until analysis. Lipocalin-2 levels were determined in the supernatants using the duoset murine Lcn-2 ELISA kit(R&D Systems, Minneapolis, MN) following the manufacturers instructions FITC Dextran Assay to assess intestinal permeability. mice were injected with either one dose or two doses of anti-CTLA4 or isotype control mAbs(administration as detailed above) and were water-starved overnight, 2 days following their last i.p. mAb administration. The following day, mice were orally administered with 0.44 mgg body weight of a 100 mg/ml solution of FITC-dextran(FD4, Sigma)in PBS (pH 7.4). Four hrs later, blood was collected from each mouse by cardiac puncture. Blood was allowed to clot overnight at 4C, then subsequently centrifuged at 3,000 rpm for 20 minutes to collect the serum. Dilutions of FITC dextran in PBS, and separately in pooled mouse serum were used as a standard curve, with serum from mice not administered FITC-dextran used to determine the background. absorbance of 100ul serum(diluted in PBS) was measured by microplate reader with excitation and emission filters set at 485 nm(20 nm band width) and 528 nm(20 nm band width), respectively(19) Experiments were performed at least twice, independently, with each read performed in duplicate Histology of gut tissue. The whole small intestine(duodenum, jejunum and ileum) and the colon were removed, cleaned from feces and fixed in 4% PFA for 2h. Rehydratation of the tissue was performed in 15% sucrose for Ih and in 30% sucrose overnight. Small intestines or colons were cut longitudinally, with the resulting ribbons rolled, then embedded in optimum cutting temperature(OCT) compound(Sakura), snap frozen, and longitudinal 6 um sections were prepared. For histological analysis, longitudinal sections were counterstained with hematoxilin and eosin. For histological quantitative analysis, inflammatory foci, appearance of the submucosa, length of villi, and the thickness of lamina propria were scored for each section by a pathologist. Score distribution between the groups were compared by proportional odds logistic regression using r software Intestinal MUC2 and Ki67 staining and evaluation. Intestinal tissue was fixed in freshly prepared Methacarn solution, subsequently incubated in methanol and toluene and embedded in paraffin For immunohistochemistry, 5 um-thick tissue sections were placed on Superfrost Plus8 until analysis. Lipocalin-2 levels were determined in the supernatants using the Duoset murine Lcn-2 ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions. FITC Dextran Assay to assess intestinal permeability. Mice were injected with either one dose or two doses of anti-CTLA4 or isotype control mAbs (administration as detailed above), and were water-starved overnight, 2 days following their last i.p. mAb administration. The following day, mice were orally administered with 0.44 mg/g body weight of a 100 mg/ml solution of FITC-dextran (FD4, Sigma) in PBS (pH 7.4). Four hrs later, blood was collected from each mouse by cardiac puncture. Blood was allowed to clot overnight at 4°C, then subsequently centrifuged at 3,000 rpm for 20 minutes to collect the serum. Dilutions of FITC￾dextran in PBS, and separately in pooled mouse serum were used as a standard curve, with serum from mice not administered FITC-dextran used to determine the background. Absorbance of 100ȝl serum (diluted in PBS) was measured by microplate reader with excitation and emission filters set at 485 nm (20 nm band width) and 528 nm (20 nm band width), respectively (ͷͿ). Experiments were performed at least twice, independently, with each read performed in duplicate. Histology of gut tissue. The whole small intestine (duodenum, jejunum and ileum) and the colon were removed, cleaned from feces and fixed in 4% PFA for 2h. Rehydratation of the tissue was performed in 15% sucrose for 1h and in 30% sucrose overnight. Small intestines or colons were cut longitudinally, with the resulting ribbons rolled, then embedded in optimum cutting temperature (OCT) compound (Sakura), snap frozen, and longitudinal 6 μm sections were prepared. For histological analysis, longitudinal sections were counterstained with hematoxilin and eosin. For histological quantitative analysis, inflammatory foci, appearance of the submucosa, length of villi, and the thickness of lamina propria were scored for each section by a pathologist. Score distribution between the groups were compared by proportional odds logistic regression using R software. Intestinal MUC2 and Ki67 staining and evaluation. Intestinal tissue was fixed in freshly prepared Methacarn solution, subsequently incubated in methanol and toluene and embedded in paraffin. For immunohistochemistry, 5 μm-thick tissue sections were placed on Superfrost Plus