Ria Baumgrass

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.

Methylation matters: binding of Ets-1 to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in regulatory T cells

The forkhead-box protein P3 (Foxp3) is a key transcription factor for the development and suppressive activity of regulatory T cells (Tregs), a T cell subset critically involved in the maintenance of self-tolerance and prevention of over-shooting immune responses. However, the transcriptional regulation of Foxp3 expression remains incompletely understood. We have previously shown that epigenetic modifications in the CpG-rich Treg-specific demethylated region (TSDR) in the Foxp3 locus are associated with stable Foxp3 expression. We now demonstrate that the methylation state of the CpG motifs within the TSDR controls its transcriptional activity rather than a Treg-specific transcription factor network. By systematically mutating every CpG motif within the TSDR, we could identify four CpG motifs, which are critically determining the transcriptional activity of the TSDR and which serve as binding sites for essential transcription factors, such as CREB/ATF and NF-κB, which have previously been shown to bind to this element. The transcription factor Ets-1 was here identified as an additional molecular player that specifically binds to the TSDR in a demethylation-dependent manner in vitro. Disruption of the Ets-1 binding sites within the TSDR drastically reduced its transcriptional enhancer activity. In addition, we found Ets-1 bound to the demethylated TSDR in ex vivo isolated Tregs, but not to the methylated TSDR in conventional CD4+ T cells. We therefore propose that Ets-1 is part of a larger protein complex, which binds to the TSDR only in its demethylated state, thereby restricting stable Foxp3 expression to the Treg lineage.

Autoregulation of Th1-mediated inflammation by twist1

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohns disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-γ, IL-2, and tumor necrosis factor-α, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, 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 γβ 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‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β 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.

Novel inhibitors of the calcineurin/NFATc hub - alternatives to CsA and FK506?

The drugs cyclosporine A (CsA) and tacrolimus (FK506) revolutionized organ transplantation. Both compounds are still widely used in the clinic as well as for basic research, even though they have dramatic side effects and modulate other pathways than calcineurin-NFATc, too. To answer the major open question - whether the adverse side effects are secondary to the actions of the drugs on the calcineurin-NFATc pathway - alternative inhibitors were developed. Ideal inhibitors should discriminate between the inhibition of (i) calcineurin and peptidyl-prolyl cis-trans isomerases (PPIases; the matchmaker proteins of CsA and FK506), (ii) calcineurin and the other Ser/Thr protein phosphatases, and (iii) NFATc and other transcription factors. In this review we summarize the current knowledge about novel inhibitors, synthesized or identified in the last decades, and focus on their mode of action, specificity, and biological effects.

Digital NFATc2 Activation per Cell Transforms Graded T Cell Receptor Activation into an All-or-None IL-2 Expression

The expression of interleukin-2 (IL-2) is a key event in T helper (Th) lymphocyte activation, controlling both, the expansion and differentiation of effector Th cells as well as the activation of regulatory T cells. We demonstrate that the strength of TCR stimulation is translated into the frequency of memory Th cells expressing IL-2 but not into the amount of IL-2 per cell. This molecular switch decision for IL-2 expression per cell is located downstream of the cytosolic Ca2+ level. Here we show that in a single activated Th cell, NFATc2 activation is digital but NF-κB activation is graded after graded T cell receptor (TCR) signaling. Subsequently, NFATc2 translocates into the nucleus in an all-or-none fashion per cell, transforming the strength of TCR-stimulation into the number of nuclei positive for NFATc2 and IL-2 transcription. Thus, the described NFATc2 switch regulates the number of Th cells actively participating in an immune response.

Human regulatory T cells rapidly suppress T cell receptor-induced Ca(2+), NF-κB, and NFAT signaling in conventional T cells.

