Hyun-Dong Chang

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 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.

1,25-dihydroxyvitamin D3 promotes IL-10 production in human B cells

1,25‐dihydroxyvitamin D3 (calcitriol) regulates immune responses, e.g., inhibits expression of IgE by B cells and enhances expression of IL‐10 by dendritic cells and T cells. We report here that activation of human B cells by B cell receptor, CD40 and IL‐4 signals induces expression of the gene for 25‐hydroxyvitamin‐D3–1α‐hydroxylase (CYP1α). Accordingly, these B cells generate and secrete significant amounts of calcitriol. In activated B cells calcitriol induces expression of the genes Cyp24, encoding a vitamin D hydroxylase, and Trpv6, encoding a calcium selective channel protein. Calcitriol enhances IL‐10 expression of activated B cells more than threefold, both by recruiting the vitamin D receptor to the promoter of Il‐10, and to lesser extent by modulation of calcium‐dependent signaling. The molecular link in activated B cells between vitamin D signaling, expression of IgE and IL‐10, and their ability to produce calcitriol from its precursor, suggest that pro‐vitamin D (25‐hydroxyvitamin D3) can be used as a modulator of allergic immune responses.

Th memory for interleukin‐17 expression is stable in vivo

Based on the memory for the re‐expression of certain cytokine genes, different subsets of Th cells have been defined. In Th type 1 (Th1) and Th2 memory lymphocytes, the genes for the cytokines interferon‐γ and interleukin (IL)‐4 are imprinted for expression upon restimulation by the expression of the transcription factors T‐bet and GATA‐3, respectively, and epigenetic modification of the cytokine genes. In Th17 cells, IL‐17 expression is dependent on the transcription factors RORγt and RORα. Here, we analyze the stability and plasticity of IL‐17 memory in Th17 cells. We have developed a cytometric IL‐17 secretion assay for the isolation of viable Th cells secreting IL‐17. For Th17 cells generated in vitro, IL‐17 expression itself is dependent on continued TGF‐β/IL‐6 or IL‐23 signaling and is blocked by interferon‐γ and IL‐4 signaling. In response to IL‐12 and IL‐4, in vitro generated Th17 cells are converted into Th1 or Th2 cells, respectively. Th17 cells isolated ex vivo, however, maintain their IL‐17 memory upon subsequent in vitro culture, even in the absence of IL‐23. Their cytokine memory is not regulated by IL‐12 or IL‐4. Th17 cells generated in vivo are a stable and distinct lineage of Th cell differentiation.

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.

IFN-γ and IL-12 synergize to convert in vivo generated Th17 into Th1/Th17 cells

Th1 and Th17 cells are distinct lineages of effector/memory cells, imprinted for re‐expression of IFN‐γ and IL‐17, by upregulated expression of T‐bet and retinoic acid‐related orphan receptor γt (RORγt), respectively. Apparently, Th1 and Th17 cells share tasks in the control of inflammatory immune responses. Th cells coexpressing IFN‐γ and IL‐17 have been observed in vivo, but it remained elusive, how these cells had been generated and whether they represent a distinct lineage of Th differentiation. It has been shown that ex vivo isolated Th1 and Th17 cells are not interconvertable by TGF‐β/IL‐6 and IL‐12, respectively. Here, we show that ex vivo isolated Th17 cells can be converted into Th1/Th17 cells by combined IFN‐γ and IL‐12 signaling. IFN‐γ is required to upregulate expression of the IL‐12Rβ2 chain, and IL‐12 for Th1 polarization. These Th1/Th17 cells stably coexpress RORγt and T‐bet at the single‐cell level. Our results suggest a molecular pathway for the generation of Th1/Th17 cells in vivo, which combine the pro‐inflammatory potential of Th1 and Th17 cells.

Expression of IL-10 in Th memory lymphocytes is conditional on IL-12 or IL-4, unless the IL-10 gene is imprinted by GATA-3.

