Nonlittermate Rip2^−/−Rag1^−/− mice are not protected from pathology by regulatory CD45RB^Low T cells. Nonlittermate Rip2^−/−Rag1^−/− and Rag1^−/− mice were injected i.p. with 0.5 × 10⁶ CD45RB^High with or without 0.25 × 10⁶ CD45RB^Low CD4 T cells and sacrificed at week 4. (A) Colons were fixed, H&E stained, and scored for colitis severity. Data from three experiments were pooled. (B and C) Spleen, colon LP, and MLNs were harvested, and isolated cells were counted, restimulated with plate bound anti-CD3/anti-CD28 antibody, and analyzed by flow cytometry to determine tissue Foxp3⁺ (C) and colon IFN-γ⁺ T cell numbers (B). Data from two experiments were pooled with 4–12 mice per group. (D) Proximal colon explants were cultured for 18 h, and supernatants were analyzed by ELISA for cytokine production. Data from two experiments were pooled with three to seven samples per group. (A–D) Mean ± SEM is shown with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 using a one-way ANOVA and Tukey’s post-hoc analysis. (E) Proximal colon explants were cultured for 18 h, and supernatants were analyzed by Bio-Plex assay for cytokine production. Data are one representative of three experiments with two samples per group. *, samples below minimum level of detection; #, samples above maximal level of detection.