Colonic tissue sections. Colonic tissue sections from (A) a normal BALB/c mouse and (B) an IL-10^−/− mouse with grade 3 histopathology. (C) Presence of crypt abscess (arrow) in colonic tissue section from an IL-10^−/− mouse with intestinal pathology. (D) Colonic weight and (E) numbers of IELs from normal BALB/c mice and age-matched IL-10^−/− mice with intestinal pathology; mean values ± sem of 18 mice.

Percent CD69 and ICOS expression. (A) Percent CD69 and ICOS expression between CD4⁺ and (B) CD8α⁺ cIELs in fresh isolates from BALB/c and IL-10^−/− mice. (C) Note that ICOS is expressed on few cIELs in normal mice despite the expression of CD69 but is up-regulated in IL-10^−/− mice. (D) IL-17⁺ and IFN-γ⁺ cells among CD4⁺ and CD8α⁺ cIELs in fresh isolates from normal BALB/c and IL-10^−/− mice. Pearson correlation analysis of (E) ICOS expression versus pathology score and (F) IL-17 expression versus ICOS expression in cIELs from IL-10^−/− mice with intestinal pathology. Data are representative of findings from five to 12 mice.

The effects of IL-23 and IL-10 exposure on cIELs from IL-10^−/− mice were retained following recall stimulation. For recall stimulation, cIELs from IL-10^−/− mice stimulated in vitro for 24 h with 1 μg/ml plate-bound anti-CD3 antibody in the presence of medium, medium plus rIL-23, or medium plus rIL-10. Recall stimulation in the presence of rIL-23 increased the percent of IL-17⁺ cells in the ICOS⁺ population, whereas IL-10 exposure decreased the percent of IL-17⁺ cells. Similarly, the MFI of ICOS expression was increased by exposure to rIL-23 and decreased by IL-10 exposure relative to cells cultured in medium alone. The high percentage of IL-17⁺ IFN-γ⁺-positive cells in the ICOS^– population may be a result of autofluorescence of dead cells. Data are representative of two independent experiments.

ICOS and IL-17 expression are up-regulated by IL-23 and suppressed by IL-10. cIELs from IL-10^−/− mice were cultured in the absence of CD3 stimulation for 24 h in the presence of media, media plus rIL-23, or media plus rIL-10. (A) rIL-23 increased the percent of IL-17-producing cells and the (C) MFI of ICOS expression. IL-10 supplementation resulted in (A and B) a decrease in ICOS expression and (C) a decrease in the MFI of ICOS expression. (D) rIL-23 supplementation resulted in an overall increase in the percent of ICOS⁺, IL-17⁺ cells, whereas IL-10 supplementation resulted in a decrease in the percent of ICOS⁺, IL-17⁺ cells. (E) Levels of ICOS and IL-17 gene expression were lower in cIELs from IL-10^−/− mice following culture with rIL-10. Data are derived from three to five independent experiments.

IL-17 expression and pathology are regulated by ICOS signaling. (A) Bone marrow DCs express the B7RP-1 ICOS ligand. (B) cIELs from normal BALB/c mice cultured with IL-17-inducing cocktail in the presence of DCs (media plus DCs) produced more IL-17A as determined by ELISA than cIELs cultured with IL-17-producing cocktail alone (media). No IL-17A was produced by DCs by themselves when cultured with IL-17-inducing cocktail (DCs plus media); data are mean values of three replicate samples. (C) cIELs cultured in media plus DCs resulted in a statistically significant increase in IL-17A production. Culture of cIELs from ICOS^−/− mice with B7RP-1⁺ DCs failed to result in elevated levels of IL-17A production. Data for A–C are representative of two to three independent experiments. (D) IL-10^−/− mice treated for 10 days with 10 μg anti-ICOS antibody (n=4) have statistically significant, lower pathology scores compared with mice treated with 10 μg isotype control Ig (n=4).

The B7RP-1 ICOS ligand is up-regulated on nonhematopoietic cells and leukocytes in the intestinal epithelium of IL-10^−/− mice. (A) Freshly isolated cells consisting of cIELs and enterocytes from the colonic epithelium of normal BALB/c and IL-10^−/− mice were stained for expression of CD45 LCA and B7RP-1. Cells were defined as hematopoietic cells based on CD45 LCA expression or nonhematopoietic cells based on a lack of CD45 LCA expression. Three populations (Gates 1, 2, and 3) in cell isolates from each animal type were defined by properties of forward or side scatter (FSC/SSC, respectively) by flow cytometry. Two notable differences were observed in normal versus IL-10^−/− mice. First, there was a large percent of large granular LCA⁺ cells in the Gate 2 population of IL-10^−/− mice that did not exist in normal animals. Second, approximately one-half of all of the cells in the Gate 3 population of IL-10^−/− mice was B7RP-1⁺ cells, which consisted of roughly equivalent percentages of hematopoietic and nonhematopoietic cells. To characterize the cells in Gate 2, freshly isolated cIELs from IL-10^−/− mice were stained for CD3 and ICOS expression. (B) The majority of those cells included CD3⁺, ICOS⁺, and LCA⁺ cells, indicating that they were activated T cells. Additionally, cells from normal and IL-10^−/− mice were stained using the G8.8 antibody that reacts with Ep-CAM on epithelial cells and some APCs [31, 32]. Cells from normal mice in Gate 3 were almost entirely G8.8⁺, LCA^– epithelial cells, whereas some cells in Gate 3 from IL-10^−/− mice were G8.8⁺, LCA⁺ leukocytes that were probably macrophages or DCs. This implies that there is a loss of epithelial cell homeostasis in IL-10^−/− with colonic pathology. Data are representative of two to three independent experiments.