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The Journal of Immunology, 2003, 171: 4131-4139.
Copyright © 2003 by The American Association of Immunologists

Clonal Expansion of Double-Positive Intraepithelial Lymphocytes by MHC Class I-Related Chain A Expressed in Mouse Small Intestinal Epithelium 1

Eun Jeong Park*,||,#, Ichiro Takahashi*,{dagger}, Junko Ikeda{dagger}, Kazuko Kawahara{dagger}, Tetsuji Okamoto{dagger}, Mi-Na Kweon*,#, Satoshi Fukuyama||,#, Veronika Groh{ddagger}, Thomas Spies{ddagger}, Yuichi Obata§, Jun-Ichi Miyazaki and Hiroshi Kiyono2,*,||,#

* Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; {dagger} Departments of Preventive Dentistry and Molecular Oral Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; {ddagger} Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA 98109; § Department of Biological System, RIKEN BioResource Center, Tsukuba, Japan; Division of Stem Cell Regulation Research, Osaka University Medical School, Osaka, Japan; || Core Research for Engineering, Science, and Technology, Japan Science and Technology, Tokyo, Japan; and # Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of a distant homologue MHC class I molecule, MHC class I-related chain A (MICA), has been found to be stress inducible and limited to the intestinal epithelium. This nonclassical MHC molecule is associated with various carcinomas in humans. To understand the biological consequences of MICA expression in the gut, we generated transgenic (Tg) mice (T3b-MICA Tg) under the control of the T3b promoter. The T3b-MICA Tg mice expressed MICA selectively in the intestine and had an increased number of TCR{alpha}{beta} CD4CD8{alpha}{alpha}, double-positive (DP) intraepithelial lymphocytes (IELs) in the small bowel. These MICA-expanded DP IELs exhibited a bias to V{beta}8.2 and overlapped motifs of the complementarity-determining region 3 region among various Tg mice. Hence, the overexpression of MICA resulted in a clonal expansion of DP IELs. Studies in model of inflammatory bowel disease showed that transgenic MICA was able to attenuate the acute colitis induced by dextran sodium sulfate administration. Therefore, this unique in vivo model will enable investigation of possible influences of stress-inducible MICA on the gut immune surveillance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Intestinal epithelia contain a developmentally and functionally specialized T cell pool, the so-called intraepithelial lymphocyte (IEL) 3 (1, 2). Because of their specific and unique location in the mucosal epithelium, IELs are often regarded as a first line of mucosal barrier against enteric flora (3). With respect to IEL ontogeny, it can be divided into {alpha}{beta} T cells bearing CD4 or CD8{alpha}{beta} coreceptors (thymus dependent) and {gamma}{delta} or {alpha}{beta} T cells bearing CD8{alpha}{alpha} coreceptors (thymus independent) (4, 5, 6, 7, 8). However, a recent report suggests that the thymus is critical for generation of TCR{alpha}{beta} CD8{alpha}{alpha} IELs also (9). Little is known about locally expressed key molecules that may be involved in the selection and maturation of extrathymic IEL; reportedly though, some integrins or chemokine receptors facilitate the migration of TCR{gamma}{delta} IELs or their precursors (10).

A nonclassical MHC class I chain A (MICA) molecule is stress inducible and mainly expressed on intestinal epithelium and various epithelial tumors (11, 12, 13). Because of the discovery that intraepithelial V{delta}1 T cells recognize MICA (14), interaction of the NKG2D receptor on these T cells with MICA has been suggested to be important for activation of NK and T cell responses against MICA-bearing tumors (15). In addition, bacterial infection has enhanced the expression of MICA on target cell surface and up-regulated V{gamma}2{delta}2 T cell activation by nonpeptide Ags (16).

The increase in MICA expression induced by various stresses, including heat shock, oxidative stress, and virus or bacteria, together with the expression of MICA locally in gut epithelium prompted us to consider the possibility that interaction of MICA with IEL is important for the development and effector functions of IEL. In particular, because the gut is in a permanent state of mild inflammation or immunological stress due to exposure to commensal microflora and food Ags, lymphocytes struggling against damaged cells on the frontline may be necessary for maintaining host homeostasis (17, 18). However, the function of MICA expressed in the human intestinal tracts and consequences of the increase in MICA observed in vivo are undefined.

