Fig. 5 | Nature Communications

Fig. 5

From: Live-cell imaging reveals the relative contributions of antigen-presenting cell subsets to thymic central tolerance

Fig. 5

Both conventional DC subsets induce negative selection of OT-I and OT-II SPs responding to RIP-mOVA and RIP-OVAhi TRAs. a Sequential gating of live CD45+ cells from a digested CD11c-EYFP thymus to detect the indicated APC subsets. Histograms show EYFP and MHC-II levels for the indicated APC subsets. b EYFP mean fluorescence intensity (MFI) of Sirpα+ cDC2 and Sirpα cDC1. Flow-cytometry data averaged from three CD11c-EYFP mice, stained independently. Data points represent mice, and bars show mean ± SEM. c 2PM volume from three perspectives, showing two activated OT-I CD8SPs (red mask, SP1 and SP2) interacting with cDC2 (yellow) and cDC1 (gray), respectively. Scale bar is 10 µm. d Frequency of activated OT-I CD8SP and OT-II CD4SP thymocytes contacting DCs, that interacted with cDC2 or cDC1 on RIP-mOVA or RIP-OVAhi thymic slices. Graph shows mean + SEM. Data are compiled from experiments analyzed in Fig. 4. Analyzed by t tests with multiple comparison correction, p-values: * < 0.05, *** < 0.001. ns not significant. e mTEChi (green), cDC2 (red), and cDC1 (blue) were sorted from RIP-mOVA and RIP-OVAhi thymi and cultured with CFSE-labeled splenic OT-I CD8+ T cells. WT splenocytes ± OVAp served as positive and negative control APCs. Histograms show CFSE dilution in Vα2+Vβ5+CD8+ cells after incubation with APCs for 72 h, the gate shows the percent of cells that proliferated. Dashed line shows the CFSE profile for T cells cultured with WT splenocytes in the absence of OVAp, and gray shading shows that of T cells cultured with OVAp-pulsed WT splenocytes. Data are representative of two independent experiments per condition, and graphs depict mean ± SEM of the percent proliferation after incubation with indicated APCs for triplicate wells. Source data are provided as a Source Data file. See also Supplementary Figs. 7, 8

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