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Spontaneous Large-Scale Lymphoid Neogenesis and Balanced Autoimmunity versus Tolerance in the Stomach of H⁺/K⁺-ATPase-Reactive TCR Transgenic Mouse¹

Tomoya Katakai^*, Takashi Nomura, Hiroyuki Gonda^*,¶, Manabu Sugai^*, Yasutoshi Agata, Akiyoshi Nishio, Tohru Masuda^||, Shimon Sakaguchi and Akira Shimizu^2,*,¶

^* Center for Genomic Medicine, Graduate School of Medicine, Department of Experimental Pathology, Institute for Frontier Medical Sciences, Horizontal Medical Research Organization, Kyoto University, Kyoto, Japan; Department of Gastroenterology and Hepatology, ^¶ Translational Research Center, Kyoto University Hospital, Kyoto, Japan; and ^|| Uji Takeda Hospital, Uji, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Autoimmunity is often accompanied by the development of ectopic lymphoid tissues in the target organ, and these tissues have been believed to have close relevance to the severity of the disease. However, the true relationship between the extent of such lymphoid structures and the intensity or type of immune responses mediated by self-reactive T cells has remained unclear. In the present study, we generated transgenic mice expressing TCR from an autoimmune gastritis (AIG)-inducing Th1 cell clone specific for one of the major stomach self-Ags, H⁺/K⁺-ATPase subunit. The transgenic mice spontaneously develop massive lymphoid neogenesis with a highly organized tissue structure in the gastric mucosa, demonstrating Ag-specific, T cell-mediated induction of the lymphoid tissues. Nevertheless, the damage of surrounding tissue and autoantibody production were considerably limited compared with those in typical AIG induced by neonatal thymectomy. Such a moderate pathology is likely due to the locally restricted activation and Th2 skewing of self-reactive T cells, as well as the accumulation of naturally occurring regulatory T cells in the target organ. Altogether, the findings suggest that lymphoid neogenesis in chronic autoimmunity does not simply correlate with the destructive response; rather, the overall activation status of the T cell network, i.e., the balance of self-reactivity and tolerance, in the local environment has an impact.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Autoimmunity occurs as a consequence of inappropriate responses of the immune system toward the body’s own components. The processes leading to severe pathophysiological conditions in autoimmune diseases are complicated and depend on various genetic and/or environmental factors (1, 2, 3). Most autoimmune disorders can be classified into roughly two categories, organ-specific and systemic (1). In the former category, the self-reactivity is directed against a restricted set of organ-specific Ags expressed in the affected cells or tissues, whereas in the latter, widely distributed self-components seem to be the target. In many autoimmune diseases, T cells expressing self-reactive TCR play a central role in the triggering and progression of the disease pathology (1, 2), although the root cause of the initial sensitization of T cells against the self-components remains largely unclear. During the last decade, however, accumulating evidence has demonstrated that CD4⁺CD25⁺ FoxP3⁺ naturally occurring regulatory T cells (Tregs)³ play a crucial role in peripheral tolerance via suppressing the priming of self-reactive T cells (4, 5). This indicates a possible mechanism by which autoimmunity can be triggered if the activity and/or numbers of Tregs are reduced (5, 6) and, in addition, indicates the presence of a complicated interaction network in the T lymphocyte compartment for maintaining the homeostasis of the immune system.

Once immune cells’ reactivity to certain self-components is permitted, organs expressing the target Ags undergo infiltration of activated lymphocytes and, in most cases, suffer resultant functional destruction of the tissue. In such a situation, the release of self-Ags from the target site might be a driving force for sustaining autoimmunity, namely, the chronic phase of the immune reaction. Occasionally, ectopically induced lymphoid aggregates with well- organized tissue structure, i.e., histologically resembling secondary lymphoid organs (SLOs) such as lymph nodes (LNs) and Peyer’s patches, appear in the chronically inflamed tissues (7, 8, 9, 10). Such newly generated lymphoid structures are defined as "tertiary lymphoid tissues (TLTs)" (11). Although the significance of the ectopic lymphoid structures and detailed events occurring within such local sites are still largely unknown, they may support a vicious cycle of self-reactivity for maintaining chronic autoimmunity, eventually resulting in dysfunction of the target organ. The relationship between TLT development and the activation status of lymphocytes with respect to a certain self-Ag also remains obscure.

Autoimmune gastritis (AIG) is an organ-specific autoimmune disease that occurs in 1–2% of people over 60 years old in Western populations (12, 13, 14). AIG is characterized by the extensive loss of parietal cells in the gastric mucosa (GM) and is associated with pernicious anemia as a consequence of vitamin B₁₂ deficiency. Production of serum autoantibody against the gastric parietal cells is a typical index of AIG; one of the major molecular targets is H⁺/K⁺-ATPase (proton pump) expressed in the parietal cells. AIG can be induced in BALB/c mouse at high frequency by neonatal thymectomy (at 3 days after birth, d3-Tx), and the disease pathology closely resembles many features of human AIG (13, 15, 16, 17). The initiation of self-reactivity in this model is thought to be due to the retardation of normal Treg differentiation together with lymphopenia caused by d3-Tx (6, 13, 18). Self-reactive CD4⁺ T cells, especially Th1 cells, are the major effectors in the GM tissue destruction (19, 20, 21, 22, 23, 24, 25). Moreover, tertiary lymphoid aggregates are often observed in the inflamed GM, with histological characteristics similar to the SLOs: a clear segregation of T and B cells, well-developed lymphoid stromal cell network, and follicle-like B cell areas supported by follicular dendritic cells producing a homeostatic chemokine CXCL13 (25). On the other hand, these ectopic lymphoid structures lack some important components common in the SLOs, e.g., high endothelial venule (HEV)-like blood vessels, expression of another homeostatic chemokine, CCL21, and naive T cell homing (25).

