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Clonal Expansion of CD4⁺ TCRßß⁺ T Cells in TCR -Chain- Deficient Mice by Gut-Derived Antigens¹

Ichiro Takahashi^2,*, Hideki Iijima^*, Rumi Katashima, Mitsuo Itakura and Hiroshi Kiyono^*

^* Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; and Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Tokushima, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
A population of CD4^{+ -}ß⁺ T cells increases in the mucosal and peripheral lymphoid tissues of TCR-chain-deficient mice with inflammatory bowel disease. The ^-ß⁺ T cells, which produce predominantly IL-4, mediate the proliferation of colonic epithelial crypts and the infiltration of large numbers of IgA-producing plasma cells into the lamina propria of the colon. To examine whether enteric Ags were recognized by a population of monoclonal ^-ß⁺ T cells leading to the intestinal inflammation, we examined the usage and clonotypes of TCR expressed by the ^-ß⁺ T cells in TCR-chain-deficient mice with inflammatory bowel disease. Analyses of immunoprecipitates by two dimensional electrophoresis and single-cell RT-PCR revealed that TCR of the ^-ß⁺ T cells was a homodimer of ß-chains that was capable of recognizing luminal bacterial Ags. PCR single-strand conformation polymorphism analysis of TCR Vß transcripts revealed monoclonal accumulation of the ^-ß⁺ T cells in the colonic lamina propria of the diseased mice. DNA sequencing revealed the accumulation of the ^-ß⁺ T cells with the same CDR3 sequences in the colon. These findings suggest that the pathogenic CD4^{+ -}ß⁺ T cells expressing a homodimeric form of the TCRß-chains can be clonally expanded upon the stimulation with gut-derived Ags.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Although the etiology and pathogenesis of human inflammatory bowel disease (IBD)³ are not well understood, several lines of evidence obtained from experimental IBD models indicate that either destruction of homeostasis in regulatory T cells, or the emergence of forbidden T cells, plays a crucial role in the development of intestinal immune disorders (1, 2).

TCR -chain-deficient (TCR^-/-) mice, created by gene targeting of the first exon of the TCR C gene in embryonic stem cells, spontaneously develop IBD-like lesions (3). An expanded population of T cells and a unique population of T cells expressing CD4 and TCR^-ß⁺ are observed both in the mucosal and peripheral lymphoid tissues in TCR^-/- mice with IBD (3, 4, 5, 6). The CD4⁺ TCR^-ß⁺ T cells purified from the intestine of the IBD mice exclusively produce IL-4, use limited subsets (Vß6, Vß8, Vß14, and Vß15) of TCR, and massively proliferate upon stimulation with bacterial Ags (6, 7, 8).

The lack of ß T cells, and the increases of ^-ß⁺ T cells and T cells, are associated with a Th2 cytokine imbalance (8), the alteration of humoral immune responses to enteric bacterial Ags (9) and the development of autoantibodies (6, 8, 10) in TCR^-/- mice. The pathological role of the Th2-driven T cells was also supported by the findings that the CD4⁺ TCR^-ß⁺ T cells can sustain the aberrant humoral immune responses in Peyer’s patches (PP) B cell cultures (6). Furthermore, the depletion of the ^-ß⁺ T cells with mAb against TCRß chain completely suppressed the onset of IBD in the TCR^-/- mice (6).

Others have reported that deviation in TCR Vß repertoire occurs in some autoimmune diseases (11, 12). Similar lines of approach have also been applied to human IBD in which clonal prevalence of a particular TCR Vß has also occurred, implicating a response to specific luminal bacterial Ags (13, 14). Other studies have identified an oligoclonal gut-homing CD4⁺ ß T cell population in murine colitis using aging scid mice expressing a transgene-encoded ß TCR (15). However, it was shown that the clonality of gut-infiltrating CD4⁺ T cells is polyclonal in other CD4⁺ T cell-transplanted scid mice (16). Thus, the clonotypes of pathogenic CD4⁺ T cells for the development of IBD remain controversial and need to be elucidated.

