The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes.

After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor-mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell-mediated immune responses and open new therapeutic possibilities.

[How T lymphocytes coordinate rheumatic inflammation]. / Wie T-Lymphozyten rheumatische Entzündung koordinieren.

Helper T (Th) cells play a decisive role in triggering and maintaining chronic rheumatic inflammation. Via secretion of proinflammatory cytokines and expression of costimulatory cell surface molecules, Th lymphocytes coordinate the recruitment and activation of effector cells, which are ultimately responsible for the immunopathology and tissue destruction. However, therapeutic approaches aimed at eliminating Th cells were unsuccessful due to their lack of selectivity. At the German Rheumatism Research Center (Deutsches Rheuma-Forschungszentrum, DRFZ), we are working to improve the understanding of the Th cells involved in chronic inflammatory reactions. Based on this understanding, our aim is to develop novel treatment strategies that selectively target the pathogenic Th lymphocytes causing rheumatic inflammation. The current article summarizes the DRFZ's research activities on this subject.

Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells.

Anti-VEGF-directed therapies have been a milestone for treating retinal vascular diseases. Depletion of monocyte lineage cells suppresses pathological neovascularization in the oxygen-induced retinopathy mouse model. However, the question whether myeloid-derived VEGF-A expression is responsible for the pathogenesis in oxygen-induced retinopathy remained unknown. We analyzed LysMCre-driven myeloid cell-specific VEGF-A knockout mice as well as mice with complete depletion of circulating macrophages through clodronate-liposome treatment in the oxygen-induced retinopathy model by immunohistochemistry, qPCR, and flow cytometry. Furthermore, we analyzed VEGF-A mRNA expression in MIO-M1 cells alone and in co-culture with BV-2 cells in vitro. The myeloid cell-specific VEGF-A knockout did not change relative retinal VEGF-A mRNA levels, the relative avascular area or macrophage/granulocyte numbers in oxygen-induced retinopathy and under normoxic conditions. We observed an insignificantly attenuated pathology in systemically clodronate-liposome treated knockouts but evident VEGF-A expression in activated Müller cells on immunohistochemically stained sections. MIO-M1 cells had significantly higher expression levels of VEGF-A in co-culture with BV-2 cells compared to cultivating MIO-M1 cells alone. Our data show that myeloid-derived cells contribute to pathological neovascularization in oxygen-induced retinopathy through activation of VEGF-A expression in Müller cells.

Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene.

E- and P-selectin ligands (E- and P-ligs) guide effector memory T cells into skin and inflamed regions, mediate the inflammatory recruitment of leukocytes, and contribute to the localization of hematopoietic precursor cells. A better understanding of their molecular regulation is therefore of significant interest with regard to therapeutic approaches targeting these pathways. In this study, we examined the transcriptional regulation of fucosyltransferase 7 (FUT7), an enzyme crucial for generation of the glycosylated E- and P-ligs. We found that high expression of the coding gene fut7 in murine CD4+ T cells correlates with DNA demethylation within a minimal promoter in skin/inflammation-seeking effector memory T cells. Retinoic acid, a known inducer of the gut-homing phenotype, abrogated the activation-induced demethylation of this region, which contains a cAMP responsive element. Methylation of the promoter or mutation of the cAMP responsive element abolished promoter activity and the binding of CREB, confirming the importance of this region and of its demethylation for fut7 transcription in T cells. Furthermore, studies on human CD4+ effector memory T cells confirmed demethylation within FUT7 corresponding to high FUT7 expression. Monocytes showed an even more extensive demethylation of the FUT7 gene whereas hepatocytes, which lack selectin ligand expression, exhibited extensive methylation. In conclusion, we show that DNA demethylation within the fut7 gene controls selectin ligand expression in mice and humans, including the inducible topographic commitment of T cells for skin and inflamed sites.

Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells.

Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/ßC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.

Transcription factor co-occupied regions in the murine genome constitute T-helper-cell subtype-specific enhancers.