In this study, to understand the self-reactivity and tolerance mediated by the complicated network of T cells in organ-specific autoimmunity, we generated transgenic mice expressing H⁺/K⁺-ATPase subunit-specific TCR isolated from a gastritogenic CD4⁺ Th1 cell clone (26, 27). The transgenic mice spontaneously show massive lymphoid neogenesis in the stomach. However, target tissue destruction and autoantibody production are considerably less extensive or milder than those in the d3-Tx AIG model. Several lines of evidence suggest that autoimmunity and tolerance are delicately balanced within the local target site, providing some insights into self-reactivity-driven, T cell-mediated lymphoid neogenesis in chronic autoimmune diseases.

	Materials and Methods

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T2-6AB transgenic mice and d3-Tx AIG

H⁺/K⁺-ATPase-reactive CD4⁺ T cell clone II-6 was maintained in vitro as described previously (26). Genomic fragments encoding the rearranged variable regions of TCR- and -chains along with endogenous promoter regions were isolated from II-6 cells. Cloned fragments were ligated with the respective constant region genomic cassettes (28, 29). After plasmid sequences were removed, the linearized constructs were injected into fertilized BALB/c oocytes, and a transgenic founder bearing both and genes was obtained. This transgenic line was named T2-6AB. Transgenes were detected by genomic PCR using the following specific primer pairs: for V-J, 5'-GACTCGAGCTGCAGTGTAACTTTTCC-3' (V-V2) and 5'-TTGGATGGACCCTAAGGATG-3' (J-c5a); for V-J, 5'-GAGGATCCATCAATGGCCAGTTGCC-3' (V-14-1) and 5'-AACCAGACTGACTGTTCTCG-3' (J-2.3-1). T2-6AB mice under the age of 6 mo were used for experiments. Neonatal thymectomy of BALB/c mice 3 days after birth was performed as described previously (15, 23). Serum autoantibody was detected by ELISA and the microsome fraction prepared from normal mice glandular stomach was used as Ag (23). pH in the stomach lumen was measured with test papers (Macherey-Nagel). Mice were maintained in specific pathogen free or conventional conditions at the animal facility in the Center for Genomic Medicine, Kyoto University. Procedures involving animals and their care were conducted according to the guidelines for animal treatment of the Institute of Laboratory Animals, Kyoto University.

Antibodies

Abs used for immunohistochemistry or flow cytometry were as follows: as primary reagents, unlabeled, biotin-, FITC-, or PE-labeled anti-B220, anti-CD3, anti-CD4, anti-CD25, anti-CD35, anti-CD45RB, anti-CD62L, anti-CD69, anti-V14, anti-mucosal addressin cell adhesion molecule (MAdCAM)-1, anti-peripheral node addressin (PNAd), anti-IFN-, anti-IL-4, anti-IL-10 (BD Pharmingen), anti-FoxP3 (eBioscience), anti-CCL21, anti-CXCL13 (R&D Systems), ER-TR7 (BMA), and anti-H⁺/K⁺-ATPase (1H9, hybridoma supernatants) (16); as secondary reagents, Texas Red-anti-mouse IgG, HRP-anti-mouse IgG, PE-anti-rat IgG, biotin-anti-rat IgG (Caltag Laboratories), biotin-anti-mouse IgM (BD Pharmingen), Alexa488-anti-rabbit IgG, PE-streptavidin, and allophycocyanin-streptavidin (Molecular Probes). To produce II-6 TCR/hIgG1-Fc chimeric protein, a variable region fragment was amplified by PCR and ligated to an hIgG1-Fc fragment in pMKIT-neo vector. X63.653 myeloma cells were transfected with the expression vector to obtain a stable transfectant, and the chimeric protein was purified from the ascites fluid. Rabbit anti-sera were obtained by the immunization of animals with the chimeric protein.

Immunohistochemistry

Glandular stomachs and LNs were isolated from the mice, embedded in OCT compound (Sakura Finetechnical) and then frozen in liquid nitrogen. Frozen sections (10 µm) were fixed with cold acetone for 5 min and were stained with hematoxylin (Harris modified; Sigma-Aldrich) or with Abs by direct or indirect methods. Nuclear DNA was stained with 4',6'-diamino-2-phenylindole (DAPI; Sigma-Aldrich). Stained sections were then examined using a confocal laser scanning microscope (TSC-SP2; Leica Microsystems) or a fluorescence microscope (TND330; Nikon). Digital images obtained were prepared using Adobe Photoshop software (Adobe Systems).

Flow cytometry

Cell suspensions were prepared from the thymus, spleen, and LNs. Infiltrating cells in the GM were collected as described previously (23). Cells were stained with Abs by direct or indirect methods and analyzed using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences). Detection of intracellular cytokines was done as described previously (23). FoxP3 was detected after cells were stained for cell suface markers, fixed, and permeabilized using a staining set (eBioscience).

In vitro proliferation assay

Five hundred thousand spleen cells were incubated with various concentrations of 890–904 synthetic peptides for 48 h, which were pulsed with 1 µCi of [³H]thymidine during the last 6 h before harvest. The radioactivity was measured using a scintillation counter.