Another important issue is the potential role of infectious, dietary, or autoantigenic elements in the pathogenesis of IBD (1, 2). Recently, reactivity of CD4⁺ T cells isolated from C3H/HeJBir with chronic colitis to enteric bacterial Ags was studied (17). Adoptive transfer of CD4⁺ T cells stimulated with MHC-class-II-restricted enteric bacterial Ags resulted in the development of Th1-mediated chronic colitis. However, the clonality and repertoire usage of TCR expressed on the CD4⁺ T cells was unknown.

In this study, we assessed the Ag recognition by the infiltrating CD4⁺, ^-ß⁺ T cells, and the molecular form of TCR and clonotypes of these cells in TCR^-/- mice with IBD by using a combination of two-dimensional electrophoretic, single-cell RT-PCR and PCR-single-strand conformation polymorphism (SSCP) analyses. Our results provided strong suggestive evidence that the structure of TCR on these CD4⁺, ^-ß⁺ T cells is ß-ß homodimeric form. In addition, PCR-SSCP analysis of TCR Vß chain transcripts indicated the prevalent usage and clonal accumulation of limited TCR Vß subfamilies in mucosal CD4⁺, ^-ß⁺ T cells from the IBD mice, suggesting the clonal expansion of CD4⁺, ^-ß⁺ T cells upon the stimulation with enteric Ags.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice

TCR^-/- mice with a background of 129 x C57BL/6 were obtained from The Jackson Laboratory (Bar Harbor, ME). TCR^-/- mice were originally developed by Mombaerts et al. (3). C57BL/6 mice were purchased from Charles River, Japan (Kanagawa, Japan). The mice were housed in the Experimental Animal Facility at the Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

Reagents

Staphylococcal enterotoxin B (SEB) was purchased from Sigma (St. Louis, MO). Heat shock protein 60 (HSP 60) from Escherichia coli was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). LPS from E. coli O55:B5 was purchased from List Biological Laboratories (Campbell, CA). Lyophilized cells of coagulase-negative, but mannitol-fermenting, staphylococci (Staphylococcus xylosus) recovered from the stool of the IBD mice (6) were also used as a stimulant of T cell proliferation assay.

Isolation of ^-ß⁺ T cells of the IBD mice

Single cell suspensions were prepared from the spleen (SP) or mesenteric lymph nodes (MLN) of the TCR^-/- mice. Lymphocytes of lamina propria (LP) of the intestine were isolated as previously described (6). In brief, intestinal tissues freed of their epitheilal cell layers were digested by collagenase type IV (Sigma). Cells in the supernatants were harvested, washed, filtered through a glass wool column, and placed on a discontinuous 40%/70% Percoll gradient. After centrifugation for 20 min at 600 x g, lymphoid cells were collected from the interface.

Immunoprecipitation

Immunoprecipitation of TCR Vß molecules expressed on the ^-ß⁺ T cells was performed according to the Manual of Cellular Labeling and the immunoprecipitation kit developed by Boehringer Mannheim Biochemicals. In brief, lymphoid cells were washed twice with PBS, and were suspended in labeling buffer containing 50 mM sodium borate (pH 8.0) and 150 mM NaCl at a concentration of 1 x 10⁷ cells/ml. D-Biotinoyl--aminocaproic acid-N-hydroxysuccinimide ester dissolved in DMSO was added to the cell suspension at a final concentration of 50 µg/ml, then incubated for 15 min at room temperature. Biotinylated cells were washed twice with PBS. Cells were lysed with a lysis buffer containing 50 mM sodium borate (pH 8.0), 150 mM NaCl, 1 µg/ml of aprotinin, 1 µg/ml of leupeptin, 100 µg/ml of PMSF, 1% Nonidet P-40, and 0.5% of sodium deoxycholate, at a concentration of 10⁷ cells/ml. Immunoprecipitation was performed either with anti-TCRß mAb (H57-597) or anti-TCR mAb (H28-710) (18). Immunoprecipitates were separated either on single-dimensional SDS-10–15% gradient PAGE under nonreducing/reducing condition or on immobilized pH gradient electrophoresis (IPHGE) and SDS-PAGE under reducing conditions (19) and transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). The biotinylated proteins were detected using alkaline phosphatase-conjugated anti-biotin Abs (New England Biolabs, Beverly, MA).

