MHCII-mediated antigen presentation and Claudin 1 expression by DC1 lineage cells are important for the establishment of central tolerance. (A) Histograms of MHCII expression within DC1 lineage subsets from I-ab^fl/fl (control; black) and XCR1^iCreI-ab^fl/fl (varying shades of green) mice. (B) Frequency of MHCII⁺ cells within DC1 subsets from I-ab^fl/fl (control; solid circle) and XCR1^iCreI-ab^fl/fl (empty circle) mice, related to Fig. S4 A (mean ± SEM, n = 5–9 mice from two independent experiments). (C) Flow cytometry gating strategy of thymic T cells. T cells were gated as CCR7⁺TCRB⁺ to analyze medullary T cells only. These were gated as CD4⁺ (helper T cells), which were further separated into CD25⁺FOXP3⁻ Treg precursors (CD25⁺ prec), CD25⁻FOXP3⁺ Treg precursors (FOXP3⁺ prec), and CD25⁺FOXP3⁺ Treg. Recirculating Tregs were distinguished from newly generated Tregs as CD73⁺. The color code of thymic T-cell subsets is indicated. (D) Frequency of CD73⁻ and CD73⁺ cells within Treg subset from thymi of I-ab^fl/fl (control; solid circle) and XCR1^iCreI-ab^fl/fl (empty circle) mice (mean ± SEM, n = 13–14 mice from four independent experiments). (E) Flow cytometry gating strategy of splenic T cells, which were gated as TCRB⁺CD4⁺ and further separated into Tregs (SP Treg) based on the expression of CD25 and FOXP3. The color code of splenic Tregs is shown. (F) Flow cytometry gating strategy to analyze thymic DC1 lineage reconstitution within competitive BM chimeras from Fig. 6, D and E. Note that Cldn1^+/+MHCII^−/− and Cldn1^−/−MHCII^+/+ BMs (or Cldn1^+/+MHCII^+/+ BM in the controls) were all CD45.2⁺; thus, we quantified the reconstitution of each mixture of these BMs based on the expression of MHCII within thymic DC1 lineage. (G) Quantification of reconstitution of thymic DC1 lineage related to Fig. S4 F (mean ± SEM, n = 7–14 mice from three independent experiments). The color code is the same as described in Fig. 6 E. (H) Flow cytometry gating strategy of OTII thymic T cells. CD45.1⁺ cells were gated as Vα2⁺Vβ5⁺ to obtain OTII cells. These cells were then gated as CD4⁺ and separated into the same populations as in Fig. S4 C and conventional CD25⁻FOXP3⁻OTII cells. (I) Frequency of newly generated CD73⁻ (left panel) and recirculating CD73⁺ (right panel) cells within OTII Tregs from thymi of competitive BM chimeras described in Fig. 6, D and E (mean ± SEM, n = 5–10 mice from two independent experiments). Color code as in Fig. S4 G. (J) Flow cytometry gating strategy of OTII cells from skin-draining lymph nodes. CD45.1⁺ OTII cells were gated as Vα2⁺Vβ5⁺. These were then gated as CD4⁺ and separated into CD25⁺FOXP3⁺ OTII Tregs and conventional CD25⁻FOXP3⁻ OTII cells. (K) Flow cytometry gating strategy of developing polyclonal T cells from the thymus. CD4⁺ polyclonal T cells were pregated as non-Vα2⁺Vβ5⁺ to leave out OTII cells from further analysis. These cells were further gated as polyclonal Tregs, which were further distinguished into newly generated (CD73⁻) and recirculating Tregs (CD73⁺). Statistical analysis in G was performed using one-way ANOVA with Tukey’s multiple comparisons test and in B, D, and I was performed using unpaired, two-tailed Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, ns = not significant. All mice were bred on the B6 background. Littermates were used as controls.