Volume 131, Issue 2, Pages 510-524 (August 2006) Autoimmune-Mediated Intestinal Inflammation–Impact and Regulation of Antigen- Specific CD8+ T Cells  Astrid Maria Westendorf, Diana Fleissner, Stefanie Deppenmeier, Achim Dieter Gruber, Dunja Bruder, Wiebke Hansen, Roland Liblau, Jan Buer  Gastroenterology  Volume 131, Issue 2, Pages 510-524 (August 2006) DOI: 10.1053/j.gastro.2006.05.015 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Phenotype of CD8+Vβ8+ T cells in the periphery of VILLIN-HA × TCR-HA mice. (A) Lymphocytes from MLN and spleen of VILLIN-HA × CL4-TCR and CL4-TCR control mice were isolated and stained for CD8 and Vβ8 expression to measure the percentage of transgenic T cells in the different compartments. (B) HA specificity of CD8+Vβ8+ T cells from MLN were identified by using an H-2Kd: HA512-520 pentamer (H-2Kd IYSTVASSL). (C) For analysis of activation status, MLN cells were additionally stained with CD25, CD45RB, CD62L, and CD69 antibodies. Cells were gated on CD8 and Vβ8 expression and analyzed regarding the expression of the HA-specific TCR and the different activation markers by FACS. (D) Lymphocytes from the MLN of CL4-TCR and VILLIN-HA × TCR-HA transgenic mice were isolated and used for in vitro proliferation assays in the presence or absence of 10 μg/mL HA512-520 peptide. Proliferation was measured by 3[H] thymidine incorporation. For A, B, and C, 1 representative experiment from 3 independent experiments (each group n = 2 per experiment) with similar results is shown. For C, mean values of 3 independent experiments (each group n = 6 total) are depicted. Gastroenterology 2006 131, 510-524DOI: (10.1053/j.gastro.2006.05.015) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 VILLIN-HA × CL4-TCR transgenic mice are characterized by infiltration of lymphocytes into the lamina propria and intestinal epithelium. (A) Intestinal villi were distended (arrow) by an increased number of lymphocytes (right panel) when compared with intestinal villi of CL4-TCR transgenic mice (left panel). Insets show anti-CD3 immunohistochemistry on paraffin-embedded tissues. Avidin-Streptavidin-Complex (ABC) method with diaminobenzidine as substrate (brown color) and hematoxylin counterstain (blue nuclei). (B) Increased number of lamina propria lymphocytes and intraepithelial lymphocytes. Lymphocytes were counted in H&E-stained sections and normalized per 100 enterocytes. Individual animals are indicated (▴) for double transgenic VILLIN-HA × CL4-TCR mice, (■) for CL4-TCR, and (•) for VILLIN-HA control mice. (C) The intensity of intestinal inflammation is expressed as a histological score that is defined in Table 1. The results of 2 independent experiments (each group n = 4 total) are depicted. Gastroenterology 2006 131, 510-524DOI: (10.1053/j.gastro.2006.05.015) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Phenotype of CD8+ T cells in the intestinal epithelium of VILLIN-HA × TCR-HA mice. IELs of VILLIN-HA × CL4-TCR and CL4-TCR control mice were isolated and stained for CD8α and Vβ8 expression to measure the percentage of transgenic T cells. One representative experiment from 3 independent experiments (each group n = 2 per experiment) with similar results is shown. Gastroenterology 2006 131, 510-524DOI: (10.1053/j.gastro.2006.05.015) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Increased Foxp3 expression in CD8+ and CD4+ T cells from VILLIN-HA × CL4-TCR transgenic mice. Lymphocytes from the MLN of CL4-TCR, VILLIN-HA, and VILLIN-HA transgenic mice were isolated and stained for the expression of CD8 and CD4 as well as for intracellular expression of Foxp3. Cells were gated for CD8 or CD4 expression and analyzed regarding the expression of Foxp3 by FACS. One representative experiment from 2 independent experiments (each group n = 3 per experiment) is shown. Gastroenterology 2006 131, 510-524DOI: (10.1053/j.gastro.2006.05.015) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Intestinal inflammation after adoptive transfer of CD8+Vβ8+ T cells. A total of 3–4 × 106 transgenic CD8+ T cells was adoptively transferred intravenously into VILLIN-HA transgenic mice and nontransgenic littermates. (A) Disease development was measured daily and is expressed in terms of body weight loss. (B) H&E staining of the paraffin-embedded tissues from the small intestine, cecum, and the colon was performed at day 4 after transfer and analyzed by histology. Gastroenterology 2006 131, 510-524DOI: (10.1053/j.gastro.2006.05.015) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Transgenic CD8+Vβ8+ T cells expand in the intestine and show an activated phenotype. Adoptive transfer was performed as described in Figure 5. At day 4 after transfer cells were isolated from the MLN, PPs, and intestinal epithelium and stained for the expression of CD8 and Vβ8, HA specificity of CD8+Vβ8+ T cells was identified by using a HA512-520 specific MHC class I pentamer (H-2Kd IYSTVASSL). For analysis of activation status, cells were additionally stained for the expression of CD25, CD69, CD45RB, and CD62L surface antigens. Cells were gated for CD8 and Vβ8 expression and analyzed regarding the previously indicated molecules by FACS. One representative experiment from 3 independent experiments (each group n = 2 per experiment) is shown. Gastroenterology 2006 131, 510-524DOI: (10.1053/j.gastro.2006.05.015) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Cotransfer of antigen-specific regulatory CD4+ T cells reduces the impact of intestinal inflammation. VILLIN-HA transgenic mice received 3 × 106 transgenic CD8+Vβ8+ T cells with 3 × 106 antigen-specific regulatory CD4+T cells from IG-HA × TCR-HA transgenic mice, 3 × 106 polyclonal regulatory CD4+ T cells from BALB/c mice, or 3 × 106 antigen-specific naive CD4+ T cells from TCR-HA transgenic mice. (A) At day 4 after transfer, H&E stainings of the paraffin-embedded tissues from the small intestine were analyzed. (B) Histological evaluations were performed as described in Table 1. Mean values of 1 independent experiment (each group n = 6 total) are depicted. (NS, not significant; **P < .01). Gastroenterology 2006 131, 510-524DOI: (10.1053/j.gastro.2006.05.015) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 8 Cotransfer of antigen-specific regulatory CD4+ T cells decreases the expansion of transgenic CD8+Vβ8+ T cells. Adoptive transfer was performed as described in Figure 7. Lymphocytes from MLN of VILLIN-HA transgenic recipient mice and nontransgenic littermates were isolated and stained for CD8 and Vβ8 expression to analyze the percentage of transgenic T cells. For measurement of activation status, cells were additionally stained for CD25 and CD62L surface molecules. Cells were gated on CD8 and Vβ8 expression and analyzed regarding the expression of the different activation markers by FACS. One representative experiment from 2 independent experiments (each group n = 3 per experiment) with similar results is shown. Gastroenterology 2006 131, 510-524DOI: (10.1053/j.gastro.2006.05.015) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions