Biochemistry and Pharmacology - Research Publications
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ItemIncreased endogenous antigen presentation in the periphery enhances susceptibility to inflammation-induced gastric autoimmunity in mice(WILEY-BLACKWELL, 2017-01)How the immune system maintains peripheral tolerance under inflammatory conditions is poorly understood. Here we assessed the fate of gastritogenic T cells following inflammatory activation in vivo. Self-reactive T cells (A23 T cells) specific for the gastric H+ /K+ ATPase α subunit (HKα) were transferred into immunosufficient recipient mice and immunised at a site distant to the stomach with adjuvant containing the cognate HKα peptide antigen. Activation of A23 T cells by immunisation did not impact on either immune tolerance or protection from gastric autoimmunity in wild-type BALB/c mice. However, increased presentation of endogenously derived HKα epitopes by dendritic cells (DCs) in the gastric lymph node of IE-H+ /K+ β transgenic mice (IEβ) reduces A23 T-cell tolerance to gastric antigens after inflammatory activation, with subsequent development of gastritis. While HKα-specific A23 T cells from immunised wild-type mice were poorly responsive to in vitro antigen specific activation, A23 T cells from immunised IEβ transgenic mice were readily re-activated, indicating loss of T-cell anergy. These findings show that DCs of gastric lymph nodes can maintain tolerance of pathogenic T cells following inflammatory stimulation and that the density of endogenous antigen presented to self-reactive T cells is critical in the balance between tolerance and autoimmunity.
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ItemA Convenient Model of Severe, High Incidence Autoimmune Gastritis Caused by Polyclonal Effector T Cells and without Perturbation of Regulatory T Cells(PUBLIC LIBRARY SCIENCE, 2011-11-09)Autoimmune gastritis results from the breakdown of T cell tolerance to the gastric H(+)/K(+) ATPase. The gastric H(+)/K(+) ATPase is responsible for the acidification of gastric juice and consists of an α subunit (H/Kα) and a β subunit (H/Kβ). Here we show that CD4(+) T cells from H/Kα-deficient mice (H/Kα(-/-)) are highly pathogenic and autoimmune gastritis can be induced in sublethally irradiated wildtype mice by adoptive transfer of unfractionated CD4(+) T cells from H/Kα(-/-) mice. All recipient mice consistently developed the most severe form of autoimmune gastritis 8 weeks after the transfer, featuring hypertrophy of the gastric mucosa, complete depletion of the parietal and zymogenic cells, and presence of autoantibodies to H(+)/K(+) ATPase in the serum. Furthermore, we demonstrated that the disease significantly affected stomach weight and stomach pH of recipient mice. Depletion of parietal cells in this disease model required the presence of both H/Kα and H/Kβ since transfer of H/Kα(-/-) CD4(+) T cells did not result in depletion of parietal cells in H/Kα(-/-) or H/Kβ(-/-) recipient mice. The consistency of disease severity, the use of polyclonal T cells and a specific T cell response to the gastric autoantigen make this an ideal disease model for the study of many aspects of organ-specific autoimmunity including prevention and treatment of the disease.
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ItemThe Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm(PORTLAND PRESS LTD, 2011-12)BACKGROUND INFORMATION: Acid-secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H+,K+ ATPase-containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane-dense cytoplasm of parietal cells. RESULTS: Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta-nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis- and trans-Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H+,K+ ATPase-deficient mice that lack tubulovesicular membranes. CONCLUSIONS: These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.
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ItemAN AUTOIMMUNE-DISEASE WITH MULTIPLE MOLECULAR TARGETS ABROGATED BY THE TRANSGENIC EXPRESSION OF A SINGLE AUTOANTIGEN IN THE THYMUS(ROCKEFELLER UNIV PRESS, 1993-08-01)Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.
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ItemHelios defines T cells being driven to tolerance in the periphery and thymus(WILEY, 2014-07)The expression of the Ikaros transcription factor family member, Helios, has been shown to be associated with T-cell tolerance in both the thymus and the periphery. To better understand the importance of Helios in tolerance pathways, we have examined the expression of Helios in TCR-transgenic T cells specific for the gastric H(+) /K(+) ATPase, the autoantigen target in autoimmune gastritis. Analysis of H(+) /K(+) ATPase-specific T cells in mice with different patterns of H(+) /K(+) ATPase expression revealed that, in addition to the expression of Helios in CD4(+) Foxp3(+) regulatory T (Treg) cells, Helios is expressed by a large proportion of CD4(+) Foxp3(-) T cells in both the thymus and the paragastric lymph node (PgLN), which drains the stomach. In the thymus, Helios was expressed by H(+) /K(+) ATPase-specific thymocytes that were undergoing negative selection. In the periphery, Helios was expressed in H(+) /K(+) ATPase-specific CD4(+) T cells following H(+) /K(+) ATPase presentation and was more highly expressed when T-cell activation occurred in the absence of inflammation. Analysis of purified H(+) /K(+) ATPase-specific CD4(+) Foxp3(-) Helios(+) T cells demonstrated that they were functionally anergic. These results demonstrate that Helios is expressed by thymic and peripheral T cells that are being driven to tolerance in response to a genuine autoantigen.
