Ludovica Bruno

T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR

Regulatory T (Treg) cells safeguard against autoimmunity and immune pathology. Because determinants of the Treg cell fate are not completely understood, we have delineated signaling events that control the de novo expression of Foxp3 in naive peripheral CD4 T cells and in thymocytes. We report that premature termination of TCR signaling and inibition of phosphatidyl inositol 3-kinase (PI3K) p110α, p110δ, protein kinase B (Akt), or mammalian target of rapamycin (mTOR) conferred Foxp3 expression and Treg-like gene expression profiles. Conversely, continued TCR signaling and constitutive PI3K/Akt/mTOR activity antagonised Foxp3 induction. At the chromatin level, di- and trimethylation of lysine 4 of histone H3 (H3K4me2 and -3) near the Foxp3 transcription start site (TSS) and within the 5′ untranslated region (UTR) preceded active Foxp3 expression and, like Foxp3 inducibility, was lost upon continued TCR stimulation. These data demonstrate that the PI3K/Akt/mTOR signaling network regulates Foxp3 expression.

ESCs Require PRC2 to Direct the Successful Reprogramming of Differentiated Cells toward Pluripotency

Embryonic stem cells (ESCs) are pluripotent, self-renewing, and have the ability to reprogram differentiated cell types to pluripotency upon cellular fusion. Polycomb-group (PcG) proteins are important for restraining the inappropriate expression of lineage-specifying factors in ESCs. To investigate whether PcG proteins are required for establishing, rather than maintaining, the pluripotent state, we compared the ability of wild-type, PRC1-, and PRC2-depleted ESCs to reprogram human lymphocytes. We show that ESCs lacking either PRC1 or PRC2 are unable to successfully reprogram B cells toward pluripotency. This defect is a direct consequence of the lack of PcG activity because it could be efficiently rescued by reconstituting PRC2 activity in PRC2-deficient ESCs. Surprisingly, the failure of PRC2-deficient ESCs to reprogram somatic cells is functionally dominant, demonstrating a critical requirement for PcG proteins in the chromatin-remodeling events required for the direct conversion of differentiated cells toward pluripotency.

Genome-wide identification of Ikaros targets elucidates its contribution to mouse B-cell lineage specification and pre-B-cell differentiation

Ikaros family DNA-binding proteins are critical regulators of B-cell development. Because the current knowledge of Ikaros targets in B-cell progenitors is limited, we have identified genes that are bound and regulated by Ikaros in pre-B cells. To elucidate the role of Ikaros in B-cell lineage specification and differentiation, we analyzed the differential expression of Ikaros targets during the progression of multipotent to lymphoid-restricted progenitors, B- and T-cell lineage specification, and progression along the B-cell lineage. Ikaros targets accounted for one-half of all genes up-regulated during B-cell lineage specification in vivo, explaining the essential role of Ikaros in this process. Expression of the Ikaros paralogs Ikzf1 and Ikzf3 increases incrementally during B-cell progenitor differentiation, and, remarkably, inducible Ikaros expression in cycling pre-B cells was sufficient to drive transcriptional changes resembling the differentiation of cycling to resting pre-Bcells in vivo. The data suggest that Ikaros transcription factor dosage drives the progression of progenitors along a predetermined lineage by regulating multiple targets in key pathways, including pre-B–cell receptor signaling, cell cycle progression, and lymphocyte receptor rearrangement.Our approachmay be of general use to map the contribution of transcription factors to cell lineage commitment and differentiation.

Runx proteins regulate Foxp3 expression

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes.

IL4 blockade of inducible regulatory T cell differentiation: The role of Th2 cells, Gata3 and PU.1

Naive CD4 T cells differentiate into functionally distinct T helper (Th) cells subsets or into regulatory T (Treg) cells in response to the cytokine milieu in which they encounter antigen. A recurring theme in post-thymic CD4 T cell differentiation is the cross-regulation of lineage choice by cytokines and transcription factors that are expressed in alternative lineages. For example, TGFbeta induces the de novo expression of the Treg cell signature transcription factor Foxp3, but iTreg differentiation is blocked by high concentrations of the Th2 cytokine IL4. However, whether IL4 can antagonise Foxp3 induction in more physiological settings remains to be addressed. Here we use a co-culture system to demonstrate that IL4 provided by Th2 cells in vitro is sufficient to block Foxp3 induction in naive CD4 T cells. In addition, we find that Foxp3 induction is efficiently blocked not only by the Th2 transcription factor Gata3, but also by PU.1, which is transiently induced during Th2 differentiation. These data suggest that iTreg differentiation may be affected by the polarity of immune responses.