To evaluate these issues, in this work, we generated a transgenic model ectopically expressing human MICA, under the control of the T3b promoter in the mouse intestine. The transgenic mice expressed human MICA specifically on their intestinal epithelium and possessed numerous CD4CD8{alpha}{alpha} (double-positive (DP)) IELs in their small intestine. We thus inquired into the characteristics of this expanded DP subset, examining the clonotype and DNA sequence of complementarity-determining region 3 (CDR3) to determine whether MICA exposure biased these cells toward a unique V{beta} repertoire. Moreover, we introduced an experimental inflammatory bowel disease model into the transgenic (Tg) mice and showed a substantial attenuation of the development of intestinal disorder.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DNA construct and the generation of T3b-MICA Tg mice

The 2.8-kb promoter region of the T3b gene was shown to direct transgene expression exclusively in the epithelial cells of the small and large intestines (19). The upstream end of the T3b promoter region in the T3b transgene vector was originally an SphI site, but was changed to a KpnI site using a linker, because of the presence of an SphI site in the MICA cDNA. The human MICA cDNA, including 1.2 kb of the whole MICA coding sequence, was cloned by PCR, and its sequence was confirmed (11). The T3b-MICA transgene was constructed by inserting this MICA cDNA into the unique EcoRI site of the T3b transgene vector, which contains the T3b promoter and the rabbit {beta}-globin gene sequences from the second exon to the third exon, including the polyadenylation signal. The transgene vector was digested with KpnI and XhoI, and the resulting 5.5-kb fragment of the T3b-MICA transgene was isolated and used for microinjection into the pronuclei of one-cell embryos of BDF1 mice to produce T3b-MICA transgenic mice, as described previously (20). ICR (recipient) and C57BL/6 (backcrossing partner) mice purchased from Japan SLC (Shizuoka, Japan) were used throughout this study. The mice were maintained under specific pathogen-free conditions in the animal facility of the Research Institute for Microbial Diseases, Osaka University. For screening of founder mice, tail DNA was isolated by use of the SDS-proteinase K method. Founders were genotyped by PCR using specific primers for the transgene, MICA cDNA. The oligonucleotide 5'-GCTGGTTATTGTGCTGTCTC-3' was used as a forward primer, and 5'-GGATCTCACAGACCCTAATC-3' was used as a backward primer.

RT-PCR

Total RNA was extracted from various tissues by using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. DNase digestion of extracted RNA was performed before cDNA synthesis. A total of 1 µg of total RNA was reverse transcribed into cDNA using Omniscript reverse transcriptase (Qiagen, Valencia, CA). PCR amplification (GeneAmp PCR system 9700) (PerkinElmer, Foster City, CA) was performed initially at 95°C for 5 min and then in sequential cycles at 95°C for 30 s, 61°C for 30 s, and 72°C for 40 s, followed by an extension for 10 min at 72°C. The oligonucleotide primers used for the determination of MICA expression were 5'-CTCGAGGAGCCCCACAGTCTTCGTTATAAC-3' as a forward primer and 5'-CTCGAGCTAGTGATTCCCCCTGTGTTCCATGTAG-3' as a backward primer. The equal amount of PCR products was used to electrophoresis on 1% agarose gel.

Immunoblotting

Whole cell lysates (20 µg of proteins) were separated on 1-mm-thick 4–20% Tris-glycine gels and then transferred to nitrocellulose. Equal protein loading in each of the lanes was confirmed by staining the same gel with GelCode Blue Stain Reagent (Pierce, Rockford, IL). The filters were blocked with 5% (w/v) nonfat dry milk powder in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.5% Tween 20). Anti-MICA mAb 2C10 (mouse IgG3) (13) was diluted 1/2000 in TBST containing 3 mg/ml BSA and incubated with the filter at 2 h. As a loading control, anti-{beta}-actin mAb (Sigma-Aldrich, St. Louis, MO) was also incubated with a separate filter. After washing with TBST, the filters were incubated for 1 h with HRP-conjugated goat anti-mouse IgG3 (1/3000 in TBST) (Southern Biotechnology Associates, Birmingham, AL). Filters were washed extensively with TBST, and immunoreactive bands were visualized by chemiluminescence reagent (NEN Life Science, Boston, MA).

