Prevention of Autoimmune Gastritis in Mice Requires Extra-Thymic T-Cell Deletion and Suppression by Regulatory T Cells

GASTROENTEROLOGY 2007;133:547–558

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, and IAN R. VAN DRIEL

Background & Aims: Autoimmune gastritis is one of the most common autoimmune diseases and is caused by a CD4ⴙ T-cell response to the gastric Hⴙ/Kⴙ ATPase encoded by Atp4a and Atp4b (Hⴙ/Kⴙ ATPase). Here, we have elucidated events that result in immunological tolerance to the Hⴙ/Kⴙ ATPase and thus the prevention of autoimmune gastritis. Methods: T cells from Hⴙ/Kⴙ ATPase– deficient mice and Hⴙ/Kⴙ ATPase–specific T-cell receptor transgenic mice were purified and transferred to wild-type (WT) or Hⴙ/Kⴙ ATPase– deficient recipients to assess the impact of exposure to antigen on pathogenicity. Results: The CD4ⴙ T-cell population from Hⴙ/Kⴙ ATPase– deficient mice was highly effective at inducing gastritis when compared with T cells from WT mice and, as a population, was comparatively resistant to the suppressive activity of regulatory T cells. Exposing T cells from Hⴙ/Kⴙ ATPase– deficient mice to Hⴙ/Kⴙ ATPase in WT mice decreased their ability to induce gastritis and resulted in a population that could be more easily suppressed by Treg cells. Transfer of clonotypic antigen-inexperienced Hⴙ/Kⴙ ATPase– specific T cells into WT mice resulted in extra-thymic clonal deletion. Conclusions: Prevention of autoimmune gastritis requires the extra-thymic purging of highly autoaggressive Hⴙ/Kⴙ ATPase–specific T cells to produce a T-cell repertoire that is more susceptible to the suppressive activity of regulatory T cells. Taken together with recent published data describing the role of T-cell receptor signalling in the maintenance of regulatory T-cell populations, we propose that exposure of T cells to antigen in the periphery is able to both delete autoaggressive specificities and maintain regulatory T-cell activity, establishing a balance between pathogenicity and regulation.

ability of transgenic mice expressing T-cell receptors directed to the gastric autoantigens4,5 and mice deficient in the H⫹/K⫹ ATPase6 – 8 has allowed a thorough investigation of the events associated with the induction of immunological tolerance to this important autoantigen. A great deal of progress has been made in understanding the processes that lead to T-cell tolerance to selfantigens. Expression of antigens in the thymus may lead to deletion of autoreactive T cells.9,10 The range of antigens that are expressed in the thymus encompasses many antigens that are primarily expressed elsewhere, thus thymic negative selection plays a more substantial role in immunological tolerance than was originally thought.9,10 Still, not all T cells directed to self-antigens are disposed of in the thymus simply because not all self-antigens are presented in the thymus and negative selection itself may not be complete.11–13 Therefore, the activity of autoreactive T cells leaving the thymus must be regulated in secondary lymphoid tissues. Various extra-thymic tolerance mechanisms have been described including deletion, anergy, and regulation. The relative importance of these mechanisms has rarely been determined for an antigen that is targeted in an autoimmune disease. T cells from H⫹/K⫹ ATPase– deficient mice are able to cause more severe autoimmune gastritis when transferred to T-cell–lymphopenic mice and have heightened immune responses to peptides derived from the H⫹/K⫹ ATPase, when compared to the equivalent population from wild-type (WT) mice.7 This indicates that antigenspecific tolerance is at work in normal animals to quell the activity of H⫹/K⫹ ATPase–specific T cells.1 The H⫹/K⫹ ATPase is expressed in epithelia and in bone marrow-derived cells in the thymus.10,11 However, our recent data indicate that thymic events play little role in shaping the T-cell receptor repertoire directed to this

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Abbreviations used in this paper: Hⴙ/Kⴙ ATPase, gastric Hⴙ/Kⴙ ATPase encoded by Atp4a and Atp4b; H/K␣, Hⴙ/Kⴙ ATPase ␣ subunit encoded by Atp4a; H/K␤, the Hⴙ/Kⴙ ATPase ␤ subunit encoded by Atp4b; Treg cells, CD4ⴙCD25ⴙFoxp3ⴙ regulatory T cells; H/K␣ⴚ/ⴚ, H/K␣ⴚdeficient; H/K␤ⴚ/ⴚ, H/K␤ⴚdeficient; BALB/c-CD90.1, CByJ. PL(B6)-Thy1a/ScrJ; BALB/cnu, athymic CByJ.Cg-Foxn1nu; CFSE, carboxyfluoroscein succinimydyl ester; CFSE, carboxyfluoroscein succinimydyl ester; TCR, T cell receptor; WT, wild-type. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.05.050

utoimmune gastritis is one of the most common autoimmune diseases in man, and in its advanced stages, leads to pernicious anemia, which in turn is the most common cause of vitamin B12 deficiency.1,2 It is well established that autoimmune gastritis is the result of a CD4⫹ T-cell response to the ␣ and ␤ subunits of the gastric H⫹/K⫹ ATPase encoded by Atp4a and Atp4b (H⫹/K⫹ ATPase; H/K␣ and H/K␤) in both man3 and mouse models.1 This information coupled with the avail-

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Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia

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autoantigen12 raising the possibility that antigen expressed in extra-thymic tissue may play a role. Autoimmune gastritis was one of the first immune phenomena found to be influenced by CD4⫹CD25⫹Foxp3⫹ regulatory T cells (Treg cells), so clearly these cells play some role in protection from this disease.14,15 Since those initial observations, it has become apparent that Treg cells play important roles in the regulation of responses to autoantigens, tumor antigens, and foreign antigens.16 –19 In this article, we demonstrate that Treg cells are able to tame H⫹/K⫹ ATPase–specific T cells but only if the most highly pathogenic T cells are first purged from the repertoire by encounter with antigen derived from extra-thymic tissues.