Transcription factors (TFs) regulate cell-type-specific gene expression programs by combinatorial binding to cis-genomic elements, particularly enhancers, subsequently leading to the recruitment of cofactors, and the general transcriptional machinery to target genes. Using data integration of genome-wide TF binding profiles, we defined regions with combinatorial binding of lineage-specific master TFs (T-BET, GATA3, and ROR-γt) and STATs (STAT1 and STAT4, STAT6, and STAT3) in murine T helper (Th) 1, Th2, and Th17 cells, respectively. Stringently excluding promoter regions, we revealed precise genomic elements which were preferentially associated with the enhancer marks p300 and H3K4me1. Furthermore, closely adjacent TF co-occupied regions constituted larger enhancer domains in the respective Th-cell subset (177 in Th1, 141 in Th2, and 266 in Th17 cells) with characteristics of so-called super-enhancers. Importantly, 89% of these super-enhancer regions were Th-cell subtype-specific. Genes associated with super-enhancers, including relevant Th-cell genes (such as Ifng in Th1, Il13 in Th2, and Il17a in Th17 cells), showed strong transcriptional activity. Altogether, the discovered catalog of enhancers provides information about crucial Th-cell subtype-specific regulatory hubs, which will be useful for revealing cell-type-specific gene regulation processes.

miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim.

Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T-bet induce expression of microRNA-148a (miR-148a). miR-148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR-148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR-148a antagomir-treated cells restores viability of the Th1 cells, demonstrating that miR-148a controls survival by regulating Bim expression. Thus, Twist1 and T-bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.

Nuclear factor of activated T cells regulates the expression of interleukin-4 in Th2 cells in an all-or-none fashion.

Th2 memory lymphocytes have imprinted their Il4 genes epigenetically for expression in dependence of T cell receptor restimulation. However, in a given restimulation, not all Th cells with a memory for IL-4 expression express IL-4. Here, we show that in reactivated Th2 cells, the transcription factors NFATc2, NF-kB p65, c-Maf, p300, Brg1, STAT6, and GATA-3 assemble at the Il4 promoter in Th2 cells expressing IL-4 but not in Th2 cells not expressing it. NFATc2 is critical for assembly of this transcription factor complex. Because NFATc2 translocation into the nucleus occurs in an all-or-none fashion, dependent on complete dephosphorylation by calcineurin, NFATc2 controls the frequencies of cells reexpressing Il4, translates analog differences in T cell receptor stimulation into a digital decision for Il4 reexpression, and instructs all reexpressing cells to express the same amount of IL-4. This analog-to-digital conversion may be critical for the immune system to respond to low concentrations of antigens.

NK cells gain higher IFN-γ competence during terminal differentiation.

NK cells are the main cells of the innate immune system that produce IFN-γ, and they express this cytokine at early stages of maturation in response to cytokine stimulation. Conversely, acquisition of IFN-γ-competence in CD4(+) T helper cells requires a differentiation process from naïve toward type 1 (Th1) cells, which is associated with epigenetic remodeling at the IFNG locus. In the present study, we show that the ability of NK cells to produce IFN-γ in response to activating receptor (actR) engagement is gradually acquired during terminal differentiation and is accompanied by progressively higher NF-κB activation in response to actR triggering. Moreover, during the differentiation process NK cells gradually display increasing expression of IFNG and TBX21 (encoding T-bet) transcripts and demethylation at the IFNG promoter. This study provides new insights in the molecular mechanisms underlying NK-cell ability to express IFN-γ upon actR engagement. Thus, we propose that in order to efficiently produce IFN-γ in response to infected or transformed cells, NK cells gain Th1-like features, such as higher IFN-γ competence and epigenetic remodeling of the IFNG promoter, during their terminal differentiation.

Repression of cyclic adenosine monophosphate upregulation disarms and expands human regulatory T cells.

The main molecular mechanism of human regulatory T cell (Treg)-mediated suppression has not been elucidated. We show in this study that cAMP represents a key regulator of human Treg function. Repression of cAMP production by inhibition of adenylate cyclase activity or augmentation of cAMP degradation through ectopic expression of a cAMP-degrading phosphodiesterase greatly reduces the suppressive activity of human Treg in vitro and in a humanized mouse model in vivo. Notably, cAMP repression additionally abrogates the anergic state of human Treg, accompanied by nuclear translocation of NFATc1 and induction of its short isoform NFATc1/αA. Treg expanded under cAMP repression, however, do not convert into effector T cells and regain their anergic state and suppressive activity upon proliferation. Together, these findings reveal the cAMP pathway as an attractive target for clinical intervention with Treg function.

Stable IL-2 decision making by endogenous c-Fos amounts in peripheral memory T-helper cells.

