Functional characterization and localization of cLP MP/DC subsets and T cell priming capacities of subsets 1–4. (A and B) Phagocytic capacities of subsets 1–4. cLP cells from C57BL/6 mice were incubated in the presence of fluorescent microbeads for 45 min and analyzed for their bead content. (A) Percentage of each subset containing at least one bead. (B) Percentage of each subset containing one bead, two beads, three beads, four beads, or more after pregating on total bead+ cells. Results are mean ± SD and are representative of two independent experiments with three mice each. (C) Allo-stimulatory capacities of C57BL/6 cLP subsets 1–4 and control splenic DCs and MPs co-cultured with CD4+ T cells from the spleen of BALB/c at the indicated ratios. (D and E) Proliferation of OT-II (D) and OT-I (E) cells after co-culture with graded doses of OVA protein–pulsed cLP subsets 1–4 and control splenic (SPL) DCs and MPs. Proliferation was determined by thymidine incorporation assay after 4 d (C and D) or 3 d (E) of culture. Results are expressed as mean ± SD of cpm triplicates and are representative of at least two independent experiments using pooled cells from 10 mice per experiment. Unpaired Student’s t tests were performed to compare subset 2 versus 3 (blue statistics) and subset 2 versus 4 (green). The comparison of subset 2 versus 1 never reached statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) In situ expression of CD11c (red) and F4/80 (green; top and middle) or CD11c (red) and CD11b (green; bottom) on C57BL/6 mouse colon sections. Hoechst counterstain is blue. Three mice were analyzed in three separate experiments. Bars, 50 µm.