Materials and Methods Mice

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BALB/cCrSlc,20 CByJ.PL(B6)-Thy1a/ScrJ (BALB/cCD90.1) congenic (jaxmice.jax.org, Thy1a encodes CD90.1 [Thy1.1]), H/K␣⫺/⫺ 8 and H/K␤⫺/⫺ 6 and A23 T cell receptor (TCR) transgenic5 mice have been previously described. All strains had been backcrossed at least 10 times to BALB/cCrSlc. H/K␤⫺/⫺ mice were crossed with BALB/ c-CD90.1 congenic mice to produce H/K␤⫺/⫺-CD90.1 mice. A23 TCR transgenic mice were crossed to H/K␣⫺/⫺ mice to generate A23.H/K␣⫺/⫺ mice. All experimental animals were maintained in conventional animal facilities at the University of Melbourne and the Bio21 Molecular Science and Biotechnology Institute. Athymic CByJ.Cg-Foxn1nu (BALB/cnu) mice (jaxmice.jax.org) were obtained from the Animal Resources Centre (Perth, Australia). The experiments described herein were approved by the University of Melbourne Animal Experimentation Ethics Committee.

Antibodies and Flow Cytometry Conjugated antibodies were purchased from BD PharMingen (San Diego, CA), except for the anti-Foxp3 Staining Set, which was obtained from eBioscience (San Diego, CA). Single-cell suspensions were analyzed for expression of cell surface markers, using a combination of the following antibodies: PerCP-conjugated anti-CD4 (RM4.5), fluorescein isothiocyanate conjugated antiCD25 (7D4), PE-conjugated anti-CD25 (PC61), biotinylated anti-CD90.1 (Thy1.1, encoded by Thy1a, OX-7), biotinylated anti-CD90.2 (Thy1.2, encoded by Thy1b, 53-2.1), and APC-conjugated streptavidin. Cytometric analyses were performed on a FACSort (BD Biosciences) using CellQuest Pro software.

Transfer of CD4ⴙ T Cells: Parking Experiments CD4⫹ cells were isolated from spleen and lymph nodes of H/K␤⫺/⫺ or H/K␣⫺/⫺ mice. Briefly, single cell suspensions were incubated with antibodies directed to CD8, B220, and F4/80, and the antibody-bound cells

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were removed using anti-rat IgG-coated magnetic beads (Dynal; Invitrogen, Carlsbad, CA). The CD4-enriched population from individual H/K␤⫺/⫺ mice was transferred to sublethally irradiated (600 rad) BALB/c-CD90.1 congenic mice (WT with respect to H/K␤) or H/K␤⫺/⫺CD90.1 mice. Mice received 5 ⫻ 107 CD4⫹ cells, and on some occasions, they also received 2 ⫻ 106 CD4⫹ cells that had been labelled with carboxyfluoroscein succinimidyl ester (CFSE) as described.21 In some experiments, CD4-enriched cells from H/K␣⫺/⫺ mice were transferred to nonirradiated BALB/c-CD90.1 mice. Six to 8 weeks later, recipient mice were euthanized, spleen and lymph node cell suspensions prepared and stained with PEconjugated anti-CD90.2, followed by anti-PE–labelled microbeads (Miltenyi Biotec GmbH, Gladbach, Germany). Labelled cells were recovered using an AutoMACS separator (Miltenyi Biotec) in accordance with the manufacturer’s instructions. The purity of the recovered CD90.2⫹ population was assessed by flow cytometry. To test for tolerance to H/K␣ and H/K␤, the repurified CD90.2⫹ cells were then transferred to BALB/cnu recipients. BALB/cnu mice received 4 ⫻ 106 CD4⫹ cells originally from H/K␤⫺/⫺ mice that had been parked in irradiated recipients or in some experiments, 5 ⫻ 105 CD4⫹ cells from H/K␣⫺/⫺ mice that had been parked in nonirradiated recipients. After an additional 8 weeks, the BALB/cnu recipients were sacrificed and stomachs were taken for histological examination. T cells from H/K␤⫺/⫺ donor mice were not pooled before transfer to either irradiated BALB/c-CD90.1 or H/K␤⫺/⫺-CD90.1 mice or to athymic BALB/cnu recipients so that each data point in these experiments could be considered independently. The yield of H/K␣⫺/⫺ 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.

Suppression of Autoimmune Disease by Treg Cells CD25⫹ and CD25⫺ populations were generated using an AutoMACS separator as previously described.22 To induce autoimmune gastritis, female BALB/cnu mice received an inoculum of CD25⫺ cells from either WT or H/K␤⫺/⫺ mice that contained 2 ⫻ 106 CD4⫹CD25⫺ T cells, injected intraperitoneally. Some mice also received 1 ⫻ 106 CD4⫹CD25⫹ cells isolated from WT mice. Eight weeks later, recipient mice were killed, and the stomachs and ovaries were taken for histological examination.

Transfer of A23 TCR Transgenic T Cells Enriched CD4⫹ cells were prepared from A23.H/K␣⫺/⫺ mice as described previously. The resulting population was labelled with CFSE,21 and 2 ⫻ 106 CD4⫹ cells were injected intravenously into WT (BALB/c-CD90.1 congenic) or H/K␣⫺/⫺ mice. Recipient mice were later euthanized, and single cell suspen-

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sions were prepared from spleen, inguinal, and paragastric lymph nodes. Single cell suspensions were prepared from the stomach by digestion with collagenase and DNAase. The resulting populations were stained with antibodies to CD4, the congenic marker CD90.2, and were analyzed by flow cytometry.

Assessment of Gastritis and Oophoritis Stomach and ovary sections were examined to assess the presence of autoimmune pathology. The methodology for scoring stomach sections was based on our previous detailed pathological studies of autoimmune gastritis.23–26 Stomachs were removed and cut along the lesser curvature, the contents were rinsed out with phosphate buffered saline, fixed overnight in 10% buffered formalin, and processed in an automated tissue processor. Tissue was then sliced lengthwise, and pieces were mounted in paraffin wax so that when sectioned, the gastric mucosa could be viewed along its entire length from the forestomach to the pyloric antrum and across its entire width from the stomach wall from the luminal face of the mucosa, in each section. Sections (5 to 10 ␮m) were cut and stained with hematoxylin and eosin and 6 to 8 discontinuous sections of mucosa were observed for each mouse. Autoimmune gastritis was graded on a scale of 0 to 6. Examples of mucosae with these scores are shown in Figure 1 and the criteria used for assignment are described in the legend. Autoimmune ovarian disease was defined as a loss of ovarian follicles, ovarian atrophy, and the presence of a mononuclear cell infiltrate. Samples were coded, and histological analyses were performed blind by 2 individuals with agreement of scoring in the great majority of cases. To ensure consistency in scoring, one experienced investigator (I.V.D.) was one of the scorers for all samples.