Phosphatidylinositol-specific phospholipase C (PIPLC) digestion

Cells (10⁶) were washed with PBS and then resuspended in 100 µl RPMI 1640 containing HEPES (pH 6.8), 2 mg/ml BSA, 5 x 10^-5 M 2-ME with or without the addition of 6 µl of PIPLC from Bacillus cereus (Boehringer Mannheim). After 1-h incubation at 37°C in a humidified 5% CO₂ incubator, cells were washed with the incubation medium followed by PBS before flow cytometric analysis.

Flow cytometric analysis

Immunofluorescent analysis was performed using a FACScan flow cytometry (Becton Dickinson, Mountain View, CA). Cells stained with single-color reagent were used to set the appropriate compensation levels, and at least 10,000 events were analyzed. The following mAbs from PharMingen (San Diego, CA) were used: anti-CD4 (RM4-5) and anti-TCRß (H57-597). For two-color flow cytometry, 1 x 10⁶ cells in 20 µl PBS containing 2% FCS and 0.02% sodium azide were first incubated with anti-FcR mAb (2.4G2) to prevent nonspecific staining, and then stained with FITC- and phycoerythrin-conjugated mAbs (6). Flow cytometric analysis was done with a FACScan flow cytometer using CellQuest software (Becton Dickinson). Results are shown as log-log dot plots.

Single-cell RT-PCR

For the analysis of TCRß-chain usage in CD4⁺, ^-ß⁺ T cells at the single-cell level, a single-cell RT-PCR was performed. A single TCR^-ß⁺ T cell was distributed into individual wells of U-bottom 96 wells of a microtiter plate (Falcon 3077; Becton Dickinson, Lincoln Park, NJ) by flow cytometric sorting using FACS Vantage (Becton Dickinson). The individual well containing a single cell was treated with 18 µl of lysing buffer composed of 10 mM Tris-HCl (pH 8.3) and 50 mM KCl, 5 mM MgCl₂, 1 mM each dNTP, 10 U of RNase inhibitor (Promega, Madison, WI), 0.1 µg of oligo(dT₁₆), and 2.5% of Nonidet P-40. Following 10-min incubation at room temperature, 2 U of Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) were added and incubated at 42°C for 60 min. After the synthesis of cDNA, 80 µl of PCR buffer containing 10 mM Tris-HCl (pH 8.3) and 50 mM KCl, 2 mM MgCl₂, 2 µM random primer (Perkin-Elmer, Branchburg, NJ), and 2.5 U of Taq DNA polymerase (Perkin-Elmer) were added. The cDNA were amplified for 30 cycles, each cycle consisting of 20 s at 94°C, 37°C to 65°C by increasing gradually for 1 min, and for 3 min at 68°C. For the detection of positive wells, 4 µl of the amplified cDNA from individual wells were subjected to the standard PCR method as described previously (6) by using ß-actin-specific primers (Clontech Laboratories, Palo Alto, CA). After the selection of wells that contained ß-actin-specific mRNA, the randomly amplified cDNA was then polymerized by a panel of TCR Vß-specific PCR as described previously (6). The frequency of a certain TCR Vß gene expressed per total ß-actin gene expressed (n = 100) in ^-ß⁺ T cells was calculated.

In vitro T cell proliferative responses

MLN were aseptically removed from TCR^-/- mice with IBD, single cell suspensions of the tissues were prepared by mechanical disruption, washed extensively, and resuspended in PBS containing 2% FBS. CD3⁺ T cells were enriched by negative panning with goat anti-mouse IgG F(ab')₂ (20 µg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA). CD4⁺ T cells were further purified by magnetic cell sorting beads (6). The CD4⁺ T cells (1 x 10⁵/well) suspended in RPMI 1640 (Sigma) supplemented with sodium bicarbonate, HEPES, L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (100 µg/ml) were cultured in flat-bottom 96-well microculture plates (Corning, Corning, NY) in the presence of mitomycin C-treated feeder spleen cells (1 x 10⁴ cells/well) for 3 days. CD4⁺ T cell proliferative responses upon the stimulation with bacterial products (SEB, LPS, HSP 60, and whole cells of enteric staphylococci) were assessed by addition of 0.5 µCi of [³H]TdR (ICN, Costa Mesa, CA) 6 h prior to the cell harvest. The level of proliferation was determined by liquid scintillation counting.

