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

Cord Blood Derived CD4+CD25high T Cells Become Functional Regulatory T Cells upon Antigen Encounter

Background: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these “excessive” responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself

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

T helper (Th) 2 predominance in atopic diseases is due to preferential apoptosis of circulating memory/effector Th1 cells

T cells constitute a large population of cellular infiltrate in atopic/allergic inflammation and a dysregulated, Th2-biased peripheral immune response appears to be an important pathogenetic factor. In atopic dermatitis, circulating cutaneous lymphocyte-associated antigen-bearing (CLA(+)) CD45RO(+) T cells with skin-specific homing property represent an activated memory/effector T cell subset. They express high levels of Fas and Fas ligand and undergo activation-induced apoptosis. The freshly purified (CLA(+)) CD45RO(+) T cells of atopic individuals display distinct features of in vivo-triggered apoptosis such as procaspase degradation and active caspase-8 formation. In particular, the Th1 compartment of activated memory/effector T cells selectively undergoes activation-induced cell death, skewing the immune response toward surviving Th2 cells in atopic dermatitis patients. The apoptosis of circulating memory/effector T cells was confined to atopic individuals whereas non-atopic patients such as psoriasis, intrinsic-type asthma, contact dermatitis, intrinsic type of atopic dermatitis, bee venom allergic patients, and healthy controls showed no evidence for enhanced T cell apoptosis in vivo. These results define a novel mechanism for peripheral Th2 response in atopic diseases

CB-derived CD4⁺CD25⁺ T cells are deficient with regard to their regulatory function harboring a subset with limited net suppressive effects.

<p>(<b>A</b>) Specificity of proliferation to BLG and BLG+LPS is displayed via CFSE labeling experiments. (<b>B</b>) Inhibition experiments were performed using and increasing amount of MACS-sorted CD4⁺CD25⁺ T cells to the CD4⁺CD25⁻ fraction in presence of irradiated APCs. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ cells. Graphs indicate the means of 5–8 independent experiments with means +/− SEM. Wilcoxon sign rank test was applied. P-values of less than 0.05 were considered significant. (<b>C</b>) CBMCs were separated via FACS on day 0 according to their CD25 and CD127 expression. Four subgroups (CD4⁺CD25^highCD127^low (F1), CD4⁺CD25^intermediateCD127^low (F2), CD4⁺CD25^intermediateCD127^high (F3), and CD4⁺CD25⁻ T cells (CD25⁻)) were obtained. An inhibition assay was performed with the first three fractions using the CD4⁺CD25⁻ T cells as effector cells. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ subgroup. Graphs indicate the means of 4 independent experiments and SEM. (<b>D</b>) Real-time PCR analysis was performed to analyze the FOXP3, TGF-β, and IL-10 expression for further examination of the four fractions. Data represents three independent experiments showing the normalized expression of means +/− SEM.</p

Upon priming with BLG, cord blood derived CD4⁺CD25⁺ T cells become highly suppressive.

<p>(<b>A</b>) Cord blood-derived mononuclear cells were co-cultured for 6 days with the antigen BLG. At day six CD4⁺CD25⁺ T cells were MACS-sorted and an inhibition assay was performed with the CD4⁺CD25⁻ (frozen on day 0, without prior antigen exposure) and stimulated with α-CD3 or BLG. The inhibitory potential is expressed as relative proliferation compared to the CD4⁺CD25⁻ cells. Results are representative for 6–9 independent experiments. Wilcoxon signed rank test was applied. P-values of less than 0.05 were considered significant (*); p<0.005 (**); p<0.001 (***). (<b>B</b>) Real-time PCR analysis was performed to quantify the gene expression of Treg-related markers (FOXP3, GARP, RUNX 1, and RUNX 3) after allergen exposure. Results are representative for three independent experiments showing the normalized expression. CD4⁺CD25⁻ T cells are displayed with white and CD4⁺CD25⁺ T cells with black squares.</p

Comparable Treg-related transcription factor and marker expression in CB and PB on day 0.

<p>(<b>A</b>) Cord and peripheral blood derived mononuclear cells were analyzed via flow cytometry on day 0 without prior stimulation. Surface and intracellular staining was performed and one out of three representative experiments is shown. (<b>B</b>) Real-time PCR analysis was performed to quantify the gene expression of Treg-related markers (FOXP3, GARP, TGF-β, RUNX 1, RUNX 3, and IL-10). Results are representative for three independent experiments showing the normalized expression. CD4⁺CD25⁻ T cells are displayed with white and CD4⁺CD25⁺ T cells with black squares.</p