Fig. 2: Thymocytes that survive negative selection develop a CD8αα IEL precursor phenotype.

a–e OT-I thymocytes were depleted of the most mature CD8 single positives and stained with cell proliferation dye eFluor450 (CPD450) prior to overlay onto the indicated thymic slices, which were harvested for flow cytometric analysis 1–4 days later. Data are representative of 2 independent experiments. a Representative flow cytometry plots of CD122, T-bet, PD-1, α4β7 integrin, CPD450, and CD8α versus CD8β expression in gated OT-I thymocytes cultured on RIPmOVA thymic slices, or WT thymic slices with or without OVAp for 4 days. Gray histograms and dots represent OT-I on slices without OVA, black lines and dots represent OT-I on slices with OVAp, and dark gray lines represent OT-I on RIPmOVA slices. b Expression of CD8α and CD8β by OT-I thymocytes cultured on WT thymic slices with OVAp for the indicated times, displayed as the ratio of the Mean Fluorescence Intensity (MFI) of sample relative to no OVA control slices. c Relative CD8β/α ratio of OT-I thymocytes on WT thymic slices with OVAp over time, displayed as the ratio of CD8β MFI/CD8α MFI, normalized to no OVA control slices. d Expression of T-bet, CD122, and PD-1 expression over time in thymocytes on WT slices with OVAp, displayed as the ratio of the MFI of the indicated marker relative to no OVA control slices. Data are compiled from two independent experiments, with mean and SEM shown. e Representative flow cytometry plot displaying dilution of CPD450 in OT-I thymocytes on slices with OVAp after the indicated days of culture. f, g OT-I thymocytes depleted of the most mature CD8 single positives or OT-I T cells collected from the lymph nodes were overlaid onto thymic slices to which OVAp was added, and analyzed 96 h later. f Fold change in CD8β/α ratio of OT-I thymocytes (thy) or T cells (T) after 96 h on thymic slices, displayed as the ratio of CD8β MFI/CD8α MFI relative to no OVA samples, where each dot represents an individual slice. Data are compiled from 2 independent experiments. g Production of IFNγ by OT-I thymocytes (thy) or OT-I T cells (T) after restimulation with OVA-loaded DC, displayed as percent of IFNγ + OT-I. Dashed line represents the average percent of IFNγ + cells in culture with unloaded DC. Data are pooled from 3 independent experiments, with mean and SEM of n = 9 thymic slices, where each dot represents an individual slice. ns not significant (p > 0.05), ****p < 0.0001 (one-way ANOVA with Bonferroni’s correction).