Stefan Klein-Hessling

Epigenetic Control of the foxp3 Locus in Regulatory T Cells

Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4+ regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3+CD25+CD4+ Tregs, but not in naïve CD25−CD4+ T cells. Partial DNA demethylation is already found within developing Foxp3+ thymocytes; however, Tregs induced by TGF-β in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-β–induced Foxp3+ Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions.

The role of NF-AT transcription factors in T cell activation and differentiation.

The family of genuine NF-AT transcription factors consists of four members (NF-AT1 [or NF-ATp], NF-AT2 [or NF-ATc], NF-AT3 and NF-AT4 [or NF-ATx]) which are characterized by a highly conserved DNA binding domain (is designated as Rel similarity domain) and a calcineurin binding domain. The binding of the Ca(2+)-dependent phosphatase calcineurin to this region controls the nuclear import and exit of NF-ATs. This review deals (1) with the structure of NF-AT proteins, (2) the DNA binding of NF-AT factors and their interaction with AP-1, (3) NF-AT target genes, (4) signalling pathways leading to NF-AT activation: the role of protein kinases and calcineurin, (5) the nuclear entry and exit of NF-AT factors, (6) transcriptional transactivation by NF-AT factors, (7) the structure and expression of the chromosomal NF-AT2 gene, and (8) NF-AT factors in Th cell differentiation. The experimental data presented and discussed in the review show that NF-AT factors are major players in the control of T cell activation and differentiation and, in all likelihood, also of the cell cycle and apoptosis of T lymphocytes.

IL-9 and IL-13 Production by Activated Mast Cells Is Strongly Enhanced in the Presence of Lipopolysaccharide: NF-κB Is Decisively Involved in the Expression of IL-9

Mast cells, due to their ability to produce a large panel of mediators and cytokines, participate in a variety of processes in adaptive and innate immunity. Herein we report that in primary murine bone marrow-derived mast cells activated with ionomycin or IgE-Ag the bacterial endotoxin LPS strongly enhances the expression of IL-9 and IL-13, but not IL-4. This costimulatory effect of LPS is absent in activated mast cells derived from the LPS-hyporesponsive mouse strain BALB/c-LPSd, although in these cells the proinflammatory cytokine IL-1 can still substitute for LPS. The enhanced production of mast cell-derived IL-13 in the presence of IL-1 is a novel observation. Coactivation of mast cells with LPS leads to a synergistic activation of NF-κB, which is shown by an NF-κB-driven reporter gene construct. In the presence of an inhibitor of NF-κB activation, the production of IL-9 is strongly decreased, whereas the expression of IL-13 is hardly reduced, and that of IL-4 is not affected at all. NF-κB drives the expression of IL-9 via three NF-κB binding sites within the IL-9 promoter, which we characterize using gel shift analyses and reporter gene assays. In the light of recent reports that strongly support critical roles for IL-9 and IL-13 in allergic lung inflammation, our results emphasize the potential clinical importance of LPS as an enhancer of mast cell-derived IL-9 and IL-13 production in the course of inflammatory reactions and allergic diseases.

Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression

T cell–specific NFAT2 deletion results in reduced CXCR5+ follicular regulatory T cells, leading to uncontrolled germinal center responses and humoral autoimmunity.

Specific and Redundant Roles for NFAT Transcription Factors in the Expression of Mast Cell-Derived Cytokines

By virtue of their ability to express a plethora of biologically highly active mediators, mast cells (MC) are involved in both adaptive and innate immune responses. MC-derived Th2-type cytokines are thought to act as local amplifiers of Th2 reactions, including chronic inflammatory disorders such as allergic asthma, whereas MC-derived TNF-α is a critical initiator of antimicrobial defense. In this study, we demonstrate that the transcription factors NFATc1 and NFATc2 are part of a MC-specific signaling network that regulates the expression of TNF-α and IL-13, whereas NFATc3 is dispensable. Primary murine bone marrow-derived MC from NFATc2−/− mice, activated by either ionomycin or IgE/Ag cross-link, display a strong reduction in the production of these cytokines, compared with bone marrow-derived MC from wild-type mice. Detailed analyses of TNF-α and IL-13 expression using small interfering RNA-mediated knockdown reveals that both NFATc2 and NFATc1 are able to drive the expression of these cytokines, whereas neither degranulation nor the expression of IL-6 depends on NFAT activity. These results support the view that high NFAT activity is necessary for TNF-α and IL-13 promoter induction in MC, irrespective of whether NFATc2 or NFATc1 or a combination of both is present.