The cytokine IL-2 performs opposite functions supporting efficient immune responses and playing a key role in peripheral tolerance. Therefore, precise fine-tuning of IL-2 expression is crucial for adjusting the immune response. Combining transcription factor analysis at the single cell and the single nucleus level using flow cytometry with statistical analysis, we showed that physiological differences in the expression levels of c-Fos and NFATc2, but not of c-Jun and NF-κBp65, are limiting for the decision whether IL-2 is expressed in a strongly activated human memory T-helper (Th) cell. Variation in the expression of c-Fos leads to substantial diversity of IL-2 expression in ∼40% of the memory Th cells. The remaining cells exhibit an equally high c-Fos expression level, thereby ensuring robustness in IL-2 response within the population. These findings reveal how memory Th cells benefit from regulated variation in transcription factor expression to achieve a certain stability and variability of cytokine expression in a controlled manner.

Differential regulation and impact of fucosyltransferase VII and core 2 ß1,6-N-acetyl-glycosaminyltransferase for generation of E-selectin and P-selectin ligands in murine CD4+ T cells.

Ligands for E-selectin and P-selectin (E-lig and P-lig) are induced on CD4+ T cells upon differentiation into effector T cells. Glycosyltransferases, especially α 1,3-fucosyltransferase VII (FucT-VII) and core 2 ß1,6-N-acetyl-glycosaminyltransferase I (C2GlcNAcT-I), are critical for their synthesis. We here analysed the signals that control the expression of E-lig, P-lig and mRNA coding for FucT-VII and C2GlcNAcT-I. In line with previous reports, we found that P-lig expression correlates with the regulation of C2GlcNAcT-I, whereas E-lig expression can occur at low levels of C2GlcNAcT-I mRNA but requires high FucT-VII mRNA expression. Interestingly, the two enzymes are regulated by different signals. Activation-induced C2GlcNAcT-I up-regulation under permissive (T helper type 1) conditions was strongly reduced by cyclosporin A (CsA), suggesting the involvement of T-cell receptor-dependent, calcineurin/NFAT-dependent signals in combination with interleukin-12 (IL-12) -mediated signals in the regulation of C2GlcNAcT-I. In contrast, expression of FucT-VII mRNA was not significantly inhibited by CsA. Interleukin-4 inhibited the expression of FucT-VII but IL-2 and IL-7 were found to support induction of FucT-VII and E-lig. E-selectin, P-selectin and their ligands initially appeared to have rather overlapping functions. These findings however, unravel striking differences in the regulation of E-lig and P-lig expression, dictated by the dominance of FucT-VII and C2GlcNAcT-I, respectively, and their dependency on signals from either promiscuous or homeostatic cytokines (FucT-VII) or a strong T-cell receptor signal in combination with inflammatory cytokines in case of C2GlcNAcT-I.

Dephosphorylation of Bcl-10 by calcineurin is essential for canonical NF-κB activation in Th cells.

Antigen-specific stimulation of T helper (Th) cells initiates signaling cascades that ultimately result in the activation of the transcription factors NF-κB, NFAT, and AP-1 which regulate, together with other factors, many T-cell functions such as cytokine production, proliferation, and differentiation. Ordered assembly and different phosphorylation events, along with subcellular translocation of the CARMA1/Bcl-10/MALT1 complex, determine NF-κB activation after T-cell receptor (TCR) triggering. We now provide evidence that inhibition of the Ser/Thr phosphatase calcineurin (CaN) prevents dephosphorylation of Bcl-10. CaN, in constant interaction with the Bcl-10/MALT1 complex, is able to dephosphorylate Bcl-10. The CaN inhibitor cyclosporine A (CsA) converts a transient phosphorylation of Bcl-10 Ser138 during the immediate early phase of T-cell activation into a persistent state. Thus, subsequent processes such as IKKß phosphorylation, IκBα degradation, p65 nuclear translocation, and DNA binding are diminished. Consistently, CsA treatment does not affect the phosphorylation pattern of the upstream kinase PKCθ. Together, our findings demonstrate that CaN functions as a critical signaling molecule during Th cell activation, regulating Bcl-10 phosphorylation and thereby NF-κB activation.

Evaluation of calcineurin/NFAT inhibitor selectivity in primary human Th cells using bar-coding and phospho-flow cytometry.

