Tolerance and autoimmunity to a gastritogenic peptide in TCR transgenic mice

Frank Alderuccio, Valenzio Cataldo, Ian R. van Driel, Paul A. Gleeson and Ban Hock Toh

Department of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, Victoria 3181, Australia

Correspondence to: F. Alderuccio


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The catalytic {alpha} and glycoprotein ß subunits of the gastric H/K ATPase are major molecular targets in human and mouse autoimmune gastritis. We have previously shown that the H/K ATPase ß subunit is required for the initiation of mouse gastritis and identified a gastritogenic H/K ATPase ß subunit peptide (H/Kß253–277). Here we report the generation of MHC class II-restricted TCR transgenic mice using V{alpha}9 and Vß8.3 TCR chains with specificity for the gastritogenic H/Kß253–277 peptide. We found an 8-fold reduction in CD4+ T cells in the thymus of the transgenic mice. Despite the reduction in intrathymic CD4+ T cells, Vß8.3-expressing T cells comprised the majority (>90%) of peripheral spleen and lymph node T cells. These peripheral T cells retained their capacity to proliferate in vitro to the H/Kß253–277 peptide. Using the responsive T cells, we have restricted the gastritogenic T cell epitope to H/Kß261–274. Despite the capacity of the peripheral T cells to proliferate in vitro to the peptide, the majority (~80%, 13 of 16) of transgenic mice remained free of gastritis while a minority (20%, three of 16) spontaneously developed an invasive and destructive gastritis. Our results confirm that H/Kß261–274 is a gastritogenic peptide. The data also suggest that CD4 T cell tolerance to the gastritogenic peptide in the transgenic mice is maintained by a combination of intrathymic and peripheral tolerance mechanisms.

Keywords: autoimmunity, T lymphocytes, TCR, transgenic mice


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
About 2% of persons >60 years of age have undiagnosed pernicious anemia, a condition associated with significant morbidity including neurological disorders (reviewed in 1). The anemia is the consequence of vitamin B12 deficiency resulting from an underlying autoimmune gastritis. The gastritis is characterized by a chronic inflammatory infiltrate in the submucosa which extends into the gastric mucosa and is associated with the loss of parietal and zymogenic cells and circulating autoantibodies to gastric parietal cells, and to intrinsic factor, a secretary product of the parietal cells. The gastric H/K ATPase, the enzyme responsible for acidification of gastric juices, is the major molecular target in human autoimmune gastritis and pernicious anemia. The gastric H/K ATPase comprises a catalytic {alpha} and a glycoprotein ß subunit. Parietal cell autoantibodies directed to both subunits of the gastric H/K ATPase are found in all patients with autoimmune gastritis and >90% of patients with pernicious anemia (2).

Our knowledge of the pathogenesis of autoimmune gastritis is derived largely from studies of mouse models (3). Gastritis can be induced in genetically susceptible BALB/c mouse by neonatal thymectomy (46), adult thymectomy combined with cyclophosphamide treatment (7), immunization with mouse H/K ATPase (8,9) or in single TCR {alpha} chain transgenic mice (10). Autoimmune gastritis also develops spontaneously in C3H/He mice (11). Mouse gastritis, like human gastritis, is characterized by a chronic inflammatory infiltrate which extends into the gastric mucosa with loss of parietal and zymogenic cells. Adoptive transfer studies have shown that mouse gastritis induced by neonatal thymectomy or by immunization with the gastric H/K ATPase is mediated by CD4 T cells (1214). The morphology of mouse gastritis is similar regardless of the method of induction. Mouse gastritis is also accompanied by circulating anti-parietal cell autoantibodies to the {alpha} and ß subunits of the gastric H/K ATPase (15,16). Given the remarkable similarity between mouse and human gastritis, the mouse diseases are excellent animal models of the human disease.

We have previously shown that the ß subunit of the gastric H/K ATPase is necessary for the initiation of mouse gastritis. Transgenic mice expressing the gastric H/K ATPase ß subunit in the thymus (IE-H/Kß transgenic) under the control of the MHC class II I-E{alpha}k promoter are resistant to the development of gastritis induced by neonatal thymectomy (17), adult thymectomy combined with cyclophosphamide treatment (7) or immunization with mouse H/K ATPase (16). Tolerance to the gastric H/K ATPase ß subunit appears to have been induced in the thymus because adoptive transfer of thymocytes from these transgenic mice to naive recipients failed to initiate gastritis.

These observations led us to search for gastritogenic epitopes in the ß subunit of the gastric H/K ATPase. Using a series of overlapping peptides designed from the deduced amino acid sequence of the H/K ATPase ß subunit, we have identified a single gastritogenic peptide (H/Kß253–277) which stimulated proliferation of T cells from BALB/c mice rendered gastritic by immunization with mouse gastric H/K ATPase in complete Freund's adjuvant. Immunization of BALB/c mice with this peptide initiated autoimmune gastritis (18). A MHC class II-restricted, CD4+ T cell hybridoma specific for H/Kß253–277 has been generated, and shown to use V{alpha}9 and Vß8.3 TCR gene elements (19).

Understanding the mechanisms associated with the initiation and pathogenesis of particular autoimmune diseases is hindered by the inability to follow the fate and/or actions of antigen-specific pathogenic lymphocytes. These can be overcome by the production of TCR transgenic mice. Here we describe the generation of MHC class II-restricted, 1E4-TCR{alpha}ß transgenic mice with TCR specificity for the gastritogenic H/Kß253–277 peptide. We show that while a minority of these transgenic mice succumb to gastritis, the majority remain tolerant in vivo despite the capacity of transgenic T cells to proliferate to peptide in vitro.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
BALB/c and (BALB/cxC57BL/6)F1 mice used for transgenic mice production and BALB/cCrSlc were maintained at Monash University Medical School animal facilities. TCR {alpha} chain gene knockout mice (TCR atm1M°m, referred to here as TCR{alpha}–/–) on a C57BL/6 background were from Jackson Laboratories (Bar Harbor, ME) and maintained at Monash University Central Animal facilities. TCR{alpha}–/– mice were backcrossed to BALB/cCrSlc as heterozygotes and screened for neomycin gene insertion by PCR (see below). 1E4-TCR{alpha}ß transgenic mice were backcrossed to BALB/cCrSlc mice a minimum of 3 times and maintained as heterozygotes.

