Inducible CD4+LAP+Foxp3- regulatory T cells suppress allergic inflammation

Abstract

Regulatory T cells (Tregs) play a critical role in the maintenance of airway tolerance. We report that inhaled soluble Ag induces adaptive Foxp3(+) Tregs, as well as a regulatory population of CD4(+) T cells in the lungs and lung-draining lymph nodes that express latency-associated peptide (LAP) on their cell surface but do not express Foxp3. Blocking the cytokine IL-10 or TGF-β prevented the generation of LAP(+) Tregs and Foxp3(+) Tregs in vivo, and the LAP(+) Tregs could also be generated concomitantly with Foxp3(+) Tregs in vitro by culturing naive CD4(+) T cells with Ag and exogenous TGF-β. The LAP(+) Tregs strongly suppressed naive CD4(+) T cell proliferation, and transfer of sorted OVA-specific LAP(+) Tregs in vivo inhibited allergic eosinophilia and Th2 cytokine expression in the lung, either when present at the time of Th2 sensitization or when injected after Th2 cells were formed. Furthermore, inflammatory innate stimuli from house dust mite extract, nucleotide-binding oligomerization domain containing 2 ligand, and LPS, which are sufficient for blocking airway tolerance, strongly decreased the induction of LAP(+) Tregs. Taken together, we concluded that inducible Ag-specific LAP(+) Tregs can suppress asthmatic lung inflammation and constitute a mediator of airway tolerance together with Foxp3(+) Tregs.

Figure 1. Inhalation of soluble antigen induces LAP+Foxp3− CD4+ T cells

Naive CD4+CD25− T cells isolated from Thy1.2 OT-II TCR transgenic Foxp3/GFP reporter mice were transferred into Thy1.1 recipient mice. Recipient mice were then tolerized by exposure to soluble OVA (100 μg) in PBS given i.n. for 3 consecutive days. Control (non-tolerized) mice were exposed to PBS without OVA. (a) Representative flow dot plot of LAP and Foxp3 (GFP) expression on gated Thy1.2+ OT-II CD4+ T cells in lung-draining LN from an individual mouse at day 5 after the initial exposure to OVA. (b) Representative flow dot plot of LAP and Foxp3 (GFP) co-staining (bottom), isotype IgG (top) on gated Thy1.2+ OT-II CD4+ T cells in pooled lung-draining LN from an individual mouse at day 5 after the initial exposure to OVA. (c) Total numbers of LAP+Foxp3− and Foxp3+LAP− OT-II CD4+ T cells in lung draining LN populations on different days after the initial exposure to OVA. Data are mean numbers ± SEM from 4 individual mice per group. Data are representative of 3 independent experiments.

Figure 2. TGF-β and IL-10 are required for induction of LAP+ T cells in vivo

Thy1.2 OT-II LAP+ and Foxp3+ T cells were tracked in vivo as described in Fig. 1. A single dose of anti-IL-10R (200 μg), anti-TGF-β (200 μg), or control IgG, was given i.p. at the time of initial exposure to i.n. OVA. (a) Representative flow cytometry dot plots of LAP and Foxp3 (GFP) expression on gated Thy1.2+ OT-II CD4+ T cells in lung-draining LN from individual mice. (b) Numbers of LAP+Foxp3− and Foxp3+LAP− OT-II CD4+ T cells, and total OT-II CD4+ cells, were calculated in lung draining lymph nodes on day 5. All results are means ± SEM from 4 individual mice per group. Data are representative of 3 independent experiments. *P < 0.05.

Figure 3. LAP+Foxp3− T cells inhibit naïve CD4+ T cell proliferation in vitro

Naïve CD4+ T cells from OT-II TCR transgenic GFP/Foxp3 reporter mice were cultured with T-depleted splenocytes and OVA peptide (1 μM) in the presence of exogenous TGF-β (10 ng/ml). Cells were analyzed and sorted on day 4. (a) Top: Foxp3 versus LAP staining on CD4 T cells before and after culture. Bottom: Purity of the sorted LAP+Foxp3− and LAP−Foxp3+ CD4+ T cell populations. (b) Foxp3 mRNA levels in sorted LAP+Foxp3− and LAP−Foxp3+ populations, analyzed using real-time PCR. Data are normalized to the housekeeping gene GAPDH. (c–d) FACS sorted LAP+Foxp3− and LAP−Foxp3+ OT-II T cells (1 × 10⁵) were cultured, alone or at varying ratios with naïve CD4+CD25− OT-II T cells, plus irradiated (3000 rads) syngeneic splenocytes in the presence of 0.5 μM OVA peptide. (c) Proliferation was assessed after a pulse with [³H] thymidine for the last 16 h of a 72-h incubation period. Data are presented as means ± SEM. (d) Responder naïve T cells were labeled with PKH26 dye and division was assessed by loss of the dye after 3 days. Data represent one out of three independent experiments. *P < 0.05.

Figure 4. Phenotype of TGF-β-induced LAP+Foxp3− T cells

LAP+Foxp3− and LAP−Foxp3+ OT-II T cells were generated as described in Fig. 3. (a) Representative expression of CD25, GITR, CD103, Granzyme B and CTLA-4 (black line), with isotype controls (gray shade), at day 4 on gated LAP+Foxp3− and LAP−Foxp3+ CD4+ T cells. (b) Cytokine secretion profiles of TGF-β-induced sorted LAP+Foxp3−, LAP−Foxp3+, and LAP−Foxp3− T cells. Positive control populations (Effector cells) were from cultures of OT-II splenocytes stimulated with OVA peptide in non-skewing conditions for 4 days. T cells were restimulated with PMA (50 ng/ml) and Ionomycin (1 μg/ml) for 24h. IL-2, IL-4 and IFNγ levels were measured using mouse cytokine multiplex kits. Similar data were obtained in 3 independent experiments. *P < 0.05.

Figure 5. LAP+ T cells prevent induction of asthmatic lung inflammation in vivo

LAP+Foxp3− or Foxp3+LAP− OT-II T cells, generated in vitro as described in Fig. 3, were sorted and transferred into naïve BL/6 recipient mice. One day later, the mice were then sensitized with OVA (20 μg) in Alum (4 mg) and subsequently challenged with OVA aerosol to induce lung inflammation. (a) Protocol timeline. (b) Eosinophils in BAL. (c) Airway hyperresponsiveness to Methacholine, assessed using a FlexiVent. (d) Cytokines in BAL by ELISA. (e) Representative H&E staining of lung sections. Data are means ± SEM from 3 mice per group. Similar data were generated from 3 independent experiments. *P < 0.05.

Figure 6. LAP+ T cells do not convert to Foxp3+ T cells in vivo

Sorted Thy1.2+LAP+Foxp3− T cells, generated in vitro as described in Figs. 3 and 5, were transferred into Thy1.1 recipient mice. One day later, the recipient mice were immunized with OVA (20 μg) and alum (4 mg). (a) Protocol timeline. (b) The expression of surface LAP and intracellular Foxp3 on gated Thy1.2+ T cells was analyzed in pooled LN and spleen on day 6 after the immunization. Representative dot plot of T cells before and 6 days after the transfer. Isotype control staining for LAP shown in bottom plot. (c) Total numbers of recovered Thy1.2+ T cells in pooled lymph nodes and spleens on day 2 and day 6, compared to recovered Thy1.2+ Foxp3+ T cells. All results are means ± SEM from 4 individual mice per group. Data are representative of 3 independent experiments. *P < 0.05.

Figure 7. LAP+ T cells suppress asthmatic lung inflammation in sensitized mice

FACS sorted LAP+Foxp3− or Foxp3+LAP− OT-II T cells, generated as described in Fig. 3, were transferred into BL/6 mice that had been sensitized with OVA and Alum for 7 days. The recipients were then challenged with OVA aerosol to induce lung inflammation starting one day later. (a) Protocol timeline. (b) Eosinophils in BAL. (c) Cytokines in BAL by ELISA. (d) Representative H&E staining of lung sections. Data are means ± SEM from 3 mice per group. Data are representative of 3 independent experiments. *P < 0.05.

Figure 8. Allergens and pro-inflammatory microbial-associated molecular patterns suppress the generation of LAP+ Treg cells

The generation of LAP+Foxp3− and Foxp3+LAP− OT-II T cells was tracked in vivo as described in Fig. 1. HDM extract, MDP, or LPS, were given i.n. concurrently with soluble OVA. (a–f) Numbers of LAP+Foxp3−, LAP−Foxp3+, and total OT-II CD4+ T cells in lung draining lymph nodes (a–c) and lung tissue (d–f). Data are means ± SEM from 3 mice per group. Data are representative of 3 independent experiments. *P < 0.05.