Immunohistochemical assay

Blocks of intestine, spleen, and thymus were removed, fixed in 4% paraformaldehyde, embedded, and snap frozen in OCT compound (Tissue-Tek, Torrance, CA). Sections (5 µm) were cut in a cryostat and air dried. The sections were quenched with H2O2, treated with 10% FBS, and then incubated with a titrated dilution of anti-MICA mAb 6D4 (mouse IgG2a) (21). For the detection of bound Abs, Vectorstain ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine substrate kit (Funakoshi, Tokyo, Japan) were used. Slides were then counterstained with hematoxylin. Control sections without the primary Ab or with an isotype control were run in parallel.

Isolation of splenocytes and IELs and flow cytometry analysis

Lymphocytes were isolated from spleens and IELs, as described previously (22). In case of IELs, after Peyer’s patches and fatty tissues were removed, a standard mechanical dissociation method was performed and followed by a Percoll discontinuous density gradient separation. After blocking with anti-CD16/CD32 FcR mAb (2.4G2), the cells were stained using following labeled mAb conjugates: FITC-conjugated anti-CD4 mAb (L3T4; RM4-5), V{beta}4 (KT4), V{beta}5.1, 5.2 (MR9-4), V{beta}6 (RR4-7), V{beta}7 (TR310), V{beta}8.1, 8.2 (MR5-2), V{beta}11 (RR3-15), V{beta}12 (MR11-1), V{beta}13 (MR12-3), V{beta}14 (MR14-2), TCR{gamma}{delta} (GL3), PE-conjugated anti-TCR{beta} mAb (H57-597), TCR{gamma}{delta} (GL3), CD69 (H1.2F3), CD44 (Ly-24; IM7), CD62L (MEL-14), and allophycocyanin (APC)-conjugated anti-CD8{alpha} (Ly-2; 53-6.7) mAb. All mAbs were purchased from BD PharMingen (San Diego, CA). The stained cells were then washed and analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Immunoscope analysis for V{beta} repertoire

As previously described, cDNA synthesized from total RNA of the sorted cells was used for V{beta}-C{beta} amplification (23). The oligonucleotide primers used for these reactions were: forward (V{beta}8.2), 5'-CATTATTCATATGGTGCTGGC-3'; reverse (C{beta}145), 5'-CACTGATGTTCTGTGTGACA-3'. The amplified products were then used to run off reactions with an oligonucleotide primer labeled with a fluorescent tag (C{beta}5-6-carboxyfluorescein; 5'-6-carboxyfluorescein-CTTGGGTGGAGTCACATTTCTC-3'). The runoff products were subjected to capillary electrophoresis in an automated DNA sequencer (PE Applied Biosystems, Foster City, CA), and CDR3 size distribution and signal intensities were analyzed with GeneScan software (PE Applied Biosystems).

Cloning and sequencing of selected V{beta}-J{beta} rearrangements

Each V{beta}-J{beta}-amplified product was shotgun cloned with the pGEM-T Easy TA cloning kit (Promega, Madison, WI) (23). Resulting colonies were randomly selected for plasmid DNA isolation by using ABI Prism Miniprep kits (PE Applied Biosystems). Sequencing reactions were performed with an IRDye AFLP Kit (LI-COR, Lincoln, NE) and analyzed on an LI-COR4000 sequencer (LI-COR).

Animal experiment for induction and analysis of dextran sodium sulfate (DSS)-induced colitis

Colitis was induced by the administration of DSS (2.5% w/v; m.w., 40,000; ICN Biomedicals, Irvine, CA) in the drinking water for 5 days (24). DSS water consumption and weight were recorded daily. For the assessment of the severity of colitis, animals were sacrificed on days 5, 9, and 17 after beginning of the DSS treatment, and the colons of the Tg mice and C57BL/6 mice were examined histologically. Tissue samples obtained from the proximal and distal colon were fixed in 4% paraformaldehyde in PBS, embedded in paraffin, and sectioned at a thickness of 6 µm. The tissue sections were stained with H&E. Severity of colitis was evaluated by the standard scoring system, as described previously (25). Each region of the colon (ascending proximal and descending distal colon) was graded semiquantitatively from 0 (no change) to 3 (most severe change) per examination item. The grading represents a degree of monocyte and/or neutrophil infiltration, goblet cell and/or mucous loss, epithelial erosion, and ulceration. The scoring was performed by a blinded manner.