Reagents
Pig gastric H/K ATPase was purified by tomato-lectin chromatography as described (20). Allophycocyanin–anti-CD4 (clone RM4.5), phycoerythrin (PE)–anti-CD8a (clone 53-6.7), FITC–anti-Vß8.3 (clone 1B3.3), PE–anti-V{alpha}2 (clone B20.1) and PE–anti-V{alpha}8 (clone B21.14) mAb were from PharMingen (San Diego, CA). Mouse mAb 1H9 and 2B6 specific for the {alpha} and ß subunits of the gastric H/K ATPase respectively were used as positive controls in ELISA and immunofluorescence assays.

The peptides H/Kß169–193 (SFGFEEGKPCFIIKMNRIVKFLPSN), H/Kß253–277 (LLNVPKNMQVSIVCKILADHVTFNN), H/Kß256–269 (VPKNMQVSIVCKIL), H/Kß261–274 (QVSIVCKILADHVT) and H/Kß266–279 (CKILADHVTFNNPH), based on the deduced amino acid sequence of mouse gastric H/K ATPase ß subunit (18) were synthesized by Auspep (Parkville, Australia) or Chiron Technologies (Clayton, Australia). Peptides were resuspended in sterile water at a concentration of 10 mg/ml and stored at –20°C.

Identification and cloning of TCR {alpha}ß genes from T cell hybridoma 1E4.C1 and production of TCR transgenic mice
The CD4 T cell hybridoma 1E4.C1 which proliferates in vitro to the H/Kß253–277 gastritogenic peptide (19) was derived from a mouse immunized with mouse gastric H/K ATPase in complete Freund's adjuvant (8). The TCR V{alpha} and Vß gene usage of the T cell hybridoma 1E4.C1 was determined by antibody staining and RT-PCR analysis using a panel of V{alpha} and Vß oligonucleotide primers paired with oligonucleotides specific for the TCR {alpha} chain and ß chain constant regions respectively. The RT-PCR products were subcloned into pGEMT (Promega, Annadale, Australia), and sequenced across the V(D)J region to confirm identity and integrity of V{alpha} and Vß gene usage. The TCR V{alpha} and Vß gene usage for 1E4.C1 was V{alpha}9J{alpha}21 and Vß8.3Dß1Jß2.1.

The strategy used to generate TCR transgenic mice was based on the use of plasmid cassettes obtained from Benoist and Mathis (21) in which the V(D)J regions of the desired TCR are inserted between the plasmid-encoded rearranged promoter and constant regions of HY-specific TCR {alpha} and ß chain genes. Genomic DNA for the 1E4.C1 TCR {alpha} chain variable region was amplified by PCR from the T cell hybridoma. Oligonucleotide primer specific for the 5' region of the TCR V{alpha}9 gene 5'-GCC TTC TCC CGG GCT AGC CAT GTT CCC AGT GAC C-3' was designed from the published sequence (22) with an introduced XmaI site (underlined). The 3' oligonucleotide 5'-ACA TTA ATA AAG CGG CCG CGC AGA TGC ATA AGA TTA AAG-3' specific for the 3' region of the J{alpha}21 genomic sequence was designed from published sequence with an introduced NotI site (underlined). The predicted PCR product is 657 bp.

Genomic DNA for the 1E4.C1 TCR ß chain variable region was amplified by PCR. Oligonucleotide primer specific for TCR Vß8.3 gene 5'-CGA CGC TCG AGT GGT CGC GAG ATG GGC TCC AGG-3' was designed from the published sequence with an introduced XhoI site (underlined). The 3' oligonucleotide primer 5'-GGA TAG TTA AAT ATC GAT ACT GCT AAG G-3' was designed from the published sequence of the Jß2.1 sequence with an introduced ClaI site (underlined). The predicted PCR product is 607 bp.

PCR reactions were performed in 25 µl reactions containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.1% gelatin, 200 mM dATP, dCTP, dGTP and dTTP, 50 pmol oligonucleotide primers and 1 U of Taq (Gibco/BRL, Melbourne, Australia), and 3x10–3 U Pfu DNA polymerase (Stratagene, La Jolla, CA). The reaction mixture was incubated at 95°C for 2 min, 30 cycles of 92°C for 30 s, 60°C for 30 s and 72°C for 1 min, and a final cycle at 72°C for 5 min. PCR products were subcloned into pBluescript and sequenced. DNA encoding genomic V(D)J sequences of 1E4 TCR {alpha} and ß chains were excised by XmaI–NotI and XhoI–ClaI restriction enzyme digestion respectively, and subcloned into pT{alpha}cass and pTßcass respectively (21). Plasmids were sequenced across insertion junctions to confirm the presence and orientation of DNA, and subjected to restriction enzyme digestion analysis to confirm the integrity of vectors following subcloning procedures.

For production of transgenic mice, transgenes with introduced V(D)J and plasmid derived TCR promoter and constant regions were excised from pT{alpha}1E4cass and pTß1E4cass by SalI and KpnI restriction enzyme digestion respectively, purified on a nucleic acid chromatography system 52 column (Gibco/BRL Life Technologies, Gaithersburg, MD) or Qiagen QIAquick gel extraction kit (Qiagen, Clifton Hill, Australia), and resuspended in 10 mM Tris–HCl, 1 mM EDTA, pH 8.0, at a concentration of 2–5 ng/µl. To produce 1E4-TCR{alpha}ß transgenic mice, 1E4-TCR {alpha} and ß chain transgenes were mixed in equal amounts, and microinjected into the pronuclei of fertilized (BALB/cxC57BL/6)xBALB/c oocytes and transferred to oviducts of pseudopregnant BALB/c mice according to the method of Hogan et al. (23). Transgenic founders were identified by PCR analysis as described below and transgenic lines were established by backcrossing to BALB/cCrSlc mice.