Adoptive transfer

CD4⁺ T cells were enriched from spleen cell suspensions by panning to deplete B cells, CD8⁺ T cells, and adherent cells as described previously (4). CD25⁺ T cells were depleted by treatment with anti-CD25 (7D4) and rabbit complement (Cedarlane Laboratories). Equal numbers (3 x 10⁶–1.2 x 10⁷) of CD4⁺ or CD4⁺CD25^– T cells from the same donor were i.v. injected into BALB/c nu/nu mice. The recipient mice were histologically and serologically examined 3 mo later.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Generation of TCR transgenic mice specific to H⁺/K⁺-ATPase subunit 890–904 epitope

We previously reported that a gastritogenic CD4⁺ T cell clone II-6 expresses TCR responsive to H⁺/K⁺-ATPase 890–904 (mouse) epitope in an MHC class II (I-A^d)-restricted manner (26, 27). The variable regions of II-6 TCR are composed of V13.2 (we initially reported it as V10 (27), but it was recently reclassified; National Center for Biotechnology Information accession no. AAL08186) and V14. To produce transgenic mice expressing this TCR in T cells, we cloned genomic fragments encoding rearranged VJ and VDJ with each promoter region from II-6 cells and ligated them with genomic fragments containing constant region genes and enhancer sequences, respectively (Fig. 1A). The constructs were injected into BALB/c oocytes. We then obtained a mouse line named T2-6AB bearing both TCR and transgenes, which were probably incorporated into a single locus of the genome. Flow cytometric analysis of T2-6AB thymocytes revealed that the ratio of CD4- and CD8-single-positive cells was strongly biased toward the CD4 compartment compared with that of nontransgenic littermates (Fig. 1B). T cells from the spleen and LNs also showed clear skewing toward the CD4⁺ population in T2-6AB mice (Fig. 1B). To detect the expression of transgenic TCR- and -chains on the cell surface, we used mAb to V14 and rabbit anti-sera developed against a chimeric protein of the variable region of II-6 TCR fused with human IgG1-Fc. Over 70% of CD4⁺ cells in T2-6AB mice expressed both II-6-type V and V14, while <2% of CD4⁺ cells in nontransgenic mice were positive for these V regions (Fig. 1C). Spleen cells from the transgenic mice exhibited a strong proliferative response to the specific peptide in vitro, whereas nontransgenic cells showed no obvious proliferation (Fig. 1D). These findings indicate that T cells expressing transgenic TCR can be efficiently selected and differentiated in the thymus and that the functional CD4⁺ T cells are exported to the periphery in T2-6AB mice.

View larger version (47K): [in this window] [in a new window]   FIGURE 1. Establishment of T2-6AB transgenic mice. A, II-6 TCR constructs used for generating the transgenic mice. A genomic fragment encoding rearranged II-6 TCR (2.8 kb) or (11 kb) variable region was joined to a fragment containing constant region (C or C) and enhancer (E or E). Italic letters indicate the positions of representative restriction enzyme recognition sites. B, T cells are efficiently produced and biased toward CD4 compartment in T2-6AB mice. Thymus, spleen, or LN cells isolated from 3-mo-old T2-6AB mice (Tg+) or nontransgenic littermates (Tg–) were stained with Abs to CD4 and CD8 and analyzed by flow cytometry. C, A majority of CD4+ T cells in T2-6AB mice express II-6-type TCR. LN cells were stained with Abs to CD4, V14, and rabbit antiserum raised against II-6 TCR variable region, and were analyzed by flow cytometry. CD4+-gated cells are shown. D, Transgenic T cells show specific proliferative response to H+/K+-ATPase 890–904 peptide in vitro. Spleen cells from T2-6AB mice or nontransgenic littermates were cultured with the indicated concentrations of 890–904 peptide and proliferation was determined by [3H]thymidine incorporation. The results are shown as mean ± SD.

We next examined the histology of the stomach of T2-6AB mice at various ages. Strikingly, the stomachs of all mice examined (8 wk to 6 mo, n >50) suffered from obvious lymphocyte infiltration, but interestingly, a large part of the cells clearly formed aggregates within the mucosal layer (Fig. 2A). The accumulation of lymphocytes often extended from the basal part to nearly the apical region surface of the GM (Fig. 2B). Immunohistochemistry revealed that T (CD3⁺) and B (B220⁺) cells were clearly segregated to form distinct subregions: T cells were mostly localized in the basal part, while B cells were relatively concentrated in the apical side (Fig. 2, B and C). All the aggregates were supported by a typical reticular stromal network visualized by immunohistochemical staining for ER-TR7, and in most cases, closely associated with large blood vessels expressing MAdCAM-1 (Fig. 2C). Lymphoid aggregates or smaller clusters could be seen in the GM of T2-6AB mice as early as 4 wk after birth (data not shown), whereas no discernable lymphocyte infiltration was observed in nontransgenic littermates or syngeneic BALB/c mice. These findings suggest that H⁺/K⁺-ATPase-specific T cells actually can recognize the target in a T2-6AB environment, infiltrate into the stomach, and eventually organize tissue structures categorized as TLTs.