NFATc1 affects mouse splenic B cell function by controlling the calcineurin--NFAT signaling network.

Mouse B cells lacking NFATc1 exhibit defective proliferation, survival, isotype class switching, cytokine production, and T cell help.

NFAT transcription factors in control of peripheral T cell tolerance

The Ca++‐regulated calcineurin/NFAT cascade is one of the crucial signalling pathways that controls adaptive immunity. However, a number of novel experimental data suggest that, in addition to their role in T cell activation, NFATc transcription factors play also a decisive role in the generation of peripheral tolerance against self‐antigens. This function of NFATc factors is mediated by controlling activation‐induced cell death and clonal anergy of T helper cells and the activity of regulatory T cells. The multi‐functional role of NFATc proteins characterize these transcription factors as key regulators of immunological tolerance and, if dysregulated, of development of autoimmune diseases.

Protein Kinase A Regulates GATA-3-Dependent Activation of IL-5 Gene Expression in Th2 Cells

Treatment of Th cells with compounds that elevate cAMP levels augments Th2-type lymphokine expression, in particular the synthesis of IL-5. Using primary murine CD4+ T lymphocytes, we show in this study that inhibition of protein kinase A (PKA) activity in Th2 effector cells impairs IL-5 synthesis, whereas the expression of PKA catalytic subunit α enhances IL-5 synthesis in Th0 cells. In addition, we observed by coexpression of PKA catalytic subunit and GATA-3 in Th1 cells that the stimulatory effect of PKA is dependent on GATA-3 activity. These data demonstrate that activation of PKA in Th effector cells induces the IL-5 gene expression in a GATA-3-dependent manner.

C/EBPβ enhances IL‐4 but impairs IL‐2 and IFN‐γ induction in T cells

C/EBP transcription factors have been described to control the activity of the human IL‐4 promoter. The C/EBP binding sites within the IL‐4 promoter overlap with composite NF‐AT and AP‐1 binding motifs. We show here that similar binding sites are part of the murine IL‐4 promoter. Retroviral overexpression of C/EBPβ in murine EL‐4 thymoma cells led to a strong induction of endogenous IL‐4 and a reduction in IL‐2 and IFN‐γ expression. Similarily, in primary murine T cells C/EBPβ induction resulted in an increase in IL‐4 levels, whereas in human Jurkat T cells a decrease in IL‐2 RNA was detected. Like AP‐1, C/EBP factors belong to the large class of basic leucine zipper proteins. However, unlike AP‐1, C/EBPβ does not act in synergy with NF‐AT in the induction of the murine IL‐4 promoter. Instead, both factors compete in their binding to the P4/Pu‐bD site, one of the most important sequence elements of the IL‐4 promoter. Whereas NF‐AT factors require high levels of free Ca2+ and calcineurin activity for induction, C/EBP induction in T cells is Ca2+/calcineurin independent. These observations suggest that various induction conditions lead to the activation of transcription factors, inducing IL‐4 promoter activity at specific developmental stages of T cells.

SUMOylation Interferes with CCAAT/Enhancer-Binding Protein β-Mediated c-myc Repression, but Not IL-4 Activation in T Cells

The transcription factor C/EBPβ transactivates the IL-4 gene in murine T lymphocytes and facilitates Th2 cell responses. In this study, we demonstrate that C/EBPβ also acts as a repressor of T cell proliferation. By binding to the c-myc promoter(s), C/EBPβ represses c-Myc expression and, therefore, arrests T cells in the G1 phase of the cell cycle. For C/EBPβ-mediated repression, the integrity of its N-terminal transactivation domain is essential whereas the central regulatory domain is dispensable. This central regulatory domain is sumoylated in vivo which leads to an alteration of the activity of C/EBPβ. Whereas sumoylation does not affect the C/EBPβ-mediated activation of the IL-4 gene, it relieves its repressive effect on c-Myc expression and T cell proliferation. Similar to several other transcription factors, sumoylation redistributes nuclear C/EBPβ and targets it to pericentric heterochromatin. These results suggest an important role of sumoylation in adjusting the finely tuned balance between proliferation and differentiation in peripheral T cells which is controlled by C/EBPβ.