PCR screening
1E4-TCR transgenic mice were identified by PCR analysis of mouse tail genomic DNA. DNA for PCR was prepared as previously described (23). PCR reactions were performed using three pairs of oligonucleotides to identify the TCR {alpha} and ß transgenes and the insulin gene as an internal control. Oligonucleotides 5'-GCC TTC TCC CGG GCT AGC CAT GTT CCC AGT GAC C-3' and 5'-ACA TTA ATA AAG CGG CCG CGC AGA TGC ATA AGA TTA AAG-3' were designed to span the promoter region of pT{alpha}cass and the inserted V(D)J region of the 1E4 TCR {alpha} chain respectively to give a product of 655 bp. Oligonucleotides 5'-CTC AAT ACA GCC ATC TCC-3' and 5'-GTC TTC TTG CGT TGT TCT GG-3' were designed to span the promoter region of pTßcass and the inserted V(D)J region of the 1E4-TCR ß chain respectively to give a product of 570 bp. Oligonucleotides 5'-CGA GCT CGA GCC TGC CTA TCT TTC AGG-3' and 5'-CGG GAT CCT AGT TGC AGT AGT TCT CCA G-3' designed from the mouse insulin gene were used to generate a PCR product of 374 bp and included as a DNA quality control. PCR was performed in 25 µl reaction volumes containing amplification buffer 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.1% gelatin, 200 µM dATP, dCTP, dGTP and dTTP, 50 pmol oligonucleotide primers, and 1.5 U of Taq DNA polymerase (Gibco/BRL). The reaction mixture was incubated at 95°C for 2 min, 30 cycles of 92°C for 30 s, 60°C for 30 s and 72°C for 1 min, and a final cycle at 72°C for 5 min. Samples of PCR product (15 µl) were separated by agarose gel electrophoresis and visualized using UV illumination. Images were captured by digital camera and inverted for publication.

TCR{alpha}–/– screening
TCR {alpha} chain mutant mice (C57BL/6J- Tcratm1M°m ) (24) were obtained as homozygotes from Jackson Laboratories. Mice were screened for the TCR {alpha} chain mutation using a PCR touchdown protocol to detect the inserted neomycin gene. Details of this procedure can be found at the Jackson web site; http://www.jax.org/pub-cgi/imrpub.sh?.

Detection of H/K ATPase autoantibodies
Gastric H/K ATPase antibodies were detected by ELISA as previously described (17). Anti-parietal cell autoantibodies were detected by indirect immunofluorescence on frozen or paraffin-embedded sections of normal mouse stomach as previously described (17).

Flow cytometry
Single-cell suspensions (1–2x106) of thymocytes, splenocytes or lymph node cells were incubated in HBSS/1% FCS, 0.02% sodium azide with Fc block (clone 2.4G2; PharMingen) followed by 45 min incubation on ice with directly conjugated antibodies outlined above. Cells were washed in HBSS/1% FCS and passed through a 100 µm nylon membrane. Dead cells were excluded by propidium iodide staining, and lymphocytes gated on forward and side scatter profiles. Cells were analyzed on a FACScan or FACSCalibur using CellQuest software (Becton Dickinson, San Jose, CA).

In vitro proliferation assay
Single-cell suspensions of whole splenocytes were treated with ammonium chloride solution (0.9%) to lyse red blood cells. Normal BALB/cCrSlc irradiated splenocytes (3000 rad) were used as antigen-presenting cells (APC). Cells were resuspended in RPMI 1640 culture media supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), 2 mM L-glutamine and 2-mercaptoethanol (50 mM). Proliferation assays were performed in 96-well tissue culture plates (Greiner Laboratories, Austria) in a total volume of 200µl containing 5x105 splenocytes, 5x105 irradiated APC and peptide at various concentrations. Cell were incubated for 48 h at 37°C/10% CO2 followed by an additional overnight incubation in the presence of 1 µCi [3H]thymidine (NEN, Boston, MA). Cell were harvested onto glass filters (Skatron, Sterling, VA) suspended in scintillant and [3H]thymidine incorporation determined on a LKB rackbeta scintillation counter. Control wells included splenocytes alone, APC alone or proliferation in the absence of antigen. Stimulation indexes were determined by dividing c.p.m. in the presence of peptide with c.p.m. of responders and APC in the absence of peptide.

Histology
Mouse stomachs and tissues were fixed in 10% formalin in PBS and embedded in paraffin. Sections (5 µm) were cut and stained with hematoxylin & eosin. Gastritis was assessed by the presence of mononuclear cell infiltrate within the gastric mucosa, cellular destruction and tissue hypertrophy.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of TCR transgenic mice
1E4-TCR transgenic mice were produced which harbored TCR V{alpha}9 and Vß8.3 transgenes, specific for a gastritogenic peptide of the H/K ATPase ß subunit. Transgenic mice were identified by PCR to identify the TCR {alpha} and ß transgenes (Fig. 1AGo). The 1E4-TCR{alpha} and 1E4-TCRß transgenes appear to have been inserted at a single locus because both transgenes remained associated without segregation in the offspring. Analysis of 1E4-TCR{alpha}ß mice by flow cytometry with mAb to Vß8.3 was used to identify the cell surface expression of the TCR Vß8.3 transgene. As expected, there was a dramatic increase in the number of Vß8.3 staining lymphocytes in lymphoid organs from 1E4-TCR{alpha}ß transgenic mice compared to non-transgenic littermates (Fig. 1BGo).



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Fig. 1. Transgene expression in TCR {alpha}ß transgenic mice. (A) PCR demonstration of transgenes. Tail DNA was extracted and subjected to PCR using primers specific for 1E4-TCR {alpha} (655 bp) chain and 1E4-TCR ß (570 bp) chain transgenes. Primers designed to amplify a 374 bp product from the insulin gene were included as control. Tg, 1E4-TCR{alpha}ß transgenic mice; N-transgenic, non-transgenic littermates. (B) Vß8.3 expression in transgenic mice. Single-cell suspensions from thymus, spleen, inguinal and paragastric lymph nodes were stained with FITC-conjugated anti-Vß8.3 mAb. Shaded area, 1E4-TCR{alpha}ß transgenic mouse; open area, non-transgenic mouse. (C) Vß8.3+ CD4+ T cells in mice lacking endogenously rearranged TCR {alpha} chains. (1E4-TCR{alpha}ßxTCR{alpha}–/–)F1 mice were crossed to produce mice lacking endogenously rearranged TCR {alpha} chains. Splenocytes were stained with CD4, Vß8.3 and V{alpha}2 + V{alpha}8 antibodies. TCR{alpha}–/– mice were identified by absence of V{alpha}2 + V{alpha}8 staining cells (right panel). Mice expressing endogenously rearranged TCR {alpha} chains produced comparable CD4+V{alpha}2/V{alpha}8 positive populations in 1E4-TCR{alpha}ß transgenic (middle panel) and in non-transgenic mice (left panel). CD4+ T cells were gated (illustrated in left panel) and Vß8.3 staining shown below corresponding dot-plots. The Vß8.3+ population observed in CD4+ T cells from 1E4-TCR{alpha}ß transgenic mice without endogenously rearranged TCR {alpha} chains confirms the expression of 1E4-TCR {alpha} chain in transgenic mice.