View larger version (85K): [in this window] [in a new window]   FIGURE 2. T2-6AB mice show large-scale lymphoid neogenesis but with moderate histopathology in the stomach. A, Infiltrating lymphocytes form large aggregates within the glandular stomach of T2-6AB mice. Horizontal section of the stomach from a 5-mo-old T2-6AB mouse was stained with hematoxylin and examined by light microscopy. Multiple isolated lymphoid aggregates were located in a scattered pattern in the GM (arrows). B, T and B cells are segregated within a lymphoid aggregate that is adjacent to H+/K+-ATPase-expressing parietal cells. Perpendicular section was stained with Abs to CD3 (T cells), B220 (B cells), and H+/K+-ATPase subunit (parietal cells) and examined by cofocal microscopy. C, An example of a huge lymphoid aggregate with ER-TR7+ lymphoid stromal reticulum and MAdCAM-1+ blood vessels. Serial sections were stained for the indicated markers. D, Histological comparison of the stomach in normal, T2-6AB, and d3-Tx AIG mice. Sections were stained with hematoxylin (upper panel) or fluorescent-labeled Abs to B220 and H+/K+-ATPase (lower panel). Arrows indicate the locations of lymphoid aggregates. Large lymphoid aggregates are present within significantly expanded and slightly disturbed GM tissue in T2-6AB mice. Bars, 200 µm for A and D; 100 µm for B and C.

Highly organized architecture of the TLTs that support naive T cell homing in T2-6AB stomach

HEV endothelial cells and stromal cells in the T cell area of the SLOs express PNAd and CCL21, both of which are crucial for naive T cell recruitment and localization (30, 31, 32). As we have shown previously, however, TLTs induced in the stomach of d3-Tx AIG mice lack these essential characteristics of lymphoid tissue (25). We therefore examined whether these tissue components are observed in the TLTs of the T2-6AB stomach. It was evident that vessels with high endothelial morphology were often associated with GM lymphoid tissues (Fig. 3A). Moreover, the expression of both PNAd and CCL21 was readily detected and was restricted to the vessels and stromal cell network within the TLT, particularly in the T cell area, and was not seen in other sites of the stomach (Fig. 3B). These structural features closely resembled those of the SLOs such as regional gastric LNs (GLNs) (Fig. 3B). In the SLOs, different stromal populations expressing CCL21 and CXCL13 constitute two lymphocyte compartments, i.e., the T cell area and B cell area (follicles), respectively (31). Likewise, a mutually exclusive pattern of CCL21- and CXCL13-producing stromal cells with an intricate filamentous network in each compartment of TLTs was observed (Fig. 3C). Therefore, TLTs developed in the T2-6AB stomach are highly organized and show clearly different features from those induced in d3-Tx AIG.

View larger version (63K): [in this window] [in a new window]   FIGURE 3. Ectopic lymphoid tissues in the stomach of T2-6AB mice are highly organized with typical lymphoid tissue architecture. A, HEV-like vessels inside ectopic lymphoid tissues. Hematoxylin staining. High magnification view (right panel) shows vessels with typical high endothelial cell morphology (arrows). B, HEV-like vessels in the GM lymphoid tissues express PNAd and CCL21. Serial sections from GM and regional LN (GLN) were stained for the indicated markers. C, Mutually exclusive pattern of CXCL13- and CCL21-expressing stromal components were colocalized with B (B220+) and T (CD3+) cells, respectively, within GM lymphoid tissue. Characteristic networks of reticular stromal cells (ER-TR7+CCL21+) in T cell area and follicular dendritic cells (CD35+CXCL13+) in B cell area were observed. Dotted lines represent the border of T and B cell areas. Bars, 100 µm for A (left panel) and B; 50 µm for A (right panel) and C.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Ectopic lymphoid tissues in the stomach of T2-6AB mice support naive T cell homing. A and B, Naive CD4+ T cells defined as CD45RBhigh and CD62Lhigh are present in the GM of T2-6AB mice, whereas they are almost absent from the GM of d3-Tx AIG mice. Representative flow cytometric profiles of cells isolated from GM or GLN stained for CD4 and CD45RB (A) or CD62L (B). Percentages of CD62L+ cells in CD4+ fractions are shown in (C). , individual mice; bars, mean ± SD.

In general, d3-Tx treatment induces AIG in 5080% of BALB/c mice, and the animals with severe disease concomitantly show high titers of anti-parietal cell autoantibody in their sera, which reflects the status of activation of lymphocytes to the self-Ags, whereas some d3-Tx mice do not develop AIG and show no autoantibody production, possibly due to operational error or some resistance to neonatal thymectomy (Fig. 5, A and B). To evaluate the intensity of anti-self-responses in T2-6AB mice, we next checked the serum autoantibody. Despite the fact that substantial amounts of autoantibody were constantly detected in sera from the transgenic animals compared with nontransgenic mice, the titers were much lower than those in sera from d3-Tx mice with AIG (Fig. 5, A and B). No remarkable changes in the autoantibody production were observed depending on the age of animals (Fig. 5C). Among the T2-6AB mice, a few (<10%) displayed relatively higher levels of autoantibody comparable to those in d3-Tx AIG mice with the lowest titer, and sera from such animals only weakly stained the parietal cells of the normal stomach (Fig. 5D). These findings are consistent with the above observations that disease severity is moderate in T2-6AB mice, supporting the notion that the activation level of lymphocytes is not so intense.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Autoantibody production in T2-6AB mice is limited. A and B, Production of autoantibody to GM Ags in T2-6AB mice is markedly lower than in d3-Tx mice bearing AIG. Reactivity to GM Ags of serially diluted sera from T2-6AB, nontransgenic littermate, and d3-Tx-treated mice (2–3 mo old) was measured by ELISA. Each reaction curve corresponds to an individual mouse and a representative result obtained from a group of mice is shown. Autoantibody titers (serum dilution point that yields OD over 0.05) from a larger number of mice are shown in B. C, Autoantibody production in T2-6AB mice does not change significantly with age. Time course of serum autoantibody production (absorbance in x40 dilution point) in a Tg+ littermate group was determined. Results at different time points in individual mice are connected by lines. D, Sera from T2-6AB mice that showed relatively higher autoantibody titers only weakly stained the parietal cells, whereas sera with lower titers did not stain them. Serial sections from normal mice stomach were stained with diluted sera (x40) and examined by fluorescent microscopy.