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ItemThymic expression of a gastritogenic epitope results in positive selection of self-reactive pathogenic T cells(AMER ASSOC IMMUNOLOGISTS, 2004-05-15)Intrathymic expression of tissue-specific self-Ags can mediate tolerance of self-reactive T cells. However, in this study we define circumstances by which thymic expression of a tissue-specific autoepitope enhances positive selection of disease-causing, self-reactive T cells. An immunodominant gastritogenic epitope, namely the gastric H/K ATPase beta subunit(253-277) (H/Kbeta(253-277)), was attached to the C terminus of the invariant chain (Ii) and the hybrid Ii (Ii-H/Kbeta(253-277)) expressed in mice under control of the Ii promoter. The Ii-H/Kbeta(253-277) fusion protein was localized to MHC class II-expressing cells in the thymus and periphery of Ii-H/Kbeta(253-277) transgenic mice. In one transgenic line the level of presentation in the periphery (spleen) was insufficient to activate naive, low affinity H/Kbeta(253-277)-specific transgenic T cells (1E4-TCR), whereas thymic presentation of H/Kbeta(253-277) enhanced positive selection of 1E4-TCR cells in Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice. Furthermore, Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice had an increased incidence of autoimmune gastritis compared with 1E4-TCR single-transgenic mice, demonstrating that the 1E4 T cells that seeded the periphery of Ii-H/Kbeta(253-277) mice were pathogenic. Therefore, low levels of tissue-specific Ags in the thymus can result in positive selection of low avidity, self-reactive T cells. These findings also suggest that the precise level of tissue-specific Ags in the thymus may be an important consideration in protection against autoimmune disease and that perturbation of the levels of self-Ags may be detrimental.
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ItemPromiscuous thymic expression of an autoantigen gene does not result in negative selection of pathogenic T cells(AMER ASSOC IMMUNOLOGISTS, 2005-11-01)"Promiscuous" thymic expression of peripheral autoantigens can contribute to immunological tolerance in some cases. However, in this study we show that thymic mRNA expression alone cannot predict a contribution to thymic tolerance. Autoimmune gastritis is caused by CD4+ T cells directed to the alpha (H/Kalpha) and beta (H/Kbeta) subunits of the gastric membrane protein the H+/K+ ATPase. H/Kalpha mRNA is expressed in the thymus, but H/Kbeta expression is barely detectable. In this study, we demonstrate that thymic H/Kalpha in wild-type mice or mice that overexpressed H/Kalpha did not result in negative selection of pathogenic anti-H/Kalpha T cells. However, negative selection of anti-H/Kalpha T cells did occur if H/Kbeta was artificially overexpressed in the thymus. Given that H/Kalpha cannot be exported from the endoplasmic reticulum and is rapidly degraded in the absence of H/Kbeta, we conclude that H/Kalpha epitopes are unable to access MHC class II loading compartments in cells of the normal thymus. This work, taken together with our previous studies, highlights that thymic autoantigen expression does not necessarily result in the induction of tolerance.
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ItemCD4+CD25+ regulatory T cells inhibit the antigen-dependent expansion of self-reactive T cells in vivo(AMER ASSOC IMMUNOLOGISTS, 2006-02-01)A deficiency of CD4+CD25+ regulatory T cells (CD25+ Tregs) in lymphopenic mice can result in the onset of autoimmune gastritis. The gastric H/K ATPase alpha (H/Kalpha) and beta (H/Kbeta) subunits are the immunodominant autoantigens recognized by effector CD4+ T cells in autoimmune gastritis. The mechanism by which CD25+ Tregs suppress autoimmune gastritis in lymphopenic mice is poorly understood. To investigate the antigenic requirements for the genesis and survival of gastritis-protecting CD25+ Tregs, we analyzed mice deficient in H/Kbeta and H/Kalpha, as well as a transgenic mouse line (H/Kbeta-tsA58 Tg line 224) that lacks differentiated gastric epithelial cells. By adoptive transfer of purified T cell populations to athymic mice, we show that the CD25+ Treg population from mice deficient in either one or both of H/Kalpha and H/Kbeta, or from the H/Kbeta-tsA58 Tg line 224 mice, is equally effective in suppressing the ability of polyclonal populations of effector CD4+ T cells to induce autoimmune gastritis. Furthermore, CD25+ Tregs, from either wild-type or H/Kalpha-deficient mice, dramatically reduced the expansion of pathogenic H/Kalpha-specific TCR transgenic T cells and the induction of autoimmune gastritis in athymic recipient mice. Proliferation of H/Kalpha-specific T cells in lymphopenic hosts occurs predominantly in the paragastric lymph node and was dependent on the presence of the cognate H/Kalpha Ag. Collectively, these studies demonstrate that the gastritis-protecting CD25+ Tregs do not depend on the major gastric Ags for their thymic development or their survival in the periphery, and that CD25+ Tregs inhibit the Ag-specific expansion of pathogenic T cells in vivo.