Figure 3. Cellularity of stomach-draining and non-draining lymph nodes. (A) The number of cells in the paragastric and inguinal lymph nodes from recipient mice 8 weeks after transfer of either wildtype or H/Ka2/2 CD4+ T cells. (B) The percentage of effector/ memory T cells in the donor population with the phenotype CD44hiCD62Llo in CD90.2+ CD4+ T cells. Data pooled from four independent experiments. Each circle represents the data from one mouse. Mann-Whitney U test was used; bars, mean, **, P,0.01 and ***, P,0.001. doi:10.1371/journal.pone.0027153.g003 since gastritic mice contained similar proportions of Treg cells in the paragastric lymph node compared to non-gastritic controls (Figure 4A, 4B). In fact, significantly more Treg cells were found in the stomach of mice with autoimmune gastritis (Figure 4A, 4C). H/Ka2/2 CD4+ T cells rapidly induced tissue damage in the stomach The severity of autoimmune gastritis was analysed at various time points after the transfer of H/Ka2/2 CD4+ T cells. Moderate depletion of parietal cells and zymogenic cells, as indicated by a gastritis score of 4 [24], could be observed as early as 2 weeks after the transfer and the disease progressed rapidly with most of the mice showing severe gastritis, as shown by a gastritis score of 5 and 6, 4 weeks after the transfer (Figure 5). Stomach weight can be used as a quantitative measure of autoimmune gastritis We have demonstrated that mice with autoimmune gastritis had a greater stomach weight than disease-free mice due to the hypertrophy of the gastric mucosa (Figure 2D). Therefore, we investigated if stomach weight could be used as a quantitative measure for the severity of autoimmune gastritis. To further test if stomach weight could differentiate among different severities of autoimmune gastritis, we examined neonatally-thymectomised A High-Incidence Model for Autoimmune Gastritis mice as greater numbers of mice with variable disease scores could be obtained. We found that there were significant differences in the mean stomach weight between each of the first five levels of autoimmune gastritis (score 0–4) (Figure 6). However, the mean stomach weights of gastritis scores 4, 5 and 6 were not significantly different from each other (Figure 6). This is likely due to the fact that the degree of hypertrophy of gastric mucosa does not increase as the disease progresses to the late stages. Induction of autoimmune gastritis by H/Ka2/2 CD4+ T cells was dependent on the presence of H/Ka and H/Kb We hypothesised that the pathogenicity of CD4+ T cells from H/Ka2/2 mice was caused by the lack of tolerance to H/Ka in the absence of this gastric autoantigen. To test this, CD4+ T cells were transferred from H/Ka2/2 mice into sublethally-irradiated H/Ka2/2 and wildtype mice. It has been demonstrated that H/ Ka, in the absence of H/Kb, cannot be presented by MHC molecules [25] (S Allen, personal communication). We therefore transferred CD4+ T cells from H/Ka2/2 mice into sublethally- irradiated H/Kb2/2 mice and compared them with H/Ka2/2 and wildtype recipient mice. The analysis of autoimmune attack on the gastric mucosa is complicated in H/Ka2/2 and H/Kb2/2 mice by the changes that occur in the gastric mucosa since a lack of gastric acid in these mice results in elevated levels of the trophic hormone gastrin. This leads to constitutive hypertrophy and depletion of zymogenic cells [26,27]. Therefore, in order to assess if an autoimmune response attacks the gastric mucosa in these mice, we instead determined the prevalence of parietal cells, which are normally depleted in autoimmune gastritis but are found in abundance in H/Ka2/2 and H/Kb2/2 mice, and also the presence of gastric autoantibodies. The stomach morphology in H/Ka2/2 mice or H/Kb2/2 mice was not affected by the transfer of H/Ka2/2 CD4+ T cells (Figure 7A, 7B). In particular, there was no depletion of parietal cells indicating the lack of an autoimmune response. In contrast, complete depletion of parietal cells was seen in wildtype recipient mice that received H/Ka2/2 CD4+ T cells (Figure 7C). Since stomachs of H/Ka2/2 and H/Kb2/2 mice appeared different to both normal and gastritic mice, we could not assess these mice using our standard gastritis scoring system. However, H+/K+ ATPase-specific autoantibodies, another hallmark of autoimmune gastritis, were not detected in the sera of both H/Ka2/2 and H/ Kb2/2 recipient mice, but were detected in wildtype recipient mice (Figure 7D). These results demonstrated that the presence of both a and b subunits was required for the depletion of parietal cells by H/Ka2/2 CD4+ T cells. Discussion Autoimmune gastritis can be induced in BALB/c mice by several means, including neonatal thymectomy, adult thymectomy with cyclophosphamide [19,21], immunisation with gastric H+/K+ ATPase [5] and generation of H+/K+ ATPase-specific TCR transgenic mice [7,8]. However, an immune system dominated by T cells with a single specificity as observed in TCR transgenic mice does not resemble normal physiological conditions. Furthermore, thymectomy and immunisation do not confer consistent disease severity and they are technically demanding, which makes it inconvenient to use these models for the analysis of immunother- apies. We therefore developed a new disease model that relied on the transfer of T cells from H/Ka2/2 mice, a polyclonal population that contained highly gastritogenic T cells. We found that immunisation of H/Ka2/2 mice with purified H+/K+ ATPase induced a vigorous T cell response as the result of PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27153