In the regional GLNs of T2-6AB mice, the fraction of CD4⁺ T cells expressing activation marker CD69 was <30%, which was the same as the fraction in normal mice GLNs, while 50–70% were CD69⁺ in the same LNs in d3-Tx AIG mice, indicating that only a fraction of T cells are activated even in the regional LNs of T2-6AB mice (Fig. 6). In contrast, 40–60% of CD4⁺ T cells in the infiltrates from T2-6AB mouse stomachs expressed CD69, which was comparable to the fraction in d3-Tx AIG mice, suggesting that many T cells accumulated in the GM are in an activated state (Fig. 6). To address whether the cells in the T2-6AB stomach show effector functions, we examined the ability of cytokine production. As also shown in previous studies (23, 24, 25), GM infiltrates from d3-Tx AIG mice, not only CD4⁺ T cells but also other cell fractions (mainly CD8⁺ T cells) strongly produced IFN- (Fig. 7A). By contrast, only a few cells from GLNs and GM in T2-6AB were IFN- producers. However, it was surprising that CD4⁺ T cells present in the GM, but not in the GLNs, of T2-6AB mice were largely skewed to IL-4-producing cells (Fig. 7, B and C). This situation is in contrast to that in d3-Tx AIG, in which GM lesions were largely Th1-biased, while both Th1 and Th2 coexisted in the GLNs (Fig. 7, B and C) (23, 24, 25). Upon in vitro stimulation, T2-6AB T cells were able to differentiate into both Th1 and Th2 cells (data not shown), indicating that the Th2-bias is not an intrinsic predisposition in T2-6AB T cells. Collectively, these findings suggest that although T2-6AB T cells localized in the GM have undergone a kind of activation, they do not generate potentially destructive Th1 effector cells, but rather brings about a Th2-dominated local microenvironment.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. T cell activation is restricted to the stomach of T2-6AB mice. A and B, Many CD4+ T cells in the GM, but a fraction of cells in the GLN, express early activation marker CD69 in T2-6AB mice. Cells from GLN and GM were stained with anti-CD69 Ab and examined by flow cytometry. Percentages of CD69+ cells in CD4+ fractions are shown in B. , individual mice; bars, mean ± SD.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. CD4+ T cells are biased to Th2 in the stomach of T2-6AB mice. A, Few GM-infiltrating CD4+ T cells in T2-6AB stomach produce IFN-. After isolated cells were transiently stimulated in vitro coupled with blockage of the secretory pathway, the ability to produce IFN- was assessed by intracellular staining and flow cytometry. Lymphocyte-gated cells are shown. B and C, Cytokine production in GM-infiltrating CD4+ T cells is skewed to IL-4. Intracellular IFN- and IL-4 in CD4+ fractions are shown (B) and the percentages of IFN-+ or IL-4+ cells are shown in C.

Naturally occurring CD4⁺CD25⁺FoxP3⁺ Tregs have been shown to be the critical T cell subset suppressing the development of various autoimmune diseases, including the AIG model (4, 6, 33). We observed that the number of Tregs in the periphery of adult T2-6AB mice was nearly the same as the number in normal BALB/c mice (Fig. 8, A and B). However, Tregs were significantly increased in the CD4⁺ cell fraction in the GM infiltrates compared with the GLNs. Importantly, Tregs in the GM, but not in the GLNs, exhibited marked IL-10-producing ability, although the other cells that could produce higher amounts of IL-4 and/or IL-10 (presumably the conventional Th2 cells) were mostly included in the CD25-intermediate fraction (Fig. 8C). In tissue sections, CD4⁺CD25⁺FoxP3⁺ cells were readily detected within the TLTs and selectively localized in the T cell area (Fig. 8, D and E). These observations are consistent with the idea that the accumulation and locally restricted activation of Tregs as well as Th2 dominancy prevent the induction of destructive responses in the T2-6AB stomach.

View larger version (27K): [in this window] [in a new window]   FIGURE 8. Tregs are accumulated in the GM lymphoid tissues of T2-6AB mice stomach. A, Number of Tregs present in the GLN of T2-6AB mice is comparable to that present in the peripheral LN (PLN) of normal BALB/c mice, while it is significantly increased in the GM. Cells from the indicated tissues were stained for CD4, CD25, and intracellular FoxP3 and examined by flow cytometry. Lymphocyte-gated cells are shown in the upper panels, and the expression of CD25 and FoxP3 in CD4+-gated cells (upper boxes) is shown in the lower panels. Percentages of CD25+FoxP3+ cells in CD4+ fractions are shown in B. , individual mice; bars, mean ± SD. C, Tregs in the T2-6AB GM produce IL-10. Cells were stained for CD4 and CD25, followed by intracellular cytokine staining for IL-4 (upper panel) or IL-10 (lower panel). D and E, Tregs are selectively localized in the T cell area of GM lymphoid tissue. Sections from GLN and GM of T2-6AB mice were stained for the indicated markers. Four-color staining reveals that FoxP3 signals are localized in the nucleus (DAPI staining) of CD4+CD25+ cells (E). Higher magnification views of insets in upper panels are shown in lower panels. Bars, 30 µm.

