Fig 2: PKD is required for CD4 lineage commitment.(a) Analysis of CD8-redirection. BM cells from WT and PKD2/3ΔT mice were transferred into irradiated TAP−/− recipient mice. At 8 weeks after BMT, CD4 and CD8 expression levels on TCRβhi thymocytes were analysed by flow cytometry. The percentage of gated population is indicated. (b) Real time PCR analysis of ThPOK, Runx3 and Runx3d mRNA expressions in CD4+CD8int thymocytes. Results are presented as relative expression of β-actin. **P<0.01, NS, not significant. (c) Flow cytometry analysis of CD4 and CD8 expression on TCRβhi thymocytes from WT, PKD2/3ΔT and PKD2/3ΔT × ThPOK Tg mice. (d) CD5 expression on CD4 SP thymocytes. Data are presented as mean±s.d. of triplicate assays (b) and representative of two (a,b) or three (c,d) independent experiments. Unpaired two-tailed Student's t test is used to calculate P values.

Fig 3: TG6, F5, and OT-1 CD8SP cells express CD5 and Nur77 across the polyclonal spectrum.CD8SP cells were harvested from the thymus and lymph nodes of wild-type (B6), TG6, F5, and OT-1 TCRtg mice and analyzed by flow cytometry. (a, b) Representative (left) and quantified by geometric mean fluorescence intensity (right) CD5 surface expression on (a) CD8SP thymocytes and (b) CD8SP lymph node cells. (c, d) Representative (left) and quantified (right) intracellular Nurr77 expression gated on (c) CD8SP thymocytes and (d) CD8SP lymph node cells. Data are presented as average ± SD and analyzed using an ordinary one-way ANOVA followed by a Tukey’s multiple comparisons (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data are compiled from three or more experiments.

Fig 4: Role of IL-17 and IL-17+?dT Cells in C. rodentium Infection of CD18-/- Mice(A and B) Eight-week-old CD18-/- and CD18+/+ littermates were injected intravenously (i.v.) with either anti-IL-17 antibody or isotype control IgG (IC) on a daily basis for 7 days starting 1 day before C. rodentium infection.(A) Survival rates at the indicated time points.(B) On day 5 post-infection, LPMCs were isolated from the large intestine of the indicated mouse groups and analyzed by flow cytometry; shown are representative FACS plots (left) and absolute numbers (right) of IL-22-expressing ROR?t+ ILCs (gated on CD3-CD5-B220-NK1.1-F4/80-Gr-1-CD127+CD90.2+ROR?t+).(C and D) Eight-week-old CD18-/-/?dT-/- mice and CD18-/-/?dT+/+ littermates (as well as CD18-sufficient mice with or without ?dT cell deficiency) were orally inoculated with C. rodentium.(C) Survival rates at the indicated time points.(D) On day 5 post-infection, LPMCs were isolated from the large intestine of the indicated mouse groups and analyzed by flow cytometry; shown are representative FACS plots (left) and absolute numbers (right) of IL-22-expressing ROR?t+ ILCs (gated on on CD3-CD5-B220-NK1.1-F4/80-Gr-1-CD127+CD90.2+ROR?t+).Data are means ± SDs and are pooled from 2 independent experiments with 5 mice/group in each experiment, for a total of 10 mice/group (A and C); or are from 2 independent experiments with 3 mice/group, for a total of 6 mice/group (B and D). ***p < 0.001 (B and D: 1-way ANOVA with Tukey’s multiple-comparisons test). NS, non-significant.

Fig 5: Thymocytes with lower self-reactivity have delayed development in steady state TCRtg and wild-type mice.TG6, F5, or OT-1 (a–c) or B6 (d–f) mice were injected with two doses of 1 mg of EdU intraperitoneally (i.p.) at 0 and 4 hr post-injection (p.i.). (a) TCRtg experimental schematic. (b) EdU incorporation into TCRtg thymuses. (c) (Left three graphs) Percent CD8SP and DP thymocytes out of gated EdU+ cells at various days p.i. in TG6, F5, and OT-1 tg thymuses. (Right) The percent of EdU+CD8 SP relative to max within each experiment. Data are presented as the average within each transgenic and analyzed using an ordinary one-way ANOVA at each time point (*p<0.05, **p<0.01). Tukey’s multiple comparisons showed significant differences between OT-1 and TG6 at day 4 (**), day 5 (*), and day 6 (*); OT-1 and F5 at day 4 (*); and a difference between F5 vs TG6 at Day 6 (p=0.069). (d) B6 experimental schematic. (e) Representative gating strategy and histograms showing B6 day 10 p.i. CD5 expression on EdU+ and EdU- mature SP cells. (f) CD5 expression of EdU+TCRß+CD8 SP and EdU+CD4 SP thymocytes at 4, 7, and 10 days p.i. CD5 expression shown relative to total TCRb+CD8 SP or CD4SP CD5 expression. In (f), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA, Tukey’s multiple comparisons (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data are compiled from three or more experiments.