PCR-SSCP

Total RNA was extracted from ^-ß⁺ T cells of LP of the colon, ileum, and SP of the TCR^-/- mice by using TRIzol reagent (Life Technologies). The total RNA (50 ng) was converted to cDNA with reverse transcriptase (Superscript II, Life Technologies) and oligo(dT₁₆) nucleotide (25 µM; Perkin-Elmer) at 42°C for 30 min in 10 µl. cDNA was amplified by PCR with a Cß primer labeled with 6-carboxyfluorescein (6-FAM; Perkin Elmer), one of the Vß subfamily-specific primers, dNTP, and Taq DNA polymerase (Perkin-Elmer) for 30 cycles (94°C for 0.5 min and 60°C for 1 min) in a thermal cycler 9600 (Perkin-Elmer). The sequences of the Vß and Cß primers were the same as described previously (6). Amplified DNA was diluted (1:50), heat denatured with a loading buffer consisting of 95% formamide and 50 mM EDTA, and electrophoresed in 6% polyacrylamide gel at 20°C set to an automated DNA sequencer (model 373A; Applied Biosystems, Foster City, CA) equipped with a temperature-controlling system (20, 21). The 6-FAM-labeled PCR products emit blue fluorescence at 517 nm when excited by an argon laser. Red-colored double-strand GENESCAN Rox-500 was used as an internal DNA size marker. Electrophoresis was performed in 1x tris-borate/EDTA buffer at constant 30 W for 3 h, and the data were collected and analyzed using GENESCAN 672 software (Applied Biosystems).

DNA Sequencing

For determination of DNA sequences of TCR Vß8 PCR products, cDNAs were PCR amplified in a reaction mixture containing primers for Cß and Vß8, dNTP, 1 µCi of [-³²P]dCTP (NEN, Boston, MA), and Taq DNA polymerase in the same cycles described above. Amplified DNA was diluted (1:5), heat denatured, electrophoresed in 6% polyacrylamide slab gel at 20°C, and visualized by autoradiogram. Corresponding SSCP bands with the identical mobility on the SSCP gel set to Applied Biosystems 373A sequencer were excised from the SSCP gel. DNA was extracted in boiling water for 20 min. The extracted DNA was amplified by PCR with Cß and Vß8 primers, and cloned into a plasmid pGEMT-Easy (Promega). DNA sequences were determined by an Applied Biosystems prism dye terminator cycle-sequencing method using an automated DNA sequencer (Applied Biosystems model 310).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Structure of TCR of the ^-ß⁺ T cells

TCR^-/- mice with IBD harbor a unique population of CD4⁺ T cells that express TCR ß-chains without TCR-chains on the cell surface (3, 4, 6, 8). Depletion of the Th2-biased ^-ß⁺ T cells by the treatment of TCR^-/- mice with mAbs against TCRß completely inhibits the development of the pathological changes in the colon (6). However, the exact mechanisms of Ag recognition by the ^-ß⁺ T cells and the selection of these cells in the mucosal and peripheral lymphoid tissues are still not known.

