Autoimmune manifestations of Claudin 1–deficient mice. (A) Representative flow cytometry gating strategy of activated T cells from MLN and spleen. CD4⁺ T cells were gated as TCRB⁺CD4⁺CD8⁻ and further distinguished using congenic markers into OTII (CD45.1⁺CD45.2⁻) and polyclonal (Poly; CD45.1⁻CD45.2⁺) cells. Both OTII and polyclonal cells were further distinguished into conventional T cells (FOXP3⁻) and Tregs (FOXP3⁺). These populations were analyzed for CD62L and CD44 expression, with CD62L⁻CD44⁺ cells representing effector memory T cells. (B and C) Frequency of OTII cells within CD4⁺ T cells (B) and OTII Tregs within OTII cells (C) isolated from MLN of BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer, gated as in Fig. S5 A (mean ± SEM, n = 3–6 mice from two independent experiments). (D) Frequency of effector memory CD62L⁻CD44⁺ cells within conventional OTII cells (FOXP3⁻) isolated from MLN (left panel) and spleen (right panel) of BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer gated as in Fig. S5 A (mean ± SEM, n = 3–8 mice from two independent experiments). (E) Representative immunofluorescence images of ileal PCs stained by lysozyme (red) and DAPI (blue) of BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer. (F) Quantification of average PC count per crypt within ilea of BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer related to Fig. S5 E (mean ± SEM, n = 6–9 mice from two independent experiments). (G) Representative colon lengths of BM chimeras from Fig. 6, D and E, around 40 wk after BM transfer. (H) Detection of autoantibodies from sera of XCR1^iCreCldn1^fl/fl mice and age-matched Cldn1^fl/fl controls with an average age of ∼35 wk at the serum harvest. 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 individual tissues (K = kidney; L = liver; and S = stomach) that make up the composite slide. 6-week-old B6 WT and ∼40-wk-old OVA⁺Cldn1^−/− competitive BM chimeras from Fig. 6, D and E, were used as negative and positive controls, respectively. 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. (I) MFI of serum autoantibody detection within the entire image of the composite tissue and individual tissues from Fig. S5 H (mean ± SEM, n = 3–10 mice from two independent experiments). (J) Representative colon lengths of XCR1^iCreCldn1^fl/fl mice and age-matched Cldn1^fl/fl controls with an average age of ∼35 wk. (K) Weight-to-length ratio of colons isolated from XCR1^iCreCldn1^fl/fl mice and age-matched Cldn1^fl/fl controls with an average age of ∼35 wk (n = 6 mice from two independent experiments). (L) Survival curve of XCR1^iCreCldn1^fl/fl mice and age-matched Cldn1^fl/fl controls (n = 5–7 mice from two independent experiments). Color code as in Fig. S5 K. Statistical analysis in B–D and F was performed using one-way ANOVA with Tukey’s multiple comparisons test, in I and K using unpaired, two-tailed Student’s t test, and in L using the log-rank (Mantel–Cox) test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns = not significant. All mice were bred on the B6 background. Littermates were used as controls. In H and I, 6-wk-old WT B6 mice from JAX were used as negative controls and ∼4-wk-old competitive BM chimeras from Fig. 6, D and E were used as positive controls. MLN, mesenteric lymph node.