Fig. 6: Innate-like T cells can mediate elimination of autoreactive CD8⁺ thymocytes through direct T cell-to-T cell antigen presentation.

a, RTOCs with sorted Jedi-TCRαβ DP thymocytes and iNKT cells were established and analyzed as in Fig. 4b but using iNKT cells sorted from WT mice (H2^b/d background) or IL-4–GFP mice on H2^b/d and H2^b/b backgrounds. Results from one experiment (n = 2 RTOCs per group) are shown. b, CB6F1 WT recipient mice (H2^b/d) were irradiated and transferred intravenously (i.v.) with a mix of BM cells from Jedi-TCRβ mice on a CB6F1 background (H2^b/d) and WT H2^b/d, IL-4–GFP H2^b/d or IL-4–GFP H2^b/b mice (on mixed C57BL/6J and BALB/c genetic backgrounds). Thymi of chimeras were analyzed 6 weeks after reconstitution. Representative plots demonstrate the frequencies of GFP-expressing thymocytes, frequencies of H2-K^d GFP_200–208 tetramer-binding thymocytes (quantification shown on the right side) and expression of the indicated cell-surface markers by the latter cells (n = 3 chimeras per group). Data are representative of two independent experiments. c, Schematic representation of RTOC experiments shown in d–g; O/N, overnight. d–g, Confocal microscopy analysis of RTOCs assembled with sorted Jedi-TCRαβ DP thymocytes (labeled with CellTrace Violet (CTV) and Cal-520 AM Ca²⁺ sensor dye) and thymic GFP⁺ iNKT cells from IL-4–GFP mice or total iNKT cells from WT thymi (labeled with CellTrace Yellow (CTY)). d, Representative images showing interactions between Jedi thymocytes and IL-4–GFP iNKT cells (top and middle) and a WT iNKT cell (bottom); scale bars, 2 µm. e, Quantification of Ca²⁺ signaling events in Jedi thymocytes contacting WT or IL-4–GFP iNKT cells. The numbers in the circles indicate the total number of interactions analyzed. f, Normalized Ca²⁺ signaling intensity in Jedi thymocytes interacting with iNKT cells. Quantification was performed as described in the Methods. Mean and 95% confidence interval are shown (n = 10 Jedi cells per group). Individual curves for each cell are shown in Extended Data Fig. 6g. g, Quantification of the interaction time with iNKT cells for 90 Jedi thymocytes from RTOCs with WT iNKT cells (gray), 35 Jedi cells that underwent Ca²⁺ flux in RTOCs with IL-4–GFP iNKT cells (orange) and 44 Jedi cells that did not undergo Ca²⁺ flux in RTOCs with IL-4–GFP iNKT cells (blue). Data are presented as mean ± s.d. (**P < 0.01, ***P < 0.001 and ****P < 0.0001) and were analyzed by one-way ANOVA with a Holm–Sidak multiple comparisons test (b) or Kruskal–Wallis test (g).

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