The results described above raise the possibility that Treg function plays an important role in the moderate pathology of T2-6AB mice. To address this possibility as well as to evaluate the potential pathogenicity of T2-6AB T cells, we performed adoptive transfer experiments in which two groups of CD4⁺ T cells (CD25⁺ depleted and nondepleted) prepared from a donor T2-6AB mouse were transferred to syngeneic nu/nu mice. Consequently, six independent experiments revealed that the simple transfer of T2-6AB CD4⁺ T cells to nude mice induced an apparently progressive gastritis more severe than the gastritis in the donor T2-6AB mice, although the degree of the parietal cell loss was not as intensive as that in the d3-Tx AIG (Fig. 9). Autoantibody production in five of the six recipients was, however, still weak and similar to the level in T2-6AB mice, except in one case that displayed a relatively high level of serum autoantibody possibly due to the aberrant activation of the transferred cells (Fig. 10). Remarkably, nude mice that received CD25⁺-depleted T cells developed much more severe gastritis that was indistinguishable from that in d3-Tx AIG, i.e., with gross and diffuse lymphocyte infiltration, drastic mucosal hyperplasia, and extensive loss of parietal cells throughout the mucosal layer of the stomach (Fig. 9). The tissue invasion of CD4⁺ T cells into the lamina propria was very severe (Fig. 9B). These recipients also showed significantly higher autoantibody production than those that received CD25⁺-containing cells from the identical donors, except for the one case described above that showed higher autoantibody titer despite the presence of Treg cells (Fig. 10).

View larger version (77K): [in this window] [in a new window]   FIGURE 9. Adoptive transfer of T2-6AB CD4+ T cells to nude mice triggers progressive gastritis, which is further exacerbated by CD25+ cell depletion but does not result in large-scale lymphoid neogenesis. A and B, Comparative histology of the GM in normal, d3-Tx AIG, T2-6AB mice, and nude mice recipients that received T2-6AB T cells. CD4+ or CD4+CD25– cells isolated from T2-6AB mice were transferred to nude mice and examined 3 mo later. Sections were stained with hematoxylin (A) or fluorescent Abs to indicated markers and DAPI (B). Bars, 200 µm.

View larger version (18K): [in this window] [in a new window]   FIGURE 10. Adoptive transfer of T2-6AB CD4+CD25– T cells induces autoantibody production in nude mice. A and B, CD25+ cell depletion augments the production of autoantibody. Autoantibody to GM Ags in sera was determined by ELISA. Each reaction curve corresponds to the results of an individual mouse. Absorbance at the x40 dilution point of each recipient is shown in B, and the same symbols connected by a line correspond to a pair of recipients that received cells (CD4+ or CD4+CD25–) from an identical T2-6AB donor.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In this study, we established a TCR transgenic mouse line, T2-6AB, in which T cells expressing H⁺/K⁺-ATPase subunit-specific TCR construct a complicated network that leads to a unique immunomicroenvironment in the stomach, i.e., an environment in which self-reactivity and tolerance are locally balanced. It is noteworthy that autoimmune responses, including target tissue destruction and autoantibody production, are clearly limited even though T2-6AB T cells exhibit substantial self-reactivity, as most evident from the fact that large-scale lymphoid neogenesis occurs in the GM. Some intrinsic limitations on the activity of effector T cells and the function of naturally occurring Tregs are likely to give rise to such a situation.

Three transgenic mouse lines expressing H⁺/K⁺-ATPase-reactive TCR have been reported so far (34, 35, 36, 37). All those TCRs are MHC class II-restricted, but two of them recognize the subunit, while one recognizes the subunit of H⁺/K⁺-ATPase. Highly gastritogenic Th1 clone TxA23 was derived from the GLN of a d3-Tx AIG mouse and recognizes the 630–641 epitope of H⁺/K⁺-ATPase subunit (38). Using the TCR genes from TxA23, transgenic mouse line A23 was established and, strikingly, all of the mice in that line spontaneously develop severe gastritis with extremely early onset (35). As few as 10³ A23 thymocytes can transfer disease to immunocompromised mice, indicating the high pathogenic potential of A23 T cells. TxA51 is another clone isolated from a d3-Tx AIG mouse, but it shows Th2 character and recognizes the 889–901 epitope (38). A51 transgenic mice expressing the TxA51 TCR also develop spontaneous gastritis with a lower frequency (58%: 40% of the animals show mild gastritis, and 18% have a more severe form) and relatively later onset of disease than A23 mice (36). Interestingly, A51 mice show eosinophilic infiltration in the GM as well as clearly Th2-biased responses in the GLNs, indicating that T cells produced in this transgenic mouse not only expressed A51 TCR but also showed the Th2 tendency, and, more importantly, indicating that even Th2 effector cells can induce severe gastritis. On the other hand, H⁺/K⁺- ATPase 253–277-specific 1E4 T cells are a hybridoma generated from a normal mouse immunized with the target peptide (37). Only a small percentage (<20%) of 1E4 TCR-expressing transgenic mice develop gastritis, probably due to low affinity of 1E4 TCR and consequently inefficient positive selection in the thymus. However, when 1E4 mice are crossed with transgenic mice expressing the epitope peptide sequence attached to the C terminus of the invariant chain (Ii-H/K253–277) in the thymus, the positive selection of 1E4 T cells is enhanced and the incidence of AIG is increased (39). Collectively, disease incidence and severity are quite diverse among various transgenic lines, including T2-6AB. Such phenotypic variety is most likely owing to differences in the affinity and expression level of transgenic TCRs used, by which the development and activation of not only effector but also Tregs are affected. However, no large-scale lymphoid neogenesis was reported in the above three transgenic mice other than T2-6AB.

