FIGURE 3.

The 12MO thymic environment does not support efficient negative selection of OT‐II CD4SP thymocytes responding to ubiquitous self‐antigens or TRAs. (a) The percent of OT‐II CD4SP cellularity following incubation overnight in 1MO or 12MO thymic slices with the indicated concentrations of OVAp_323‐339 relative to OT‐II CD4SP cells remaining in slices incubated without OVAp. Plots show mean ± SEM of compiled data from four independent experiments. Analyzed by two‐way ANOVA with Šídák's correction for multiple comparisons. (b) Negative selection of OT‐II CD4SP thymocytes and (c) induction of CD25⁺ Treg‐P responding to the indicated TRAs on 1MO or 12MO thymic slices were quantified. (b) The percent of OT‐II CD4SP cells remaining in RIP‐mOVA and RIP‐OVA^hi thymic slices relative to OVA‐ slices. Addition of 10 μM of OVAp_323‐339 (OVAp) served as a positive control for OT‐II negative selection. (c) Normalized cell numbers (left) and frequencies (right) of CD25+ OT‐II CD4SP cells. Data in (b‐c) show mean ± SEM of thymic slices, compiled from 3‐4 independent experiments, with three thymic slices per experiment. Each data point represents an individual thymic slice. Analyzed by two‐way ANOVA with Šídák's correction for multiple comparisons, p‐values: * <0.05, ** <0.01, *** <0.001, **** <0.0001, ns: not significant. See also Figure S2