Volume 118, Issue 5, Pages 880-891 (May 2000) Intestinal inflammation observed in IL-2R/IL-2 mutant mice is associated with impaired intestinal T lymphopoiesis  Philippe Poussier*,‡,§, Terri Ning*, Jun Chen*, Diponkar Banerjee∥, Michael Julius§  Gastroenterology  Volume 118, Issue 5, Pages 880-891 (May 2000) DOI: 10.1016/S0016-5085(00)70174-0 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Surface phenotype of T-cell subsets developing in 6–7-week-old (A, C, E, and G) IL-2Rβ+/− and (B, D, F, and H) IL-2Rβ−/− mice. Numbers represent the proportion of cell subsets defined by the differential expression of the various surface markers in these 2 animals and are representative of those observed in other IL-2Rβ+/− (n = 7) and IL-2Rβ−/− (n = 3) mice. Thymocytes and iIELs were isolated; stained with fluorochrome-conjugated MAbs specific for CD4, CD8α, CD8β, TCRαβ, and TCRγδ; and analyzed by 3-color immunofluorescence and flow cytometry using a FACScan, as described by Poussier et al.1 Gastroenterology 2000 118, 880-891DOI: (10.1016/S0016-5085(00)70174-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Phenotypic analysis of splenocytes and iIELs derived from representative euthymic (middle column) and athymic (left and right columns) irradiated RAG-2−/− recipients of IL-2Rβ−/− (middle and right columns) and IL-2Rβ+/− (left column) bone marrow. The numbers of chimeric animals of each type that were studied varied from 6 to 10. The preparation of radiation chimeras, isolation of MNCs, their staining with fluorochrome-conjugated MAbs, and flow-cytometric analysis were performed as described in Materials and Methods and by Poussier et al.1 Gastroenterology 2000 118, 880-891DOI: (10.1016/S0016-5085(00)70174-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Phenotypic analysis of CD3−, mIg− MNCs derived from the intestinal epithelium and LP of representative euthymic (n = 12) and athymic (n = 9) animals. The preparation of fetal liver radiation chimeras, isolation of MNCs and their staining with fluorochrome-conjugated MAbs, and flow-cytometric analysis using 4-color immunofluorescence and a FACScalibur were performed as described in Materials and Methods. (A) CD3−, mIg− LP MNCs (top) and iIELs (bottom) isolated from a euthymic mouse (left), and an athymic fetal liver radiation chimera (right), stained with phycoerythrin (PE)-conjugated anti-CD4 and biotinylated anti-CD8 followed by streptavidin–PE/Texas red tandem. (B) Unfractionated LP MNCs (left), isolated from a euthymic animal and stained with PE-conjugated anti-Kit and a combination of fluorescein isothiocyanate–labeled anti-CD3 and anti-mouse Ig. Kit+, CD3−, mIg− LP MNCs (right) isolated from the same animal and stained with allophycocyanin-conjugated anti-CD4 and biotinylated anti-CD8 followed by streptavidin–PE/Texas red tandem. Numbers represent the proportion of cell subsets defined by the differential expression of the various surface markers. Gastroenterology 2000 118, 880-891DOI: (10.1016/S0016-5085(00)70174-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Histological and immunohistochemical analysis of CPs in unmanipulated 6-week-old IL-2Rβ+/− (left) and IL-2Rβ−/− (right) mice. (A and B) H&E stain of a CP. (C and D) CD3 expression in CP MNCs. (Original magnifications 200×.) Gastroenterology 2000 118, 880-891DOI: (10.1016/S0016-5085(00)70174-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Histological analysis of the small and large bowel obtained from unmanipulated 6-week-old (A, C, and G) IL-2Rβ+/− and (B, D, and H ) IL-2Rβ−/− mice, and from (E and I ) athymic and (F and J ) euthymic irradiated RAG-2−/− mice reconstituted with bone marrow from IL-2Rβ−/− donors. (A and B) Small bowel villi and crypts. (C and D) Paneth cells (arrow) at crypt bases. Absence of red granules, increased mitotic activity, and presence of apoptotic cells (arrow) in the IL-2Rβ−/− mouse. (E and F) Small bowel mucosa of irradiated RAG-2−/− chimeras reconstituted with bone marrow cells from IL-2Rβ−/− donors. (G and H ) Large bowel histology. Mucosal hyperplasia, increased number of lymphocytes, and crypt abscess formation (arrow) in the LP of the IL-2Rβ−/− mouse. (I and J ) Large bowel mucosa of RAG-2−/− chimeras reconstituted with bone marrow cells from IL-2Rβ−/− donors. LP infiltration by lymphocytes. (H&E stain; original magnifications: A, B, E, and F, 100×; C and D, 400×; G–J, 200×.) Gastroenterology 2000 118, 880-891DOI: (10.1016/S0016-5085(00)70174-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Histological analysis of the small and large bowel obtained from (A, C, and E ) euthymic and (B, D, and F) athymic irradiated RAG-2−/− mice reconstituted with bone marrow from (A and B) IL-2−/− and (C–F) IL-2Rα−/− donors. (A and B) Large bowel mucosa. Mucosal hyperplasia, increased number of lymphocytes, and crypt abscess formation in the LP of the euthymic chimera. (C and D) Small bowel mucosa. Crypt hyperplasia and increased number of lymphocytes in the LP of the euthymic chimera. (E and F) Large bowel histology. Mucosal hyperplasia and the LP infiltration by lymphocytes in the euthymic chimera. (H&E stain; original magnifications: A–F, 100×.) Gastroenterology 2000 118, 880-891DOI: (10.1016/S0016-5085(00)70174-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 CD3ϵ expression in CP MNCs (original magnification 200×) of (A, C, and E ) euthymic and (B, D, and F) athymic irradiated RAG-2−/− mice reconstituted with bone marrow from (A and B) IL-2−/−, (C and D) IL-2Rα−/−, and (E and F) IL-2Rβ−/− donors. Gastroenterology 2000 118, 880-891DOI: (10.1016/S0016-5085(00)70174-0) Copyright © 2000 American Gastroenterological Association Terms and Conditions