The above facts raise the possibility that II-6-type TCR expressed in T2-6AB mice might possess an intermediate affinity among the four H⁺/K⁺-ATPase-specific TCRs. Because T2-6AB mice efficiently produce MHC class II-restricted T cells, positive selection takes place normally in the thymus. Moreover, mature T cells can respond well to antigenic peptide in vitro and probably in vivo, as suggested by the fact that all adult mice display the infiltration of lymphocytes with activated phenotypes in the stomach. Therefore, T2-6AB TCR seems to be more efficient than 1E4 TCR because thymic selection in the 1E4 mice is very inefficient and the incidence of gastritis is low (37). However, the overall disease severity in T2-6AB mice is clearly milder compared with that observed in the d3-Tx AIG model and transgenic lines such as A23 and A51 (35, 36). Treg-depleted T cells from T2-6AB mice are able to transfer severe disease into nude mice, demonstrating the sufficient gastritogenicity of T2-6AB T cells, particularly in the absence of Tregs. However, even though functional Tregs are also produced in A23 transgenic mice, all A23 mice develop severe gastritis, and only a small number of A23 thymocytes can induce gastritis in nude mice, suggesting that, if TCR has a high potential, it overwhelms the influence of Tregs (35).

Noteworthy, TxA51 and II-6 recognize overlapping epitopes, 889–901 and 890–904, respectively (26, 38). T cell clones recognizing nearly the same region of the subunit are also isolated from the GM biopsies of human AIG patients (40), suggesting that this part might be one of the dominant subunit epitopes conserved in mouse and human AIG. It is interesting that A51 and T2-6AB show some similarities with regard to bias toward Th2 and relatively mild symptoms, although A51 T cells seem to be able to exert more intensive and destructive responses. Candon et al. (36) pointed out that the presentation of the epitope recognized by A51 T cells is inefficient in vivo, which may result in the preferential induction of Th2 differentiation of A51 T cells during priming. Likewise, the local skewing of T cells toward Th2 in the GM of T2-6AB mice could be explained by the low availability of the target epitope in vivo and the relatively low affinity of II-6-type TCR, concomitant with the suppressive effect of Tregs (33).

The unique in vivo situation in T2-6AB mice might also constitute a predisposition to promote large-scale lymphoid neogenesis. In general, a continuous supply of Ags and various stimuli owing to long-lasting inflammation could be causative for inducing ectopic lymphoid structures. Thus, in the case of autoimmune disorders, self-Ags could be the driving force of lymphoid neogenesis. Thorough destruction of target tissue might spread a large amount of self-Ags and lead to a temporary enhancement of TLT development, however, which would in turn result in the exhaustion of the Ags and eventually the contraction of TLTs. If self-reactivity and tolerance are delicately balanced, responses directed to the self-component might conversely facilitate large-scale lymphoid neogenesis in the target organ because the continuous supply of the self-components results in the continued accumulation of T cells in a low-level activation state forming a positive feedback loop in the local site.

In several respects, TLTs in T2-6AB mice resemble intestinal lymphoid structures such as isolated lymphoid follicles (ILFs). The ILFs are mucosal-associated lymphoid tissues that have been suggested to be inducible in response to commensal bacterial flora (41). Therefore the magnitude of ILFs varies depending on the composition of intestinal flora. Importantly, although lymphocytes in the ILFs are continuously stimulated with the bacterial flora and foodstuff constituents, the host has to be tolerant to most of such Ags. In contrast, obvious lymphoid structures are normally absent in the stomach, presumably due to a strongly acidic environment in the stomach lumen that keeps most microbes away. Lymphoid tissues developed in the stomach of T2-6AB mice therefore might be regarded as an ILF-analog maintained by self-reactivity and provide an opportunity to address a unique immunomicroenvironment in the GM.

H. pylori infection in the stomach often induces follicular gastritis in patients, in which lymphoid follicles with large germinal centers are organized in the GM and are associated with lymphoma (42). It has reported that T cell clones recognizing both H⁺/K⁺-ATPase- and H. pylori-derived peptide are present in infected patients with gastric autoimmunity, suggesting that H. pylori can activate cross-reactive gastric T cells via molecular mimicry and lead to AIG (43, 44). Interestingly, when d3-Tx BALB/c mice bearing AIG were infected with H. pylori, the tissue destruction in the GM was markedly ameliorated with concomitant recovery of parietal cells and increased IL-4 production, as well as germinal center formation (45). Thus, H. pylori infection of T2-6AB mice might be informative about the relationship of this microbe and the gastric immune system with self-reactivity. It is worth noting that T2-6AB mice maintained under conventional conditions often develop gastritis of various degrees of severity and are also short-lived (our unpublished data). Some of the aged mice (>6 mo) under specific pathogen-free conditions also suffer from progressive gastritis (our unpublished data). These facts imply that a variety of infectious stimuli or alterations by aging might perturb the balance of the immune system in this animal, triggering destructive autoimmunity. It would be interesting to determine what kinds of stimuli disturb the balance.