Statistical Analysis Gastritis severity data were analyzed using the two-tailed Mann–Whitney U test. P ⬎ .05 was considered not significant.

Results CD4ⴙ Effector Cells From H/K␤ⴚ/ⴚ Mice Are Highly Effective at Inducing Gastritis and Comparatively Resistant to Suppression by Treg Cells CD4⫹

Our previous work had shown that the T-cell population from H⫹/K⫹ ATPase-deficient mice was highly effective at inducing gastritis when transferred to lymphopenic recipients.7 In WT mice, CD4⫹ T cells capable of causing autoimmune gastritis are present in the CD25⫺ fraction, but the pathogenic potential of these cells is suppressed by Treg cells.14,15,22 Therefore, one possible explanation for the ability of CD4⫹ T cells from H⫹/K⫹ ATPase-deficient mice to cause gastritis is that

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Treg cells are unable to efficiently suppress the CD4⫹CD25⫺ effector cells of these mice. To test this directly, CD4⫹CD25⫺ cells were purified from WT and H/K␤⫺/⫺ mice and transferred alone or together with Treg cells from WT mice into athymic recipients. The ability of these cells to cause gastritis was determined by semiquantitative analysis of tissue sections using a scoring system (Figure 1) based on our previous extensive studies of the pathology of autoimmune gastritis in mice.23–26 The sections in Figure 1 were derived from mice in the experiments that contributed to Figure 2. As described previously,7 gastritis occurred in mice that received CD25⫺ T cells from either H/K␤⫺/⫺ or WT mice, although disease induced by the former was significantly more severe (Figure 2). Again, as we have observed in the past, gastritis was completely suppressed in mice that received both CD4⫹CD25⫺ cells from WT mice and Treg cells from WT mice.22 In contrast, gastritis was found in 9 of 11 mice that received CD4⫹CD25⫺ cells from H/K␤⫺/⫺ mice and Treg cells from WT mice, albeit less severe than that observed in mice that received CD4⫹CD25⫺ cells from H/K␤⫺/⫺ mice transferred without WT Treg. In addition, we examined the ovaries of the recipient mice, as these organs are also a target of autoimmune attack in athymic BALB/c mice after transfer of CD25⫺ cells (Figure 2). Almost all mice (22 of 23, filled circles) that received CD25⫺ cells alone from either WT or H/K␤⫺/⫺ mice developed autoimmune ovarian disease as evidenced by depletion of ovarian follicles, ovarian atrophy, and infiltration of mononuclear cells into the ovaries. In contrast, all mice that received Treg cells regardless of whether they received CD4⫹CD25⫺ cells from WT or H/K␤⫺/⫺ mice were free of significant ovarian inflammation (empty circles). Significantly, the mice that received WT Treg cells but CD25⫺ cells from H/K␤⫺/⫺ mice were protected from ovarian but not gastric autoimmunity, indicating that the more pathogenic H⫹/K⫹ ATPase– specific T cells taken from the H/K␤⫺/⫺ donors could not be efficiently suppressed by Treg cells, but that the proclivity of T cells of other specificities to be suppressed was unaltered.

CD4ⴙ T Cells From Hⴙ/Kⴙ ATPase–Deficient Mice Were Unable to Induce Gastritis After Exposure to Hⴙ/Kⴙ ATPase The results in Figure 2 and our previous data7 demonstrate that the H⫹/K⫹ ATPase–specific T cells present in H⫹/K⫹ ATPase– deficient mice are highly effective at inducing gastritis relative to the equivalent population found in WT mice. Recently, we have also shown that events in the thymus do not shape the H⫹/K⫹ ATPase–specific T-cell repertoire.12 Here, we develop a protocol to determine the impact of exposing the gastritis-inducing T cells from H⫹/K⫹ ATPase-deficient mice to H⫹/K⫹ ATPase expressed in extra-thymic tissues (Figure

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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 ␮m and is applicable to A through C. Bar in D ⫽ 100 ␮m and is applicable to D through J. Bar in K ⫽ 100 ␮m and is applicable to K through Q.

3A). Initial experiments used T cells from H/K␤⫺/⫺ mice. CD4⫹ T cells from H/K␤⫺/⫺ mice were transferred to lightly irradiated WT (BALB/c-CD90.1 congenic) mice. The irradiation enhanced the survival of the transferred T cells such that after a “parking” period of 8 weeks, the transferred cells constituted 50% to 80% of the peripheral CD4⫹ cells (Figure 3B). In some experiments, the T cell inoculum was spiked with a small number of CD4⫹ cells labelled with CFSE, and from analysis of the fluorescence of those cells, it is clear that after transfer, the vast majority of T cells underwent only 1 or 2 cell divisions

(data not shown). At 8 weeks, the transferred H/K␤⫺/⫺ CD4⫹ cells were reisolated using the expression of the congenic marker, CD90.2, and the gastritis-inducing potential of these “parked” cells was assayed in a secondary transfer to athymic recipients. After an additional 8 weeks, the athymic mice were assessed for evidence of gastritis (Figure 4). None of the mice in which T cells were parked developed gastritis (data not shown). We also assessed the proportions of Foxp3⫹ Treg cells in 3 mice before and after parking. We found that the levels of Foxp3⫹ Treg cells in the CD4⫹ T cell population after

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Figure 2. Treg cells are less capable of suppressing CD4⫹CD25⫺ effector cells from H/K␤⫺/⫺ mice. CD25⫺ and CD25⫹ cells were prepared from WT BALB/c and H/K␤⫺/⫺ mice. Female athymic BALB/cnu mice were injected intraperitoneally with 2 ⫻ 106 CD4⫹CD25⫺ T cells from either WT or H/K␤⫺/⫺ 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.