In the first experiments, we analyzed the TCR structure on the ^-ß⁺ T cells. The ^-ß⁺ T cells isolated from the colonic LP of the diseased mice were surface-biotinylated and immunoprecipitated by using mAbs against TCRß-chain or TCR-chain coupled to protein A-Sepharose. The mAb specific for TCRß-chain (clone H57-597) but not mAb reactive to TCR-chain (clone H28-710) precipitated a protein with a molecular mass of 88 kDa under nonreducing conditions and of 44 kDa under reducing conditions (Fig. 1A), and of almost identical mobility to the band given by immunoprecipitation of ß T cells with the mAb specific for TCRß or TCR. In this regard, others have reported that there were at least two forms of the TCRß protein on immature thymocytes in TCRß transgenic mice, i.e., a monomer of 40 kDa, as well as a disulfide-linked form of 80 kDa (22). It was unlikely that the TCRß protein on the ^-ß⁺ T cells from the diseased TCR^-/- mice was a monomer of 40 kDa, because the immunoprecipitation showed that the TCR expressed on the ^-ß⁺ T cells was a protein with a molecular mass of 88 kDa under nonreducing conditions and 44 kDa under reducing conditions, respectively (Fig. 1A).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Structure of TCR of the -ß+ T cells from TCR-/- mice with IBD. In A, surface expression of TCRß-chain dimer on a -ß+ T cells from TCR-/- mice with IBD. TCRß-specific or TCR-specific immunoprecipitates from lysates of surface-biotinylated -ß+ T cells from the diseased TCR-/- mice and ß T cells from TCR+/+ mice were analyzed under nonreducing and reducing conditions by SDS-PAGE on a 10–15% gradient gel. TCR bands in nonreduced and reduced immunoprecipitates can be seen as 88 kDa and 44 kDa, respectively. B, Lack of expression of pre T and TCR C of -ß+ T cells. CD4+, -ß+ T cells were isolated from LP of the colon of the diseased TCR-/- mice. As a positive control for the expression of pre T and TCR C mRNA, CD4+CD8+ T cells were isolated from thymi of C57BL/6 mice. The left end column shows a 100-bp DNA ladder. Primers for pre T and TCR C were constructed from published sequences (34 35 36 ). C, Single TCRß-chain usage in -ß+ T cells by allelic exclusion. A single-cell RT-PCR was done for the analysis of TCRß-chain usage in -ß+ T cells as described in the text. RNA isolated from ß T cells of C57BL/6 mice was also used for the analysis. The frequency of a certain TCR Vß gene expressed per total ß-actin gene expressed (n = 100) in -ß+ T cells or ß T cells was calculated. D, Two-dimensional IPHGE/PAGE analyses of H57-597 (anti-TCRß) and H28-710 (anti-TCR) immunoprecipitates from surface biotinylated -ß+ T cells from TCR-/- mice with IBD. E, Flow cytometric analysis of lymphocytes isolated from LP of the colon in TCR-/- mice before and after incubation with PIPLC. Cell suspensions were stained with mAbs specific for FITC-TCRß and phycoerythrin-CD4.

To further determine assembly patterns (i.e., homodimer or heterodimer) of TCRß-chain on the ^-ß⁺ T cells, TCR Vß expression was analyzed by RT-PCR at the single-cell level. For this purpose, individual LP lymphocytes reactive with mAb against pan TCRß-chain (H57-597) were sorted into individual wells of 96-well microtiter plates, then RT-PCR analysis was performed to detect the expression of TCR Vß mRNA by using each Vß-specific sense-primer (Vß1 to Vß19) and Cß-specific antisense primer. As shown in Fig. 1C, individual ^-ß⁺ T cells isolated from the colon expressed a single kind of TCR ß-chain mRNA, mainly TCR Vß8, presumably by the rule of allelic exclusion. Coexpression of two distinct TCRß-chain was not seen at all. Together, these results provide strong evidence that the TCR of the ^-ß⁺ T cells was composed of TCRß-chain homodimer.

The TCRß-chain could be expressed together with an unidentified polypeptide of the same m.w. In order to determine whether the TCR expressed on the ^-ß⁺ T cells contained not only TCRß-chain but also unknown polypeptide, we analyzed the immunoprecipitates of ^-ß⁺ T cells by immobilized pH gradient gel electrophoresis (IPHGE) followed by SDS-PAGE. Two-dimensional IPHGE-PAGE analysis of TCRß immunoprecipitates of the ^-ß⁺ T cells gave a slightly basic spot in the reducing gel (Fig. 1D). No bands with intermediate isoelectric point were found in the TCRß immunoprecipitates. In addition, TCR immunoprecipitates of the ^-ß⁺ T cells confirmed the absence of acidic TCR in the gels. These results clearly rule out the possibility that a monomer of TCRß-chain is expressed together with an unidentified polypeptide of the same m.w. Instead, these biochemical data further support the possibility that TCR expressed on the ^-ß⁺ T cells is a ß-ß homodimer.

