Department of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, Victoria 3181, Australia
Correspondence to: F. Alderuccio
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Abstract |
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Keywords: autoimmunity, T lymphocytes, TCR, transgenic mice
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Introduction |
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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 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
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-Ek 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/Kß253277) 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/Kß253277 has been generated, and shown to use V9 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-TCRß transgenic mice with TCR specificity for the gastritogenic H/Kß253277 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.
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Methods |
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Reagents
Pig gastric H/K ATPase was purified by tomato-lectin chromatography as described (20). Allophycocyaninanti-CD4 (clone RM4.5), phycoerythrin (PE)anti-CD8a (clone 53-6.7), FITCanti-Vß8.3 (clone 1B3.3), PEanti-V2 (clone B20.1) and PEanti-V
8 (clone B21.14) mAb were from PharMingen (San Diego, CA). Mouse mAb 1H9 and 2B6 specific for the
and ß subunits of the gastric H/K ATPase respectively were used as positive controls in ELISA and immunofluorescence assays.
The peptides H/Kß169193 (SFGFEEGKPCFIIKMNRIVKFLPSN), H/Kß253277 (LLNVPKNMQVSIVCKILADHVTFNN), H/Kß256269 (VPKNMQVSIVCKIL), H/Kß261274 (QVSIVCKILADHVT) and H/Kß266279 (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 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/Kß253277 gastritogenic peptide (19) was derived from a mouse immunized with mouse gastric H/K ATPase in complete Freund's adjuvant (8). The TCR V 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
and Vß oligonucleotide primers paired with oligonucleotides specific for the TCR
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
and Vß gene usage. The TCR V
and Vß gene usage for 1E4.C1 was V
9J
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 and ß chain genes. Genomic DNA for the 1E4.C1 TCR
chain variable region was amplified by PCR from the T cell hybridoma. Oligonucleotide primer specific for the 5' region of the TCR V
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
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 TrisHCl, 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 3x103 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 and ß chains were excised by XmaINotI and XhoIClaI restriction enzyme digestion respectively, and subcloned into pT
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 pT1E4cass 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 TrisHCl, 1 mM EDTA, pH 8.0, at a concentration of 25 ng/µl. To produce 1E4-TCR
ß transgenic mice, 1E4-TCR
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 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
cass and the inserted V(D)J region of the 1E4 TCR
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 TrisHCl, 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/ screening
TCR chain mutant mice (C57BL/6J- Tcratm1M°m ) (24) were obtained as homozygotes from Jackson Laboratories. Mice were screened for the TCR
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 (12x106) 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.
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Results |
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Intrathymic and peripheral T cells in TCR ß transgenic mice
Flow cytometric analysis of the CD4/CD8 thymocyte profiles revealed that virtually all CD4+CD8 and CD4CD8+ thymocytes from 1E4-TCRß transgenic mice expressed the Vß8.3 transgene (Fig. 2A
). 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
ß transgene in cells of the
T cell lineage (25).
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T cell populations were also examined from the spleens of TCR ß transgenic and non-transgenic mice at 1 and 6 weeks of age (representative data, Fig. 2B
). Overall, splenoctye numbers from 6-week-old TCR
ß transgenic mice were significantly greater (TCR
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
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
ß transgenic mice was not as marked (Fig. 2B
). The splenic CD4+ T cell population from TCR
ß transgenic mice was reduced by a factor of 1.6 (TCR
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
ß transgenic, 2.2 ± 0.5%; non-transgenic 5.1 ± 0.8%; P = 3.5x105). Similar reductions were observed in neonatal mice (data not shown). In contrast with the thymus, if the increased cellularity of spleens from TCR
ß transgenic was taken into account, there was not a significant difference in the total number of CD4+ T cells recovered from TCR
ß transgenic (TCR
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
ß transgenic remained significantly lower (TCR
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 253277 as a major gastritogenic T cell epitope associated with gastritis induced by immunization with the gastric H/K ATPase (18). Splenocytes from 1E4-TCRß transgenic mice proliferated in vitro to the gastritogenic H/Kß253277 peptide and not to a non-gastritogenic H/Kß169193 peptide, whereas splenocytes from non-transgenic littermates did not respond to either peptide (Fig. 3A
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The T cell proliferative response to the H/Kß261274 peptide was examined in 1E4-TCRß transgenic mice compared to non-transgenic mice. All five transgenic mice tested responded to H/Kß261274 peptide in vitro (Fig. 3C
), 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/Kß256271 peptide required pairing of the transgenic TCR
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/Kß256269 peptide (data not shown).
Gastritis in TCR 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 ß transgenic mice (13 of 16, 81%, eight females and five males) did not develop spontaneous gastritis (Fig. 4A
). However, spontaneous gastritis did develop in three of 16 (19%) 1E4-TCR
ß transgenic mice but not in 12 (four females and eight males) non-transgenic littermates (Fig. 4B
). All three TCR
ß transgenic mice that developed autoimmune gastritis were males of 1121 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
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. 4D
). 1E4-TCR
ß transgenic and non-transgenic mice which did not develop gastritis did not have circulating parietal cell autoantibodies (Fig. 4C
).
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Discussion |
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Our findings from analysis of 1E4-TCRß 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
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
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
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
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
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
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-TCRß 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 caseclonal 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-TCRß 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
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/Kß261274 epitope is a gastritogenic peptide. But why has spontaneous gastritis developed only in a minority of TCR
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 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
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.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cell |
PE phycoerythrin |
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Notes |
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Received 4 August 1999, accepted 22 November 1999.
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References |
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