Beate Rückert

Molecular Mechanisms Underlying FOXP3 Induction in Human T Cells

FOXP3 is playing an essential role for T regulatory cells and is involved in the molecular mechanisms controlling immune tolerance. Although the biological relevance of this transcription factor is well documented, the pathways responsible for its induction are still unclear. The current study reveals structure and function of the human FOXP3 promoter, revealing essential molecular mechanisms of its induction. The FOXP3 promoter was defined by RACE, cloned, and functionally analyzed using reporter-gene constructs in primary human T cells. The analysis revealed the basal, T cell-specific promoter with a TATA and CAAT box 6000 bp upstream the translation start site. The basal promoter contains six NF-AT and AP-1 binding sites, which are positively regulating the trans activation of the FOXP3 promoter after triggering of the TCR. The chromatin region containing the FOXP3 promoter was bound by NF-ATc2 under these conditions. Furthermore, FOXP3 expression was observed following TCR engagement. Promoter activity, mRNA, and protein expression of T cells were suppressed by addition of cyclosporin A. Taken together, this study reveals the structure of the human FOXP3 promoter and provides new insights in mechanisms of addressing T regulatory cell-inducing signals useful for promoting immune tolerance. Furthermore, the study identifies essential, positive regulators of the FOXP3 gene and highlights cyclosporin A as an inhibitor of FOXP3 expression contrasting other immunosuppressants such as steroids or rapamycin.

In vivo switch to IL-10-secreting T regulatory cells in high dose allergen exposure.

High dose bee venom exposure in beekeepers by natural bee stings represents a model to understand mechanisms of T cell tolerance to allergens in healthy individuals. Continuous exposure of nonallergic beekeepers to high doses of bee venom antigens induces diminished T cell–related cutaneous late-phase swelling to bee stings in parallel with suppressed allergen-specific T cell proliferation and T helper type 1 (Th1) and Th2 cytokine secretion. After multiple bee stings, venom antigen–specific Th1 and Th2 cells show a switch toward interleukin (IL) 10–secreting type 1 T regulatory (Tr1) cells. T cell regulation continues as long as antigen exposure persists and returns to initial levels within 2 to 3 mo after bee stings. Histamine receptor 2 up-regulated on specific Th2 cells displays a dual effect by directly suppressing allergen-stimulated T cells and increasing IL-10 production. In addition, cytotoxic T lymphocyte–associated antigen 4 and programmed death 1 play roles in allergen-specific T cell suppression. In contrast to its role in mucosal allergen tolerance, transforming growth factor β does not seem to be an essential player in skin-related allergen tolerance. Thus, rapid switch and expansion of IL-10–producing Tr1 cells and the use of multiple suppressive factors represent essential mechanisms in immune tolerance to a high dose of allergens in nonallergic individuals.

GATA3-Driven Th2 Responses Inhibit TGF-β1–Induced FOXP3 Expression and the Formation of Regulatory T Cells

Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-β–mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.

IgG4 production is confined to human IL-10–producing regulatory B cells that suppress antigen-specific immune responses

BACKGROUND IL-10-producing regulatory B cells suppress immune responses, and lack of these cells leads to exacerbated symptoms in mouse models of chronic inflammation, transplantation, and chronic infection. IgG4 is a blocking antibody isotype with anti-inflammatory potential that is induced in human high-dose antigen tolerance models. OBJECTIVE We sought to characterize human inducible IL-10-secreting B regulatory 1 (BR1) cells and to investigate their immunoregulatory capacity through suppression of cellular immune responses and production of anti-inflammatory immunoglobulins. METHODS Highly purified IL-10-secreting B cells were phenotypically and functionally characterized by means of whole-genome expression analysis, flow cytometry, suppression assay, and antibody production. B cells specific for the major bee venom allergen phospholipase A2 (PLA) were isolated from beekeepers who displayed tolerance to bee venom antigens and allergic patients before and after specific immunotherapy. RESULTS Human IL-10+ BR1 cells expressed high surface CD25 and CD71 and low CD73 levels. Sorting of CD73-CD25+CD71+ B cells allowed enrichment of human BR1 cells, which produced high levels of IL-10 and potently suppressed antigen-specific CD4+ T-cell proliferation. IgG4 was selectively confined to human BR1 cells. B cells specific for the major bee venom allergen PLA isolated from nonallergic beekeepers show increased expression of IL-10 and IgG4. Furthermore, the frequency of IL-10+ PLA-specific B cells increased in allergic patients receiving allergen-specific immunotherapy. CONCLUSION Our data show the characterization of IL-10+ BR1 cells and in vivo evidence for 2 essential features of allergen tolerance: the suppressive B cells and IgG4-expressing B cells that are confined to IL-10+ BR1 cells in human subjects.

Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells.

Forkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-β (TGF-β) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-β (CBFβ) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2−/− mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4+ CD25high CD127− T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-β mRNA compared with CD4+CD25− cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-β–induced Foxp3 expression in iT reg cell differentiation and function.

Increased activation-induced cell death of high IFN-γ–producing TH1 cells as a mechanism of TH2 predominance in atopic diseases

BACKGROUND A dysregulated and T(H)2-biased immune response appears to be a key pathogenetic factor in atopic diseases. Increased activation and massive infiltration of T cells in the dermis without any evidence for the expansion of their numbers in peripheral blood characterize atopic dermatitis. OBJECTIVE To investigate differences and mechanisms of T(H)1 and T(H)2 cell activation-induced cell death (AICD) in atopic disease. METHODS Naive (CD4(+)CD45(+)RA) and memory (CD4(+)CD45(+)RO) T cells were isolated from healthy and atopic individuals. T(H)1 and T(H)2 subsets were in vitro differentiated. High IFN-gamma-producing T cells and CXCR3(+) T cells were purified, and AICD of isolated cells was determined in addition to expression of apoptosis receptors and caspase activation. RESULTS T(H)1 cells, particularly their high IFN-gamma-producing fraction, and CXCR3(+) T cells showed significantly increased apoptosis in atopic individuals. During their in vitro differentiation, both T(H)1 and T(H)2 cells of atopic individuals showed increased apoptosis compared with the healthy control group, with a significantly high apoptosis in T(H)1 cells. Increased expression of Fas, Fas-ligand, tumor necrosis factor receptor-II, and caspase activation was detected on T(H)1 cells that underwent apoptosis. Neutralization experiments demonstrated a dominant role of IFN-gamma and Fas-Fas-ligand interaction-mediated suicide in T(H)1 cell AICD. CONCLUSION Predominant T(H)2 profile in atopic diseases might be a result of the increased tendency to activation and apoptosis of high IFN-gamma-producing T(H)1 cells.

Unique Phenotype of Human Tonsillar and In Vitro-Induced FOXP3+CD8+ T Cells

Forkhead box p3 (FOXP3) is known to program the acquisition of suppressive capacities in CD4+ regulatory T cells (Treg), whereas its role in CD8+ T cells is unknown. The current study investigates whether FOXP3 also acts as a Treg master switch in peripheral blood and tonsillar CD8+ T cells. Single-cell analyses reveal the existence of a FOXP3+CD8+ population in human tonsils, whereas FOXP3+CD8+ T cells are rarely detected in peripheral blood. Tonsillar FOXP3+CD8+ T cells exhibit a Treg phenotype with high CTLA-4 and CD45RO and low CD127 and CD69 expression. Interestingly, the tonsillar FOXP3+CD8+ T cells are mostly CD25negative and some cells also express the proinflammatory cytokines TNF-α, IFN-γ, or IL-17A. Particularly, IL-17A-expressing cells are present among FOXP3+CD8+ T cells. Even though FOXP3 expression is at the detection limit in peripheral blood CD8+ T cells ex vivo, it can be induced in vitro in naive CD8+ T cells by polyclonal stimulation. The induced FOXP3+CD8+ T cells are predominantly CD25high and CD28high and similar to tonsillar cells, they produce high levels of TNF-α, IFN-γ, and granzyme B. However, IL-4 expression is mutually exclusive and IL-17A expression is not detectable. These FOXP3+CD8+ T cells suppress the proliferation of CD4+ T cells in cocultures, while showing no direct cytotoxic activity. In conclusion, the current study characterizes FOXP3-expressing CD8+ T cells from human tonsils and shows that in vitro activation leads to FOXP3 expression in CD8+ T cells and gain of suppressive activity.

