High incidence of spontaneous autoimmune gastritis is observed in transgenic mice expressing a TCR specific for H/Ka [8]. However, these mice do not represent an ideal disease model for all experiments because the monoclonal nature of the pathogenic T cells does not recapitulate the polyclonal response that occurs in pathophysiological conditions. From this perspective, we developed a disease model which relied on the transfer of polyclonal T cells from H/Ka-deficient (H/Ka2/2) mice into sublethally-irradiated wildtype mice. We demonstrated that mice that received unfractionated T cells from H/Ka2/2 mice all succumbed to the most severe autoimmune gastritis with pathological changes observed throughout the whole stomach. Furthermore, these pathological changes significantly affected the physiology of stomach. We demonstrated the usefulness of this model by showing that the development of autoimmune gastritis in this model was strictly dependent on the presence of both H+/K+ ATPase a and b subunits. Results Elevated T and B cell response in H/Ka2/2 mice following immunisation with H+/K+ ATPase In order to have a system that is suitable for the study of different aspects of autoimmunity, we established a disease model in which the disease is caused by pathogenic polyclonal T cells and the disease severity is consistently severe. A T cell response to H/ Ka is required for the onset of autoimmune gastritis and may be the dominant source of gastric autoreactive epitopes [7,8,22]. As T cell tolerance to H/Ka was unlikely to occur in H/Ka2/2 mice, we investigated if T cells that were specific to H+/K+ ATPase and capable of causing the gastritis were present in H/Ka2/2 mice. + To determine if there were elevated immune responses to H / K+ ATPase in H/Ka2/2 mice relative to wildtype mice, mice were immunised twice with purified gastric H+/K+ ATPase. After the immunisation, autoantibodies specific for H+/K+ ATPase were detected in the immunised H/Ka2/2 mice, but not in wildtype + mice (Figure 1A). T cell response to in vitro restimulation with H / K+ ATPase was also significantly greater in H/Ka2/2 mice than in wildtype mice (Figure 1B). Therefore, as has been previously demonstrated in H/Kb2/2 mice [23], there is also a significant T and B cell response to H+/K+ ATPase in H/Ka2/2 mice that is not present in wildtype mice. CD4+ T cells from H/Ka2/2 mice were highly pathogenic To determine if adoptive transfer of T cells from H/Ka2/2 mice would be able to cause autoimmune gastritis, we enriched CD4+ T cells from either H/Ka2/2 or wildtype mice to ,85% purity and transferred 56107 of these cells into sublethally irradiated wildtype mice. Since it was previously demonstrated that H+/K+ ATPase-specific CD4+ T cells were rapidly deleted in the periphery and unable to induce autoimmune gastritis in non- irradiated wildtype mice [24], all recipient mice used in this study were lightly irradiated at 600 rads to enhance the survival of transferred T cells from H/Ka2/2 mice. Eight weeks after the transfer, mice that received H/Ka2/2 CD4+ T cells all developed the most severe forms of autoimmune gastritis (score 5 and 6). This was demonstrated by the complete depletion of parietal cells and zymogenic cells, and hypertrophy of the gastric mucosa (Figure 2A, 2B). In contrast, mice that received wildtype CD4+ T cells were completely free of gastritis (Figure 2A, 2B), which indicated that autoimmune gastritis in mice receiving H/Ka2/2 CD4+ T cells was not caused by irradiation per se, but rather, the T cell inoculum. A High-Incidence Model for Autoimmune Gastritis + Figure 1. Elevated immune response to immunisation with H / K+ ATPase in H/Ka2/2 mice. WT and H/Ka2/2 mice were immunised with H+/K+ ATPase as described in ‘‘Materials and Methods’’. (A) H+/K+ ATPase-specific autoantibodies in serum were detected by ELISA using serial 2-fold dilutions of mouse sera, with a starting dilution factor of 5. Data is representative of three independent experiments. Error bars, standard error. (B) Cells from inguinal lymph nodes were cultured for 72 hrs with splenic DC, and T cell proliferation was assessed by incorporation of 3H-thymidine. For each mouse, T cells were cultured in triplicate with either DC with gastric membrane or with DC alone. The mean cpm of T cells cultured with DC with antigen was divided by the mean cpm of the same T cells cultured with DC alone to calculate the stimulation index. Data pooled from four independent experiments. In (B), each circle represents the data from one mouse. Mann-Whitney U test was used; bars, mean and ***, P,0.001. doi:10.1371/journal.pone.0027153.g001 Pathological changes in the stomach of gastritic mice signifi- cantly affected their stomach physiology. Hypertrophy of the gastric mucosa resulted in the enlargement of stomach rugae (Figure 2C) and an increase in stomach weight (Figure 2D). Secondly, depletion of parietal cells caused an increase in stomach pH due to the lack of acid secretion (Figure 2E). In addition, autoantibodies specific to the gastric H+/K+ ATPase could be detected in all gastritic mice (Figure 2F). Autoantibodies reached high levels in all mice 6 weeks after the transfer (Figure 2G). Together, these results demonstrated the presence of highly pathogenic H+/K+ ATPase-specific T cells in H/Ka2/2 mice. Autoimmune gastritis was caused by CD4+ T cell- mediated inflammation Mice with autoimmune gastritis induced by transfer of H/Ka2/2 CD4+ T cells had significantly more cells in their stomach-draining paragastric lymph nodes compared to normal mice and mice that received CD4+ T cells from wildtype donors. In contrast, there was PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27153