In Th1 and Th2 memory lymphocytes, the genes for the cytokines interleukin (IL)‐4 and interferon‐γ (IFN‐γ) are imprinted for expression upon restimulation. This cytokine memory is based on expression of the transcription factors T‐bet for IFN‐γ, and GATA‐3 for IL‐4, and epigenetic modification of the cytokine genes. In Th2 cells, expression of the cytokine IL‐10 is also induced by GATA‐3. Here, we show that this induction is initially not accompanied by epigenetic modification of the IL‐10 gene. Only after repeated restimulation of a memory Th2 cell in the presence of IL‐4, extensive histone acetylation of the IL‐10 gene is detectable. This epigenetic imprinting correlates with the development of a memory for IL‐10 in repeatedly restimulated Th2 cells. In Th1 cells, IL‐10 expression is induced by IL‐12, but the IL‐10 gene lacks detectable histone acetylation. Accordingly, IL‐10 expression in restimulated memory Th1 cells remains conditional on the presence of IL‐12. This finding defines a potential anti‐inflammatory role for IL‐12 in Th1 recall responses. While in primary Th1 responses IL‐12 is required to induce expression of the pro‐inflammatory cytokine IFN‐γ, in secondary Th1 responses IFN‐γ re‐expression is independent of IL‐12, which still is able to induce expression of the anti‐inflammatory cytokine IL‐10.

Human cytomegalovirus drives epigenetic imprinting of the IFNG locus in NKG2Chi natural killer cells.

Memory type 1 T helper (TH1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8+ T cells or TH1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2Chi NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.

Leptin: A Critical Regulator of CD4+ T-cell Polarization in Vitro and in Vivo

Besides being mandatory in the metabolic system, adipokines like leptin directly affect immunity. Leptin was found to be necessary in T helper 1 (Th1)-dependent inflammatory processes, whereas effects on Th2 cells are rarely understood. Here, we focused on leptin in T-helper cell polarization and in Th2-mediated intestinal inflammation in vivo. The induction of cytokine-producing Th1 or Th2 cells from naive CD4(+) T cells under polarizing conditions in vitro was generally decreased in cells from leptin-deficient ob/ob mice compared with wild-type mice. To explore the in vivo relevance of leptin in Th2-mediated inflammation, the model of oxazolone-induced colitis was employed in wild-type, ob/ob, and leptin-reconstituted ob/ob mice. Ob/ob mice were protected, whereas wild-type and leptin-reconstituted ob/ob mice developed colitis. The disease severity went in parallel with local production of the Th2 cytokine IL-13. A possible explanation for the protection of ob/ob mice in Th1- as well as in Th2-dependent inflammation is provided by a decreased expression of the key transcription factors for Th1 and Th2 polarization, T-bet and GATA-3, in naive ob/ob T cells. In conclusion, these results support the regulatory function of the adipokine leptin within T-cell polarization and thus in the acquired immune system and support the concept that there is a close interaction with the endocrine system.

Human memory T cells from the bone marrow are resting and maintain long-lasting systemic memory

Significance Memory T cells are essential components of immunological memory. In the apparent absence of antigen, numbers of recirculating antigen-specific memory T cells dwindle, provoking the question of whether there is immunological memory without memory T cells. Here we show that human memory T cells can reside in the bone marrow as resting cells in terms of proliferation, transcription, and mobility. The repertoire of bone marrow memory T cells is enriched for systemic pathogens representing persistent, recent, and childhood challenges. In terms of absolute numbers, memory T cells specific for systemic antigens are maintained predominantly in the bone marrow, in particular those representing historic encounters. In the bone marrow, a population of memory T cells has been described that promotes efficient secondary immune responses and has been considered to be preactivated, owing to its expression of CD69 and CD25. Here we show that human bone marrow professional memory T cells are not activated but are resting in terms of proliferation, transcription, and mobility. They are in the G0 phase of the cell cycle, and their transcriptome is that of resting T cells. The repertoire of CD4+ bone marrow memory T cells compared with CD4+ memory T cells from the blood is significantly enriched for T cells specific for cytomegalovirus-pp65 (immunodominant protein), tetanus toxoid, measles, mumps, and rubella. It is not enriched for vaccinia virus and Candida albicans-MP65 (immunodominant protein), typical pathogens of skin and/or mucosa. CD4+ memory T cells specific for measles are maintained nearly exclusively in the bone marrow. Thus, CD4+ memory T cells from the bone marrow provide long-term memory for systemic pathogens.