Fig. 4: Only very high affinity agonists drive CD8αα IEL development.

OT-I thymocytes depleted of mature CD8 single positives were overlaid onto WT thymic slices to which OVAp (SIINFEKL) or lower-affinity Q4 peptide was added. In some cases (a, b) a reference population was added with the OT-I thymocytes. Slices were harvested at 16 (a, b) or 96 (c–f) hours for flow cytometric analysis. a Negative selection displayed as the ratio of live OT-I thymocytes relative to live reference thymocytes, normalized to no OVA controls. b Antigen recognition of OT-I thymocytes displayed as expression of the activation marker CD69. Data are representative of b or pooled from (a) 3 independent experiments, with mean and SEM of n = 11–15 total slices per condition, where each dot represents an individual slice. c–e Expression of T-bet (c), CD122 (d), or PD-1 (e) in OT-I thymocytes presented as fold induction displayed as MFI relative to levels in no peptide controls (top), or representative flow cytometry plots (bottom). f Fold change in CD8β/α ratio displayed as the ratio of CD8α MFI/CD8β MFI relative to no peptide samples, where each dot represents an individual slice. Data are representative of (bottom panels) or pooled from (top panels) 2 independent experiments, with mean and SEM of n = 6 total slices per condition, where each dot represents an individual slice. ns not significant (p > 0.05), **p < 0.01, ****p < 0.0001 (one-way ANOVA with Bonferroni’s correction).