Adoptive transfer of induced-Treg cells effectively attenuates murine airway allergic inflammation

Abstract

Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4(+)FoxP3(+)) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.

Figure 1. In vitro induction of regulatory T (iTreg) cells by TGF-β.

Naive CD4⁺CD25⁻ cells were stimulated with anti-CD3/CD28 coated beads with IL-2 in the presence (CD4_TGF-β) and absence (CD4_med) of TGF-β for 5–6 days. nTreg cells were splenic CD4⁺CD25⁺ cells that were sorted and expanded with anti-CD3/CD28 coated beads with IL-2 for 6–7 days. (A). FoxP3 expression was determined by flow cytometry with anti-Foxp3 antibody. cIgG, control IgG. (B). T cells labeling with CFSE were stimulated with anti-CD3 with or without CD4 condition cells (ratio of CD4 condition to T responder = 1∶2) for three days and CFSE dilution was identified on the CD4⁺ cell gate. (C). T cell proliferation was determined by ³H-thymidine incorporation assay. (D). The T cell proliferation was determined in the different ratios of CD4 conditioned cells and T responder cells. Data was representative or mean ± SEM of three independent experiments.

Figure 2. Attenuated allergic inflammation in lung tissues by adoptive transferring of iTreg cells prior to OVA challenge.

(A) Lung tissue sections from the mice with indicated treatments were stained with H&E. (B) Overall lung inflammation were graded with scores 0 to 15 (none to severe inflammation, see Materials and Methods for details). (C) Eosinophil, detected by Discombe's staining (red intracellular granules), was the major type of cells that were infiltrated in small airways and adjacent vasculature. (D) Total proteins in BAL fluids from mice with different treatments were quantified. (E) IgE level in serum from different groups of mice was quantified by an ELISA. *P<0.05, **P<0.01, n = 5.

Figure 3. Abnormal airway wall remodeling and AHR were subsided with iTreg cell treatment.

(A) Excessive mucin expression in small airway epithelial cells was detected by PAS staining (red color). (B) The numbers of airways with PAS-positive epithelial cells per lung tissue section were quantified in different experimental groups (n = 5). (C) Clara cells in small airway epithelia were stained by CCSP immunofluorescence staining (green) and the surrounding airway smooth muscle cells were immunostained by SMA (red). (D) Airway resistance was measured upon Mch (40mg/ml) aerosol delivery. Although the airway resistance was still significantly higher in iTreg cell-treated group than that in normal control group, significant reduction of airway resistance was achieved in iTreg cell-treated group compared to non-treated (OVA) or control T cell-treated group. *P<0.05, **P<0.01.

Figure 4. Comparison of infused CFSE-labeled iTreg cells in the absence and presence of OVA-induced allergic inflammation.

No significant difference was detected in mediastic draining lymph nodes and lung tissues for infused iTreg cells between normal control mice and mice that had OVA-induced allergic inflammation.

Figure 5. Increased total CD4⁺FoxP3⁺ Treg cells in the spleen and draining lymph nodes of the mice that received exogeneous iTreg cells.

In contrast, the frequencies of CD4⁺FoxP3⁺ Treg cells did not show significant changes between normal mice and OVA mice, as well as control T cell-treated mice. The experiments were repeated with consistent results.

Figure 6. Adoptive transfer of iTreg cells significantly diminished Th1/Th2/Th17 cell frequencies in draining lymph nodes in asthmatic mice.

Intracellular expression of IFN-γ, IL-5, and IL-17A in CD3⁺ T cells were determined by FACS. (A) A representative of 9 mice in each group. Cells were gated on CD3⁺ cells. (B) Results were mean ± SEM of values of 9 mice in each group. *P<0.05, **P<0.01, ***P<0.001. (C) Significant reductions of T cell differentiation markers, including T-bet1 and Gata-3, but not RORγT, were detected in iTreg-treated OVA mice compared to iTreg-untreated OVA control.

Figure 7. Adoptive transfer of iTreg cells altered cytokine production.

(A) iTreg cells inhibited OVA-induced increase of Th2 cytokines. IL-5 and IL-13 in mouse serum were quantified by specific ELISA. Significant reduction of IL-5 and IL-13 in the group receiving iTreg treatment was detected compared to OVA challenge only control group. **P<0.01. (B) Expression of IRF-4 and IL-10 at the mRNA level of lung lymphocytes was significantly increased in iTreg-treated OVA mice compared to untreated OVA mice. *P<0.05.

Figure 8. Alteration of dentritic cells and related cytokine IL-23.

Significant reduction of both CD11c⁺CD86⁺ and CD11c⁺CD80⁺ subsets of DCs in mediastinal lymph nodes were detected in iTreg-treated OVA mice compared to untreated OVA mice. (B) IL-23 expression at the mRNA level in draining lymph nodes was also significantly reduced by iTreg treatment compared to untreated OVA mice (Fig. 8B).

Figure 9. Adoptive transfer of Treg cells after first OVA challenge still effectively suppressed airway inflammation and AHR, as well as Th1/Th2 cell frequencies.

(A) Histopathological changes of lungs from the mice that received no cell, iTreg, nTreg, or control T cells after first OVA challenge (day 25). Lung specimens were taken on day 28 after another two daily OVA challenges on day 26 and 27. (B) Accumulated inflammatory scores of peri-vascular, peri-bronchiolar and alveolar regions of different groups. (C) Airway resistance was measured to evaluate lung functional changes among different groups. (D) Altered frequencies of Treg, Th1, and Th2 subsets in the spleens of the mice receiving different treatments. *P<0.05, **P<0.01.