Volume 133, Issue 2, Pages 547-558 (August 2007) Prevention of Autoimmune Gastritis in Mice Requires Extra-Thymic T-Cell Deletion and Suppression by Regulatory T Cells  Simon Read, Thea V. Hogan, Tricia D. Zwar, Paul A. Gleeson, Ian R. van Driel  Gastroenterology  Volume 133, Issue 2, Pages 547-558 (August 2007) DOI: 10.1053/j.gastro.2007.05.050 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Semiquantiative histopathological assessment of autoimmune gastritis. Examples of gastric mucosae with gastritis scores 0 to 6 are shown. The following criteria were used for scoring: score 0, (A, D, K) normal gastric mucosa, A is a low-magnification micrograph illustrating overall structure of normal oxyntic mucosa. D shows the structure of normal gastric units. K is a higher magnification of a portion of D showing the large pink-staining parietal cells predominantly in the middle of the gastric units (asterisk) and the blue/purple-stained zymogenic cells predominantly toward the base of the gastric units (arrowhead); score 1, (E, L) has very mild submucosal mononuclear cell infiltration throughout the glandular mucosa (arrow in L) or more substantial infiltration restricted to the glandular mucosa adjacent to the forestomach (not shown); score 2 (F, M) is characterized by mild submucosal mononuclear cell infiltration throughout the glandular mucosa accompanied by focal aggregates of mononuclear cells that impinge into the mucosal area (arrows in F, M) but no widespread depletion of differentiated cell types; score 3 (G, N) characterized by submucosal infiltration and mild, disseminated mononuclear cell infiltration of the mucosa accompanied by marked depletion of zymogenic cells in some areas of the oxyntic mucosa, although many areas retain zymogenic cells (B, G, arrowheads). Parietal cells still abound (N); score 4 (H, O), as for score 3 except that depletion of zymogenic cells is nearly complete and low-level hyperplasia is often seen; Score 5 (C, I, P), submucosal and mucosal mononuclear cell infiltrates, almost complete depletion of zymogenic cells, severe but not complete depletion of parietal cells and a very marked increase in immature cell types or mucous-rich cells usually resulting in substantial hyperplasia. Asterisks indicate residual parietal cells and arrowheads indicate immature cells; score 6 (J, Q), as for score 5 except near-complete loss of zymogenic and parietal cell types. Bar in A = 100 μm and is applicable to A through C. Bar in D = 100 μm and is applicable to D through J. Bar in K = 100 μm and is applicable to K through Q. Gastroenterology 2007 133, 547-558DOI: (10.1053/j.gastro.2007.05.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Treg cells are less capable of suppressing CD4+CD25− effector cells from H/Kβ−/− mice. CD25− and CD25+ cells were prepared from WT BALB/c and H/Kβ−/− mice. Female athymic BALB/cnu mice were injected intraperitoneally with 2 × 106 CD4+CD25− T cells from either WT or H/Kβ−/− mice either alone or together with 106 CD4+CD25+ T cells from WT BALB/c mice, as indicated. Mice were killed 8 weeks later, and their stomachs and ovaries were removed and examined for histological evidence of autoimmune gastritis and autoimmune ovarian disease. Gastritis scores of individual mice are shown. Filled circles indicate mice that developed autoimmune ovarian disease, and open circles indicate mice that had normal ovarian morphology. The plot shows data pooled from 2 independent experiments. Gastroenterology 2007 133, 547-558DOI: (10.1053/j.gastro.2007.05.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Protocol used for parking CD4+ T cells. (A) CD4+CD90.2+ cells were isolated from spleen and lymph node preparations from H/Kα−/− or H/Kβ−/− mice. In some experiments, CD4+ cells (5 × 107) from H/Kα−/− mice were transferred to nonirradiated BALB/c-CD90.1 congenic mice. In others, an equivalent number of CD4+ cells from H/Kβ−/− mice were transferred to sublethally irradiated (600 rad) BALB/c-CD90.1 congenic or H/Kβ−/−-CD90.1 mice as described in Materials and Methods. Six to 8 weeks later, CD90.2+ cells were re-purified from the recipients and transferred to athymic BALB/cnu recipients. After a further 8 weeks, the athymic recipients were killed, and stomachs were taken for histological examination. The numbers in the figure indicate the order of procedures. (B) Representative analysis of CD4+ cells from the BALB/c-CD90.1 mice that had received cells from H/Kβ−/− donors and were stained with antibodies to CD90.1 and CD90.2 and analyzed by flow cytometry. After 8 weeks, CD90.2+ cells constituted 50% to 80% of peripheral CD4+ T cells. T cells from H/Kβ−/− donor mice were not pooled before transfer to either the BALB/c-CD90.1, H/Kβ−/−-CD90.1, or BALB/cnu athymic recipients in these experiments so that each data point could be considered independently. The yield of H/Kα−/− T cells from the BALB/c-CD90.1 recipients was low because the recipients were not irradiated, therefore, it was necessary to pool T cells before transfer to the BALB/cnu athymic recipients. (C) Representative analysis of Foxp3+ Treg cells in parked CD4+ T cells. CD4+ T cells were isolated and analyzed for CD4 and Foxp3 expression before parking in a WT host (Before). After 8 weeks’ parking, the cells were reisolated and stained to detect CD4, CD90.2 and Foxp3 (After). The percentages indicate the proportion of Foxp3+ cells in the CD4+CD90.2+ population. These data are representative of the analysis of 3 separate transfers. Gastroenterology 2007 133, 547-558DOI: (10.1053/j.gastro.2007.05.