Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition - PubMed (original) (raw)

Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition

Brian M Weist et al. Nat Immunol. 2015 Jun.

Abstract

The thymic production of regulatory T cells (Treg cells) requires interleukin 2 (IL-2) and agonist T cell antigen receptor (TCR) ligands and is controlled by competition for a limited developmental niche, but the thymic sources of IL-2 and the factors that limit access to the niche are poorly understood. Here we found that IL-2 produced by antigen-bearing dendritic cells (DCs) had a key role in Treg cell development and that existing Treg cells limited new development of Treg cells by competing for IL-2. Our data suggest that antigen-presenting cells (APCs) that can provide both IL-2 and a TCR ligand constitute the thymic niche and that competition by existing Treg cells for a limited supply of IL-2 provides negative feedback for new production of Treg cells.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1

Limited Treg cell development in thymic slices regardless of the route of antigen delivery. Thymocytes from OT-II TCR transgenic Rag2 −/− mice (OT-II) were cultured on thymic slices in the presence or absence of the cognate antigen ovalbumin for 3 days prior to flow cytometric analysis. (a-c) Left hand panels show representative flow cytometry plots gated on OT-II donor CD4SP thymocytes and right panels shows total number of OT-II Treg cells (CD25+Foxp3+) recovered per slice. (a) Wild type mice were injected i.v. with 0.2 or 2 mg of ovalbumin protein 1h prior to sacrifice, preparation of thymic slices, and addition of OT-II thymocytes. n=12,10, and 13 slices pooled from 3 experiments. (b) Thymic slices were prepared from mice expressing a RIPmOVA transgene. n=3, 13 slices. (c) Bone marrow derived DCs loaded with the indicated concentrations of OVA protein were added to thymic slices after addition of OT-II thymocytes. n=5, 6, 6, and 6 slices respectively. (d) Treg cell development efficiency shown as a ratio of number of OT-II FoxP3+ CD4 single positive thymocytes recovered per slice relative to the number of resident CD4 single positive thymocytes within the slice for various routes and concentration of antigen. Each dot represents a value from an individual slice compiled from multiple experiments: PBS (3 exp.); 0.2mg (2 exp.); 2mg (2 exp.); mOVA (3 exp.); none (1 exp.); 0.01mg/ml (2 exp.); 0.1mg/ml (2 exp.); 1mg/ml (2 exp.); resident Treg cells (4 exp.). Error bars represent +/− SEM. *p<0.01 and **p<0.001 by unpaired student’s _t_-test (b), or one-way ANOVA using Tukey’s post test analysis (a,c). Panels b-c show representative data from 3 (b) or 5 (c) independent experiments.

Figure 2

Existing thymic Treg cells reduce the capacity of the Treg developmental niche. (a,b) OT-IIx_Rag2_−/− (OT-II) thymocytes overlaid onto thymic slices from WT or AND TCRx_Rag2_−/− (AND) mice in the presence of 1μM OVA peptide. Treg cell development measured by flow cytometric analysis at d3. (a) Representative flow cytometry plots of CD25 and Foxp3 expression on gated OT-II donor CD4SP thymocytes. (b) Number of OT-II Treg cells (CD25+Foxp3+) recovered per thymic slice, n=3,3,6,7 slices. (c-d) Thymic Treg cells (1x105) sorted from Foxp3-GFP mice were added to thymic slices from WT or AND mice that were injected with 2mg OVA protein. OT-II Treg cell development measured at d3. (c) Representative flow cytometry plots gated on OT-II donor CD4SP thymocytes. (d) Number of OT-II Treg cells (CD25+Foxp3+) recovered per thymic slice, n=7 slices. Data representative of 3 (a, b) and 5 (c, d) independent experiments. Error bars represent +/− SEM. *p<0.01,**p<0.001, ***p< 0.0001 by one-way ANOVA using Tukey’s post test analysis (b) or unpaired student’s t-test (d).

Figure 3

Existing thymic Treg cells do not prevent TCR signaling within Treg precursors. (a-c) OT-II thymocytes cultured in the presence of 1μm OVA peptide for 6h within WT or AND TCR (Treg deficient) thymic slices and analyzed by flow cytometry. (a) Representative flow cytometry plots showing gated OT-II CD4SP thymocytes. (b) Percent of CD25+ Nur77+ OT-II thymocytes. (c) Nur77 MFI of OT-II single positive thymocytes. (d) Analysis of OT-II Treg cell development 3d after culture on WT or –Treg thymic slices in the presence of 1μm OVA peptide. (e) Comparison of Nur77 levels between the indicated CD4SP populations at d3. Error bars represent +/− SEM. All figures representative of 3 independent experiments. n=6 slices per condition. *p<0.001 by one-way ANOVA using Tukey’s post test analysis (b,c).

