Figure S3.
Mouse models used to study the role of Claudin in CAT. (A) Histograms show CLAUDIN 1 expression within thymic DC1 subsets from Cldn1fl/fl (control; black) and XCR1iCreCldn1fl/fl (varying shades of green) mice. (B) Frequency of CLAUDIN 1+ cells within DC1 subsets related to Fig. S3 A (mean ± SEM, n = 8 mice from three independent experiments). (C) Frequency of DC1 lineage subsets within parent populations from Cldn1fl/fl (control; solid circle) and XCR1iCreCldn1fl/fl (empty circle) mice (mean ± SEM, n = 8 mice from three independent experiments). (D) Representative flow cytometry plot shows the reconstitution of BMs that give rise to Ly5.1 DC1 (CD45.1+) and XCR1iCreCldn1fl/fl DC1 (CD45.2+) within BM chimeras from Fig. 3 D. (E) Quantification of BM reconstitution from Fig. S3 D (mean ± SEM, n = 17 mice from four independent experiments). (F) MFI of TdTOMATO expression within Claudin 1–sufficient and Claudin 1–deficient DC1 subsets from Fig. 3, D and E (mean ± SEM, n = 10 mice from three independent experiments). (G) Violin plots from scRNAseq analysis (Fig. 1) show the expression of DC1 lineage markers used for flow cytometry gating strategy of DC1 subsets in Bosteels et al. (2023). (H) Violin plots from scRNAseq analysis (Fig. 1) show the expression of selected marker genes of homeostatic (Homeo.) and immunogenic (Immuno.) maturation and genes associated with both maturation programs (Common) from Bosteels et al. (2023). The subsets analyzed are the same as in Fig. S3 G. (I) Representative flow cytometry plots show CLAUDIN 3 positivity within mTECs, cTECs, CD45+ cells (hematopoietic cells), and EPCAM–CD45− cells (fibroblasts, endothelial cells, etc.). FMO controls are shown. (J) Representative flow cytometry plots show CLAUDIN 3 positivity within individual mTEC subsets gated as in Fig. 4 A. FMO controls are shown. (K) Heat map showing the relative expression of genes encoding cathepsins across individual sequenced samples. The heat map color scale corresponds to z scores of regularized log data values. The numbers below each column in the heat map correspond to three independent biological replicates, each derived from an individual mouse. Statistical analysis in B and C was performed using unpaired, while statistical analysis in E and F was analyzed by paired, two-tailed Student’s t test, ***P ≤ 0.001, ****P < 0.0001, ns = not significant. All mice were bred on the B6 background. Littermates were used as controls.

Mouse models used to study the role of Claudin in CAT. (A) Histograms show CLAUDIN 1 expression within thymic DC1 subsets from Cldn1fl/fl (control; black) and XCR1iCreCldn1fl/fl (varying shades of green) mice. (B) Frequency of CLAUDIN 1+ cells within DC1 subsets related to Fig. S3 A (mean ± SEM, n = 8 mice from three independent experiments). (C) Frequency of DC1 lineage subsets within parent populations from Cldn1fl/fl (control; solid circle) and XCR1iCreCldn1fl/fl (empty circle) mice (mean ± SEM, n = 8 mice from three independent experiments). (D) Representative flow cytometry plot shows the reconstitution of BMs that give rise to Ly5.1 DC1 (CD45.1+) and XCR1iCreCldn1fl/fl DC1 (CD45.2+) within BM chimeras from Fig. 3 D. (E) Quantification of BM reconstitution from Fig. S3 D (mean ± SEM, n = 17 mice from four independent experiments). (F) MFI of TdTOMATO expression within Claudin 1–sufficient and Claudin 1–deficient DC1 subsets from Fig. 3, D and E (mean ± SEM, n = 10 mice from three independent experiments). (G) Violin plots from scRNAseq analysis (Fig. 1) show the expression of DC1 lineage markers used for flow cytometry gating strategy of DC1 subsets in Bosteels et al. (2023). (H) Violin plots from scRNAseq analysis (Fig. 1) show the expression of selected marker genes of homeostatic (Homeo.) and immunogenic (Immuno.) maturation and genes associated with both maturation programs (Common) from Bosteels et al. (2023). The subsets analyzed are the same as in Fig. S3 G. (I) Representative flow cytometry plots show CLAUDIN 3 positivity within mTECs, cTECs, CD45+ cells (hematopoietic cells), and EPCAM–CD45− cells (fibroblasts, endothelial cells, etc.). FMO controls are shown. (J) Representative flow cytometry plots show CLAUDIN 3 positivity within individual mTEC subsets gated as in Fig. 4 A. FMO controls are shown. (K) Heat map showing the relative expression of genes encoding cathepsins across individual sequenced samples. The heat map color scale corresponds to z scores of regularized log data values. The numbers below each column in the heat map correspond to three independent biological replicates, each derived from an individual mouse. Statistical analysis in B and C was performed using unpaired, while statistical analysis in E and F was analyzed by paired, two-tailed Student’s t test, ***P ≤ 0.001, ****P < 0.0001, ns = not significant. All mice were bred on the B6 background. Littermates were used as controls.

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