Inhibition of calcium signaling is critical for the suppression of T cell responses by regulatory T cells. Suppressing Calcium Suppresses T Cells Regulatory T cells (Tregs) are required to keep conventional T cells in check, and disruption of the generation or function of Tregs leads to autoimmunity. Conversely, Tregs can have a deleterious effect by dampening antitumor responses of T cells. Thus, improved understanding of the mechanisms by which Tregs inhibit T cell receptor (TCR)–induced responses in conventional T cells would help to develop better therapies against autoimmune disorders and cancer. Schmidt et al. found that TCR-induced, Ca2+-dependent signaling in human conventional T cells that were incubated with Tregs was inhibited compared to that in nonsuppressed T cells, which led to defective activation of the transcription factors NFAT and NF-κB. In contrast, Ca2+-independent signaling was unaffected. Suppressed signaling persisted after the Tregs were removed from cocultures. Increasing the intracellular concentration of Ca2+ in conventional T cells reversed the inhibitory effects of Tregs. Together, these data suggest that inhibition of Ca2+ signaling is critical for the suppressive effects of Tregs. CD4+CD25hiFoxp3+ regulatory T cells (Tregs) are critical mediators of self-tolerance, which is crucial for the prevention of autoimmune disease, but Tregs can also inhibit antitumor immunity. Tregs inhibit the proliferation of CD4+CD25− conventional T cells (Tcons), as well as the ability of these cells to produce effector cytokines; however, the molecular mechanism of suppression remains unclear. Here, we showed that human Tregs rapidly suppressed the release of calcium ions (Ca2+) from intracellular stores in response to T cell receptor (TCR) activation in Tcons. The inhibition of Ca2+ signaling resulted in decreased dephosphorylation, and thus decreased activation, of the transcription factor nuclear factor of activated T cells 1 (NFAT1) and reduced the activation of nuclear factor κB (NF-κB). In contrast, Ca2+-independent events in Tcons, such as TCR-proximal signaling and activation of the transcription factor activator protein 1 (AP-1), were not affected during coculture with Tregs. Despite suppressing intracellular Ca2+ mobilization, coculture with Tregs did not block the generation of inositol 1,4,5-trisphosphate in TCR-stimulated Tcons. The Treg-induced suppression of the activity of NFAT and NF-κB and of the expression of the gene encoding the cytokine interleukin-2 was reversed in Tcons by increasing the concentration of intracellular Ca2+. Our results elucidate a previously unrecognized and rapid mechanism of Treg-mediated suppression. This increased understanding of Treg function may be exploited to generate possible therapies for the treatment of autoimmune diseases and cancer.

Interferon-α suppresses cAMP to disarm human regulatory T cells

IFN-α is an antineoplastic agent in the treatment of several solid and hematologic malignancies that exerts strong immune- and autoimmune-stimulating activity. However, the mechanisms of immune activation by IFN-α remain incompletely understood, particularly with regard to CD4(+)CD25(high)Foxp(+) regulatory T cells (Treg). Here, we show that IFN-α deactivates the suppressive function of human Treg by downregulating their intracellular cAMP level. IFN-α-mediated Treg inactivation increased CD4(+) effector T-cell activation and natural killer cell tumor cytotoxicity. Mechanistically, repression of cAMP in Treg was caused by IFN-α-induced MAP-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK)-mediated phosphodiesterase 4 (PDE4) activation and accompanied by downregulation of IFN receptor (IFNAR)-2 and negative regulation of T-cell receptor signaling. IFN-α did not affect the anergic state, cytokine production, Foxp3 expression, or methylation state of the Treg-specific demethylated region (TSDR) within the FOXP3 locus associated with a stable imprinted phenotype of human Treg. Abrogated protection by IFN-α-treated Treg in a humanized mouse model of xenogeneic graft-versus-host disease confirmed IFN-α-dependent regulation of Treg activity in vivo. Collectively, the present study unravels Treg inactivation as a novel IFN-α activity that provides a conceivable explanation for the immune-promoting effect and induction of autoimmunity by IFN-α treatment in patients with cancer and suggests IFN-α for concomitant Treg blockade in the context of therapeutic vaccination against tumor antigens.

Substitution in Position 3 of Cyclosporin A Abolishes the Cyclophilin-mediated Gain-of-function Mechanism but Not Immunosuppression

Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine3]CsA was shown to inhibit calcineurin, either in its free form with an IC50 value of 10 μm, or in its complex form with cyclophilin 18 with an IC50 of 500 nm. [Dimethylaminoethylthiosarcosine3]CsA ([Dat-Sar3]CsA) was found to inhibit calcineurin on its own, with an IC50 value of 1.0 μm, but was not able to inhibit calcineurin after forming the [Dat-Sar3]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar3]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar3]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Δ12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar3]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Δ12 strain.

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 IKKβ 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.