IL-10–overexpressing B cells regulate innate and adaptive immune responses

BACKGROUND Distinct human IL-10-producing B-cell subsets with immunoregulatory properties have been described. However, the broader spectrum of their direct cellular targets and suppressive mechanisms has not been extensively studied, particularly in relation to direct and indirect IL-10-mediated functions. OBJECTIVE The aim of the study was to investigate the effects of IL-10 overexpression on the phenotype and immunoregulatory capacity of B cells. METHODS Primary human B cells were transfected with hIL-10, and IL-10-overexpressing B cells were characterized for cytokine and immunoglobulin production by means of specific ELISA and bead-based assays. Antigen presentation, costimulation capacity, and transcription factor signatures were analyzed by means of flow cytometry and quantitative RT-PCR. Effects of IL-10-overexpresing B cells on Toll-like receptor-triggered cytokine release from PBMCs, LPS-triggered maturation of monocyte-derived dendritic cells, and tetanus toxoid-induced PBMC proliferation were assessed in autologous cocultures. RESULTS IL-10-overexpressing B cells acquired a prominent immunoregulatory profile comprising upregulation of suppressor of cytokine signaling 3 (SOCS3), glycoprotein A repetitions predominant (GARP), the IL-2 receptor α chain (CD25), and programmed cell death 1 ligand 1 (PD-L1). Concurrently, their secretion profile was characterized by a significant reduction in levels of proinflammatory cytokines (TNF-α, IL-8, and macrophage inflammatory protein 1α) and augmented production of anti-inflammatory IL-1 receptor antagonist and vascular endothelial growth factor. Furthermore, IL-10 overexpression was associated with a decrease in costimulatory potential. IL-10-overexpressing B cells secreted less IgE and potently suppressed proinflammatory cytokines in PBMCs, maturation of monocyte-derived dendritic cells (rendering their profile to regulatory phenotype), and antigen-specific proliferation in vitro. CONCLUSION Our data demonstrate an essential role for IL-10 in inducing an immunoregulatory phenotype in B cells that exerts substantial anti-inflammatory and immunosuppressive functions.

Human CD8 T cells of the peripheral blood contain a low CD8 expressing cytotoxic/effector subpopulation

Heterogeneity of lymphocyte populations demonstrates the diversity of cellular immune responses and provide a better understanding of the immune system. CD3+ CD8+ T cells exhibit a low CD8 expressing (CD8low) population in flow cytometric analysis of peripheral blood T cells. In healthy donors, this population consists of 0·2–7·0% of all CD8 T cells. The majority of the CD8low T cell population showed an elevated expression of CD25, CD45RA, and CD95L, and low levels of CD28, CD62L and CD45RO. Circulating CD8low T cells resemble cytotoxic effector cells because they express cytolytic mediators and are able to execute cytotoxicity. A restricted T cell receptor profile with increased Vβ9, Vβ14 and Vβ23 expression was observed and the CD8low T cell population contain Epstein–Barr virus‐specific T cells. Therefore, the CD8low population represent a subset of activated CD8 effector T cells, resulting most probably from a continous and/or balanced immune response to intracellular pathogens.

Regulation of bronchial epithelial barrier integrity by type 2 cytokines and histone deacetylases in asthmatic patients

Background: Tight junctions (TJs) form a barrier on the apical side of neighboring epithelial cells in the bronchial mucosa. Changes in their integrity might play a role in asthma pathogenesis by enabling the paracellular influx of allergens, toxins, and microbes to the submucosal tissue. Objective: The regulation of bronchial epithelial TJs by TH2 cells and their cytokines and their involvement in epigenetic regulation of barrier function were investigated. Methods: The expression, regulation, and function of TJs were determined in air‐liquid interface (ALI) cultures of control and asthmatic primary human bronchial epithelial cells (HBECs) by means of analysis of transepithelial electrical resistance, paracellular flux, mRNA expression, Western blotting, and immunofluorescence staining. Results: HBECs from asthmatic patients showed a significantly low TJ integrity in ALI cultures compared with HBECs from healthy subjects. TH2 cell numbers and levels of their cytokines, IL‐4 and IL‐13, decreased barrier integrity in ALI cultures of HBECs from control subjects but not in HBECs from asthmatic patients. They induced a physical separation of the TJs of adjacent cells in immunofluorescence staining of the TJ molecules occludin and zonula occludens‐1. We observed that expression of histone deacetylases (HDACs) 1 and 9, and Silent information regulator genes (sirtuins [SIRTs]) 6 and 7 were significantly high in HBECs from asthmatic patients. IL‐4 and IL‐13 significantly increased the expression of HDACs and SIRTs. The role of HDAC activation on epithelial barrier leakiness was confirmed by HDAC inhibition, which improved barrier integrity through increased synthesis of TJ molecules in epithelium from asthmatic patients to the level seen in HBECs from control subjects. Conclusion: Our data demonstrate that barrier leakiness in asthmatic patients is induced by TH2 cells, IL‐4, and IL‐13 and HDAC activity. The inhibition of endogenous HDAC activity reconstitutes defective barrier by increasing TJ expression.