 
Whereas we could readily detect the TCR Vß8.3 transgene in 1E4-TCR{alpha}ß transgenic mice with the Vß8.3-specific antibody, we do not have a specific antibody to the TCR V{alpha}9 gene product. Therefore we used other methods to identify expression of the TCR V{alpha}9 transgene. Firstly, we observed a strong RT-PCR product specific for the TCR {alpha} transgene with the spleens of 1E4-TCR{alpha}ß transgenic mice but not with the spleens of non-transgenic littermates (data not shown). Secondly, we obtained more direct evidence by crossing 1E4-TCR{alpha}ß transgenic mice to TCR{alpha}–/– mice. TCR{alpha}–/– mice lack endogenously rearranged TCR {alpha} chains and do not generate mature CD4+ or CD8+ T cells. Using the expression of TCR V{alpha}2 + V{alpha}8 chains as markers for endogenously rearranged V{alpha} chains, we showed that Vß8.3+ T cells were still generated in the spleens of transgenic mice in the absence of endogenous V{alpha} chains (Fig. 1CGo). These observations indicate that CD4+ T cells have been generated and exported to the periphery in the absence of endogenously rearranged TCR {alpha} chains, which can only occur with pairing of the Vß8.3 and V{alpha}9 transgenes.

Intrathymic and peripheral T cells in TCR {alpha}ß transgenic mice
Flow cytometric analysis of the CD4/CD8 thymocyte profiles revealed that virtually all CD4+CD8 and CD4CD8+ thymocytes from 1E4-TCR{alpha}ß transgenic mice expressed the Vß8.3 transgene (Fig. 2AGo). High levels of expression were also observed in the CD4+CD8+ (double positive) and CD4CD8 (double negative) populations. Expression in the DN population is most likely due to the expression of the TCR {alpha}ß transgene in cells of the {gamma}{delta} T cell lineage (25).



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Fig. 2. CD4/CD8 profiles of thymocytes (A) and spleen cells (B) from 6-week-old 1E4-TCR{alpha}ß transgenic and non-transgenic mice. Single-cell suspensions stained with anti-CD4–allophycocyanin, anti-CD8–PE and anti-Vß8.3–FITC. Propidium iodide used to exclude dead cells. (A) Dot-plots of CD4 versus CD8 staining of thymocytes. CD4+CD8 (CD4+), CD4+CD8+ (double positive), CD4CD8+ (CD8+) and CD4CD8 (double negative) populations gated and percentage of cells in each population shown. Histograms and percentage of Vß8.3 staining cells in each population shown below dot-plots. (B) Dot-plots of CD4 versus CD8 staining of spleen cells. Quadrants used to separate CD4+, CD8+ and CD4CD8 cell populations, and corresponding percentages shown. CD4+ and CD8+ cells gated, and histograms and percentage of Vß8.3 staining cells in each population shown. Vß8.3-staining cells were not seen in CD4CD8 population of TCR transgenic or non-transgenic mice (not shown).

 
To assess T cell populations, age-matched mice were examined at 1 week (TCR {alpha}ß transgenic, n = 3; non-transgenic, n = 3) and 6 weeks [TCR {alpha}ß transgenic (n = 4), two females and two males; non-transgenic (n = 8), four females and four males] of age. Neonatal mice were littermates and 6-week-old mice were two litters from the same transgenic male. Thymocyte profiles from 6-week-old TCR {alpha}ß transgenic mice revealed a marked reduction in the CD4+CD8 and CD4CD8+ populations compared to non-transgenic mice (representative data, Fig. 2AGo). Compared to non-transgenic mice, in TCR {alpha}ß transgenic mice there was ~8- and 7-fold decrease in the percentage of CD4+CD8 (TCR {alpha}ß transgenic, 0.8 ± 0.2%, non-transgenic, 6.7 ± 1.3%; P = 2.7x10–6) and CD4CD8+ (TCR {alpha}ß transgenic, 0.6 ± 0.2% and 3.9 ± 2.5%; P = 0.006) thymocytes respectively. In neonatal TCR {alpha}ß transgenic mice, a similar reduction was observed in the CD4+CD8 cells (TCR {alpha}ß transgenic 0.7 ± 0.2%; non-transgenic, 4.1 ± 0.9%; P = 0.02) but not the CD4CD8+ (TCR {alpha}ß transgenic, 0.4 ± 0.1%; non-transgenic, 0.5 ± 0.2%) population. Thymii from 1-week-old mice were similar in total thymocyte numbers regardless of transgenic status (TCR {alpha}ß transgenic, 5.3 ± 0.1x107; non-transgenic 5.9 ± 0.8x107); however, in the 6-week-old group there appeared to be an increase in total thymocyte numbers in TCR {alpha}ß transgenic mice (TCR {alpha}ß transgenic, 2.8 ± 0.7x108; non-transgenic 1.9 ± 0.7x108), although this failed to reach significance. Nevertheless, the calculated total numbers of mature CD4+ and CD8+ T cells in the thymii of TCR {alpha}ß transgenic mice were still markedly reduced by ~6- and 4-fold respectively compared to non-transgenic mice.

