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. 2011;6(8):e23618.
doi: 10.1371/journal.pone.0023618. Epub 2011 Aug 23.

In vivo induction of Tr1 cells via mucosal dendritic cells and AHR signaling

Henry Yim Wu  1 , Francisco J Quintana, Andre Pires da Cunha, Benjamin T Dake, Thomas Koeglsperger, Sarah C Starossom, Howard L Weiner
Affiliations

In vivo induction of Tr1 cells via mucosal dendritic cells and AHR signaling

Henry Yim Wu et al. PLoS One. 2011.

Abstract

Background: Type 1 regulatory T (Tr1) cells, characterized by the secretion of high levels of the anti-inflammatory cytokine interleukin-10 (IL-10), play an important role in the regulation of autoimmune diseases and transplantation. However, effective strategies that specifically induce Tr1 cells in vivo are limited. Furthermore, the pathways controlling the induction of these cells in vivo are not well understood.

Methodology/principal findings: Here we report that nasal administration of anti-CD3 antibody induces suppressive Tr1 cells in mice. The in vivo induction of Tr1 cells by nasal anti-CD3 is dependent on IL-27 produced by upper airway resident dendritic cells (DCs), and is controlled by the transcription factors aryl hydrocarbon receptor (AHR) and c-Maf. Subsequently, IL-21 acts in an autocrine fashion to expand and maintain the Tr1 cells induced in vivo by nasally administered anti-CD3.

