Alexander G. Betz

The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3’s function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression. The suppressive activity of regulatory T cells provides the immune system with a mechanism to prevent detrimental immune responses, such as autoimmunity, attack of the beneficial commensal microbiota and rejection of the fetus. Intriguingly, expression of a single lineage factor Foxp3 is sufficient to completely reprogram T cells from a pro-inflammatory to a suppressive phenotype. Here, we show that Foxp3 alters the expression of thousands of genes through several independent mechanisms. In many cases, its own ability to bind to DNA appears to be dispensable, but rather it binds indirectly to the DNA by interaction with other transcription factors. Foxp3 then in turn recruits other proteins that affect gene expression through chromatin modification. For example, Foxp3 indirectly binds to the IL-2 promoter via interaction with the transcriptional activators c-Rel, AML-1 and NFAT. This leads to the Foxp3 mediated recruitment of class I histone deacetylases HDAC1, 2 and 3, which in turn counteracts the activation-induced hyper-acetylation of the promoter, thereby switching the gene off. In a way, Foxp3 hijacks pre-existing regulatory mechanism to reverse the transcriptional expression status of the target gene. By dissecting Foxp3 on a molecular level, we also show that this is only one of several independent mechanism utilised by Foxp3.

Regulatory T (Treg) cells are critically important for the maintenance of immunological tolerance. Both centrally arising natural nTreg cells and those emerging in the periphery in response to TGF-β, iTreg cells, play a role in the control of unwanted immune responses. Treg cells adopt multiple mechanisms to inhibit effector T cells, yet it is unclear whether these mechanisms are shared by nTreg cells and iTreg cells alike. Here, we show that iTreg cells, like nTreg cells, are able to out-compete naïve T cells in clustering around dendritic cells (DCs). However, using both a tamoxifen-responsive inducible Foxp3 retroviral construct and TGF-β-induced iTreg cells from hCD2-Foxp3 knock in reporter mice, we show that it is prior antigen-induced activation rather than Foxp3 expression per se that determines the ability of iTreg cells to competitively cluster around DCs. We found no difference in the capacity of iTreg cells to displace naïve T cells around DCs to that of Tr1, Th1, Th2, or Th9 cells. An important difference was, however, that clustering of iTreg cells around DCs, just as for naïve T cells, did not effectively activate DCs.

The immune system has the ability to suppress undesirable responses, such as those against commensal bacteria, food and paternal antigens in placenta pregnancy. The lineage-specific transcription factor Foxp3 orchestrates the development and function of regulatory T cells underlying this immunological tolerance. Despite the crucial role of Foxp3 in supporting immune homeostasis, little is known about its origin, evolution and species conservation. We explore these questions using comparative genomics, structural modeling and functional analyses. Our data reveal that key gain-of-function events occurred during the evolution of Foxp3 in higher vertebrates. We identify key conserved residues in its forkhead domain and show a detailed analysis of the N-terminal region of Foxp3, which is only conserved in mammals. These components are under purifying selection, and our mutational analyses demonstrate that they are essential for Foxp3 function. Our study points to critical functional adaptations in immune tolerance among higher vertebrates, and suggests that Foxp3-mediated transcriptional mechanisms emerged during mammalian evolution as a step-wise gain of functional domains that enabled Foxp3 to interact with a multitude of interaction partners.

Expression of the lineage-specific DNA-binding factor Foxp3 controls the development and function of naturally occurring regulatory T cells. Foxp3 has been shown to interact with a multitude of transcriptional regulators including NFAT, NF-κB (p65), Runx1 and RORγt, as well as the histone modification enzymes TIP60, HDAC7 and HDAC9. The sum of these interactions is believed to cause the change in the transcriptional program of regulatory T cells. Here we show that Foxp3 directly or as part of a multimeric complex engages with the NF-κB component c-Rel. We demonstrate that the N-terminal region of Foxp3 is required for the binding of c-Rel, but not NFAT. Conversely, deletion of the forkhead domain causes a loss of interaction with NFAT, but not c-Rel. Our findings are of particular interest, as c-Rel is crucial for the induction of Foxp3 in regulatory T cells during thymic development, but has to be repressed in mature regulatory T cells to maintain their suppressive phenotype.

T cell development occurs in waves, involving distinct stem cell populations, throughout human fetal development.

Intra vital microscopy and whole‐body imaging promise to revolutionize how we study the immune system. They compel by the intrinsic beauty of the images obtained and the undeniable direct biological relevance of the observations. However, it is important to remember that in many cases, fundamental insights into the underlying biological processes have already been obtained using ex vivo reductionist approaches. Indeed, it is likely that with the advent of microfluidics, new and exciting avenues will open up for ex vivo experimentation. Here, we give a brief but comprehensive overview of the various imaging techniques available, their relative strengths and shortcomings and how these tools have been used to get us to where we are today. The challenge for the future will be to apply the most suitable technology and to integrate the findings across various imaging disciplines to build a unified, comprehensive “big picture” of the immune system.

Forkhead box p3 (Foxp3)-expressing regulatory T cells are key mediators of peripheral tolerance suppressing undesirable immune responses. Ectopic expression of Foxp3 confers regulatory T cell phenotype to conventional T cells, lending itself to therapeutic use in the prevention of autoimmunity and transplant rejection. Here, we show that adoptive transfer of polyclonal, wild-type T cells transduced with an inducible form of Foxp3 (iFoxp3) can be used to suppress immune responses on demand. In contrast to Foxp3-transduced cells, iFoxp3-transduced cells home “correctly” into secondary lymphoid organs, where they expand and participate in immune responses. Upon induction of iFoxp3, the cells assume regulatory T cell phenotype and start to suppress the response they initially partook in without causing systemic immunosuppression. We used this approach to suppress collagen-induced arthritis, in which conventional Foxp3-transduced cells failed to show any effect. This provides us with a generally applicable strategy to specifically halt immune responses on demand without prior knowledge of the antigens involved. Autoimmune diseases come in many diverse forms—such as rheumatoid arthritis, type I diabetes, multiple sclerosis, and inflammatory bowel disease—yet all share the same underlying cause, the launch of a detrimental immune response. In healthy individuals, a specialized immune cell type called regulatory T cells prevents these undesirable immune responses. Here, we present a strategy to suppress undesirable immune responses using genetically modified proinflammatory T cells that participate in these inappropriate immune responses until they are activated with a drug. At this point, the genetic modification causes them to change their behavior to that of regulatory T cells. Using a mouse model, we demonstrate that this approach can be used to stop undesirable immune responses on demand with minimal intervention.