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parking was very similar to the levels of Foxp3⫹ Treg cells found prior to parking (proportion of CD4⫹ cells that were Foxp3⫹, percentage before parking: percentage after parking; mouse 1, 7.6%:7.5%; mouse 2, 8:1%:8.0%; mouse 3, 8.8%:6.3%). Thus, the proportion of Foxp3⫹ Treg was not greatly influenced by the parking protocol. Representative data are shown in Figure 3C. When CD4⫹ T cells from H/K␤⫺/⫺ mice were transferred directly to athymic mice (Figure 4, group A), all recipients developed autoimmune gastritis with 9 of the 14 developing gastritis with the most severe scores (scores 5 or 6), consistent with published data.7 However, if T cells from H/K␤⫺/⫺ mice were first parked in WT mice and then 8 weeks later transferred into athymic recipients (group B), 9 of 17 athymic mice remained free of autoimmune gastritis (score 0), while another 4 mice had only very mild disease (score 1). Parking CD4⫹ T cells from H/K␤⫺/⫺ mice in H/K␤⫺/⫺ mice did not prevent the development of autoimmune gastritis, as all the athymic BALB/cnu recipients receiving this population of cells developed severe gastritis (group D). However, regardless of whether the CD4⫹ T cells were parked, gastritis did

Figure 3. Protocol used for parking CD4⫹ T cells. (A) CD4⫹CD90.2⫹ cells were isolated from spleen and lymph node preparations from H/K␣⫺/⫺ or H/K␤⫺/⫺ mice. In some experiments, CD4⫹ cells (5 ⫻ 107) from H/K␣⫺/⫺ mice were transferred to nonirradiated BALB/c-CD90.1 congenic mice. In others, an equivalent number of CD4⫹ cells from H/K␤⫺/⫺ mice were transferred to sublethally irradiated (600 rad) BALB/ c-CD90.1 congenic or H/K␤⫺/⫺-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/K␤⫺/⫺ 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/K␤⫺/⫺ donor mice were not pooled before transfer to either the BALB/c-CD90.1, H/K␤⫺/⫺CD90.1, or BALB/cnu athymic recipients in these experiments so that each data point could be considered independently. The yield of H/K␣⫺/⫺ 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.

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Figure 4. The gastritis-inducing CD4⫹ T cell population from H/K␤⫺/⫺ 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/K␤⫺/⫺ or WT mice, as indicated, and were transferred to irradiated BALB/c-CD90.1 congenic mice (WT) or H/K␤⫺/⫺-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/K␤⫺/⫺ 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/K␤⫺/⫺ 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/K␤⫺/⫺ or unmanipulated WT mice resulted in many of the recipients developing gastritis with lower scores (groups G and H).

not occur to a significant degree if this population were derived from WT mice (groups C and E). We also performed similar experiments with CD4⫹ T cells derived from H/K␣⫺/⫺ mice. In contrast to the experiments with H/K␤⫺/⫺ mice, irradiated BALB/ c-CD90.1 mice that received T cells from H/K␣⫺/⫺ mice developed severe gastritis (7 of 7 recipients, disease score 5 to 6). This suggests that the CD4⫹ population found in H/K␣⫺/⫺ mice is more pathogenic than that in H/K␤⫺/⫺

mice or perhaps that H/K␣ antigen is more abundantly presented in the irradiated mice. To overcome this unexpected phenomenon, it was necessary to transfer CD4⫹ T cells from H/K␣⫺/⫺ mice into nonirradiated WT (BALB/ c-CD90.1) mice. In this case, all recipients remained gastritis free after 6 weeks (data not shown), but, as expected, the number of donor-type T cells recovered from the unmanipulated recipients was much lower than that from irradiated mice. However, sufficient cells could be

Figure 5. Gastritis-inducing T cells from H/K␣⫺/⫺ mice can be rendered tolerant in the periphery of WT mice. CD4⫹CD90.2⫹ cells from H/K␣⫺/⫺ 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.

obtained to transfer to athymic recipients (Figure 5). Eight weeks later, the athymic recipients were killed and assessed for evidence of gastritis. All mice that received CD4⫹ T cells taken directly from H/K␣⫺/⫺ mice developed severe gastritis; however, after parking in WT mice, the ability of this population to induce disease was markedly and significantly decreased. Therefore, as we had observed in our experiments with T cells taken from H/K␤⫺/⫺ mice, exposure to H/K␣ in the periphery of the recipient “parking” mice was sufficient to render T cells from H/K␣⫺/⫺ mice incapable of inducing severe autoimmune gastritis. Collectively, these data indicate that H⫹/K⫹ ATPase– specific, gastritis-inducing T cells are rendered nonpathogenic in the periphery of mice in an antigen-dependent manner.

Highly Pathogenic Hⴙ/Kⴙ ATPase–Specific T Cells Are Purged From the Repertoire by Extra-Thymic Exposure to the Hⴙ/Kⴙ ATPase As discussed previously, the CD4⫹ T cells capable of causing autoimmune gastritis are present in the CD25⫺ fraction, but, in WT mice, the pathogenicity of these cells is suppressed by Treg cells.14,15,22 We therefore wanted to determine whether the CD25⫺ fraction of the CD4⫹ T cells from H/K␤⫺/⫺ mice that had been parked

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in an H/K␤–sufficient environment retained the ability to induce gastritis (Figure 4). Athymic mice that received CD4⫹ T cells that had been depleted of CD25⫹ Treg cells after parking (Figure 4, group G) developed much more severe gastritis than athymic mice that received the unfractionated parked CD4⫹ cells (Figure 4, group B). However, the gastritis seen in these mice that received Treg cell– depleted parked CD4⫹ cells (Figure 4, group G) was significantly less severe than that seen in athymic mice that received Treg cell– depleted CD4⫹ cells directly from H/K␤⫺/⫺ mice (Figure 4, group F). Although depletion of Treg cells enhanced the pathogenicity of the parked H/K␤⫺/⫺ CD4⫹ T cell population, it did not restore it to the levels seen in the unparked precursor population. In fact, the severity of disease seen in athymic mice that received CD4⫹ cells that were parked and then Treg cell depleted from H/K␤⫺/⫺ mice (Figure 4, group G) was similar to that observed in athymic mice that received Treg cell– depleted CD4⫹ T cells directly from WT mice (Figure 4, group H). Collectively, these data demonstrate that parking in an H⫹/K⫹ ATPase–sufficient environment significantly decreased the pathogenic potential of the CD4⫹CD25⫺ population from H/K␤⫺/⫺ mice and that the level of pathogenicity approximated that of the equivalent CD4⫹CD25⫺ population from WT mice.

