Volume 120, Issue 4, Pages 914-924 (March 2001) Murine ileitis after intracellular parasite infection is controlled by TGF-β–producing intraepithelial lymphocytes  Dominique Buzoni–Gatel, Hajer Debbabi, Franck J.D. Mennechet, Valentina Martin, Anne C. Lepage, Joseph D. Schwartzman, Lloyd H. Kasper  Gastroenterology  Volume 120, Issue 4, Pages 914-924 (March 2001) DOI: 10.1053/gast.2001.22432a Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Inflammation of the small intestine after adoptive transfer of IELs and oral infection with T. gondii. Four days after adoptive transfer of (A) unprimed or (B) primed IELs (2 × 106/mouse), C57BL/6 mice were orally challenged with T. gondii and monitored for pathology 7 days after infection. Results are representative of a total of 6 animals per group. At least 4 further experiments gave similar results. Gastroenterology 2001 120, 914-924DOI: (10.1053/gast.2001.22432a) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Histologic changes in the small intestine (ileum) of C57BL/6 mice after adoptive transfer of IELs and peroral challenge with T. gondii. Primed or unprimed IELs (2 × 106) were adoptively transferred into recipient mice. Four days after the adoptive transfer of IELs, recipient C57BL/6 mice were challenged with 30 cysts. Seven days later, histologic studies were performed on the small intestines. All slides were stained with H&E. (A) Sections (3 μm) and photomicrographs of ileum from C57Bl/6 mice infected with T. gondii after they received unprimed IELs demonstrates reduced length and increased thickness of the villi, with dramatic leukocytic infiltrates into the lamina propria. (B) There is evidence of focal loss of superficial epithelial cells at the tip of villi, indicating the earliest stage of necrosis. In contrast, photomicrographs of ileum from mice infected with T. gondii after they received primed IELs reveal an almost normal epithelium (C) with a mild leukocytic infiltrate in the lamina propria compared with normal noninfected intestine (D). Six mice were used in each experimental group. Gastroenterology 2001 120, 914-924DOI: (10.1053/gast.2001.22432a) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 mRNA chemokine production by T. gondii–infected ICcl2 enterocytes. Polarized ICcl2 epithelial cells were infected at the apical surface with tachyzoites from the RH strain of T. gondii. (A) Chemokine RNA was analyzed at different times after infection using a multiprobe RNase protection. Results are expressed as relative densitometry units after normalization to GAPDH mRNA. (B) To ensure that proteins were translated, culture supernatants were assayed for MCP-1 by ELISA at different times after infection of the ICcl2 cell line. A coculture of both cell types was carried out in a Transwell system. The enterocyte cell line (ICcl2), either uninfected or infected, was plated on the lower surface of the filter, resulting in an inverted monolayer. IELs (unprimed, uIEL or primed, pIEL) were added to the upper part of the Transwell. (C) After 6 hours of coculture, the IELs were removed from the upper part of the Transwell by extensive washings and the enterocyte monolayer was examined for expression of chemokine mRNA. Results are representative of at least 4 independent experiments. Gastroenterology 2001 120, 914-924DOI: (10.1053/gast.2001.22432a) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of TGF-β on chemokine mRNA produced by ICcl2 cells infected with T. gondii. Primed IELs were incubated 2 hours before the culture with αTGFβ mAbs. Irrelevant antibodies were used to incubate the control group. IELs were then added to the upper part of the Transwell. The medium of this coculture was supplemented with αTGFβ mAbs. Chemokine mRNA expression by the ICcl2 cell line was assessed 4 hours after contact by RNase protection assay. A representative autoradiogram of RNase protection assay is shown on the left. Results are expressed as relative densitometry units after normalization to GAPDH mRNA and are representative of 2 separated experiments. Gastroenterology 2001 120, 914-924DOI: (10.1053/gast.2001.22432a) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Production of TGF-β and IFN-γ by (A and B) IELs and (C and D) splenocytes from C57BL/6 mice that were transferred with primed or unprimed IELs. Primed IELs were isolated from infected C57BL/6 mice at day 7 postinfection and were adoptively transferred to naive recipients. Control mice received unprimed IELs. The recipients were challenged at day 4 after the transfer. IELs and splenocytes from the recipient were collected at different times after the transfer and assayed for TGF-β and IFN-γ production in the absence of cocultured cells. Six mice were used as recipient mice; results are the mean of 6 wells and representative of 2 independent experiments. Gastroenterology 2001 120, 914-924DOI: (10.1053/gast.2001.22432a) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Inhibition of IFN-γ production by primed C57BL/6 splenocytes. Primed splenocytes were cultured with T. gondii tachyzoites at a ratio of 1:1 with or without homologous primed IELs. To reverse the inhibitory effect of primed IELs, αTGFβ mAbs were added to the IEL-splenocyte coculture. After 24 hours, the culture supernatant was measured for IFN-γ production. Results are the mean of 6 wells and are representative of 2 independent experiments. Gastroenterology 2001 120, 914-924DOI: (10.1053/gast.2001.22432a) Copyright © 2001 American Gastroenterological Association Terms and Conditions