Nature Immunology: doi: /ni Supplementary Figure 1. Time course of OT-II T reg cell development in thymic slices.

Transcription

1 Supplementary Figure 1 Time course of OT-II T reg cell development in thymic slices. Time course of T reg cell development following addition of OT-II TCR transgenic Rag2 -/- mice (OT-II) thymocytes to thymic slices from mice that were previously injected with 2mg OVA protein. Data are representative of 4 independent experiments, gated on OT-II CD4SP.

2 Supplementary Figure 2 T reg cell development in thymic slices recapitulates normal thymic development rather than in vitro induced T reg cell development. (a) OT-II thymocytes or mature OT-II T cells were added to thymic slices from wild type mice that were injected with OVA protein 1hr prior to sacrifice. TGF-β (5ng/ml) was added along with mature OT-II T cells in some samples as indicated. (b) Neuropilin-1 expression levels on gated OT-II Foxp3+ CD4SP from thymocytes, splenocytes, or Foxp3 negative thymic CD4SP. (c) OT-II thymocytes were depleted of CD4SP prior to introduction into thymic slices. Flow cytometric analysis of samples before and after depletion are shown. (d) OT-II CD4SP depleted thymocytes overlaid onto WT thymic slices along with 1μM OVA peptide. Flow cytometry of gated OT-II CD4SP after days 3 or 4 of culture are shown. Data representative of 2 (a,b) or 3 (c,d) independent experiments.

3 Supplementary Figure 3 Relationship between the frequency of precursors of T reg cells and T reg cell development. The % T reg cells (CD25+Foxp3+) amongst OT-II donor thymocytes plotted versus the % of donor OT-II thymocytes within a thymic slice. Each dot represents the values for an individual thymic slice and data are compiled from 3-4 independent experiments. Ovalbumin was introduced via i.v. injection of mice prior to sacrifice and preparation of thymic slices (a), use of thymic slices from mice expressing a RIPmOVA transgene (b), or addition of OVA loaded bone marrow derived DCs to thymic slices containing OT-II thymocytes (c). Data compiled from multiple experiments, (a) n=15 (4 exp.); (b) n=16 (3 exp.); (c) n=19 (4 exp.).

4 Supplementary Figure 4 T reg cell development is enhanced in the absence of existing thymic T reg cells. (a) Slices from WT or AND (T reg deficient) thymi, overlaid with 2D2 TCR transgenic thymocytes in the presence of 3.8 M MOG peptide. T reg cell development was analyzed on day 3 by flow cytometry. (b) Number of 2D2 T reg cells recovered per thymic slice after 3d. *p<0.01 by student s t-test, n=6 and 3 slices respectively, representative of 2 independent experiments. (c) Slices from WT or F5 TCR transgenic Rag2-/-(T reg deficient) thymi, overlaid with OT-II thymocytes in the presence of the indicated concentrations of OVA peptide. T reg cell development analyzed on day 3 by flow cytometry. (d) Number of OT-II T reg cells recovered per thymic slice after 3d. *p<0.01 and **p<0.001 one-way ANOVA using Tukey s post test analysis. Data pooled from 3 independent experiments, n=9 slices each.

5 Supplementary Figure 5 DCs derived from IL-2-deficient bone marrow have normal antigen-presentation capacity. DCs were generated in culture from WT and Il2 / bone marrow, incubated with 1mg/ml OVA protein, and introduced into thymic slices along with OT-II thymocytes. Flow cytometric analysis of activation markers on OT-II thymocytes was performed after 16h of culture. (a) Representative flow cytometry plots and (b) expression levels of activation markers on gated CD4SP OT-II thymocytes. Error bars represent +/- SEM, n=6 slices per condition, ns= not significant by unpaired student s t-test, data representative of 3 independent experiments..

6 Supplementary Figure 6 Localized IL-2 production by the DC population presenting antigen. Il2 / thymic slices were overlaid with OT-II thymocytes followed by the addition of WT or Il2 / DCs loaded with 1mg/ml OVA protein. OT-II T reg cell development was measured on d3. (a) Representative flow cytometry plots. (b) Quantification of the number of OT-II T reg cells recovered per thymic slice. *p<0.001 by student s t-test. Data representative of 3 independent experiments, n=8 slices per condition. (c) T reg precursors (orange) interact with niche-defining APCs that bear agonist self-peptide (orange rectangles) and also supply IL2 (black dots). As precursors upregulate CD25 and differentiate into T reg cells (blue) they limit IL2 availability from the APC, providing negative feedback and preventing future T reg cell development within the niche. Shaded stars indicate cells in the surrounding tissue that are not part of the niche.