recipient mice whilst H/Kb was expressed in these mice [26]. On the other hand, the absence of parietal cell depletion in H/Kb2/2 recipient mice that received H/Ka2/2 CD4+ T cells reinforced our previous studies suggesting the need for H/Kb in order for H/ Ka to be presented by MHC molecules [22]. Together, these results suggested that parietal cell depletion in this transfer model developed as a consequence of immune responses to H/Ka by CD4+ T cells from H/Ka2/2 mice. We note that in previous work we have found that parietal cells are not depleted in either the H/ Ka2/2 nor H/Kb2/2 mice after neonatal thymectomy, which highlights the consistency of this new model with results from this previous, well-established model. We also demonstrated here that stomach weight can be used as a quantitative measure of the disease severity since hypertrophy of the gastric mucosa was observed in mice with autoimmune gastritis and resulted in the increase in stomach weight [37]. There was a significant correlation between stomach weight and the severity of autoimmune gastritis in mice that received H/Ka2/2 CD4+ T cells. This correlation was further confirmed in neonatally-thymectomised mice, with the mean stomach weight in each gastritis score being significantly different to another. However, due to the variation within the groups, large sample size will be required if stomach weight is to be used to estimate disease severity. Therefore, histological examination combined with stomach weight can be viewed as complementary measures for assessing the severity of autoimmune gastritis. We showed that autoimmune gastritis induced by adoptive transfer of H/Ka2/2 CD4+ T cells represented an excellent disease model for the dissection of autoimmune response and the design of interventionary therapies for autoimmune diseases. In this disease model, autoimmune gastritis was caused by an unfractionated polyclonal T cell population that contained pathogenic T cells specific for a single antigen, and consistent high-grade disease severity is observed in all recipient mice. Moreover, we showed that disease severity could be easily determined by stomach weight, stomach pH and histological examination of stomach section. Materials and Methods Ethics statement This work seeks to obtain a more detailed knowledge of autoimmune disease and the immunological mechanisms that prevent autoimmunity in normal individuals. As the immune system is a highly complex, integrated network, animals are required to properly evaluate these issues. The majority of procedures were clinical tests performed on blood and tissue samples taken from mice that are maintained in excellent housing conditions. Mice were monitored closely and killed if severely affected or distressed. At the termination of each experiment, mice were killed by CO2 asphyxiation or cervical dislocation, which are associated with a minimum of distress. All experiments are carefully planned to reduce animal numbers and are performed in strict adherence to guidelines dictated by the University of Melbourne’s Animal Experimentation Ethics Com- mittee; the Prevention of Cruelty to Animals Act 1986; and the NHMRC/CSIRO/AAC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1997). This work was approved by the University of Melbourne Animal Ethics Committee under project number 0706327.3. Mice BALB/cCrSlc [38], BALB/cCrSlc.CD90.1 congenic, H/Ka2/2 [26], H/Kb2/2 [27] and BALB.B6-Gasa congenic mice [20] have A High-Incidence Model for Autoimmune Gastritis been previously described. Both H/Ka2/2 and H/Kb2/2 mice had been backcrossed greater than 10 times to BALB/cCrSlc. H/ Ka2/2 mice were crossed to BALB/c.CD90.1 mice to generate H/ Ka2/2.CD90.1 mice. All mice were maintained in a conventional animal facility at the Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne. All mice and experiments were approved by the University of Melbourne Animal Experi- mentation Ethics Committee. Antibodies and flow cytometric analysis Anti-CD3-FITC (145-2C11), anti-CD90.1-FITC (HIS51), anti- CD90.2-FITC (30H-12), anti-CD62L-PE (MEL-14), anti-CD4 PerCP (RM4-5), and anti-CD8-APC (53-6.7) were purchased from BD Pharmingen; anti-CD44-APC (IM-7) was purchased from eBioscience. Intracellular staining of Foxp3 was performed using anti-Foxp3-APC (FJK-16S) and Foxp3 Staining Kit (eBioscience) according to the manufacturer’s instruction. Flow cytometry was performed on a Becton Dickinson FACSort flow cytometer and analysed using CellQuest Pro software or Beckman Coulter CyAn and analysed using Summit software. Assay of T cell responses in mice immunised with H+/K+ ATPase Purified pig gastric H+/K+ ATPase was prepared in a 1:1 ratio with Freund’s complete adjuvant (Life Technologies). Mice were injected subcutaneously at each side of the tail base. H/Ka2/2 + and BALB/cCrSlc mice were immunised twice with 30 mg of H / K+ ATPase, with one month apart between the immunisations. One week after the second immunisation, mice were sacrificed for their sera and inguinal lymph nodes. H+/K+ ATPase-specific autoantibodies were detected by ELISA. Cells from the inguinal lymph nodes were used in a T cell proliferation assay. 16105 cells were cultured for 72 hrs with 26104 splenic DC from BALB/ cCrSlc mice. T cell proliferation was assessed by incorporation of 3H-thymidine over the final 16 hours of culture. T cells were cultured in triplicate with either DC with antigen (50 mg/ml mouse gastric membrane) or with DC alone. Gastric membranes were prepared from mouse stomachs as previously described [5]. Induction of autoimmune gastritis in mice CD4+ T cells were enriched to .85% purity from lymph nodes and spleens of H/Ka2/2 and BALB/cCrSlc mice. Single cell suspensions were incubated in a mix of F4/80 and B220 hybridoma supernatants plus anti-CD8 antibody (53.6.72, BioX- Cell). The non-CD4 cells were removed using anti-rat IgG-coated magnetic beads (Dynal, Invitrogen). CD4+ (56107) T cells from either H/Ka2/2 or BALB/cCrSlc mice were injected into sublethally irradiated (600 rads) wildtype congenic BALB/ cCrSlc.CD90.1 mice via the intraperitoneal route. In some experiments, purified H/Ka2/2 CD4+ T cells were injected into irradiated BALB/cCrSlc, H/Ka2/2 and H/Kb2/2 mice. All recipient mice were irradiated at 600 rads one day before T cell transfer. Eight weeks later, recipient mice were killed and stomachs, lymph nodes and sera were harvested. BALB/cCrSlc and BALB.B6-Gasa congenic mice were used in the thymectomy experiments. Neonatal thymectomy was per- formed as previously described [20]. Histological examination of stomachs and measurement of gastric pH Stomach sections were examined for the presence of patholog- ical changes as previously described [24]. Scoring was performed in a blind fashion and each slide was independently scored by at PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27153