Extended Data Fig. 3: aDC1s, followed by aDC2s, are highly efficient at mTEC-derived antigen presentation on MHC-I and MHC-II.
Related to Fig. 2. (a-c) Thymic DC subsets isolated from RIP-OVAhi mice were tested for their capacity to cross-present the mTEC-derived OVA TRA on MHC-I and induce OT-I proliferation. (a) Representative histograms showing the percentage of OT-I CD8+ T cells that underwent at least 1 cell division after co-culture for 3.5 days with the specified DC subsets isolated from RIP-OVAhi mice. OT-I CD8+ T cells incubated only with IL-2 served as negative controls, while those co-cultured with 50 nM OVA257-264-pulsed splenocytes served as positive controls. For (a-c), data are representative of N = 3 independent experiments. (b) Quantification of OT-I CD8+ T cells that underwent proliferation from the experiment described in (a). Bars represent mean ± SEM. Dots represent replicate wells from one representative experiment. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. Significance is relative to “IL-2 only” control wells. (c) Modeling, based on data from the experiment in (a), of the frequency of OT-I CD8+ T cells that underwent the indicated number of cell divisions. Bars represent mean ± SEM. (d-g) To determine if the different functional capacities of DCs subsets could reflect differences in their ability to process and present antigens or co-stimulate thymocytes/T cells, differential expression of genes and proteins were evaluated. (d, f) Heatmaps displaying average normalized gene expression values of select (d) genes associated with antigen processing and presentation and (f) costimulatory genes in each cDC cluster from Fig. 1a. Expression is z-score normalized per gene across different cDC clusters. (e, g) Relative cell-surface expression levels of (e) MHC-I and MHC-II, and (g) CD80 and CD86 proteins by cDC subsets, as determined by flow cytometry. Data are normalized to average MFI levels of aDC1s for each protein within an individual experiment. Data are compiled from N = 3 independent experiments; n = 8 total WT mice. Bars represent mean ± SEM and symbols represent individual mice. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. Significance is relative to the aDC1 subset.
