro- and anti-inflammatory mechanisms.

In the course of infection or autoimmunity, particular transcription factors orchestrate the differentiation of T H 1, T H 2 or T H 17 effector cells, the responses of which are limited by a distinct lineage of suppressive regulatory T cells (T reg ). T reg cell differentiation and function are guided by the transcription factor Foxp3, and their deficiency due to mutations in Foxp3 results in aggressive fatal autoimmune disease associated with sharply augmented T H 1 and T H 2 cytokine production [1][2][3] . Recent studies suggested that Foxp3 regulates the bulk of the Foxp3-dependent transcriptional program indirectly through a set of transcriptional regulators serving as direct Foxp3 targets 4,5 . Here we show that in mouse T reg cells, high amounts of interferon regulatory factor-4 (IRF4), a transcription factor essential for T H 2 effector cell differentiation, is dependent on Foxp3 expression. We proposed that IRF4 expression endows T reg cells with the ability to suppress T H 2 responses. Indeed, ablation of a conditional Irf4 allele in T reg cells resulted in selective dysregulation of T H 2 responses, IL4-dependent immunoglobulin isotype production, and tissue lesions with pronounced plasma cell infiltration, in contrast to the mononuclear-cell-dominated pathology typical of mice lacking T reg cells. Our results indicate that T reg cells use components of the transcriptional machinery, promoting a particular type of effector CD4 + T cell differentiation, to efficiently restrain the corresponding type of the immune response.T reg cell deficiency results in activation and expansion of CD4 + and CD8 + T cells, dendritic cells, granulocytes and macrophages, and greatly increased production of a wide range of cytokines including interleukin (IL)-2, T H 1 and T H 2 cytokines 6,7 . Expression of Foxp3 is required for the establishment and maintenance of T reg lineage identity and suppressor function [8][9][10][11] . Our recent study suggested that in T reg cells Foxp3 might regulate expression of IRF4 (refs 12 -14) a transcription factor that is indispensable for T H 2 effector cell differentiation 15,16 . Furthermore, a recent study suggested a prominent role for IRF4 in T H 17Correspondence and requests for materials should be addressed to A.Y.R. (rudenska@mskcc.org). † Present address: Department of Immunology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Information Reprints and permissions information is available at www.nature.com/reprints. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Fig. 1a, b). Irf4 messenger RNA was increased in thymic and peripheral Foxp3 + T reg cells in comparison to CD25 − Foxp3 − CD4 + T cells (data not shown) 8 . Furthermore, Foxp3 knockdown using a retrovirally encoded Foxp3-specific short hairpin RNA resulted in a marked diminution in Irf4 mRNA ( Supplementary Fig. 1c 1...

Forkhead winged-helix transcription factor Foxp3 serves as the dedicated mediator of the genetic program governing CD25 + CD4 + regulatory T cell (T r ) development and function in mice. In humans, its role in mediating T r development has been controversial. Furthermore, the fate of T r precursors in FOXP3 deficiency has yet to be described. Making use of flow cytometric detection of human FOXP3, we have addressed the relationship between FOXP3 expression and human T r development. Unlike murine Foxp3 − T cells, a small subset of human CD4 + and CD8 + T cells transiently up-regulated FOXP3 upon in vitro stimulation. Induced FOXP3, however, did not alter cell-surface phenotype or suppress T helper 1 cytokine expression. Furthermore, only ex vivo FOXP3 + T r cells persisted after prolonged culture, suggesting that induced FOXP3 did not activate a T r developmental program in a significant number of cells. FOXP3 flow cytometry was also used to further characterize several patients exhibiting symptoms of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) with or without FOXP3 mutations. Most patients lacked FOXP3-expressing cells, further solidifying the association between FOXP3 deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Interestingly, one patient bearing a FOXP3 mutation enabling expression of stable FOXP3 mut protein exhibited FOXP3 mut -expressing cells among a subset of highly activated CD4 + T cells. This observation raises the possibility that the severe autoimmunity in FOXP3 deficiency can be attributed, in part, to aggressive T helper cells that have developed from T r precursors.

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.

The role of self-peptides in positive selection of CD4+ T cells has been controversial. We show that some self-peptides are presented by the MHC class II molecule I-A(b) in mice lacking Ii or H-2M but not in mice expressing a transgene-encoded peptide fused to I-A(b). In experiments using specific antibodies to block selection, these low-abundance self-peptides were implicated in the positive selection of some CD4+ T cells in H-2M-/- mice. However, all three mutant backgrounds failed to positively select two class II-restricted transgenic T cell receptors. Our findings suggest that minor components of the self-peptide repertoire can contribute to positive selection of a significant number of CD4+ T cells. In addition, the data suggest that T cell receptor repertoires selected in wild-type mice and in mice displaying limited spectra of self-peptides are distinct.

The thymus is extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood and this capacity diminishes considerably with age. Here we show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration, via their production of BMP4. ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signalling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance and regeneration; and its downstream targets such as Dll4, itself a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.

We have recently described two independent mouse models in which the administration of diphtheria toxin (DT) leads to specific depletion of regulatory T cells (Tregs) due to expression of DT receptor-enhanced GFP under the control of the Foxp3 promoter. Both mouse models develop severe autoimmune disorders when Foxp3+ Tregs are depleted. Those findings were challenged in a recent study published in this journal suggesting the expression of Foxp3 in epithelial cells as the cause for disease development. By using genetic, cellular, and immunohistochemical approaches, we do not find evidence for Foxp3-expression in nonhematopoietic cells. DT injection does not lead to a loss of epithelial integrity in our Foxp3-DTR models. Instead, Foxp3 expression is Treg-specific and ablation of Foxp3+ Tregs leads to the induction of fatal autoimmune disorders. Autoimmunity can be reversed by the adoptive transfer of Tregs into depleted hosts, and the transfer of Foxp3-deficient bone marrow into T cell-deficient irradiated recipients leads to full-blown disease development.

The enzymes that degrade proteins to peptides for presentation on MHC class II molecules are poorly understood. The cysteinal lysosomal proteases, cathepsin L (CL) and cathepsin S (CS), have been shown to process invariant chain, thereby facilitating MHC class II maturation. However, their role in Ag processing is not established. To examine this issue, we generated embryonic fibroblast lines that express CL, CS, or neither. Expression of CL or CS mediates efficient degradation of invariant chain as expected. Ag presentation was evaluated using T cell hybridoma assays as well as mass spectroscopic analysis of peptides eluted from MHC class II molecules. Interestingly, we found that the majority of peptides are presented regardless of CL or CS expression, although these proteases often alter the relative levels of the peptides. However, for a subset of Ags, epitope generation is critically regulated by CL or CS. This result suggests that these cysteinal proteases participate in Ag processing and generate qualitative and quantitative differences in the peptide repertoires displayed by MHC class II molecules.