Adhesion molecules expressed on homing lymphocytes in model intestinal epithelia  Takeshi Shibahara, Mustapha Si-Tahar, Sunil K. Shaw, James L. Madara  Gastroenterology  Volume 118, Issue 2, Pages 289-298 (February 2000) DOI: 10.1016/S0016-5085(00)70211-3 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Phenotype of homed lymphocytes is α4low, αEβ7high, and αXhigh. Lymphocytes (“total” lymphocytes; top panels) were allowed to home into the epithelial monolayers for 4 hours, and nonhomed (middle panels) and homed lymphocytes (bottom panels) were collected respectively. The expression of CD3, the integrin subunits β1, α4, αEβ7, β7, αL, αM, αX, β2, and other representative adhesion molecules ICAM-1, CD44, and CD47 were examined. The gray histograms show the expression of CD3, α4, αEβ7, αL, and αX, whereas the open histograms show the negative controls obtained with an irrelevant primary antibody. Because all lymphocytes were prelabeled with CMFDA, they were successfully separated from the contamination of debris and epithelial cells when analyzed. Homed lymphocytes predominantly contained the α4low, αEβ7high, and αXhigh populations (arrows). These histograms are representative of 5–12 experiments. (B) The diversity of phenotype between nonhomed and homed lymphocytes. Ratio of expression was calculated by dividing the mean fluorescence intensity (MFI) of each molecule on homed lymphocytes by the MFI on nonhomed lymphocytes. Although homed lymphocytes tended to highly express various adhesion molecules, high expression of αX and αEβ7 (and β7) on homed lymphocytes was the most prominent. The data are the mean ± SD obtained from 5–12 separate experiments. Gastroenterology 2000 118, 289-298DOI: (10.1016/S0016-5085(00)70211-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Lymphocytes with a high-level expression of αX or αEβ7 show a high homing capacity into the epithelial monolayers. Lymphocytes were sorted into the subpopulations of either high- or low-level expression of αX (αEβ7, or αM) using FACS Vantage. After washing out the monoclonal antibody for 5–7 days, those subpopulations still maintained their phenotypes (inset: the gray histograms represent subpopulations with high-level expression; the open histograms represent subpopulations with low-level expression). (A and B) Lymphocytes highly expressing αX or αEβ7 had a greater capacity of homing, respectively, (C) whereas no significant difference was observed between αM high and low subpopulations. Data were normalized as described previously.30 Data are the mean ± SD and are representative of 3 separate experiments. (*P < 0.05.) Gastroenterology 2000 118, 289-298DOI: (10.1016/S0016-5085(00)70211-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 αEβ7 and ICAM-1 expressions are up-regulated on lymphocytes during their retention within the epithelial monolayers. (A) CMFDA-labeled lymphocytes were allowed to home into the epithelial monolayers for 4 hours. Subsequently, the basolateral medium of monolayers was replaced with fresh medium void of lymphocytes, and monolayers were further incubated for 24 hours. Lymphocytes retained in the monolayers (“retained”; upper panels) and lymphocytes exited basolaterally (“exited”; bottom panels) were collected, and each phenotype was examined by flow cytometry. The open histograms indicate the expression of each molecule on homed lymphocytes at 4 hours, and the gray histograms indicate the expression of the same molecule on “retained” or “exited” lymphocytes after a 24-hour incubation. Expression of CD3, αEβ7, and ICAM-1 on “retained” lymphocytes was significantly increased compared with homed lymphocytes (arrows). The histograms are representative of 3–5 experiments. (B) The diversity of the phenotype between homed and “retained” lymphocytes. Ratio of expression was calculated by dividing the MFI of each molecule on retained lymphocytes by the MFI on homed lymphocytes. Note that the slight rightward change of CD3 and ICAM-1 on retained lymphocytes compared with homed lymphocytes in Figure 3A indicates a large change in MFI caused by the log scale. The data are the mean ± SD obtained from 3–5 separate experiments. Gastroenterology 2000 118, 289-298DOI: (10.1016/S0016-5085(00)70211-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 αEβ7 expression on lymphocytes is up-regulated by epithelial-derived hTGF-β1. Lymphocytes and T84 monolayers were cocultured for 48 hours without their direct contact as described in Materials and Methods. In that context, only soluble factors could diffuse through the filters. Then the alteration of lymphocyte phenotype was examined. The number in each histogram indicates the percentage of cells expressing αEβ7. The expression of αEβ7 on lymphocytes was significantly up-regulated by coculture (arrow), and this up-regulation was completely inhibited by an Ab to hTGF-β1 (2 μg/mL). The histograms are representative of 3 separate experiments. Gastroenterology 2000 118, 289-298DOI: (10.1016/S0016-5085(00)70211-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 TGF-β1 down-regulates αX on lymphocytes. Lymphocytes were incubated with 2 ng/mL hTGF-β1 for 1–5 days. The gray histogram shows αX expression after 5 days' TGF-β1 exposure. The bold open histogram show αX expression on untreated lymphocytes, and the dotted open histogram indicates the background fluorescence obtained with an irrelevant primary antibody. These histograms are representative of 3 experiments. Gastroenterology 2000 118, 289-298DOI: (10.1016/S0016-5085(00)70211-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Phenotypic comparison of fresh IELs and PBLs. Fresh human IELs and PBLs were isolated as described in Materials and Methods. Only CD3-positive cells were analyzed by double staining. The gray histograms show the expression of each molecule on lymphocytes; and the open histograms show the negative controls obtained with an irrelevant primary antibody. Freshly isolated IELs expressed high levels of αEβ7 and ICAM-1 compared with PBLs. Although the expression of αX was observed only on a subset of fresh IELs (4.5% ± 0.6%), it was remarkably up-regulated by in vitro stimulation by alloreactive cells (combination of irradiated JY cells, irradiated PBLs and phytohemagglutinin; arrow). The histograms are representative of 3 experiments. Gastroenterology 2000 118, 289-298DOI: (10.1016/S0016-5085(00)70211-3) Copyright © 2000 American Gastroenterological Association Terms and Conditions