Directing T cell differentiation and function with small molecule inhibitors.

Regulatory T (Treg) cells that express the signature transcription factor Foxp3 safeguard against autoimmunity and immune pathology. Recent studies show that a signaling network with the components phosphatidyl inositol 3 kinase (PI3K), Akt, and the mammalian target of rapamycin (mTOR) regulates the de novo expression of Foxp3 in CD4 T cells. In addition to CD4 T cell differentiation, PI3K/Akt/mTOR signaling also controls T cell migration. Here we review the new data, consider their evolutionary context and discuss their potential implications for immunotherapy.

A reappraisal of evidence for probabilistic models of allelic exclusion.

B cell development requires the coordinated rearrangement of Ig heavy (IgH) and light chain loci (IgL). Most mature B cells express a single B cell receptor of unique specificity, and a central question in immunology concerns the mechanisms that prevent the productive rearrangement of >1 IgH and IgL allele per cell. Probabilistic models of allelic exclusion maintain that simultaneous rearrangement of both alleles is rare, because the likelihood of undergoing rearrangement is low for a given Ig allele. Strong support for this idea came from studies in which a GFP marker was inserted into the Igk locus. In this system, the probability of high-level germ-line transcription and subsequent locus rearrangement appeared to be low in pre-B cells. Readdressing the validity of GFP expression as a reporter for the level of germ-line transcription, we found a striking discordance between GFP transcript and protein levels at the pre-B cell stage, which is explained at least in part by the developmentally regulated usage of 2 alternative Igk-J germ-line promoters. These results question the validity of the kappa-GFP system as evidence for probabilistic models of allelic exclusion.

microRNAs Regulate Cell-to-Cell Variability of Endogenous Target Gene Expression in Developing Mouse Thymocytes

The development and homeostasis of multicellular organisms relies on gene regulation within individual constituent cells. Gene regulatory circuits that increase the robustness of gene expression frequently incorporate microRNAs as post-transcriptional regulators. Computational approaches, synthetic gene circuits and observations in model organisms predict that the co-regulation of microRNAs and their target mRNAs can reduce cell-to-cell variability in the expression of target genes. However, whether microRNAs directly regulate variability of endogenous gene expression remains to be tested in mammalian cells. Here we use quantitative flow cytometry to show that microRNAs impact on cell-to-cell variability of protein expression in developing mouse thymocytes. We find two distinct mechanisms that control variation in the activation-induced expression of the microRNA target CD69. First, the expression of miR-17 and miR-20a, two members of the miR-17-92 cluster, is co-regulated with the target mRNA Cd69 to form an activation-induced incoherent feed-forward loop. Another microRNA, miR-181a, acts at least in part upstream of the target mRNA Cd69 to modulate cellular responses to activation. The ability of microRNAs to render gene expression more uniform across mammalian cell populations may be important for normal development and for disease.

The melanocortin receptor agonist NDP-MSH impairs the allostimulatory function of dendritic cells

As α‐melanocyte‐stimulating hormone (α‐MSH) is released by immunocompetent cells and has potent immunosuppressive properties, it was determined whether human dendritic cells (DCs) express the receptor for this hormone. Reverse transcription–polymerase chain reaction detected messenger RNA specific for all of the known melanocortin receptors in DCs. Mixed lymphocyte reactions also revealed that treatment with [Nle4, DPhe7]‐α‐MSH (NDP‐MSH), a potent α‐MSH analogue, significantly reduced the ability of DCs to stimulate allogeneic T cells. The expression of various cell surface adhesion, maturation and costimulatory molecules on DCs was also investigated. Although treatment with NDP‐MSH did not alter the expression of CD83 and major histocompatibility complex class Ι and ΙΙ, the surface expression of CD86 (B7.2), intercellular adhesion molecule (ICAM‐1/CD54) and CD1a was reduced. In summary, our data indicate that NDP‐MSH inhibits the functional activity of DCs, possibly by down‐regulating antigen‐presenting and adhesion molecules and that these events may be mediated via the extracellular signal‐regulated kinase 1 and 2 pathway.

Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults

Summary Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.