Groettrup and von Boehmer reported that a TCRß-monomer, but not a TCRß-dimer, expressed on the surface of thymocytes of TCRß-transgenic mice is glycosyl-phosphatidylinositol (GPI) linked and that the GPI-linked ß-chain monomer is expressed without CD3 complex (22). We analyzed the possibility that the TCR expressed on the CD4^{+ -}ß⁺ T cells is tethered to the membrane via a GPI anchor. Since phosphatidylinositol-specific phospholipase C (PIPLC) digestion leads to the removal of GPI-anchored proteins from the cell surface (22), ^-ß⁺ T cells isolated from the inflamed colon of TCR^-/- mice were treated with the enzyme. Flow cytometric analysis showed that GPI cleavage could not remove a substantial amount of TCRß chain from the surface of the ^-ß⁺ T cells (Fig. 1E). To confirm the validity of our PIPLC digestion assay, the expression of Thy1 molecules on the ^-ß⁺, CD4⁺ T cells before/after PIPLC digestion was analyzed. FACS analysis clearly showed that GPI-anchored Thy1 molecules on the T cells were completely removed after the PIPLC digestion (data not shown). These results clearly showed that the ß-chain of TCR expressed on the ^-ß⁺ T cells is not a GPI-linked monomer and that it retains as transmembrane and cytoplasmic domain at the carboxyl-terminal region. This is the first evidence on the surface expression of homodimer of TCRß-chains in the mature CD4⁺ T cells.

Reactivity of the CD4^{+ -}ß⁺ T cells with enteric antigens

We next analyzed whether the homodimer of TCRß-chains on the surface of the CD4⁺ T cells was capable of signaling after binding to bacterial ligands. As shown in Fig. 2, the ^-ß⁺ T cells massively proliferated in response to both whole cells of S. xylosus, a clinical isolate from the enteric flora of the IBD mice (6), and to SEB in a dose-dependent manner. The levels of [³H]TdR uptake by S. xylosus, SEB, and mAb against CD3-stimulated ^-ß⁺ T cells from the diseased mice were similar to the levels obtained from ß T cells of TCR^+/+ mice (Fig. 2). However, the CD4^{+ -}ß⁺ T cells did not respond to LPS or HSP 60 of E. coli. Thus, the type of Ags recognized by the TCRßß may be similar to those seen by TCRß. Instead, the homodimer of TCRß-chains could not recognize Ags such as glycolipids (LPS) and HSP, which are presented by MHC-class-I-related molecules, i.e., CD1, MICA, and MICB (25, 26). In terms of restriction molecules recognized by the ^-ß⁺ T cells, Mombaerts et al. reported that ^-ß⁺ T cells are not detected in TCR x MHC class II double-deficient mice, suggesting that they are dependent on MHC class II molecule expression (4). Together, these findings further suggest that the TCRßß expressed on these CD4⁺, ^-ß⁺ T cells is a biologically functional receptor that can transduce activation signals provided by peptidic Ag and superantigen.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. T cell proliferative responses to enteric proteinaceous Ags in CD4+ T cell cultures prepared from the TCR-/- mice with IBD and from the TCR+/+ mice. The [3H]TdR uptake in CD4+ T cell cultures without stimulants was 5344 ± 626 for the diseased TCR-/- mice and 3045 ± 90 in TCR+/+ mice, respectively. The results presented are representative of three separate experiments. Data are expressed as means ± SDs for triplicate cultures.

Comparison of the clonality of ^-ß⁺ T cells from the IBD mice with that of ß T cells from the background mice

Mizoguchi et al. (9) reported that the alteration of the humoral immune responses against cecal aerobic bacterial Ags such as E. coli from polyclonal to oligoclonal is strongly associated with the development of IBD in TCR^-/- mice. The ^-ß⁺ T cells sustain the formation of germinal centers that are an anatomical signature of classical Th-B interaction (5). In addition, the ^-ß⁺ T cells isolated from the diseased mice can markedly enhance the IgA and IgG Ab responses, i.e., T cell-dependent Ab responses, in PP B cell cultures (6). Thus, we speculated that these humoral immune responses reflect the deviation in the infiltrating ^-ß⁺ T cells in the colon of the diseased TCR^-/- mice.

In this context, we assessed the clonotypes of the infiltrating CD4⁺, ^-ß⁺ T cells in TCR^-/- mice with IBD by using a PCR-SSCP analysis of the TCRß chain. A profile of PCR-SSCP analysis of ^-ß⁺ T cells from IBD mice was compared with the clonotypes of the ß T cells isolated from wild-type C57BL/6 mice (Fig. 3). The ^-ß⁺ T cells used a limited number of subfamilies of the TCR Vß-chain repertoire. TCR Vß4, Vß6, Vß8, Vß10, and Vß14 gene products of the ^-ß⁺ T cells were dominant both in mucosal (LP of the colon) and systemic (SP) lymphoid tissues. The PCR amplified products of the respective TCR Vß subfamilies in the ^-ß⁺ T cells exhibited relatively invariant clonality, e.g., monoclonal to oligoclonal mobility on the SSCP gels, suggesting that the ^-ß⁺ T cells in the diseased mice might be expanded from a limited number of T cell clones reactive with specific Ags. On the other hand, PCR-SSCP analysis of Vß subfamilies of ß T cells isolated from naive, wild-type mice revealed a diversified repertoire of TCR Vß subfamilies expressed, while the cDNA products of the Vß subfamilies exhibited a smeared band pattern on the SSCP gel, consistent with a polyclonal population of ß T cells.

View larger version (62K): [in this window] [in a new window]   FIGURE 3. Comparison of T cell clonality of -ß+ T cells from TCR-/- mice with IBD with that of ß T cells from TCR+/+ mice. -ß+ T cells or ß T cells were isolated from the LP of the colon and SP of the diseased TCR-/- mice and TCR+/+ mice, respectively. RT-PCR-amplified Vß-Cß cDNAs (blue bands) of these T cells were electrophoresed in a 6% nondenaturing SSCP gel at 20°C using an automated DNA sequencer. The red bands indicate Genescan Rox-500, an internal DNA size marker. -ß+ T cells use limited subsets of TCR Vß and give a monoclonal or oligoclonal sharp band pattern. On the other hand, ß T cells use diversified TCR Vß repertoire and show a polyclonal wide band pattern.

Clonal prevalence of colon-infiltrating ^-ß⁺ T cells of the IBD mice

We also compared the clonotypes of expressed TCR Vß chains of the ^-ß⁺ T cells in the diseased colon with those of the ileum and SP. The PCR-SSCP analysis revealed that cDNA products of the respective TCR Vß subfamilies of the ^-ß⁺ T cells in the colon gave more common bands on the SSCP gel as compared with the ileum and SP; the clonality of each TCR Vß repertoire of the cells in nondiseased lesions were more divergent, i.e., multiple band pattern, as compared with the diseased sites (Fig. 4). Furthermore, the PCR products of each TCR Vß subfamily in the colon of individual mice exhibited nearly identical mobilities (data not shown). These results suggest that a limited set of T cell clones commonly accumulates in the colon of diseased mice, presumably by clonal proliferation of the ^-ß⁺ T cells upon stimulation with common enteric Ags.

View larger version (93K): [in this window] [in a new window]   FIGURE 4. PCR-SSCP analysis of -ß+ T cell clonality of TCR-/- mice with IBD. -ß+ T cells were isolated from the LP of the colon and ileum, and the SP of the diseased TCR-/- mice. PCR amplified Vß-Cß cDNAs (blue bands) of -ß+ T cells were analyzed in a nondenaturing gel using an ABI 373A sequencer. The red bands represent an internal DNA size marker. -ß+ T cells isolated from the colon give a monoclonal sharp band pattern. On the other hand, those isolated from the ileum and SP give an oligoclonal multiple band pattern.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. VDJ junctional DNA sequences and predicted amino acid sequences of common bands of TCR Vß 8 of -ß+ T cells from TCR-/- mice with IBD. PCR products exhibiting identical mobility on SSCP gel were extracted and cloned into a plasmid pGEM-T. VDJ junctional sequences of 15 independent plasmid clones were determined for each band. The sequences that frequently appeared in each band are shown. The frequency of the indicated DNA sequence in each band is shown on the left. Locations of the Vß, Dß, and Jß segments and CDR3 are shown (marked). N sequence additions flanking the germline Dß segments are underlined.