Hⴙ/Kⴙ ATPase–Specific T Cells Proliferate in the Paragastric Lymph Node The parking experiments indicate that exposure of H⫹/K⫹ ATPase–specific T cells to cognate antigen in the periphery results in the induction of tolerance. To examine the cellular mechanism of this tolerance, we turned to the use of T cells derived from the A23 TCR transgenic mouse.5 A23 mice express a transgenic TCR directed to a peptide derived from H/K␣.27 T cells derived from these mice are very effective at causing gastritis; as few as 103 thymocytes transferred to lymphopenic mice will induce autoimmune gastritis.5,22 However, autoimmune gastritis does not occur if A23 T cells are transferred to normal unmanipulated mice unless very large numbers of cells (⬎107) are transferred.5 CD4⫹ cells were purified from A23 TCR transgenic mice that lacked H/K␣ to ensure that the transgenic T cells had not encountered antigen and were thus naïve. The cells were labelled with CFSE and transferred to WT mice. Representative plots showing CFSE labelling of A23 T cells in the paragastric lymph node, inguinal lymph node, and spleen at days 4 and 7 are shown in Figure 6. At day 4 after transfer, approximately 90% of A23 T cells in the paragastric lymph node had divided; nearly all of those at least twice and the majority 3 or 4 times. At day 7, a greater proportion of cells had divided multiple times, although there were still significant numbers of cells that had divided only once or twice. A23 T cells could be found in the spleen and inguinal lymph

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Figure 6. Highly pathogenic H⫹/K⫹ ATPase–specific T cells proliferate in the paragastric lymph node. Purified CD4⫹ cells from A23.H/K␣⫺/⫺ 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.

node at day 4, and for the most part, were either nondivided or had divided 3 or more times. By day 7, far fewer cells were found in the spleen and inguinal nodes (Figure 7). These data are consistent with a situation where H/K␣-specific TCR transgenic T cells become activated in the paragastric lymph node but then may recirculate and be found at other sites. No cell division was observed in the paragastric lymph node of the H/K␣⫺/⫺ mice (data not shown) indicating that division of the A23 T cells is dependent on H/K␣, as previously observed.22,28 These data indicate that A23 T cells are stimulated to divide in response to H/K␣– derived peptide presented in the paragastric lymph node. Significantly, very few cells were present in the gastric mucosa at either day 4 or 7 (note the scale on the y-axis in Figure 7), and those cells that could be found had undergone several divisions (⬎4).

Highly Pathogenic Hⴙ/Kⴙ ATPase–Specific T Cells Are Rapidly Depleted From the Periphery We then enumerated the number of A23 TCR transgenic T cells in paragastric and inguinal lymph nodes, spleen, and gastric mucosa lymph nodes at days 4, 7, 14, and 28 after transfer (Figure 7). At day 4, the great majority of cells were present in the spleen. By day 7, the total number of A23 cells was reduced to on average 26% of the number at day 4. Similar numbers of A23 cells were found in the spleen and paragastric lymph nodes with few cells detectable in either inguinal lymph nodes or gastric mucosa. At day 14, the number of cells had again fallen (to 8% of day 4 total, 30% of day 7 total), and the majority of the remaining cells (57%) were found in the paragastric lymph node. By day 28, A23 T cells were

Figure 7. Highly pathogenic H⫹/K⫹ ATPase–specific T cells are rapidly depleted from the periphery. CD4⫹ cells from A23.H/K␣⫺/⫺ 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.

undetectable in any organ. At all time points, only very few A23 cells were evident in the gastric mucosa, although host-derived CD4⫹ cells were readily isolated from this organ. These data show that despite substantial mitotic activity in the paragastric lymph node, the number of A23 T cells declines steadily. The cells appear to be stimulated to divide in the paragastric lymph node and then disperse to other lymphoid organs with only a minority making their way to the gastric mucosa. However, few cells, if any, persist at 28 days after transfer. Significantly, there was no sign of gastric inflammation or of an accumulation of A23 T cells in the gastric mucosa.

Discussion In this article, we have continued our characterization of the immune response to the major gastric autoantigens, the ␣ and ␤ subunits of the H⫹/K⫹ ATPase1 and have demonstrated the importance of peripheral tolerance in the protection from autoimmune gastritis. Several mechanisms may be involved in the regulation of immune responses to these antigens. There have been reports that the H⫹/K⫹ ATPase is expressed in the thymus, raising the possibility of a role for central tolerance.10,11,13 In addition, autoimmune gastritis occurs in systems where Treg cells are depleted, indicating a role for this specialized regulatory population.1,7,14,15,22,29 Fi-

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nally, H⫹/K⫹ ATPase– derived peptides are constitutively presented to T cells in the lymph node draining the stomach, raising the possibility of extra-thymic deletion or anergy.30 In contrast to the unfractionated CD4⫹ population from WT mice, unfractionated CD4⫹ T cells from mice deficient in the gastric H⫹/K⫹ ATPase were able to induce gastritis in lymphopenic hosts.7 The data presented herein suggest that this phenomenon is due to the presence of highly pathogenic T cells in the H⫹/K⫹ ATPase– deficient mice that cannot be suppressed by the regulatory activity of the normal Treg cell population. Our previous work indicated that thymic events were unlikely to influence the H⫹/K⫹ ATPase–specific repertoire.12 Subsequent to that work, we performed a more extensive series of thymic transplantation experiments that support this conclusion (Read, Gleeson, and van Driel, unpublished data, 2004). These studies prompted us to investigate extra-thymic events that might influence the immune response to gastric autoantigens. As relatively little is known about the diversity of T-cell receptor affinities that are responsible for causing gastritis, we took an approach that would allow us to examine a polyclonal repertoire and not just monoclonal populations of cells. Thus, we used CD4⫹ T cells from H⫹/K⫹ ATPase– deficient mice; this population is very efficient at causing gastritis when transferred to athymic recipients as illustrated previously7 and herein. We have also previously demonstrated that T cells from H⫹/K⫹ ATPase– deficient mice are more highly reactive to H⫹/K⫹ ATPase antigen in in vitro assays and are therefore likely to be enriched in H⫹/K⫹ ATPase–specific T cells.7 The T-cell transfers into athymic mice (Figure 2) taken together with the “parking” experiments (Figures 4 and 5) indicate that the gastritis-inducing T-cell population found in H⫹/K⫹ ATPase– deficient mice is relatively resistant to suppression by Treg cells. In the T-cell transfer experiment (Figure 2), Treg cells from WT mice were able to completely suppress gastritis caused by transfer of CD4⫹CD25⫺ T cells from WT mice but could not prevent gastritis if the CD4⫹CD25⫺ population was derived from H/K␤⫺/⫺ mice. We suggest that this is most likely due to an increased frequency of gastritis-inducing T cells in the CD4⫹CD25⫺ population derived from H/K␤– deficient mice changing the ratio of pathogenic T cells to Treg cells in favor of autoimmunity. This is a significant finding because it suggests that the suppressive activity of the Treg cell population may be insufficient to prevent the activation of potentially pathogenic T cells as they exit the thymus, at least in the case of T cells capable of inducing gastritis. By parking CD4⫹ T cells from H⫹/K⫹ ATPase– deficient mice in WT mice, we demonstrated that exposure to the H⫹/K⫹ ATPase as it is normally expressed and presented in WT mice resulted in a substantial and significant decrease in the ability of the transferred CD4⫹ T-cell population to cause gastritis. A decrease in this patho-

