Figure 7.
Claudin 1 deficiency in the DC1 lineage leads to break in tolerance and premature death. (A) Detection of autoantibodies from sera of BM chimeras from Fig. 6, D and E, using composite tissue slides. Serum autoantibody levels were quantified by the MFI of the secondary anti-mouse antibody conjugated to Alexa 555 (shown in green) across the entire image and from the regions demarcated by white lines corresponding to the individual tissues (K = kidney; L = liver; and S = stomach) that make up the composite slide. 6-wk-old B6 WT and 30-wk-old Aire−/− mice on BALB/c background were used as negative and positive controls, respectively. Note that at the time of serum harvest, the competitive BM chimeras were 30–40 wk after BM transfer. Slides were imaged using the Tile Scan function in LAS X software (Leica) with a 20% overlap between adjacent tiles, which were subsequently stitched to generate the final composite images. (B) Normalized MFI of serum autoantibody detection within the entire image of the composite tissue and individual tissues from Fig. 7 A (mean ± SEM, n = 3–7 mice from two independent experiments). (C and D) Frequency of effector memory CD62L−CD44+ cells within polyclonal conventional CD4+ T cells (Poly. FOXP3−) and Tregs (Poly. FOXP3+) gated as in Fig. S5 A, isolated from MLNs (C) and spleens (D) of BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer (n = 5–9 mice from two independent experiments). (E) Weight-to-length ratio of colons isolated from BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer (n = 6–9 mice from two independent experiments). (F) Survival curve of BM chimeras from Fig. 6, D and E, (n = 5 mice from two independent experiments). Color code as in Fig. 6 E. Note that two OVA+Cldn1−/− mice died in the first experiment and one OVA+Cldn1−/− mouse died in the second experiment. Statistical analysis in B–E was performed using one-way ANOVA with Tukey’s multiple comparisons test and in F using the log-rank (Mantel–Cox) test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, ns = not significant. All mice were bred on the B6 background except for Aire−/− mice, which were bred on the BALB/c background. Littermates were used as controls. In A and B, 6-wk-old WT B6 mice from JAX were used as negative controls and ∼30-wk-old Aire−/− mice were used as positive controls. MLNs, mesenteric lymph nodes.

Claudin 1 deficiency in the DC1 lineage leads to break in tolerance and premature death. (A) Detection of autoantibodies from sera of BM chimeras from Fig. 6, D and E, using composite tissue slides. Serum autoantibody levels were quantified by the MFI of the secondary anti-mouse antibody conjugated to Alexa 555 (shown in green) across the entire image and from the regions demarcated by white lines corresponding to the individual tissues (K = kidney; L = liver; and S = stomach) that make up the composite slide. 6-wk-old B6 WT and 30-wk-old Aire−/− mice on BALB/c background were used as negative and positive controls, respectively. Note that at the time of serum harvest, the competitive BM chimeras were 30–40 wk after BM transfer. Slides were imaged using the Tile Scan function in LAS X software (Leica) with a 20% overlap between adjacent tiles, which were subsequently stitched to generate the final composite images. (B) Normalized MFI of serum autoantibody detection within the entire image of the composite tissue and individual tissues from Fig. 7 A (mean ± SEM, n = 3–7 mice from two independent experiments). (C and D) Frequency of effector memory CD62L−CD44+ cells within polyclonal conventional CD4+ T cells (Poly. FOXP3−) and Tregs (Poly. FOXP3+) gated as in Fig. S5 A, isolated from MLNs (C) and spleens (D) of BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer (n = 5–9 mice from two independent experiments). (E) Weight-to-length ratio of colons isolated from BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer (n = 6–9 mice from two independent experiments). (F) Survival curve of BM chimeras from Fig. 6, D and E, (n = 5 mice from two independent experiments). Color code as in Fig. 6 E. Note that two OVA+Cldn1−/− mice died in the first experiment and one OVA+Cldn1−/− mouse died in the second experiment. Statistical analysis in B–E was performed using one-way ANOVA with Tukey’s multiple comparisons test and in F using the log-rank (Mantel–Cox) test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, ns = not significant. All mice were bred on the B6 background except for Aire−/− mice, which were bred on the BALB/c background. Littermates were used as controls. In A and B, 6-wk-old WT B6 mice from JAX were used as negative controls and ∼30-wk-old Aire−/− mice were used as positive controls. MLNs, mesenteric lymph nodes.

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