T cell populations were also examined from the spleens of TCR {alpha}ß transgenic and non-transgenic mice at 1 and 6 weeks of age (representative data, Fig. 2BGo). Overall, splenoctye numbers from 6-week-old TCR {alpha}ß transgenic mice were significantly greater (TCR {alpha}ß transgenic 3.4 ± 0.4x108; non-transgenic, 2.4 ± 0.3x108; P = 0.004) compared to non-transgenic siblings. This was not observed in neonatal mice (TCR {alpha}ß transgenic, 3.5 ± 0.4x107; non-transgenic, 3.8 ± 0.1x107). In contrast to the thymocyte analysis, the reduction in proportion of peripheral CD4+ and CD8+ T cells in TCR {alpha}ß transgenic mice was not as marked (Fig. 2BGo). The splenic CD4+ T cell population from TCR {alpha}ß transgenic mice was reduced by a factor of 1.6 (TCR {alpha}ß transgenic, 6.1 ± 1.9%; non-transgenic 10 ± 1.4%; P = 0.02) and the CD8+ T cell population by a factor of 2.3 (TCR {alpha}ß transgenic, 2.2 ± 0.5%; non-transgenic 5.1 ± 0.8%; P = 3.5x10–5). Similar reductions were observed in neonatal mice (data not shown). In contrast with the thymus, if the increased cellularity of spleens from TCR {alpha}ß transgenic was taken into account, there was not a significant difference in the total number of CD4+ T cells recovered from TCR {alpha}ß transgenic (TCR {alpha}ß transgenic, 2.1 ± 0.6x107; non-transgenic, 2.4 ± 0.3x107) compared to non-transgenic mice. However, the total number of the CD8+ T cells recovered from TCR {alpha}ß transgenic remained significantly lower (TCR {alpha}ß transgenic, 0.8 ± 0.2x107; non-transgenic, 1.2 ± 0.2x107; P = 0.005).

T cell proliferation to H/K ATPase ß subunit peptides
We have previously identified the H/K ATPase ß subunit peptide 253–277 as a major gastritogenic T cell epitope associated with gastritis induced by immunization with the gastric H/K ATPase (18). Splenocytes from 1E4-TCR{alpha}ß transgenic mice proliferated in vitro to the gastritogenic H/Kß253–277 peptide and not to a non-gastritogenic H/Kß169–193 peptide, whereas splenocytes from non-transgenic littermates did not respond to either peptide (Fig. 3AGo).



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Fig. 3. In vitro T cell proliferation to H/K ATPase ß subunit peptides. (A) T cell proliferation to the gastritogenic H/Kß253–277 peptide compared to a non-gastritogenic H/Kß169–193 peptide. Cells cultured in the absence of peptide showed no response (data not shown). (B) T cell proliferation to H/Kß261–274 peptide compared to the overlapping H/Kß256–269 and H/Kß266–279 peptides; relationship of H/Kß253–277 peptide to the H/Kß256–269, H/Kß261–274 and H/Kß266–279 peptides shown above the plot. (C) Proliferation of T cells from 1E4-TCR{alpha}ß transgenic and non-transgenic mice to H/Kß261–274 peptide. Proliferation is shown as stimulation indexes. Transgenic status confirmed by Vß8.3 staining of splenocytes (data not shown). Splenocyte (5x105) from 1E4-TCR{alpha}ß or non-transgenic mice cultured with 5x105 irradiated splenocytes in the absence or presence of serial dilutions of peptides for 48 h at 37°C followed by overnight incubation with 1 µCi [3H]thymidine. Each point represents the mean of duplicate wells.

 
The H/Kß253–277 peptide was identified as the major gastritogenic peptide from a set of 25mer overlapping peptides spanning the entire H/K ATPase ß subunit protein (18). Since MHC class II epitopes generally comprise 12–14 amino acids, a set of smaller 14mer overlapping peptides spanning the H/Kß253–277 peptide was generated. The relationship of these peptides to the H/Kß253–277 peptide is shown in Fig. 3Go(B). T cells from 1E4-TCR{alpha}ß transgenic mice reacted to the H/Kß261–274 peptide, whereas no response was observed with the overlapping H/Kß256–269 or H/Kß266–279 peptides (Fig 3BGo). The T cell hybridoma 1E4.C1 also reacted specifically with the H/Kß261–274 peptide (19). Therefore, the gastritogenic T cell epitope resides in the 14mer amino acid sequence of H/Kß261–274.

The T cell proliferative response to the H/Kß261–274 peptide was examined in 1E4-TCR{alpha}ß transgenic mice compared to non-transgenic mice. All five transgenic mice tested responded to H/Kß261–274 peptide in vitro (Fig. 3CGo), whereas the three non-transgenic mice did not. However, we did observe that although all transgenic mice responded to peptide in vitro (stimulation index >3), the response varied between animals. The T cell proliferation response to the H/Kß256–271 peptide required pairing of the transgenic TCR {alpha} and ß chains since single-chain 1E4-TCRß transgenic mice expressing the transgenic TCR ß chain did not respond to the peptide (data not shown). Transgenic and non-transgenic mice did not respond to a control H/Kß256–269 peptide (data not shown).

Gastritis in TCR {alpha}ß transgenic mice
Mice used in this analysis were sampled over a period of time and ranged in age from 7 to 40 weeks of age. From our experience with the incidence of autoimmune gastritis in the neonatal thymectomy model, disease can be identified in mice from 6 to 8 weeks of age (26). The majority of TCR {alpha}ß transgenic mice (13 of 16, 81%, eight females and five males) did not develop spontaneous gastritis (Fig. 4AGo). However, spontaneous gastritis did develop in three of 16 (19%) 1E4-TCR{alpha}ß transgenic mice but not in 12 (four females and eight males) non-transgenic littermates (Fig. 4BGo). All three TCR {alpha}ß transgenic mice that developed autoimmune gastritis were males of 11–21 weeks of age and not the oldest transgenic mice in the analysis. At this stage, it is premature to suggest that only transgenic males will predominantly develop disease. As yet, we have not completed a systematic study to determine the incidence of autoimmune gastritis in different age groups. The gastritis is characterized by a chronic inflammatory infiltrate in the submucosa with invasion into the lamina propria of the mucosa. The mucosal invasion of the chronic inflammatory infiltrate is accompanied by degenerative changes in gastric parietal cells and zymogenic cells, and hypertrophy of the glands. The presence of H/K ATPase-reactive autoantibodies is characteristic of human and murine gastritis (6,17). Of the three 1E4-TCR{alpha}ß transgenic mice with gastritis, two developed autoantibodies which reacted with the H/K ATPase by ELISA (data not shown) and with parietal cells by indirect immunofluorescence (Fig. 4DGo). 1E4-TCR{alpha}ß transgenic and non-transgenic mice which did not develop gastritis did not have circulating parietal cell autoantibodies (Fig. 4CGo).



