Inducible and naturally occurring regulatory T cells enhance lung allergic responses through divergent transcriptional pathways

Abstract

Background: Regulatory T cells attenuate development of asthma in wild-type (WT) mice, with both naturally occurring regulatory T (nTreg) cells and inducible regulatory T (iTreg) cells exhibiting suppressive activity. When transferred into CD8-deficient (CD8^-/-) recipients, both cell types enhanced development of allergen-induced airway hyperresponsiveness.

Objective: We sought to determine whether the pathways leading to enhancement of lung allergic responses by transferred nTreg and iTreg cells differed.

Methods: nTreg cells (CD4⁺CD25⁺) were isolated from WT mice and iTreg cells were generated from WT CD4⁺CD25^- T cells after activation in the presence of TGF-β and transferred into sensitized CD8^-/- recipients before challenge. Development of airway hyperresponsiveness, cytokine levels, and airway inflammation were monitored.

Results: Transfer of nTreg cells enhanced lung allergic responses, as did transfer of iTreg cells. Although anti-IL-13 reduced nTreg cell-mediated enhancement, it was ineffective in iTreg cell-mediated enhancement; conversely, anti-IL-17, but not anti-IL-13, attenuated the enhancement by iTreg cells. Recovered iTreg cells from the lungs of CD8^-/- recipients were capable of IL-17 production and expressed high levels of signature genes of the T_H17 pathway, RORγt and Il17, whereas reduced expression of the Treg cell key transcription factor forkhead box p3 (Foxp3) was observed. In vitro exogenous IL-6-induced IL-17 production in iTreg cells, and in vivo conversion of transferred iTreg cells was dependent on recipient IL-6.

Conclusions: iTreg cells, similar to nTreg cells, exhibit functional plasticity and can be converted from suppressor cells to pathogenic effector cells, enhancing lung allergic responses, but these effects were mediated through different pathways.

Keywords: IL-13; IL-17; Inducible and naturally occurring regulatory T cells; asthma; enhancement; suppression.

Figure 1

Effect of nTreg, iTreg, and CD4⁺CD25⁻ T cell transfer from naive donors into sensitized and challenged CD8^+/+ recipients. (A) In vitro cytokine production from nTregs, iTregs, and CD4⁺CD25⁻ T cells, (B) Flow cytometry of nTregs, iTregs, and CD4⁺CD25⁻ T cells, (C) Gene expression levels of nTregs, iTregs, and CD4⁺CD25⁻ T cells prior to transfer, #p<0.05 comparing CD4⁺CD25⁻ T cells to iTregs and nTregs, (D) AHR, (E) BAL fluid inflammatory cell composition, (F) BAL fluid cytokine levels. Shown are the means±SEM from 3 independent experiments (4 mice/group, n=12). *p<0.01 comparing sensitized and challenged mice (given PBS) to mice challenged alone. †p<0.05 comparing sensitized and challenged recipients of iTregs and nTregs to recipients of PBS or CD4⁺CD25⁻ T cells.

Figure 1

Effect of nTreg, iTreg, and CD4⁺CD25⁻ T cell transfer from naive donors into sensitized and challenged CD8^+/+ recipients. (A) In vitro cytokine production from nTregs, iTregs, and CD4⁺CD25⁻ T cells, (B) Flow cytometry of nTregs, iTregs, and CD4⁺CD25⁻ T cells, (C) Gene expression levels of nTregs, iTregs, and CD4⁺CD25⁻ T cells prior to transfer, #p<0.05 comparing CD4⁺CD25⁻ T cells to iTregs and nTregs, (D) AHR, (E) BAL fluid inflammatory cell composition, (F) BAL fluid cytokine levels. Shown are the means±SEM from 3 independent experiments (4 mice/group, n=12). *p<0.01 comparing sensitized and challenged mice (given PBS) to mice challenged alone. †p<0.05 comparing sensitized and challenged recipients of iTregs and nTregs to recipients of PBS or CD4⁺CD25⁻ T cells.

Figure 2

Effect of nTreg, iTreg, and CD4⁺CD25⁻ T cell transfer from naive donors into sensitized and challenged CD8^−/− recipients. (A) AHR, (B) BAL fluid inflammatory cell composition, (C) BAL fluid cytokine levels. Shown are the means±SEM from 3 independent experiments (4 mice/group, n=12). *p<0.05 comparing sensitized and challenged mice (given PBS) to mice challenged alone. †p<0.05 comparing sensitized and challenged recipients of iTregs and nTregs to recipients of PBS or CD4⁺CD25⁻ T cells. (D) Gene expression of recovered naive CD4, iTregs, Th17, and nTregs (compared to levels of pre-transferred iTregs set at 1) #p<0.05, (increased), †p<0.05 (decreased), Xp<0.05 comparing iTregs to Th17 cells.

Figure 3

Effect of administrating anti-IL-13 or anti-IL-17 antibody on enhancement of lung allergic responses in sensitized and challenged CD8^−/− recipients of iTregs and nTregs. (A) AHR, (B) BAL fluid inflammatory cell composition, (C) BAL fluid cytokine levels. Shown are the means±SEM from 3 independent experiments (4 mice/group, n=12). *p<0.05 comparing sensitized and challenged mice (given PBS) to mice challenged alone. #p<0.05 comparing recipients of iTregs given control and anti-IL-13 antibody to recipients given anti-IL-17. †p<0.05 comparing recipients of nTregs given control and anti-IL-17 antibody to recipients given anti-IL-13 antibody.

Figure 4

Effect of iTregs from WT, IL-13^−/−, and IL-17^−/− mice on enhancement of lung allergic responses in sensitized and challenged CD8^−/− recipients. (A) AHR, (B) BAL fluid inflammatory cell composition, (C) BAL fluid cytokine levels. Shown are the means±SEM from 3 independent experiments (4 mice/group, n=12). *p<0.05 comparing sensitized and challenged mice (given PBS) to mice challenged alone. #p<0.05 comparing recipients of WT and IL13^−/− iTregs to sensitized and challenged mice (given PBS). †p<0.05 comparing recipients of IL-13^−/− to IL-17^−/− iTregs.

Figure 5

Effect of administrating Th17 cells or IL-17 on lung allergic responses in sensitized and challenged and challenged only CD8^−/− recipients. (A) Cytokine production and (B) FACS analysis of Th17 cells, (C) AHR, (D) BAL fluid cell composition, (E) BAL fluid cytokine levels. Results shown are means±SEM from 3 independent experiments, n=12. *p<0.05 comparing sensitized and challenged mice (given PBS) to challenged alone given PBS, IL-17, or Th17 cells. #p<0.05 comparing sensitized and challenged recipients of IL-17 protein and Th17 cells to recipients of PBS.

Figure 6

Effect of administrating control IgG, anti-IL-6, anti-TGF-β, anti-GITRL, or anti-OX40 antibody on lung allergic responses in sensitized and challenged CD8^−/− recipients of iTregs. (A) Effect of GITRL and OX40L on in vitro cytokine production from iTregs and Th17 cells. (B) AHR, (C) BAL fluid inflammatory cell composition, (D) BAL fluid cytokine levels, (E) Flow cytometry of recovered iTregs. Shown are the means±SEM from 3 independent experiments (4 mice/group, n=12). *p<0.05 comparing sensitized and challenged mice (given PBS) to mice challenged alone. #p<0.05 comparing recipients of iTregs given control and anti-TGF-β, anti-GITRL, and anti-OX40 antibody to recipients given anti-IL-6.