Claudia Haftmann

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.

IL-17 and GM-CSF Expression Are Antagonistically Regulated by Human T Helper Cells

GM-CSF–producing T helper cells in humans follow a distinct regulation program as compared to TH17 cells and are associated with multiple sclerosis. Cytokine Rivalry In patients with autoimmune diseases, cytokines—secreted immune mediators—are a crucial cause of tissue damage. However, the interplay between different cytokines and their individual roles in disease aggravation and resolution remain poorly defined, especially in humans. Noster et al. report that T helper (TH) cell production of granulocyte-macrophage colony-stimulating factor (GM-CSF) may play a pathogenic role in the brain of patients with multiple sclerosis (MS). They found that TH17-related cytokines—thought from mouse studies to be critical for pathogenesis—actually prevented induction of GM-CSF, whereas TH1-type cytokines promoted GM-CSF. These data provide a rationale for decreasing GM-CSF in patients with MS and suggest that, for MS at least, human may know best. Although T helper 17 (TH17) cells have been acknowledged as crucial mediators of autoimmune tissue damage, the effector cytokines responsible for their pathogenicity still remain poorly defined, particularly in humans. In mouse models of autoimmunity, the pathogenicity of TH17 cells has recently been associated with their production of granulocyte-macrophage colony-stimulating factor (GM-CSF). We analyzed the regulation of GM-CSF expression by human TH cell subsets. Surprisingly, the induction of GM-CSF expression by human TH cells is constrained by the interleukin-23 (IL-23)/ROR-γt/TH17 cell axis but promoted by the IL-12/T-bet/TH1 cell axis. IL-2–mediated signal transducer and activator of transcription 5 (STAT5) signaling induced GM-CSF expression in naïve and memory TH cells, whereas STAT3 signaling blocked it. The opposite effect was observed for IL-17 expression. Ex vivo, GM-CSF+ TH cells that coexpress interferon-γ and T-bet could be distinguished by differential chemokine receptor expression from a previously uncharacterized subset of GM-CSF–only–producing TH cells that did not express TH1, TH2, and TH17 signature cytokines or master transcription factors. Our findings demonstrate distinct and counterregulatory pathways for the generation of IL-17– and GM-CSF–producing cells and also suggest a pathogenic role for GM-CSF+ T cells in the inflamed brain of multiple sclerosis (MS) patients. This provides not only a scientific rationale for depleting T cell–derived GM-CSF in MS patients but also multiple new molecular checkpoints for therapeutic GM-CSF suppression, which, unlike in mice, do not associate with the TH17 but instead with the TH1 axis.

ICOS maintains the T follicular helper cell phenotype by down-regulating Krüppel-like factor 2

ICOS signaling is required for inhibition of the transcription factor Klf2, which controls expression of genes expressed by follicular T helper (Tfh) cells. When ICOS signaling is blocked, Tfh cells lose expression of characteristic Tfh genes and revert to an effector phenotype, resulting in disruption of the germinal center response.

miR-148a promotes plasma cell differentiation and targets the germinal center transcription factors Mitf and Bach2

B cells undergo affinity maturation and class switch recombination of their immunoglobulin receptors during a germinal center (GC) reaction, before they differentiate into long‐lived antibody‐secreting plasma cells (PCs). Transcription factors such as Bach2 and Mitf are essential during this process, as they delay premature differentiation of GC B cells by repressing Blimp‐1 and IRF4, two transcription factors required for terminal PC differentiation. Therefore, Bach2 and Mitf expression must be attenuated in activated B cells to allow terminal PC differentiation, but the precise mechanism remains enigmatic. Here, we provide evidence that miR‐148a, a small noncoding microRNA, fosters PC differentiation and survival. Next‐generation sequencing revealed that miR‐148a is the most abundant microRNA in primary human and murine PCs, and its expression is upregulated in activated murine B cells and coincides with Blimp‐1 synthesis. miR‐148a targets Bach2, Mitf and proapoptotic factors such as PTEN and Bim. When prematurely expressed, miR‐148a promotes the differentiation and survival of plasmablasts and reduces frequencies of IgG1+ cells in primary B‐cell cultures. In summary, we propose that miR‐148a is a new player in the regulatory network controlling terminal PC differentiation and could, therefore, be a therapeutic target for interfering with PC differentiation and survival.

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.

Lymphocyte signaling: regulation of FoxO transcription factors by microRNAs.