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Fig. 4. Histological and immunofluorescence staining for gastritis and circulating parietal cell autoantibodies. (A and B) Hematoxylin & eosin stain of paraffin-embedded stomach sections from non-gastritic (A) and gastritic (B) 1E4-TCR{alpha}ß transgenic mice, showing normal gastric mucosa in (A) and gastritic mucosa in (B) displaying mononuclear cell infiltrates extending into the lamina propria of the mucosa (arrowhead) with attendant gland hypertrophy. (C and D) Indirect immunofluorescence stain with sera from non-gastritic (C) and gastritic (D) 1E4-TCR{alpha}ß transgenic mice on paraffin-embedded stomach sections. Non-gastritic mice show no staining (C) while gastritic mice show staining of parietal cells (D). Bar: 100 µm.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have generated MHC class II-restricted CD4 TCR {alpha}ß transgenic mice using V{alpha}9 and Vß8.3 TCR chains with specificity for a defined gastritogenic H/K ATPase ß subunit peptide (H/Kß253–277). Based on expression of the Vß8.3 transgene, TCR {alpha}ß transgenic T cells appear to have been positively selected in the thymus and exported to the periphery where they constitute the vast majority of T cells. Although we do not have a clonotypic antibody for the V{alpha}9 gene product, we have shown that T cells harboring only the TCR transgenes can be produced in the absence of endogenous TCR {alpha} chain rearrangement. Further, the observation of specific in vitro T cell proliferation to the H/Kß253–277 peptide provides compelling evidence that functional TCR have been generated in the transgenic mice. Using the responsive T cells, we have restricted the gastritogenic T cell epitope to a 14mer (H/Kß261–274) stretch of amino acids.

Our findings from analysis of 1E4-TCR{alpha}ß transgenic mice showed a marked reduction in the CD4+ and CD8+ thymocyte populations. While the reduction in CD8+ thymocytes is expected based on the CD4+, MHC class II-restricted phenotype of the CD4 T cell hybridoma from which the TCR specificity was derived, the decrease in CD4+ thymocytes was unexpected. Our observations contrast with other MHC class II-restricted TCR {alpha}ß transgenic mice specific for self-antigens, including myelin basic protein (27), pancreatic islet antigen (28) and type II collagen (29) in which no reduction of CD4 thymocytes was observed. One possible explanation for the reduction of CD4+ T cells in our 1E4-TCR{alpha}ß transgenic mice is inefficient positive selection of this population. Since positive selection occurs at the CD4+CD8+ stage of thymocyte development (30), inefficient positive selection may result in fewer single-positive thymocytes. This is the most likely explanation for the reduction in CD8 thymocytes. In the case of CD4 thymocytes with rearranged TCR {alpha}ß transgenes, it is possible that the reduction in this population is a result of failure to positively select cells of low avidity. Alternatively, the reduction in CD4+ thymocytes may reflect deletion of high-avidity TCR {alpha}ß T cells. The level of CD4+ thymocyte reduction in our transgenic mice is similar to that observed in CD8+ thymocytes in class I-restricted TCR transgenic mice (Des-TCR ) crossed with transgenic mice (RIP-Kb) in which low levels of antigen were found in the thymus (31). The flow cytometric profile in 1E4-TCR{alpha}ß transgenic mice is also similar to that observed in C5 (fifth component of complement)-reactive TCR transgenic mice in the presence of circulating C5 (32). In both of these cases, deletional mechanisms have been invoked. If the reduction in intrathymic CD4 T cells in 1E4-TCR{alpha}ß transgenic mice is a consequence of deletion, it may have resulted from expression of the ß subunit in the thymus or its transport from the periphery into the thymus (32). There are increasing numbers of reports of expression of `peripheral' antigens in the thymus raising speculation of a role for central deletional tolerance mechanisms for these self antigens (3335). However, we and others have shown by RT-PCR that the H/K ATPase ß subunit is not expressed in the thymus (16,17). Therefore if the H/K ATPase ß subunit is expressed in the thymus, it is below this level of detection.

Although the T cells exported from the thymus to the periphery of 1E4-TCR{alpha}ß transgenic mice retained their capacity to proliferate to the gastritogenic peptide in vitro and therefore were not tolerant to the peptide; yet the majority (80%) of the transgenic mice failed to develop gastritis. These observations implicate peripheral tolerance mechanisms which prevent these T cells from initiating gastritis. At least two non-mutually exclusive mechanisms may be responsible for peripheral tolerance in this case—clonal ignorance and clonal regulation. The ability to initiate gastritis in BALB/c following immunization with antigen or peptide in adjuvant (8,18) is consistent with the operation of clonal ignorance. Studies of gastritis induced in BALB/c mice by neonatal thymectomy have implicated a subset of CD25+ CD4 T cells as regulatory T cells. These regulatory cells appear to develop as a distinct `professional lineage' in the thymus, and to be able to suppress proliferation of pathogenic CD4 T cells in vitro and in vivo (3639). CD4+ regulatory T cells have also been implicated in myelin basic protein-specific TCR transgenic mouse models of experimental autoimmune encephalomyelitis (40,41).

Approximately 20% of 1E4-TCR{alpha}ß transgenic mice developed an invasive and destructive gastritis, characterized by a chronic inflammatory infiltrate in the gastric mucosa extending into the lamina propria, with loss of parietal and zymogenic cells and mucosal hypertrophy. The invasive and destructive gastritis is similar to that seen in mice immunized with the gastric H/K ATPase and following neonatal thymectomy (8,17). In two of the three mice, autoantibodies to the H/K ATPase were also found. Although gastritis without H/K ATPase autoantibodies is rarely seen in gastritis induced by neonatal thymectomy, it has been described in gastritis following adoptive transfer with pathogenic T cell clones (42,43). The gastritis which develops in 1E4-TCR{alpha}ß transgenic mice appears to be specific because spontaneous gastritis does not develop in non-manipulated BALB/c mice. The development of spontaneous gastritis in the transgenic mice provides further confirmation that the H/Kß261–274 epitope is a gastritogenic peptide. But why has spontaneous gastritis developed only in a minority of TCR {alpha}ß transgenic mice? Studies of TCR transgenic mice specific for myelin basic protein peptide (111) (27) have shown that transgenic mice housed under sterile conditions did not develop experimental allergic encephalomyelitis, whereas almost half the mice housed under conventional conditions developed disease. Therefore, like the TCR transgenic mice in the Goverman study, environmental factors may also be associated with the development of autoimmune gastritis in our TCR transgenic model. To address this, we will need to observe the TCR transgenic mice under specific pathogen-free conditions.

