Large intestinal IgM⁺ B cells are characterized by low frequency of SHM and by production of IL-12 in response to CpG. (A) Expressions of IgA and CD138 versus B220 on large intestinal cells from day 0 (n = 6) and day 8 (n = 6) of DSS colitis are shown. (B and C) ELISPOT assay shows the number of IgA-secreting cells among total large intestinal cells at day 0 and 8 of DSS colitis (B). The data are summarized in C. (D) Expressions of activation-induced deaminase in purified B cells from the colon of day 0 (open bar, n = 16) and day 8 (gray bar, n = 15) after DSS treatment and from the spleen of day 0 (black bar, n = 8) are shown. *, P < 0.1; **, P < 0.05. (E) Analysis of sequences with mutations (splenic and colonic IgM⁺ B cells of normal WT mice) is depicted by pie charts. Numbers outside of each pie chart are the number of mutations/clone. The size of the wedge is proportional to the percentage of clones carrying that number of mutations. Inside each inner circle is the number of clones sequenced. Data from an individual spleen (as control) and two different normal large intestines are shown. (F) Mutation frequencies are calculated by the number of mutations per 10⁴ bp from splenic and large intestinal IgM⁺ B cells of WT mice. (G) Cells from the normal large intestine and spleen of WT mice were stimulated with 1 μM CpG for 17 h, including a 7-h culture period with GolgiStop. The stimulated cells were subjected to surface staining for detection of IgM and B220 and intracellular staining for detection of IL-12p70. Expression of IL-12p70 in the gated IgM⁺B220⁺ cell population is shown. (H) The mean percentage of IL-12–producing cells among IgM⁺ B cells from the large intestine (n = 6) and spleen (n = 6) of WT mice is shown. *, P < 0.001.