Fig. 3: Efficient central CD8⁺ T cell tolerance to a model inflammation self-antigen.

a, GFP expression by thymic nonstromal populations in IL-4–GFP mice. Frequency of GFP⁺ cells (n = 5 mice) and pie chart showing frequencies of different subsets within the GFP⁺ compartment (average percentage ± s.d.; n = 3 mice). See also Extended Data Fig. 2a–c. b, Images of IL-4–GFP thymus sections as analyzed by confocal immunofluorescence microscopy. The medulla was identified by staining with UEA1 lectin (blue). IL-4–GFP⁺ eosinophils were distinguished from IL-4–GFP⁺ lymphocytes (green) by co-staining with anti-Siglec-F (red). See also Extended Data Fig. 2d. c, WT and IL-4–GFP mice (on a BALB/c (H2^d) background; CD45.2) were lethally irradiated and reconstituted with syngeneic WT or IL-4–GFP BM cells mixed with BM cells from Jedi mice (B10.D2 genetic background (H2^d); CD45.1). Thymi of the chimeras were analyzed 6 weeks after reconstitution (n = 4 chimeras for analysis of thymi in WT + Jedi-TCRαβ → WT; n = 6 for the other groups). Jedi thymocytes were gated as CD45.1⁺tetramer⁺ cells. Expression of CD4 and CD8β on CD45.1⁺tetramer⁺ cells and viability dye and PD-1 staining of CD45.1⁺tetramer⁺CD4⁺CD8β⁺ (Jedi DP) cells are shown. The frequencies of CD4⁺CD8β⁺ (DP) and CD4^–CD8β⁺ (CD8SP) cells of CD45.1⁺tetramer⁺ Jedi thymocytes and the frequencies of dead Jedi DP cells were quantified (bottom). Data are representative of three independent experiments; Tet, tetramer. d, Analysis of the frequency of cleaved caspase-3⁺ cells among tetramer⁺CD4⁺CD8β⁺ (Jedi DP) cells in WT + Jedi-TCRαβ → WT and IL-4–GFP + Jedi-TCRαβ → WT mixed BM chimeras (from the experiment shown in Supplementary Fig. 2). Data are from one experiment with four chimeras per group; Casp3, caspase-3. e, Representative flow cytometric analysis of splenocytes from four groups of BM chimeras (same experiment as in c). Jedi cells were identified by H2-K^d GFP_200–208 tetramer staining, and CD8α, CD8β and CD4 expression on these cells was analyzed. The frequencies of total and CD8α^hiCD8β^hi tetramer-binding cells among splenocytes were quantified (bottom). Data are presented as mean ± s.d. (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) and were analyzed by two-tailed Student’s t-test (d) or one-way ANOVA with a Holm–Sidak multiple comparisons test (c and e).

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