This is the first report of a TCR transgenic mouse generated to a gastritogenic autoepitope. The dramatic reduction of CD4+ T cells in the thymus of the transgenic mice implicates central intrathymic tolerance mechanisms arising as a consequence of either inadequate positive selection and/or negative selection of self-reactive T cells harboring TCR {alpha}ß transgenes. However, CD4+ T cells harboring the transgenes comprised the vast majority of peripheral CD4+ T cells. Although these CD4+ T cells have retained the capacity to proliferate to the H/K ATPase ß subunit peptide in vitro, the majority of mice remain free of gastritis, implicating peripheral tolerance mechanisms. Nonetheless, a minority do develop gastritis confirming the pathogenic potential of the transgenic T cells. The 1E4-TCR{alpha}ß transgenic mice should be useful for further studies directed towards a better understanding of the mechanisms of tolerance and autoimmunity to the gastric autoantigen.


    Acknowledgments
 
This work is supported by grants from the National Health and Medical Research Council of Australia and The Alfred Healthcare Group


    Abbreviations
 
APC antigen-presenting cell
PE phycoerythrin

    Notes
 
Transmitting editor: D. Tarlinton

Received 4 August 1999, accepted 22 November 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Toh, B. H., van Driel, I. R. and Gleeson, P. A. 1997. Pernicious anaemia. N. Engl. J. Med. 337:1441.[Free Full Text]
  2. Toh, B. H., Gleeson, P. A., Simpson, R. J., Moritz, R. L., Callaghan, J. M., Goldkorn, I., Jones, C. M., Martinelli, T. M., Mu, F-T., Humphris, D. C., Pettitt, J. M., Mori, Y., Masuda, T., Sobieszczuk, P., Weinstock, J., Mantamadiotis, T. and Baldwin, G. S. 1990. The 60- to 90-kDa parietal cell autoantigen associated with autoimmune gastritis is a ß subunit of the gastric H+/K+–ATPase (proton pump). Proc. Natl Acad. Sci. USA 87:6418.[Abstract]
  3. Gleeson, P. A., Toh, B. H. and van Driel, I. R. 1996. Organ-specific autoimmunity induced by lymphopenia. Immunol. Rev. 149:97.[ISI][Medline]
  4. Kojima, A. and Prehn, R. T. 1981. Genetic susceptibility to post-thymectomy autoimmune diseases in mice. Immunogenetics 14:15.[ISI][Medline]
  5. Fukuma, K., Sakaguchi, S., Kuribayashi, K., Chen, W.-L., Morishita, R., Sekita, K., Uchino, H. and Masuda, T. 1988. Immunologic and clinical studies in murine experimental autoimmune gastritis induced by neonatal thymectomy. Gastroenterology 94:274.[ISI][Medline]
  6. Alderuccio, F., Toh, B. H., Gleeson, P. A. and van Driel, I. R. 1995. A novel method for isolating mononuclear cell from the stomachs of mice with experimental autoimmune gastritis. Autoimmunity 21:215.[ISI][Medline]
  7. Barrett, S. P., Toh, B. H., Alderuccio, F., van Driel, I. R. and Gleeson, P. A. 1995. Organ-specific autoimmunity induced by adult thymectomy and cyclosphamide-induced lymphopenia. Eur. J. Immunol. 25:238.[ISI][Medline]
  8. Scarff, K. J., Pettitt, J. M., van Driel, I. R., Gleeson, P. A. and Toh, B. H. 1997. Immunization with gastric H+/K+-ATPase induces a reversible autoimmune gastritis. Immunology 92:91.[ISI][Medline]
  9. Claeys, D., Saraga, E., Rossier, B. C. and Kraehenbuhl, J.-P. 1997. Neonatal injection of native proton pump antigens induces autoimmune gastritis in mice. Gastroenterology 113:1136.[ISI][Medline]
  10. Sakaguchi, S., Ermak, T. H., Toda, M., Berg, L. J., Ho, W., Fazekas de St.Groth, B., Peterson, P. A., Sakaguchi, N. and Davis, M. M. 1994. Induction of autoimmune disease in mice by germline alteration of the T cell receptor gene expression. J. Immunol. 152:1471.[Abstract/Free Full Text]
  11. Alderuccio, F. and Toh, B. H. 1998. Spontaneous autoimmune gastritis in C3H/He mice: a new mouse model for autoimmunity. Am. J. Pathol. 153:1311.[Abstract/Free Full Text]
  12. Smith, H., Lou, Y. H., Lacy, P. and Tung, K. S. K. 1992. Tolerance mechanisms in experimental ovarian and gastric autoimmune diseases. J. Immunol. 149:2212.[Abstract/Free Full Text]
  13. Sakaguchi, S., Fukuma, K., Kuribayashi, K. and Masuda, T. 1985. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. J. Exp. Med. 161:72.[Abstract]
  14. de Silva, H. D., van Driel, I. R., La Gruta, N., Toh, B. H. and Gleeson, P. A. 1998. CD4+ T cells, but not CD8+ T cells, are required for the development of experimental autoimmune gastritis. Immunology 93:405.[ISI][Medline]
  15. Jones, C. M., Callaghan, J. M., Gleeson, P. A., Mori, Y., Masuda, T. and Toh, B. H. 1991. The parietal cell autoantibodies recognised in neonatal thymectomy-induced murine gastritis are the {alpha} and ß subunits of the gastric proton pump. Gastroenterology 101:287.[ISI][Medline]
  16. Alderuccio, F., Gleeson, P. A., Berzins, S. P., Martin, M., van Driel, I. R. and Toh, B. H. 1997. Expression of the gastric H/K ATPase {alpha}-subunit in the thymus may explain the dominant role of the ß-subunit in the pathogenesis of autoimmune gastritis. Autoimmunity 25:167.[ISI][Medline]
  17. Alderuccio, F., Toh, B. H., Tan, S. S., Gleeson, P. A. and van Driel, I. R. 1993. An autoimmune disease with multiple molecular targets abrogated by the transgenic expression of a single autoantigen in the thymus. J. Exp.Med. 178:419.[Abstract]