Figure 4

IL2 availability from the thymic environment influences the Treg niche. WT and _Il2_−/− mice were injected with 2mg of OVA protein 1h prior to sacrifice and preparation of thymic slices. OT-II thymocytes were added to thymic slices and were treated with IL2 complexes daily. OT-II Treg cell development was assessed by flow cytometry at d3. (a) Representative flow cytometry profiles. (b) Number of OT-II Treg cells (CD25+Foxp3+) recovered per thymic slice. (c) Endogenous Treg cell population expressed as the ratio relative to slice resident CD4 single positive thymocytes. Error bars represent +/− SEM. Data representative of 5 independent experiments, n=6,7,6, and 7 slices respectively. *p<0.05 and **p<0.001 by one way ANOVA using Tukey’s post test analysis.

Figure 5

Existing Treg cells limit available IL-2/IL-15 within the thymus. (a,b) The IL-2/IL-15-dependent CTLL2 cell line was labeled with eFluor450 and cultured on either WT or Treg deficient (AND TCR) thymic slices. (a) Proliferation assessed at d4. Wild type thymocytes labeled with eFluor450 were included as a reference population to provide an indication of the level of fluorescence in a non-dividing population. (b) Survival relative to reference wild type thymocyte population following 2d of culture, n=6 slices. (c,d) OT-II Treg cell development on WT or Treg deficient slices in the presence or absence of added IL-2 complexes. Antigen was introduced by i.v. injection of mice with 2mg of OVA protein. (c) Representative flow cytometry plots. (d) Number of OT-II Treg cells (CD25+Foxp3+) recovered per slice, n=9, 11,15, and 11 slices respectively. Error bars represent +/− SEM. Data representative (a,b) or pooled (c,d) from 3 independent experiments, *p<0.05, and **p<0.001 by student’s _t_-test (b) or one-way ANOVA using Tukey’s post test analysis (d).

Figure 6

Antigen-bearing dendritic cells provide a local source of IL-2 to developing thymic Treg cells. (a-d) Impact of IL2 mutation on ability of DCs to support Treg cell development. Bone marrow derived DCs from WT or _Il2_−/− mice were incubated with 1 mg/ml OVA protein and added to WT thymic slices along with OT-II thymocytes. OT-II Treg cell development was assessed after 3 days of culture. (a) Representative flow cytometry analysis of gated OT-II CD4SP. (b) Number of OT-II Treg cells (CD25+Foxp3+) recovered per thymic slice. (c) Ratio of OT-II Treg cells to slice CD4 single positive thymocytes. (d) Quantification of endogenous slice Treg cells recovered per slice. (a-d) n=20 and 21 slices respectively, data pooled from 3 independent experiments. (e) qRT-PCR analysis of IL-2 expression. RNA was prepared from the indicated samples and analyzed by qRT-PCR using TaqMan probes. Data are normalized to GAPDH and presented as fold increase over the background values from _Il2_−/− bone marrow derived DCs. Thymi from wild type mice were dissociated with collagenase-digestion and separated into single cell suspension “whole thymus” and an adherent fraction “stroma:. For some samples, single cell suspensions were depleted of CD11c+ cells using magnetic beads: “CD11c depleted”. CD11c enriched fractions were further purified by FACS to yield > 85% CD11c+ cells: “thymic DC”. Bone marrow derived DCs from wild type or _Il2_−/− mice were included for comparison. For _Il2_−/− DCs, no IL2 signal was observed after 40 cycles of PCR, therefore values reported are upper estimates (indicated by grey shading), and the dashed line indicates the lower limit of detection of the assay. Data from thymic DC samples are included on both plots for comparison. n=12 experimental replicates from 4 biological samples except “CD11c depleted” n=9 experimental replicates, 3 biological samples or “stroma” n=3 experimental replicates, 1 biological sample. Error bars represent +/− SEM. *p<0.05, **p<0.001, and n.s. = not significant by unpaired student’s _t_-test or one-way ANOVA using Tukey’s post test analysis.

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Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition - PubMed (original) (raw)