Fig 2: The transcriptional profile and IKKa and p38 MAPK signaling pathways are impaired in mTEClo of ?CD4 mice.(A, B) Total IKKa, p38 MAPK, phospho-IKKa(Ser180)/IKKß(Ser181), and p38 MAPK (Thr180/Tyr182) (A) and the ratio of phospho/total proteins (B) analyzed by flow cytometry in mTEClo from WT and ?CD4 mice. Data are representative of two independent experiments (n = 3–4 mice per group and experiment). (C) Scatter plot of gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) of mTEClo from WT versus ?CD4 mice. Genes with fold difference =2 and p-adj<0.05 were considered as upregulated or downregulated genes (red and blue dots, respectively). RNA-seq was performed on two independent biological replicates with mTEClo derived from 3 to 5 mice. (D) Numbers of tissue-restricted self-antigens (TRAs) and non-TRAs in genes up- and downregulated (left panel) and the proportion of upregulated TRAs compared to those in the all genome (right panel). ND, not determined. (E) Numbers of induced Aire-dependent, Fezf2-dependent, Aire/Fezf2-dependent, and Aire/Fezf2-independent TRAs. (F) The expression of Aire-dependent (Meig1, Nov), Fezf2-dependent (Fcer2a, Kcnj5), Aire/Fezf2-dependent (Krt1, Reig1), and Aire/Fezf2-independent (Crp, Rsph1) TRAs measured by qPCR in WT (n = 3–4) and ?CD4 (n = 3–4) mTEClo. (G) Expression fold change in HDAC3-induced transcriptional regulators and other transcription factors significantly upregulated in WT versus ?CD4 mTEClo. The color code represents gene expression level. (H) Heatmaps of genes encoding for cell adhesion molecules and cytokines that were significantly downregulated in mTEClo from ?CD4 mice. (I) Hierarchical clustering and heatmap of mean expression of these cell adhesion molecules and cytokines in mTEC subsets identified by scRNA-seq. Error bars show mean ± SEM, *p<0.05, **p<0.01 using two-tailed Mann–Whitney test for (A), (B) and (F) and chi-squared test for (D).

Fig 3: 2PM approach for imaging antigen-specific SP thymocytes responding to TRAs presented by medullary APCs in live thymic slices. a CMTPX and Indo1AM-labeled OT-I CD8SPs or OT-II CD4SPs were incubated on live thymic slices expressing fluorescent reporters for DCs or AIRE+ mTECs, along with RIP-mOVA or RIP-OVAhi transgenes. Time-lapse imaging through 40-µm depth was carried out with lasers tuned to excite Indo1AM (730 nm) and CMTPX with EGFP/EYFP (840/875 nm). b–e Maximum intensity projections of 2PM volumes, at ×40 magnification. SP thymocytes are displayed in b, d red or in c, e pseudo-color for [Ca2+]i ratio, in b, c DC (yellow), or d, e AIRE+ mTEC (green) reporter slices. Thymocyte track times are color-encoded as indicated. Scale bar is 30 µm. f Mean cell velocity and g track straightness of individual OT-I CD8SP and OT-II CD4SP cells migrating in WT, RIP-mOVA, or RIP-OVAhi slices. Data points represent cells from all experiments, and red bars and numbers show mean values. Data from 3 to 6 experiments per condition, number of cells: WT (NOT-I = 827, NOT-II = 385), RIP-mOVA (NOT-I = 780, NOT-II = 1138), and RIP-OVAhi (NOT-I = 738, NOT-II = 1354). Analyzed by t tests, p-values: *** < 0.001, **** < 0.0001. h–j Left: 2PM image of cells undergoing h initial activation, i sustained activation as a singlet, and j activation as an aggregate. Thymocyte tracks and Indo1 ratios are color-encoded. Scale bar is 5 µm. Middle: single-cell traces of [Ca2+]i (blue) and velocity (red) over time. Right: cell distance to the nearest identified APC (DC or AIRE+ mTEC) over time. Data in j were from the thymocyte denoted by a white arrow. Source data are provided as a Source Data file. See also Supplementary Figs. 1, 2 and Movies 1–8

Fig 4: Both AIRE+ mTECs and DCs engage polyclonal SP thymocytes undergoing TCR signaling. a, b Maximum intensity projections of 2PM imaging volumes collected at ×20 magnification showing a WT CD4SP thymocytes (Indo1 ratio) migrating in AireEGFP (green) or b WT CD8SP thymocytes (Indo1 ratio) migrating in CD11c-EYFP (yellow) slices. Thymocyte tracks are color-encoded for time; scale bar is 30 µm. c Frequency of polyclonal CD8SP (red) and CD4SP (blue) thymocytes undergoing activation in WT thymic slices. Data points represent the average of all cells in an experiment, and bars show mean ± SEM. d Mean cell velocities of unactivated and activated WT CD8SPs and CD4SPs in WT thymic slices. Data points represent individual cells in all experiments, and the red bar and numbers show means. e Average [Ca2+]i of WT CD8SP (red; N = 3) and CD4SP thymocytes (blue; N = 19) undergoing initial activation on WT thymic slices. Single-cell calcium traces were aligned such that the lowest ratio value before the increase in [Ca2+]i was set at time point 0. f Average [Ca2+]i of all activated WT CD8SPs and CD4SPs on WT thymic slices. Data points represent the average of all cells in an experiment, and bars show mean ± SEM. g Frequency of activated WT CD8SP and CD4SP thymocytes contacting AIRE+ mTECs or DCs on AireEGFP (green) or CD11c-EYFP (yellow) thymic slices. Data points represent the average of all cells in an experiment, and bars show mean ± SEM. h Frequency of activated WT CD8SPs and CD4SPs contacting DCs, that interacted with cDC2 (yellow) or cDC1 (gray) on CD11c-EYFP thymic slices. Graphs show mean + SEM. Data compiled from 7 to 8 separate imaging experiments per condition, number of cells: CD8SP (Nunactivated = 399, Nactivated = 46), CD4SP (Nunactivated = 419, Nactivated = 60). Analyzed by unpaired t tests, except for h which was analyzed by t tests with Sidak–Bonferroni correction for multiple comparisons, p-values: * < 0.05, ** < 0.01, **** < 0.0001, ns: not significant. Source data are provided as a Source Data file. See also Supplementary Movies 9, 10

Fig 5: PLIC quantitative analysis of protein post-translational modification in mTECs. a A graphical scheme illustrating PLIC analysis of acetylated Aire, using mouse anti-Aire #209 antibody, and rabbit anti-lysine acetylation antibody. b PLIC analysis showing representative images of Aire acetylation in mTEChi and CD45+ cells isolated from Sirt1 +/– and Sirt1 –/– mice. Presented as (left to right): bright field (BF), PLA (green), staining of CD80 (PB), EpCAM (APC), CD45 (APC/Cy7), and BF/PLA/EpCAM overlay (merged). Scale bar 7 µM. c Quantitative analysis of Aire acetylation (presented as the percentage of PLA+ cells) in mTEChi or CD45+ cells isolated from Sirt1 +/– and Sirt1 –/– mice. Shown are values from three independent experiments. Each point on the graph (triangle for mTEChi, circle for CD45+, and cross for probes only) represents an average value of single independent experiment, calculated from 2 to 4 biological replicates, +s.e.m; **p-value = 0.0014 (independent student t-test)