TLTs induced in T2-6AB GM are highly organized with many features of typical SLOs. In particular, PNAd and CCL21 expression in HEV-like vessels and stromal cells, as well as naive T cell homing, are significant in the GM of T2-6AB, while they are not observed in d3-Tx AIG. Although the reason that gives rise to such differences in these two models remains unclear, the limited activation of the immune system and/or continuous naive T cell supply from the thymus in T2-6AB mice might be involved in the induction of the additional components. The molecular mechanism underlying the TLT development is likely to resemble that of the organogenesis and maintenance of SLOs (7, 8, 9, 10, 46). SLO development is a highly ordered but dynamic cellular process mediated by the intimate interaction of hemopoietic lymphoid tissue inducer cells and mesenchymal organizer stromal cells. Lymphotoxin (LT)-12 and to some extent TNF produced by lymphoid tissue inducer cells or mature lymphocytes are critical cytokines for the construction of SLOs, which induce several adhesion receptors and homeostatic chemokines, i.e., CXCL13, CCL21, and CCL19, in lymphoid tissue mesenchymal organizer stromal cells via NF-B-mediated signal transduction pathways. Transgenic expression of LT or homeostatic chemokines in ectopic tissues is sufficient to induce lymphoid neogenesis (47, 48, 49, 50). These models for lymphoid neogenesis are not always accompanied by the activation of lymphocytes and inflammation, but rather occur as a consequence of a nondestructive positive feedback loop, i.e., the accumulated lymphocytes induce the specific stromal cells and further attract lymphocytes. TLTs in the T2-6AB stomach are presumably in a similar situation, i.e., self-reactive T and B cells in a weakly activated state accumulating within the target organ possibly produce LT and TNF, which induce a chemokine-producing lymphoid stromal network and further promote the positive feedback loop of lymphoid neogenesis.

Extensive lymphocyte infiltration due to chronic inflammation is in most cases accompanied by the functional destruction of the affected tissue. AIG is generally diagnosed by the extent of the infiltration of mononuclear cell in the GM and the loss of parietal cells, as well as the titer of serum autoantibody (15, 19, 23). Because the loss of parietal cells is thought to directly influence gastric acidity, the pH of the stomach lumen is necessarily increased in d3-Tx AIG mice (45). In contrast, despite the presence of massive lymphocyte infiltration, a large fraction of the parietal cells are preserved, and thus, the function of the stomach seems to be maintained normally in most T2-6AB mice. In this respect, it is not straightforward to diagnose these animals with gastritis using the above criteria. On the other hand, gastric pH actually increased in some transgenic mice displaying various degrees of progressive gastritis (including mice maintained in conventional and even in specific pathogen-free conditions, as well as aged mice), demonstrating the functional destruction of the stomach in such animals.

There have been many reports on the roles of organized lymphoid aggregates in the progression of various autoimmune diseases (7, 8, 9, 10). Especially, ectopic germinal centers that often emerge within the TLTs have been extensively analyzed and several lines of evidence have suggested that these structures can support Ag-driven clonal expansion and differentiation of B cells. Terminally differentiated plasma cells derived from TLTs are likely to secrete autoantibodies and be involved in tissue injury. By contrast, since there is no clear evidence for the priming of T cells by self-Ag within TLTs, the role of the ectopic lymphoid organizations in T cell activation is not well understood. Although the functional significance of TLTs induced in T2-6AB mice remains to be clarified, the findings in this study support the notion that self-reactive T cells, including the naive population, are primed there. The induction of gastric TLTs undoubtedly depends on a kind of activation in T2-6AB T cells; however, it seems not to be a simple outcome of canonical "inflammation-mediated" events. Rather, TLT formation might be a consequence of some compensatory response of the host for immune homeostasis. From this point of view, it is plausible that the GM TLTs could substitute for the function of GLNs as regional lymphoid tissues, where newly arrived naive T cells, which have passed through ectopic HEVs, are primed by dendritic cells carrying the cognitive self-Ag from neighboring tissues and organ-specific tolerance takes place.

In summary, we propose that TLTs are a dynamic structure dependent on the balance between activation and inhibition of long-lasting immune reactivity and do not simply correlate with the severity of diseases. The T cell compartment in T2-6AB transgenic mice seems to be in a delicately balanced condition whereby largely expanded TLTs can be organized in the affected organ. Once the microenvironment is arranged, the balance can be maintained locally, which forms a kind of sustainable feedback loop in the T cell interaction network.

	Acknowledgments

 
We thank S. Habu for genomic constructs; T. Takahashi and T. Toraya for mice transfer; and T. Hara, J.W. Lee, H. Suto, K. Araki, and T. Ohfuji for technical assistance.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants-in-Aid for Science Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

² Address correspondence and reprint requests to Dr. Akira Shimizu, Translational Research Center, Kyoto University Hospital, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail address: shimizu{at}virus.kyoto-u.ac.jp

³ Abbreviations used in this paper: Treg, regulatory T cell; AIG, autoimmune gastritis; d3-Tx, day 3 thymectomy; GLN, gastric LN; GM, gastric mucosa; HEV, high endothelial venule; H⁺/K⁺-ATPase, H⁺/K⁺-adenosine triphosphatase; ILF, isolated lymphoid follicle; LN, lymph node; LT, lymphotoxin; MAdCAM-1, mucosal adressin cell adhesion molecule-1; PNAd, peripheral node addressin; SLO, secondary lymphoid organ; TLT, tertiary lymphoid tissue.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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