Figure 3. Citrobacter rodentium-induced adult CD4⁺ LTi cell responses are dependent on IL-23.

Il23a^+/+ and Il23a^−/− mice were infected with C. rodentium on day 0 and sacrificed on day 8. (A) Frequency of CD4⁺ LTi cells in the mLN of naïve and infected mice. Populations are gated on live CD3⁻ CD5⁻ CD90⁺ CD4⁺ cells. (B) Frequency of ex vivo stimulated IL-22⁺ CD4⁺ LTi cells in the mLN. Populations are gated on live CD3⁻ CD5⁻ CD90⁺ CD4⁺ cells. All data are representative of 2 or more independent experiments with a minimum of 3–4 mice per group. Naïve Rag1^−/− mouse splenocytes were cultured overnight with or without rIL-23. (C) Frequency of IL-22⁺ cells with gating on live cells. (D) Frequency of CD90⁺ CD4⁺ cells in the IL-22⁺ gated population of (C). (E) The IL-22⁺ CD90⁺ CD4⁺ population of (D) was stained with c-kit, CD127, CD25, CCR6 and RORγt antibodies (bold black lines) and corresponding isotype and negative control antibodies (solid grey histograms). (F) CD4⁺ LTi cells were purified from naïve Rag1^−/− splenocytes and stained with CD4, CD11c and B220 antibodies. Fold induction of (G) Id2 and (H) Il23r mRNA in purified CD4⁺ LTi cells relative to total unfractionated Rag1^−/− splenocytes (SPL). (I) IL-22 protein in culture supernatants from purified CD4⁺ LTi cells stimulated in the presence or absence of rIL-23. All in vitro data are representative of 2 or more independent experiments with a minimum of 2–3 replicate wells. Data shown are the mean ± SEM. * p < 0.05 ** p < 0.01. See Figure S2 for additional data.