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Figure S1 Flow cytometry staining of lymphocytes in LN and the spleen. mLN, pLN and the spleen were removed from mice, and flow cytometry was performed after OT or ST induction. Cell suspensions were prepared, and lymphocytes were identified in FSC and SSC. Cells were stained for CD3, CD4, CD19 and IgG. Furthermore, CD4, CD25 and FoxP3 positive cells were measured. CD4+ CD25+ cells were gated, and FoxP3+ cells were calculated. Isotype control and FoxP3 staining are shown in the dot plots.

Figure S2 Germinal centres are detectable in the spleen of skin tolerized mice. Immunofluorescent staining for B220 (B cells in green; A and D) and GL-7, used to identify germinal centres (red; B and E), was performed in the spleen of mice from peripherally or orally tolerized animals. The merging shows that in the spleen of peripherally tolerized mice, germinal centres are seen, whereas in orally tolerized mice, only marginal GL-7 expression is found.

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