Figure 1. CCR7⁺ DC subset in MLN was necessary for induction of gut-homing receptor expression by donor T cells.

(A) Mononuclear cells of MLN and PLN from BALB/c mice 7 days after treatment with PBS or anti-CD3 were stained for CD11c, CCR7, and CD103. Gated CD11c⁺ cells are shown as CCR7 versus CD103 staining. A representative flow cytometry pattern and the percentage and yield (mean ± SE, N=4) of CCR7⁺ DC subsets and total CD11c⁺ cell yield from 1 of 4 replicate experiments are shown. (B) CD11c⁺CD103⁺ DCs from MLN of untreated BALB/c mice were sorted into CCR7⁺ and CCR7⁻ subsets. Each subset (0.05 × 10⁶) was co-cultured with C57BL/6 T cells (0.1 × 10⁶) for 4 days. Thereafter, cultured cells were stained with CD4, CD8, α4β7, and CCR9. Gated CD4⁺ or CD8⁺ T cells are shown as CD4 or CD8 vs α4β7 or CCR9 staining. A representative flow cytometry pattern and the percentages (mean ± SE) of α4β7⁺ or CCR9⁺ cells among CD4⁺ or CD8⁺ T cells from 1 of 3 replicate experiments are shown. (C) Total RNA was isolated from sorted CCR7⁺ and CCR7⁻ DCs from MLN and PLN of untreated BALB/c mice, and RALDH expression was analyzed by real-time PCR. Relative expression levels (mean ± SE) from 3 replicate experiments are shown. (D–F) Seven days after treatment with PBS or anti-CD3, CD11c⁺ DCs from host-type BALB/c MLN were sorted. The sorted CD11c⁺ cells (0.1 × 10⁶) from PBS- or anti-CD3-treated mice were first co-cultured with splenic CD4⁺ and CD8⁺ T cells (0.2 × 10⁶) from donor-type C57BL/6 (D); then, the sorted CD11c⁺ DCs from anti-CD3-treated mice were co-cultured with the donor T cells in the presence or absence of RA (E); finally, the sorted CD11c⁺ DCs from PBS-treated control mice were cultured with the donor T cells in the presence or absence of RA antagonist LE135 (1 µM) (E). After culture for 4 days, expression of α4β7 and CCR9 by the cultured T cells was analyzed by flow cytometry. Percentages (mean ± SE, N = 4) of α4β7⁺ or CCR9⁺ cells among CD4⁺ or CD8⁺ T cells are shown.