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genic activity was not observed if the CD4⫹ T cells from H/K␤⫺/⫺ mice were parked in other H/K␤⫺/⫺ mice indicating that loss of pathogenicity was antigen specific. These observations combined with our previous data12 argue that H⫹/K⫹ ATPase–specific T cells capable of inducing gastritis arise in the thymus and are rendered tolerant in extra-thymic tissues. Scheinecker et al30 demonstrated that dendritic cells in the paragastric lymph node acquire and present H⫹/K⫹ ATPase– derived peptide to T cells. Previous work by this laboratory22 and others28 has shown that the T cells derived from A23 transgenic mice that express an H⫹/K⫹ ATPase–specific TCR proliferate in the paragastric lymph node. Here we demonstrate that these potentially pathogenic T cells fail to induce gastritis and fail to accumulate in WT mice despite undergoing a substantial amount of mitotic activity (Figures 6 and 7). By day 4, the great majority of A23 CD4⫹ T cells had divided at least once, with many having undergone 5 or more divisions. Despite this considerable amount of cell division, the number of A23 cells fell consistently from day 4 to day 14, and the cells were undetectable by day 28. Importantly, we failed to observe any accumulation of A23 T cells in the gastric mucosa. A small number of A23 T cells (1% of those A23 T cells present in paragastric lymph node at day 4, 3% at day 7, 8% at day 14, not detectable at day 28) were found in that tissue, but we did not observe any sign of gastric inflammation by histological analysis even in mice that received the A23 T cells 28 days earlier (data not shown). Hence, it appears that the A23 T cells are efficiently depleted from the lymphoid system and, critically, do not accumulate in the organ expressing the target antigen. We cannot rule out the possibility that, for some reason, the H⫹/K⫹ ATPase– specific cells are accumulating in another part of the mouse. If this is the case, it appears that such “sequestered” A23 cells are of no pathological consequence. T cells derived from A23 transgenic mice are not rendered tolerant in the thymus but are rather seeded into the secondary lymphoid organs.12 They are therefore representative of T cells in the normal repertoire and are potentially capable of causing autoimmune disease. We suggest that the depletion of the A23 cells illustrates a likely process that occurs both in the parking experiments and when H⫹/K⫹ ATPase–specific T cells exit the thymus in normal animals. That is, that exposure of T cells recognizing H⫹/K⫹ ATPase epitopes with a certain affinity to their cognate ligand in the paragastric lymph node ultimately results in the deletion of those cells. Heath et al have shown that CD8⫹ T cells proliferate in response to cross-presented antigen expressed by pancreatic ␤ cells and are then deleted.31 Similarly, the activation and induction of mitosis in T cells as a prelude to tolerance, either deletion and/or anergy, has also been reported for CD4⫹ T cells responding to systemically expressed or administered antigen.32–35 Here we show

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that deletion also occurs for CD4⫹ T cells responding to a bona fide self-antigen expressed in a tissue-specific manner. Although the depletion process was very efficient for T cells bearing the transgenic A23 TCR, it is possible that the fate of T cells expressing TCRs with other characteristics would be different. Certainly, in other systems, anergy appears to be the dominant outcome,33 and in other situations, “TCR revision” has also been observed.35 The parking experiments described here also indicate that some H⫹/K⫹ ATPase–specific T cells avoid all of these fates and remain potentially pathogenic. Exposure of T cells from H⫹/K⫹ ATPase– deficient mice to the H⫹/K⫹ ATPase did not appear to completely deplete the repertoire of gastritis-inducing T cells. Although the parked cells were unable to cause severe gastritis in athymic recipients (Figure 4, group B), if the parked CD4⫹ T-cell populations were depleted of Treg cells, the athymic recipients went on to develop gastritis (Figure 4, group G). Significantly, the severity of the disease was lower than that induced by Treg cell– depleted CD4⫹ T cells taken directly from H⫹/K⫹ ATPase– deficient mice and transferred directly to athymic mice without parking (Figure 4, group F). It is also noteworthy that the spectrum of disease severity seen in athymic mice that received Treg cell– depleted parked CD4⫹ cells from H/K␤⫺/⫺ mice was similar to that obtained if athymic mice received Treg cell– depleted CD4⫹ cells taken directly from WT mice (Figure 4, group H), supporting the notion that the parking protocol we have used mimics normal physiology. The parking experiments (Figures 4 and 5) along with the transfer data (Figure 2) suggest that extra-thymic events result in a population of T cells that can be controlled by Treg cells, thus preventing autoimmune disease. We and other investigators have shown that depletion of Treg cells leads to spontaneous autoimmunity in WT adult and neonatal mice,29,36,37 indicating that Treg cells are required to prevent the development of autoimmunity even after purging the repertoire of highly pathogenic T cells. Our data indicate that if repertoire purging does not occur, as is the case in H⫹/K⫹ ATPase– deficient mice, then the H⫹/K⫹ ATPase–specific T cells are more pathogenic than the equivalent population from WT mice and that WT Treg cells are less able to curb the development of pathology mediated by this more autoaggressive population. Therefore, we suggest that both the presence of Treg cells and repertoire purging may be critically required to prevent autoimmune disease in the presence of inflammatory stimuli38 such as infection with Helicobacter species,39 in neonatal mice,29 or under lymphopenic conditions, here mimicked by T-cell transfer to athymic or irradiated recipients. Recent work has shown that exposure to antigens in the periphery can maintain and/or generate Treg cells.40 – 43 For example, Tung et al42,43 have shown that