The Forkhead box O (FoxO) family of transcription factors is important for the maintenance of immunological homeostasis and tolerance by controlling the development and function of B and T lymphocytes. Because dysregulation in FoxO activity can result in chronic inflammation and autoimmunity, the transcriptional activity of FoxO proteins is tightly controlled and generally dependent on complex posttranslational modifications that lead either to their nuclear entry and subsequent activation or, alternatively, to their nuclear export. The phosphatidylinositol 3‐kinase (PI3K)–protein kinase B (PKB/Akt) axis represents the major pathway phosphorylating and thereby inactivating FoxO proteins. However, recent results have revealed an additional posttranscriptional mechanism of FoxO inactivation by microRNAs. The discovery of this molecular pathway may provide a new therapeutic avenue for the modulation of FoxO activity in immune‐mediated diseases using either microRNA targeting antagomirs or synthetic microRNA mimics, a topic that is addressed in this review.

Identification of T Cell-Mediated Vascular Rejection After Kidney Transplantation by the Combined Measurement of 5 Specific MicroRNAs in Blood.

Background MicroRNAs (miRNAs, miR) hold important roles in the posttranscriptional regulation of gene expression. Their function has been correlated with kidney disease, and they might represent a new class of biomarkers for frequent evaluation of renal graft status. We analyzed their potential in identifying severe T cell–mediated vascular rejection (TCMVR) (Banff 4-II/III) in kidney transplanted patients. Methods Microarray experiments and semiquantitative real-time reverse transcription polymerase chain reaction were performed with total RNA isolated from blood cells of kidney graft recipients. Initial microarray analysis revealed 23 differentially expressed miRNAs distinguishing patients with TCMVR from patients with stable grafts. From these, we validated and further determined the expression of 6 differentially expressed miRNAs and 2 control miRNAs in 161 samples from patients with T cell–mediated rejection (Banff 3-Borderline, Banff 4-I/II/III), Banff-2 antibody-mediated rejection, Banff-5 interstitial fibrosis/tubular atrophy, in samples from stable patients and in samples from patients with urinary tract infection using real-time reverse transcription polymerase chain reaction. Results Expression levels of all 6 candidate miRNAs were significantly downregulated in blood of TCMVR patients compared to the other groups and displayed high sensitivities and specificities for diagnosing TCMVR. The combination of 5 miRNAs, identified by an unbiased multivariate logistic regression followed by cross-validation, enhanced the sensitivity and specificity for the diagnosis of TCMVR after renal transplantation. Conclusions The combined measurement of miRNA-15B, miRNA-16, miRNA-103A, miRNA-106A, and miRNA-107 may help to better identify TCMVR after renal transplantation in a precise and clinically applicable way.

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

Background: Not every T helper type 2 (Th2) lymphocyte imprinted to express interleukin-4 (IL-4) does so when activated. Results: Preventing nuclear translocation of the nuclear factor of activated T cells (NFAT) reduces the number of Th2 lymphocytes reexpressing IL-4. Conclusion: NFAT is the limiting factor determining digital IL-4 expression in Th2 lymphocytes. Significance: This might help us to understand the regulation of immunopathology in allergy and asthma. 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.

Antigen receptor-mediated depletion of FOXP3 in induced regulatory T-lymphocytes via PTPN2 and FOXO1.

Regulatory T-cells induced via IL-2 and TGFβ in vitro (iTreg) suppress immune cells and are potential therapeutics during autoimmunity. However, several reports described their re-differentiation into pathogenic cells in vivo and loss of their key functional transcription factor (TF) FOXP3 after T-cell antigen receptor (TCR)-signalling in vitro. Here, we show that TCR-activation antagonizes two necessary TFs for foxp3 gene transcription, which are themselves regulated by phosphorylation. Although the tyrosine phosphatase PTPN2 is induced to restrain IL-2-mediated phosphorylation of the TF STAT5, expression of the TF FOXO1 is downregulated and miR-182, a suppressor of FOXO1 expression, is upregulated. TGFβ counteracts the FOXP3-depleting TCR-signal by reassuring FOXO1 expression and by re-licensing STAT5 phosphorylation. Overexpressed phosphorylation-independent active versions of FOXO1 and STAT5 or knockdown of PTPN2 restores FOXP3 expression despite TCR-signal and absence of TGFβ. This study suggests novel targets for stabilisation and less dangerous application of iTreg during devastating inflammation.

Direct uptake of Antagomirs and efficient knockdown of miRNA in primary B and T lymphocytes

Depending on their physiological expression level, microRNAs (miRNA) address different target genes, thus have different biological functions. To identify these, the physiological expression has to be blocked. Here, we describe the use of inhibitory cholesterol-modified oligonucleotides (Antagomirs) to inhibit miRNAs selectively in primary human and murine T and B lymphocytes. Due to their lipophilic cholesterol tag Antagomirs enter primary lymphocytes efficiently and quantitatively. We show here that at concentrations of 0.125 to 1 μM, Antagomirs selectively inhibit expression of their target miRNA up to 99.5% without affecting cell viability.