  18. de Silva, H. D., Gleeson, P. A., Toh, B. H., van Driel, I. R. and Carbone, F. R. 1999. Identification of a gastritogenic epitope of the H/K ATPase ß-subunit. Immunology 96:145.[ISI][Medline]
  19. de Silva, H. D. 1997. H+/K+-ATPase T cell autoepitope in gastric autoimmunity. PhD dissertation.
  20. Callaghan, J. M., Toh, B. H., Simpson, R. J., Baldwin, G. S. and Gleeson, P. A. 1992. Rapid purification of the gastric H+/K+-ATPase complex by tomato-lectin affinity chromatography. Biochem. J. 283:63.[ISI][Medline]
  21. Kouskoff, V., Signorelli, K., Benoist, C. and Mathis, D. 1995. Cassette vectors directing expression of T cell receptor genes in transgenic mice. J. Immunol. Methods 180:273.[ISI][Medline]
  22. Uematsu, Y. 1992. Preferential association of {alpha} and ß chains of the T cell antigen receptor. Eur. J. Immunol. 22:603.[ISI][Medline]
  23. Hogan, B., Costantini, F. and Lacy, E. 1986. Manipulating the Mouse Embryo, 1 edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  24. Monbaerts, P., Clarke, A. R., Rudnicki, M. A., Lacomini, J., Itohara, S., Lafaille, J. J., Wang, L., Ichikawa, Y., Jaenisch, R., Hooper, M. L. and Tonegawa, S. 1992. Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages. Nature 360:225.[ISI][Medline]
  25. Bruno, L., Fehling, H. J. and von Boehmer, H. 1996. The {alpha}ß T cell receptor can replace can replace the {gamma}{delta} receptor in the development of {gamma}{delta} lineage cells. Immunity 5:343.[ISI][Medline]
  26. Martinelli, T. M., van Driel, I. R., Aldernuccio, F., Gleeson, P. A. and Toh, B. H. 1996. Analysis of mononuclear cell infiltrate and cytokine production in murine autoimmune gastritis. Gastroenterology 110:1791.[ISI][Medline]
  27. Goverman, J., Woods, A., Larson, L., Weiner, L. P., Hood, L. and Zaller, D. M. 1993. Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity. Cell 72:551.[ISI][Medline]
  28. Katz, J. D., Wang, B., Haskins, K., Benoist, C. and Mathis, D. 1993. Following a diabetogenic T cell from genesis through pathogenesis. Cell 74:1089.[ISI][Medline]
  29. Osman, G. E., Cheunsuk, S., Allen, S. E., Chi, E., Liggitt, H. D., Hood, L. E. and Ladiges, W. C. 1998. Expression of a type II collagen-specific TCR transgene accelerates the onset of arthritis in mice. Int. Immunol. 10:1613.[Abstract]
  30. Sprent, J. and Kishimoto, H. 1998. T cell tolerance and the thymus. Ann. NY Acad. Sci. 841:236.[Free Full Text]
  31. Heath, W. R., Allison, J., Hoffmann, M. W., Schonrich, G., Hammerling, G. J., Arnold, B. and Miller, J. F. A. P. 1992. Autoimmune diabetes as consequence of locally produced interleukin-2. Nature 359:547.[ISI][Medline]
  32. Zal, T., Volkmann, A. and Stockinger, B. 1994. Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen. J. Exp. Med. 180:2089.[Abstract]
  33. Smith, K. M., Olson, D. C., Hirose, R. and Hanahan, D. 1997. Pancreatic gene expression in rare cells of thymic medulla: evidence for functional contribution to T cell tolerance. Int. Immunol. 9:1355.[Abstract]
  34. Hanahan, D. 1998. Peripheral-antigen-expressing cells in thymic medulla: factors in self tolerance and autoimmunity. Curr. Opin. Immunol. 10:656.[ISI][Medline]
  35. Fritz, R. B. and Zhao, M. L. 1996. Thymic expression of myelin basic protein (MBP). Activation of MBP-specific T cells by thymic cells in the absence of exogenous MBP. J. Immunol. 157:5249.[Abstract]
  36. Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. and Sakaguchi, S. 1999. Thymus and autoimmunity: Production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunological self-tolerance. J. Immunol. 162:5317.[Abstract/Free Full Text]
  37. Takahashi, T., Kuniyasu, Y., Toda, M., Sakaguchi, N., Itoh, M., Iwata, M., Shimizu, J. and Sakaguchi, S. 1998. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969.[Abstract]
  38. Suri-Payer, E., Amar, A. Z., Thornton, A. M. and Shevach, E. M. 1998. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J. Immunol. 160:1212.[Abstract/Free Full Text]
  39. Thornton, A. M. and Shevach, E. M. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
  40. Olivares-Villogomez, D., Wang, Y. and Lafaille, J. J. 1998. Regulatory CD4+ T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis. J. Exp. Med. 188:1883.[Abstract/Free Full Text]
  41. Van de Keere, F. and Tonegawa, S. 1998. CD4+ T cells prevent spontaneous experimental autoimmune encephalomyelitis in anti-myelin basic protein T cell receptor transgenic mice. J. Exp. Med. 188:1875.[Abstract/Free Full Text]
  42. Nishio, A., Hosono, M., Watanabe, Y., Sakai, M., Okuma, M. and Masuda, T. 1994. A conserved epitope on H+,K+-adenosine triphosphatase of parietal cells discerned by a murine gastritogenic T-cell clone. Gastroenterology 107:1408.[ISI][Medline]
  43. Suri-Payer, E., Amar, A. Z., Mchugh, R., Natarajan, K., Margulies, D. H. and Shevach, E. M. 1999. Post-thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti-H/K ATPase-reactive T cells. Eur. J. Immunol. 29:669.[ISI][Medline]