Conclusions/significance: Our findings identify a unique approach to generate Tr1 cells in vivo and provide insights into the mechanisms by which these cells are induced.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Nasal anti-CD3 induces suppressive Tr1 cells.
A. Tiger mice were nasally treated with IC (clear bars) or anti-CD3 (filled bars) and 72 hrs after the last nasal dose GFP(IL-10) expression by CD4+ T cells in CLN was examined by flow cytometry. This experiment was repeated 4 times with same results. B and C. CD4+CD25-GFP-, CD4+CD25+GFP- or CD4+CD25-GFP+ T cells were sorted from CLN of Tiger mice nasally treated with IC (clear bar) or anti-CD3 (filled bar). Sorted T cells were stimulated in vitro with plate bound anti-CD3 and anti-CD28 antibodies (1 µg/ml each) and IL-10 (B) and (C) IFN-γ were detected in the supernatants by ELISA. Error bars represent standard deviations and P values were calculated by t-test. D. The percentage of CD4+CD25-LAP+ T cells that express IL-10 following nasal anti-CD3 was assessed by intracellular staining. Each symbol represents an individual mouse. E. FACS-sorted Tr1 cells (CD4+CD25-GFP(IL-10)+, clear bar) from CLN of nasal anti-CD3 treated Tiger mice or IL-27 in vitro differentiated Tr1 cells (filled bar) were used in a standard suppression assay with naïve CD4+CD25-GFP- responder T cells at various ratios. To test the role of IL-10 in in vitro suppression, IC or anti-IL-10 (50 µg/ml) neutralizing antibodies were added to co-cultures at 1∶1 ratio.
Figure 2
Figure 2. In vivo induction of Tr1 cells is dependent on mucosal DC-derived IL-27 and TGF-β.
A. CD11c+ DCs or B. CD11b+ macrophages were positively selected from CLNs following nasal IC (clear bars) or anti-CD3 (filled bars) and used in quantitative RTPCR. This experiment was repeated 3 times with same results. Error bars represent standard deviations and P values were calculated by t-test. C. CD11c DTR-GFP mice were nasally treated with PBS or 500 ng of DT. CLNs and spleens were harvested 24 hrs following nasal DT. 10 µM frozen sections were stained with Toprol-3. Pictures were taken at ×40 magnification. D. CD11c DTR-GFP mice were nasally treated with anti-CD3 or DT followed anti-CD3. 72 hrs following nasal treatment, CLN cells were stained with anti-CD4, anti-CD25 and anti-LAP antibodies for FACS. LAP staining on gated CD4+CD25- T cells is shown. E. B6 mice were nasally treated with anti-CD3. CD11b+ macrophages (left column) or CD11c+ DCs (right column) were positively selected from CLN at 72 hrs after the last nasal dose. Co-cultures of macrophages or DCs and CD4+CD25-GFP- naïve Tiger T cells were stimulated with LPS (1 µg/ml) or FLT3 ligand (1 µg/ml) respectively and plate bound anti-CD3 (1 µg/ml) for 96 hrs. Cells were stained with anti-LAP antibody. FACs plots shown here were on gated CD4+ lymphocytes. Representative FACs plots of 3 independent experiments are shown here. F. B6 mice were nasally treated with anti-CD3. CD11c+ DCs were positively selected from CLN 72 hrs after the last nasal dose. Co-cultures of DCs and CD4+CD25-GFP- naïve Tiger T cells were stimulated with 1 µg of FLT3 ligand and plate bound anti-CD3 in the presence of 10 µg/ml neutralizing antibody to TGF-β and/or IL-27 for 96 hrs. Cells were stained with anti-LAP antibody. Representative FACs plots of 3 independent experiments are shown here.
Figure 3
Figure 3. Tr1 cells induced by nasal anti-CD3 express ahr, cmaf, il21 and il21r.
A. Naive CD4+ T cells (CD4+CD25-GFP(IL-10)-), nTregs (CD4+CD25+GFP(foxp3)+) sorted from Foxp3-GFP knock-in mice, ex vivo Tr1 cells (CD4+CD25-GFP(IL10)+) sorted from CLN of nasal anti-CD3 treated Tiger mice and in vitro differentiated Tr1 using plate bound anti-CD3 and anti-CD28 plus 50 ng/ml IL-27 were used in quantitative RTPCR reactions. Expressions of IL-10, IFN-γ and foxp3 mRNA were normalized to expression of β-actin. B. AHR, C. cMAF, D. IL-21 and E. IL-21R mRNA expression by CD4+GFP(IL-10)- T cells or CD4+GFP(IL-10)+ Tr1 cells. These experiments were repeated 3 times with same results.
Figure 4
Figure 4. The induction of Tr1 cells by nasal anti-CD3 requires AHR and IL-21 signaling.
A. Tiger or Ahrd/tiger mice were nasally treated with IC or anti-CD3 and 72 hrs after the last nasal dose GFP(IL-10) expression by CD4+ T cells in CLN was examined by flow cytometry. Each symbol represents an individual mouse. B. Tiger mice were nasally treated with IC or anti-CD3 alone or together with recombinant mouse IL-21 (4 µg/day). C. WT or IL-21R−/− mice were nasally treated with IC or anti-CD3.
Figure 5
Figure 5. Tr1 cells induced by nasal anti-CD3 in an IL-27-dependent manner control systemic autoimmunity.
A. 8 wks old female MRL/lpr or IL-27R−/−/lpr mice were nasally treated with 0.5 µg IC or anti-CD3. CLN cells were harvested at 72 hrs after the last nasal dose and stained with anti-CD4 and anti-LAP antibodies. B. CD4+ T cells were isolated by positive selection and stimulated with plate bound anti-CD3 and anti-CD28 antibodies (1 µg/ml each) for 96 hrs. Culture supernatant was used in detection of IL-2, IL-5, IL10, IL-12 and IFN-γ by ELISA. C. 4-wk old MRL/lpr or IL-27R−/−/lpr mice (n = 10) received three 5-day courses of 0.5 µg IC or anti-CD3 given at alternative weeks. Survival of mice following treatment was followed for 40wks. D. Serum was collected from MRL/lpr, IL-27R−/+/lpr, IL-27R−/−/lpr or IL-27R−/−/lpr recipients of CD4+LAP- or CD4+ T cells. IgG anti-dsDNA autoantibodies were detected by ELISA. E. 8 wk old female MRL/lpr mice were nasally treated with anti-CD3 for 5 consecutive days. 72 hrs after the last nasal dose CD4+ (red line) or CD4+LAP- (yellow line) T cells were sorted from CLN cells and adoptively transferred (1×106 cells/mouse) to 4-wk old IL-27R−/−/lpr recipients (n = 10). Survival of recipients was followed for 40wks and compared to MRL/lpr (black line), IL-27R−/+/lpr (blue line) or IL-27R−/−/lpr (green line) mice (n = 10) without cell transfer. F. CD4+ T cells from IL-27R−/−/lpr recipients of CD4+LAP- (clear bars) or CD4+ (filled bars) T cells were stimulated with plate bound anti-CD3 and anti-CD28 antibodies for 96 hrs. IL-12 and IFN-γ in culture supernatant was detected by ELISA.
Figure 6
Figure 6. A model for Tr1 cell generation by nasal anti-CD3.
Nasal anti-CD3 triggers T cell activation via TCR/CD3 complex. T cell activation in the presence of IL-27 secreted by local DCs in the CLN leads to activation of AHR and cMAF, which cooperate to transactivate the il10 and il21 promoters and promote IL-10 and IL-21 production. IL-21 then acts as a Tr1 growth factor in an autocrine fashion.
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