CD4+CD25+ regulatory T cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction

Abstract

T regulatory (Treg) cells play a role in the suppression of immune responses, thus serving to induce tolerance and control autoimmunity. Here, we explored whether Treg cells influence the immediate hypersensitivity response of mast cells (MCs). Treg cells directly inhibited the FcvarepsilonRI-dependent MC degranulation through cell-cell contact involving OX40-OX40L interactions between Treg cells and MCs, respectively. When activated in the presence of Treg cells, MCs showed increased cyclic adenosine monophosphate (cAMP) concentrations and reduced Ca(2+) influx, independently of phospholipase C (PLC)-gamma2 or Ca(2+) release from intracellular stores. Antagonism of cAMP in MCs reversed the inhibitory effects of Treg cells, restoring normal Ca(2+) responses and degranulation. Importantly, the in vivo depletion or inactivation of Treg cells caused enhancement of the anaphylactic response. The demonstrated crosstalk between Treg cells and MCs defines a previously unrecognized mechanism controlling MC degranulation. Loss of this interaction may contribute to the severity of allergic responses.

Figure 1. In vivo co-localization of Foxp3⁺ T_regs and mast cells and in vitro impairment of IgE-mediated degranulation of BMMCs by T_regs

(A) Inguinal lymph node sections were stained with mouse anti-rat-FcεRIβ (red) and rat anti-mouse-Foxp3 Ab (brown). Arrows indicate cell-cell contact. Original magnification 400X. (B) BMMCs sensitized with IgE anti-DNP (IgE) and challenged with Ag (IgE/Ag) in the absence or presence of equal amount of CD4⁺CD25⁺ T_regs (T_reg), or CD4⁺CD25⁻ T cells (T resting) or a-CD3- plus a-CD28-stimulated CD4⁺CD25⁻ effector T cells (T_eff), were examined for release of β-hexosaminidase expressed as percentage of the cells’ total mediator content. Shown are the means ± SD of four independent experiments, each performed in duplicate. (C) TNF-α and IL-6 levels were evaluated in the supernatants of BMMCs-T_regs co-cultures. Shown are the means ± SD of three independent experiments.

Figure 2. The contact-dependent inhibitory role of T_regs on MCs degranulation depends on T_regs OX40 expression and requires OX40L on BMMCs

(A) BMMCs sensitized with mouse IgE anti-DNP (IgE) and challenged with Ag (IgE/Ag) in the absence or presence of equal amount of WT or OX40^−/− CD4⁺CD25⁺ T_regs or separated by a transwell membrane (Transwell) were then examined for release of β-hexosaminidase. (B) Same as (A), but BMMCs were obtained from WT or OX40L^−/−mice and co-cultured with WT CD4⁺CD25⁺ T_regs. Shown are the means ± SD of three independent experiments, each performed in duplicate. (C) IgE-sensitized BMMCs were challenged with Ag in the absence or presence of membranes from K562 cells expressing OX40 (K562-OX40) or empty vector (K562). (D) IgE-sensitized BMMCs were challenged with Ag in presence of T_regs, plus blocking anti-OX40L (clone MGP34) or isotype control (rat IgG2c) antibodies.

Figure 3. T_regs do not affect FcεRI-dependent PLC-γ phosphorylation

WT or OX40L^−/− sensitized BMMCs were stimulated with Ag in the presence of WT or OX40^−/− T_regs for the indicated times. Cells were immediately fixed and stained for c-kit and phosphorylated PLC-γ2. From c-kit⁺-gated BMMCs (A), histogram overlays of phosphorylated PLC-γ2 at different time points were obtained from WT (upper panels) or OX40L^−/− (lower panels) BMMCs challenged in absence of T_regs (B). Dot plot overlay of basal phosphorylated PLC-γ2 (left, gray) and after 10 min (right, violet) is shown in panel C. Histogram overlays of phosphorylated PLC-γ2 from WT (upper panels) or OX40L^−/− (lower panels) BMMCs challenged in the presence of T_regs (D). Results shown are representative of three independent experiments. Kinetics of PLC-γ2 phosphorylation at different conditions are shown in panel E and are the mean + SD of three independent experiments.

Figure 4. Reduced FcεRI-dependent Ca²⁺ influx following BMMC-T_reg engagement, but not intracellular Ca²⁺ mobilization

(A) BMMCs loaded with FURA-2AM were stimulated via FcεRI in the absence (BMMC, black line) or presence of CD4⁺CD25⁻ T cells (T resting, green line), WT CD4⁺CD25⁺ T_regs (red line) or OX40^−/− CD4⁺CD25⁺ T_regs (blue line) and fluorescence emission was monitored. (B) FURA-2AM-loaded BMMCs were stimulated via FcεRI and co-cultured with WT CD4⁺CD25⁺ T_regs (red line) in the absence of extracellular Ca²⁺. 400 sec after Ag stimulation, 2μM Ca²⁺ was added to the medium and fluorescence emission was monitored. (C and D) At the end of each experiment, 14 minutes after Ag addition, percentage of β-hexosaminidase release from individual sample, was measured. Results shown are representative of three independent experiments.

Figure 5. Intracellular levels of cAMP are increased in BMMCs after co-culture with WT but not with OX40-deficient T_regs

Sensitized WT and OX40L^−/− BMMCs were CFSE-labeled and Ag-stimulated alone or with WT or OX40^−/− T_regs. BMMCs and T_regs were separated using FACS-based cell sorting and cytosolic cAMP concentrations were measured using a cAMP-specific ELISA. As positive control sensitized BMMCs were incubated with forskolin and challenged with Ag. (A) BMMCs baseline [cAMP] was 10 pmoles/1×10⁵. Results are expressed as fold induction over BMMCs alone. (B) T_regs baseline [cAMP] was 50 pmoles/1×10⁵. Results are expressed as fold induction over T_regs alone. (C) Sensitized BMMCs were activated with Ag plus K562 or K562-OX40 membranes. The mean ± SD of three independent experiments are shown.

Figure 6. Antagonism of cAMP effects in BMMCs reverses T_reg-mediated suppression of degranulation and restores extracellular Ca²⁺ uptake

(A) Anti-DNP IgE preloaded BMMCs were preincubated for 30 min with 1mM of the specific cAMP antagonist Rp-cAMPs. Cells were washed and activated with Ag separately (BMMC) or in co-culture with CD4⁺CD25⁺ T_regs (BMMC + T_reg). After 30 min samples were examined for release of β-hexosaminidase as described. Shown are the means ± SD of three independent experiments each performed in duplicate. (B) IgE-sensitized BMMCs were preincubated for 30 min with 1mM of the specific cAMP antagonist Rp-cAMPs and Ca2+ mobilization was assessed.

Figure 7. T_regs via OX40 reduce in vivo systemic release of histamine by MCs

(A) Plasma histamine levels were measured 1.5 minutes after challenge with PBS (mock) or DNP-HSA (Ag) in PC61-treated mice, OX40^−/− mice and control WT littermates that were pre-sensitized with IgE anti-DNP (n = 8, each group). Results shown are pooled from two independent experiments. (B) CD45⁺ c-kit⁺ peritoneal mast cells (PMC, left panel) from unsensitized mice in each experimental group were stained for FcεRI (black lines) or isotype control (shaded areas). Mean fluorescence intensity (MFI) is indicated. (C) Thymectomized and anti-CD25 treated mice were left unreconstituted or received i.v. 3 weeks after 1 ×10⁶ T_regs obtained from WT or OX40^−/− donors. After 3 days passive systemic anaphylaxis was induced and plasma histamine measured as in (A).