antigens derived from the ovaries and prostate enhance the activity of Treg populations essential for preventing autoimmune ovarian disease and prostatitis, a conclusion suggested by previous studies.44 Hence, Treg from male mice inhibited prostatitis more effectively than Treg from females.42 Here, we have shown a corollary of this finding in that the more highly pathogenic CD4⫹ CD25⫺ population found in H⫹/K⫹ ATPase– deficient mice was able to overcome suppression mediated by Treg from WT mice (Figure 2). Significantly, this phenomenon was gastritis-specific as Treg suppressed oophoritis mediated by the same CD4⫹CD25⫺ population. It is possible that the decrease in the ability of CD4⫹ T cells from H⫹/K⫹ ATPase– deficient animals to cause gastritis after parking was partly due to the induction of gastric-specific Treg cells. However, our data indicate that if the induction of gastric-specific Treg does take place, it occurs together with deletion of effector cells (unpublished observations). These data lead us to conclude that contact with antigen in the periphery is able to establish a balance between suppressor and effector T cells by maintaining Treg, coincidentally silencing the most autoaggressive effector T cells, and that this process will occur independently for different organs. Such a mechanism may be vital to maintain a Treg cell population sufficiently potent to suppress autoreactive T cells, while still allowing the generation of responses to foreign antigens. This work combined with our recent data12 has significant implications for the causes of autoimmune gastritis, which is a very common condition afflicting the alimentary system. As deletion of the H⫹/K⫹ ATPase– specific T cells in the thymus does not appear to play a significant role in specifying the repertoire that can cause gastritis, defects in thymic selection are unlikely to be the cause of gastric autoimmunity. However, circumstances that alter extra-thymic selection of T cells or alter the activity of Treg may predispose to autoimmune gastritis. Patients who are infected by Helicobacter pylori have been shown to possess activated T cells directed to the H⫹/K⫹ ATPase,45 which may alter the balance of the ratio of effector:regulatory T cells in favor of autoimmunity. However, it is not clear that such an imbalance would persist after clearance of Helicobacter and thus lead to chronic autoimmunity. It is an implication of our work that in the treatment of autoimmune gastritis, regimens that favor generation of gastric-specific Treg or, more significantly, enhance the deletion of H⫹/K⫹ ATPase– specific T cells are likely to be effective in the prevention or treatment of this disease. References 1. van Driel IR, Read S, Zwar TD, Gleeson PA. Shaping the T cell repertoire to a bona fide autoantigen: lessons from autoimmune gastritis. Curr Opin Immunol 2005;17:570 –576. 2. Toh BH, van Driel IR, Gleeson PA. Mechanisms of disease— Pernicious anemia. N Engl J Med 1997;337:1441–1448.

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3. D’Elios MM, Bergman MP, Azzurri A, Amedei A, Benagiano M, De Pont JJ, Cianchi F, Vandenbroucke-Grauls CM, Romagnani S, Appelmelk BJ, Del Prete G. H⫹,K⫹-ATPase (proton pump) is the target autoantigen of Th1-type cytotoxic T cells in autoimmune gastritis. Gastroenterology 2001;120:377–386. 4. Alderuccio F, Cataldo V, van Driel IR, Gleeson PA, Toh BH. Tolerance and autoimmunity to a gastritogenic peptide in TCR transgenic mice. Internat Immunol 2000;12:343–352. 5. McHugh RS, Shevach EM, Margulies DH, Natarajan K. A T cell receptor transgenic model of severe, spontaneous organ-specific autoimmunity. Eur J Immunol 2001;31:2094 –2103. 6. Scarff KL, Judd LM, Toh BH, Gleeson PA, van Driel IR. Gastric H⫹,K⫹-Adenosine triphosphatase ␤ subunit is required for normal function, development, and membrane structure of mouse parietal cells. Gastroenterology 1999;117:605– 618. 7. Laurie KL, van Driel IR, Zwar TD, Barrett SP, Gleeson PA. Endogenous H/K ATPase beta-subunit promotes t cell tolerance to the immunodominant gastritogenic determinant. J Immunol 2002; 169:2361–2367. 8. Spicer Z, Miller ML, Andringa A, Riddle TM, Duffy JJ, Doetschman T, Shull GE. Stomachs of mice lacking the gastric H,K-ATPase alpha-subunit have achlorhydria, abnormal parietal cells, and ciliated metaplasia. J Biological Chem 2000;275:21555–21565. 9. Mathis D, Benoist C. Back to central tolerance. Immunity 2004; 20:509 –516. 10. Kyewski B, Derbinski J, Gotter J, Klein L. Promiscuous gene expression and central T-cell tolerance: more than meets the eye. Trends Immunol 2002;23:364 –371. 11. Gotter J, Brors B, Hergenhahn M, Kyewski B. Medullary epithelial cells of the human thymus express a highly diverse selection of tissue-specific genes colocalized in chromosomal clusters. J Exp Med 2004;199:155–166. 12. Allen S, Read S, DiPaolo R, McHugh RS, Shevach EM, Gleeson PA, van Driel IR. Promiscuous thymic expression of an autoantigen gene does not result in negative selection of pathogenic T cells. J Immunol 2005;175:5759 –5764. 13. Anderson MS, Venanzi ES, Klein L, Chen Z, Berzins SP, Turley SJ, von Boehmer H, Bronson R, Dierich A, Benoist C, Mathis D. Projection of an immunological self shadow within the thymus by the aire protein. Science 2002;298:1395–1401. 14. Asano M, Toda M, Sakaguchi N, Sakaguchi S. Autoimmune disease as a consequence of developmental abnormality of a T cell subset. J Exp Med 1996;184:387–396. 15. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25)— breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995;155:1151–1164. 16. Randolph DA, Fathman CG. CD4⫹CD25⫹ regulatory T cells and their therapeutic potential. Annu Rev Med 2006;57:381– 402. 17. Rudensky AY, Campbell DJ. In vivo sites and cellular mechanisms of T reg cell-mediated suppression. J Exp Med 2006;203:489 – 492. 18. Rudge G, Gleeson PA, van Driel IR. Control of immune responses by immunoregulatory T cells. Arch Immunol Ther Exp (Warsz) 2006;54:381–391. 19. Zwar TD, van Driel IR, Gleeson PA. Guarding the immune system: suppression of autoimmunity by CD4⫹CD25⫹ immunoregulatory T cells. Immunol Cell Biol 2006;84:487–501. 20. Silveira PA, Baxter AG, Cain WE, van Driel IR. A major linkage region on distal chromosome 4 confers susceptibility to mouse autoimmune gastritis. J Immunol 1999;162:5106 –5111. 21. Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods 1994;171:131–137. 22. Zwar TD, Read S, van Driel IR, Gleeson PA. CD4⫹CD25⫹ Regulatory T cells inhibit the antigen-dependent expansion of selfreactive T cells in vivo. J Immunol 2006;176:1609 –1617.

