Fig. 4: Antigen expression exclusively by small numbers of iNKT cells is sufficient to induce elimination of autoreactive CD8+ thymocytes.
a, Schematic representation of the RTOC experiments shown in b–d. Jedi-TCRαβ and OT-I DP thymocytes were sorted as CD4+CD8+ cells, and ex vivo iNKT cells were sorted from the thymi of WT and IL-4–GFP mice as CD1d tetramer-binding cells (without additional gating on GFP). Transduced thymic iNKT cells were sorted as Thy1.1+ (c) or GFP+ (d) CD1d tetramer-binding cells; E14.5–15.5, embryonic days 14.5–15.5. b, Flow cytometric analysis of RTOCs with ex vivo thymic WT or IL-4–GFP iNKT cells 5 days after initiation of reaggregation. Representative plots show the distribution of Jedi cells (CD45.1+), iNKT cells (CD45.2+) and fetal thymocytes (CD45.1+CD45.2+), frequencies of GFP-expressing cells, frequencies of H2-Kd GFP200–208 tetramer-binding Jedi thymocytes (quantification is shown on the right) and expression of the indicated cell-surface markers by the latter cells. Results are pooled from three independent experiments (n = 4 RTOCs for WT iNKT cells and n = 7 for IL-4–GFP iNKT cells). c, Thymic iNKT cells were transduced with Thy1.1-IRES-i.c.GFP, Thy1.1-IRES-secGFP or Thy1.1-only retroviruses. Sorted Thy1.1+ iNKT cells were added to RTOCs together with Jedi-TCRαβ DP thymocytes. Flow cytometric analysis and quantification is shown as in b (n = 5 RTOCs for empty vector, n = 10 for i.c.GFP and n = 9 for secGFP). Results are pooled from two independent experiments. d, Thymic iNKT cells were transduced with cOVA-IRES-GFP retroviruses encoding the indicated versions of the OVA257–264 epitope. Sorted GFP+ iNKT cells were added to RTOCs together with OT-I DP thymocytes. RTOCs with nontransduced iNKT cells were used as a no-antigen control. Flow cytometric analysis and quantification is shown as in b (n = 6 RTOCs for uninfected, n = 3 for N4, n = 6 for Q4, n = 3 for T4 and n = 5 for V4). Representative results from two independent experiments are shown. 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 (b) or one-way ANOVA with a Holm–Sidak multiple comparisons test (c and d).