Small molecular inhibitors are excellent tools for manipulating cell reactions. They are widely used in scientific research to study molecular mechanisms of cells under physiological and pathophysiological conditions as well as in clinical applications to treat patients. However, their selectivity is often not well known. Moreover, it can vary according to cell types and the analysis methods used. Therefore, it is usually not possible to make comparisons between the data presented in the literature. Here we analyzed the selectivity of five chosen inhibitors of calcineurin/NFAT activation under the same conditions. Using a combination of fluorescent cell barcoding and phospho-specific flow cytometry we studied the inhibition of activation of NF-κBp65 and MAPK pathways in stimulated primary human Th cells. This semi-high throughput approach enabled us to demonstrate that (i) CsA and NCI3 are around 5 to 10- and 20-fold less potent, respectively, at inhibiting phosphorylation of NF-κBp65 and p38 than activation of NFAT, (ii) AM404 is at least 15-fold selective for NFAT but already toxic at concentrations above 40 µM, (iii) INCA6 is not selective at all, and (iv) BTP1 is at least 100-fold selective for inhibition of NFAT activation relative to NF-κBp65, p38 and ERK1/2 phosphorylation. Altogether, our results not only show the applicability of a semi-high throughput inhibitor test system but also that BTP1 is the most selective inhibitor of calcineurin/NFAT activation among the studied inhibitors under the used conditions.

PRI: Re-Analysis of a Public Mass Cytometry Dataset Reveals Patterns of Effective Tumor Treatments.

Recently, mass cytometry has enabled quantification of up to 50 parameters for millions of cells per sample. It remains a challenge to analyze such high-dimensional data to exploit the richness of the inherent information, even though many valuable new analysis tools have already been developed. We propose a novel algorithm "pattern recognition of immune cells (PRI)" to tackle these high-dimensional protein combinations in the data. PRI is a tool for the analysis and visualization of cytometry data based on a three or more-parametric binning approach, feature engineering of bin properties of multivariate cell data, and a pseudo-multiparametric visualization. Using a publicly available mass cytometry dataset, we proved that reproducible feature engineering and intuitive understanding of the generated bin plots are helpful hallmarks for re-analysis with PRI. In the CD4+T cell population analyzed, PRI revealed two bin-plot patterns (CD90/CD44/CD86 and CD90/CD44/CD27) and 20 bin plot features for threshold-independent classification of mice concerning ineffective and effective tumor treatment. In addition, PRI mapped cell subsets regarding co-expression of the proliferation marker Ki67 with two major transcription factors and further delineated a specific Th1 cell subset. All these results demonstrate the added insights that can be obtained using the non-cluster-based tool PRI for re-analyses of high-dimensional cytometric data.

The novel calcineurin inhibitor CN585 has potent immunosuppressive properties in stimulated human T cells.

The Ca2+/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K(i) value of 3.8 mum. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naïve B cells.

1α,25-dihydroxyvitamin D(3) (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human naïve B cells. Naïve B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naïve B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naïve B cells, namely by reducing CD40 signaling.

TLR2-activated human langerhans cells promote Th17 polarization via IL-1beta, TGF-beta and IL-23.

The cytokines IL-6, IL-1beta, TGF-beta, and IL-23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ-resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC-like cells TLR2-dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor gammabeta was detected. To clarify if IL-17(+)cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c(+)/langerin(+) monocyte-derived LC-like cells (MoLC), macrophage-activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL-6, IL-1beta, and IL-23. TGF-beta, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti-TLR2 inhibited secretion of IL-6, IL-1beta, and IL-23 by MoLC, while TGF-beta was unaffected. The amount of IL-17 and the ratio of IL-17 to IFN-gamma expression was higher in MoLC- than in monocyte-derived DC-cocultured Th cells. Anti-IL-1beta, -TGF-beta and -IL-23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN-stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.

Whole blood flow cytometric measurement of NFATc1 and IL-2 expression to analyze cyclosporine A-mediated effects in T cells.

The calcineurin inhibitor Cyclosporine A (CsA) is one of the crucial immunosuppressive drugs given after organ transplantation. The small therapeutic window of CsA generates the dilemma that efficient and toxic drug doses differ only slightly. Moreover, these threshold concentrations differ considerably between individuals; therefore, functional assays are urgently needed. We explored whether the transcription factor NFATc1, a direct as well as indirect target of CsA, can be used as a potential biomarker to determine the individual immunosuppressive activity of CsA. First, in isolated human T cells we showed that flow cytometry is practicable to measure NFATc1, the most abundant NFATc isoform in activated T cells. Second, for whole blood we developed a flow cytometric assay to determine in parallel the inducible transcription factor NFATc1 and the cytokine IL-2 in stimulated T cells. We found that added CsA inhibits both the expression of NFATc1 and IL-2 in T cells of stimulated whole blood samples with IC(50) values of 200 and 150 nM, respectively. The intra- and inter-assay variability was low, and clinical practicability was good. Further experiments have to demonstrate whether the parallel cytometric measurement of NFATc1 and IL-2 in whole blood is a good predictor of individual CsA efficacy and toxicity in CsA-treated patients.