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23. Barrett SP, Gleeson PA, Desilva H, Toh BH, van Driel IR. Interferon-gamma is required during the initiation of an organ-specific autoimmune disease. Eur J Immunol 1996;26:1652–1655. 24. Martinelli TM, van Driel IR, Alderuccio F, Gleeson PA, Toh BH. Analysis of mononuclear cell infiltrate and cytokine production in murine autoimmune gastritis. Gastroenterology 1996;110:1791– 1802. 25. Judd LM, Gleeson PA, Toh BH, van Driel IR. Autoimmune gastritis results in disruption of gastric epithelial cell development. Am J Physiol Gastointest Liver Physiol 1999;277:G209 –G218. 26. Franic TV, Judd LM, Nguyen NV, Samuelson LC, Loveland KL, Giraud AS, Gleeson PA, van Driel IR. Growth factors associated with gastric mucosal hypertrophy in autoimmune gastritis. Am J Physiol Gastrointest Liver Physiol 2004;287:G910 –G918. 27. Suri-Payer E, Amar AZ, McHugh R, Natarajan K, Margulies DH, Shevach EM. Post-thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti-H/K ATPase-reactive T cells. Eur J Immunol 1999;29:669 – 677. 28. DiPaolo RJ, Glass DD, Bijwaard KE, Shevach EM. CD4⫹CD25⫹ T cells prevent the development of organ-specific autoimmune disease by inhibiting the differentiation of autoreactive effector T cells. J Immunol 2005;175:7135–7142. 29. Laurie KL, van Driel IR, Gleeson PA. The role of CD4⫹CD25⫹ immunoregulatory T cells in the induction of autoimmune gastritis. Immunol Cell Biol 2002;80:567–573. 30. Scheinecker C, McHugh R, Shevach EM, Germain RN. Constitutive presentation of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph node. J Exp Med 2002;196: 1079 –1090. 31. Davey GM, Kurts C, Miller JF, Bouillet P, Strasser A, Brooks AG, Carbone FR, Heath WR. Peripheral deletion of autoreactive CD8 T cells by cross presentation of self-antigen occurs by a Bcl-2inhibitable pathway mediated by Bim. J Exp Med 2002;196:947– 955. 32. Lohr J, Knoechel B, Kahn EC, Abbas AK. Role of B7 in T cell tolerance. J Immunol 2004;173:5028 –5035. 33. Adler AJ, Marsh DW, Yochum GS, Guzzo JL, Nigam A, Nelson WG, Pardoll DM. CD4⫹ T cell tolerance to parenchymal self-antigens requires presentation by bone marrow-derived antigen-presenting cells. J Exp Med 1998;187:1555–1564. 34. Pape KA, Merica R, Mondino A, Khoruts A, Jenkins MK. Direct evidence that functionally impaired CD4⫹ T cells persist in vivo following induction of peripheral tolerance. J Immunol 1998;160: 4719 – 4729. 35. Ali M, Weinreich M, Balcaitis S, Cooper CJ, Fink PJ. Differential regulation of peripheral CD4⫹ T cell tolerance induced by deletion and TCR revision. J Immunol 2003;171:6290 – 6296. 36. Kim J, Rasmussen J, Rudensky A. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol 2006;8:191–197.

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37. Williams LM, Rudensky AY. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat Immunol 2007;8:277–284. 38. McHugh RS, Shevach EM. Cutting edge: depletion of CD4(⫹)CD25(⫹) regulatory T cells is necessary, but not sufficient, for induction of organ-specific autoimmune disease. J Immunol 2002;168:5979 –5983. 39. Kaparakis M, Laurie KL, Wijburg O, Pedersen J, Pearse M, van Driel IR, Gleeson PA, Strugnell RA. CD4⫹ CD25⫹ Regulatory T cells modulate the T-cell and antibody responses in Helicobacterinfected BALB/c mice. Infection and Immunity 2006;74:3519 – 3529. 40. Kretschmer K, Apostolou I, Hawiger D, Khazaie K, Nussenzweig MC, von Boehmer H. Inducing and expanding regulatory T cell populations by foreign antigen. Nat Immunol 2005;6:1219 –1227. 41. Knoechel B, Lohr J, Kahn E, Bluestone JA, Abbas AK. Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen. J Exp Med 2005;202:1375–1386. 42. Setiady YY, Ohno K, Samy ET, Bagavant H, Qiao H, Sharp C, She JX, Tung KSK. Physiologic self antigens rapidly capacitate autoimmune disease-specific polyclonal CD4⫹CD25⫹ regulatory T cells. Blood 2006;107:1056 –1062. 43. Samy ET, Parker LA, Sharp CP, Tung KSK. Continuous control of autoimmune disease by antigen-dependent polyclonal CD4⫹CD25⫹ regulatory T cells in the regional lymph node. J Exp Med 2005;202: 771–781. 44. Taguchi O, Nishizuka Y. Self tolerance and localized autoimmunity. Mouse models of autoimmune disease that suggest tissuespecific suppressor T cells are involved in self tolerance. J Exp Med 1987;165:146 –156. 45. Amedei A, Bergman MP, Appelmelk BJ, Azzurri A, Benagiano M, Tamburini C, van der Zee R, Telford JL, Vandenbroucke-Grauls CMJE, D’Elios MM, Del Prete G. Molecular mimicry between Helicobacter pylori antigens and H⫹,K⫹-adenosine triphosphatase in human gastric autoimmunity. J Exp Med 2003;198: 1147–1156.

Received May 24, 2006. Accepted May 10, 2007. Address requests for reprints to: Assoc Prof Ian R. van Driel, PhD, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia. e-mail: [email protected]; fax: (61) 3 9348 1421. The authors thank Shiralee Whitehead, Troy Taylor, and Max Walker for excellent animal husbandry.