FIGURE 2.

The 12MO thymic environment does not support efficient negative selection of OT‐I CD8SP thymocytes responding to moderate‐avidity self‐antigens. (a) Schematic of heterochronic slice deletion assays to assess negative selection of OT‐I or OT‐II thymocytes responding to ubiquitous self‐antigens or TRAs in young (1MO) versus middle‐aged (12MO) thymic slices. Slices were generated from (1) C57BL/6J wild‐type mice that did not express OVA (OVA⁻), (2) wild‐type mice followed by incubation with OVAp or lower affinity APLs, or (3) RIP‐mOVA, RIP‐OVA^hi, RIP‐mQ4R7, or RIP‐mT4 model TRA transgenic mice. (b) The percent of OT‐I CD8SP cells remaining in 1MO thymic slices incubated overnight with the indicated concentrations of SIINFEKLp, Q4R7p, or T4p, relative to OT‐I CD8SP cells in slices incubated without peptide. Data are compiled from 6 experiments. (c) The percent of OT‐I CD8SP cells remaining in 1MO or 12MO thymic slices incubated with the indicated concentrations of SIINFEKLp, Q4R7p or T4p relative to those in slices without peptide. Data are compiled from 6–7 independent experiments. Data in (b) are a composite of the 1MO data shown in (c). (d) Negative selection of OT‐I CD8SP thymocytes responding to AIRE‐dependent TRAs in 1MO and 12MO thymic slices, evaluated after 48 h. The percent of OT‐I CD8SPs remaining in RIP‐mOVA, RIP‐OVA^hi, RIP‐mQ4R7 and RIP‐mT4 thymic slices were quantified relative to OT‐I CD8SPs in OVA^‐ thymic slices. Addition of 10 μM of SIINFEKLp (OVAp) served as a positive control for OT‐I negative selection. Data show mean ± SEM compiled from 3‐4 independent experiments per genotype, with three thymic slices per experiment. Each data point represents results from an individual thymic slice. Values were normalized to the mean of triplicate OVA⁻ slices in each experiment. Data in (c) and (d) were analyzed by two‐way ANOVA with Šídák's correction for multiple comparisons, p‐values: * <0.05, ** <0.01, ns: not significant