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 The gastritis-inducing CD4+ T cell population from H/Kβ−/− mice can be rendered tolerant in the periphery of WT mice but retained the ability to induce gastritis in the absence of Treg cells. (Upper panel) CD4+CD90.2+ cells from H/Kβ−/− or WT mice, as indicated, and were transferred to irradiated BALB/c-CD90.1 congenic mice (WT) or H/Kβ−/−-CD90.1 mice as indicated. Eight weeks later, CD4+CD90.2+ cells of donor origin were repurified from the parking mice and were transferred to athymic BALB/cnu recipients. In some cases, whole CD4+ cells were transferred to athymic recipients (groups A-E), while in others, the CD4+ cells were depleted of CD25+ Treg cells before transfer to athymic mice (groups F-G). (--) indicates that CD4+ cells were transferred directly to athymic BALB/cnu mice without parking. Mice were killed 8 weeks later, and their stomachs were removed and examined for histological evidence of autoimmune gastritis. Gastritis scores of individual mice are shown. NS indicates no significant difference. P > .05 between groups indicated. (Lower panels) Representative tissue sections from mice in the experiments depicted in the upper panel. The group and score of sections are indicated. Parking of CD4+ T cells from H/Kβ−/− mice in WT mice results in most athymic recipients developing gastritis with scores of 0 (group B) rather than 6 (group A). The transfer of CD4+ T cells depleted of CD25+ Treg from H/Kβ−/− mice resulted in all mice developing a score of 6 (group F). The transfer of CD4+ T cells depleted of CD25+ Treg from parked H/Kβ−/− or unmanipulated WT mice resulted in many of the recipients developing gastritis with lower scores (groups G and H). Gastroenterology 2007 133, 547-558DOI: (10.1053/j.gastro.2007.05.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Gastritis-inducing T cells from H/Kα−/− mice can be rendered tolerant in the periphery of WT mice. CD4+CD90.2+ cells from H/Kα−/− were transferred to BALB/c-CD90.1 congenic mice (WT). Eight weeks later, CD90.2+ cells were repurified from the parking mice and transferred to athymic BALB/cnu recipients. (--) indicates that CD4+ cells were transferred directly to athymic BALB/cnu mice without parking. Mice were killed 8 weeks later, and their stomachs were removed and examined for histological evidence of autoimmune gastritis. Gastritis scores of individual mice are shown. Gastroenterology 2007 133, 547-558DOI: (10.1053/j.gastro.2007.05.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Highly pathogenic H+/K+ ATPase–specific T cells proliferate in the paragastric lymph node. Purified CD4+ cells from A23.H/Kα−/− mice were labelled with CFSE, and 2 × 106 cells were injected intravenously into BALB/c-CD90.1 congenic mice. Recipient mice were killed 4 or 7 days after transfer, as indicated, and single-cell suspensions were prepared from the spleen, inguinal lymph node, and paragastric lymph nodes. The stomachs were stained with antibodies to CD4 and the congenic marker CD90.2 and were analyzed by flow cytometry. The histograms show CFSE fluorescence and are gated on CD4+CD90.2+ lymphocytes. Percentages indicate the proportion of cells that remained undivided. The number of events displayed for each histogram was chosen to illustrate the number of divisions the cells had undergone in each organ and does not necessarily reflect the relative abundance of the A23 T cells in each organ. The plot shows data pooled from 2 independent experiments. Data are representative of approximately 7 experiments. Gastroenterology 2007 133, 547-558DOI: (10.1053/j.gastro.2007.05.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Highly pathogenic H+/K+ ATPase–specific T cells are rapidly depleted from the periphery. CD4+ cells from A23.H/Kα−/− mice were labelled with CFSE, and 2 × 106 cells were injected intravenously into BALB/c-CD90.1 congenic mice. Recipient mice were killed at the times indicated after transfer, and single-cell suspensions were prepared from the spleen, inguinal lymph node, and paragastric lymph nodes. The stomachs were stained with antibodies to CD4 and the congenic marker CD90.2 and were analyzed by flow cytometry. The number of A23 CD4+ T cells in each organ was enumerated, and the average for each organ is shown. Error bars represent the standard deviation for the total number of cells summed over all organs in the mice. ND indicates A23 cells were not detectable. The graph shows data pooled from 3 independent experiments. Gastroenterology 2007 133, 547-558DOI: (10.1053/j.gastro.2007.05.050) Copyright © 2007 AGA Institute Terms and Conditions