In this regard, we assessed the influence of diet on the development of IBD in the TCR^-/- mice. The TCR^-/- mice fed an elemental diet consisting of 17 kinds of chemically defined amino acids without antigenic proteins exhibited complete loss of colitis, however, the ^-ß⁺ T cells still increased in the mucosal lymphoid tissues (manuscript in preparation). Interestingly, the clonotype of TCR Vß of the ^-ß⁺ T cells isolated from the TCR^-/- mice fed an elemental diet gave more divergent, i.e., quiescent pattern. In addition, bacterial species resident in the TCR^-/- mice fed an elemental diet were quite different from that in TCR^-/- mice fed a nutritional diet. Thus, the clonally infiltrating ^-ß⁺ T cells stimulated by some luminal bacteria including staphylococci mediate the mucosal inflammation in these mice.

Recently, Groux et al. (31) reported that regulatory CD4⁺ T cell subsets (e.g., Tr1) prominently producing IL-10 actively down-regulate inflammatory response in Th1-mediated murine colitis. A lack of regulatory T cells that regulate the Th1/Th2 cytokine balance may also participate in the development of colitis in TCR^-/- mice. To this end, it is possible to suggest that aberrant Th2 type ^-ß⁺, CD4⁺ T cells are developed in TCR^-/- mice with IBD due to the lack of Tr1 type cells. Our previous study demonstrated that IL-4 producing Th2 type ^-ß⁺, CD4⁺ T cells are increased in the diseased TCR^-/- mice (6). Furthermore, most recent findings showed that treatment of TCR^-/- mice with anti-IL-4 mAb resulted in the inhibition of IL-4 producing ^-ß⁺ T cells and disease development (32).

The role of IL-4 producing ^-ß⁺, CD4⁺ T cells in the pathogenesis of colitis in TCR^-/- mice was also vigorously investigated by creating double-mutant mice by crossing TCR^-/- with IL-4^-/- or IFN-^-/- mice. While the lack of IL-4 markedly suppressed the onset of colitis, IFN- x TCR^-/- mice develop colitis similar to that in TCR^-/- mice (33).

In conclusion, this study demonstrates the critical role for clonally infiltrated CD4⁺, ^-ß⁺ T cells expressing TCRßß in the development of murine IBD, and suggests that blocking the development of the T cells may be a therapeutic approach worth testing in the treatment of IBD.

	Acknowledgments

 
We thank Dr. T. Nakayama (Science University of Tokyo, Tokyo, Japan) for providing H28-710. We are also grateful to Dr. S. Hamada (Osaka University, Osaka, Japan) for his encouragement of the work and to Dr. M. Kronenberg (La Jolla Institute for Allergy and Immunology, La Jolla, CA) for his helpful comments and discussions.

	Footnotes

 
¹ This work was supported by Grant-in-Aid from the Ministry of Education, Science, and Culture, and Ministry of Health and Welfare of Japan.

² Address correspondence and reprint requests to Dr. Ichiro Takahashi, Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan.

³ Abbreviations used in this paper: IBD, inflammatory bowel disease; PIPLC, phosphatidyl inositol-specific phospholipase C; HSP, heat shock protein; LP, lamina propria; MLN, mesenteric lymph nodes; PP, Peyer’s patches; SEB, staphylococcal enterotoxin B; SP, spleen; SSCP, single-strand conformation polymorphism; IPHGE, immobilized pH gradient gel electrophoresis; GPI, glycosyl-phosphatidylinositol; PE, phycoerythrin.

Received for publication July 13, 1998. Accepted for publication October 21, 1998.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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