Statistical analysis

Significant differences between mean values were determined by Student’s t test. p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selective expression of human MICA in the intestine of T3b-MICA Tg mice

Human MICA cDNA, expressed under the control of the T3b promoter, was specifically expressed in intestinal epithelial cells (Fig. 1A). From 10 founder mice, three representative lines (Tg-07-5, Tg-07-6, and Tg-09-4) were maintained by mating to C57BL/6 mice. We mainly used F2 and F3 MICA Tg mice in this study. Thus, in addition to the background effect of backcross partner, C57BL/6 strain, the effect of the DBA2 strain that was used to make a donor for BDF1 embryos also might have been involved in the phenotype of the Tg mice. To determine the tissue specificity of the transgene expression, total RNA was isolated from various tissues of the T3b-MICA Tg and wild-type (WT) mice and subjected to MICA-specific semiquantitative RT-PCR analysis (Fig. 1B). Transgenic MICA mRNA was expressed selectively in the gastrointestinal tract, i.e., small and large intestine, but not in other tissues, i.e., spleen, thymus, and mesenteric lymph node, and in none of the same tissues of WT mice. MICA production by intestinal epithelial cells was confirmed also at the protein level by immunoblotting analysis (Fig. 1C); by use of anti-MICA mAb 2C10, MICA protein was found in the small and large intestines of T3b-MICA Tg, but not in those tissues of WT mice. In situ expression of MICA protein was documented also by immunohistological analysis using anti-MICA mAb 6D4; in both the small and large intestine, MICA protein was located in the epithelium, but not in the lamina propria (Fig. 1D). MICA was present on most of the villi of the small intestine, and in the colonic epithelium it was concentrated in tip regions; this expression pattern was almost identical in all the lines of Tg mice examined. These combined results established that the human MICA was specifically expressed in the intestinal tracts of the T3b-MICA Tg mice.



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  		FIGURE 1. Selective expression of MICA in the intestinal tracts of T3b-MICA Tg mice. A, DNA construct. Human MICA cDNA was designed to be expressed in mouse intestinal tracts under the control of the T3b promoter. B, RT-PCR. MICA expression in intestinal tissue of the T3b-MICA Tg mice was determined by standard RT-PCR, as described in Materials and Methods. The expected size of MICA-specific cDNA (0.8 kb) was shown in the lanes of small and large intestines. C, Immunoblotting. Left, Matured MICA proteins were expressed in small (SI) and large intestinal (LI) tissues, but not in spleen, thymus, and mesenteric lymph node of T3b-MICA Tg mice. As a loading control, {beta}-actin blot was also used in a separate membrane containing the same amount of whole cell lysates as those of MICA blot. Right, MICA was specifically expressed only in small and large intestines from all three lines of Tg mice, but not in non-Tg C57BL/6 mice. D, Immunohistochemical assay. The analyses by using anti-MICA mAb (6D4) exhibited that MICA resides in villous of the small intestine (SI) and tip and villous regions of the large intestine (LI). a, Tg-09-4; b, Tg-07-5; c, magnified photographs of Tg-09-4 (upper, SI; lower, LI; left, proximal; right, distal region). d, C57BL/6 (left, SI; right, LI). e, Tg-09-4 (left, spleen; right, thymus). MICA protein was expressed both in proximal and distal regions, although the differential pattern of transgenic expression showed large intestine and proximal region were slightly more than small intestine and distal region, respectively. Bars indicate 100 µm.

 
Increase in TCR{alpha}{beta} CD4CD8{alpha}{alpha} (DP) IELs in the small intestine

IELs from the WT mice and three Tg lines were isolated and analyzed for possible alterations induced by MICA expression in the intestinal epithelium. In the presence of the MICA, the total number of IEL and the numbers of {gamma}{delta} T cells and the CD4 and CD8{alpha}{beta} subsets were not changed (Table I). However, the absolute number as well as the percentage of TCR{alpha}{beta} CD4CD8{alpha}{alpha} (DP) IELs in the total IEL population were increased 7–10 times, whereas the number of TCR{alpha}{beta} CD8{alpha}{alpha} IELs was decreased by about one-half (Table I and Fig. 2A).


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  		Table I. Enumeration of cells for each SI-IEL population in T3b-MICA Tg linesa

 


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  		FIGURE 2. Analysis of TCR{alpha}{beta} IEL subsets in WT mice and T3b-MICA Tg lines. IELs were stained with FITC anti-CD4 or anti-CD8{beta}, PE anti-TCR{beta}, and APC anti-CD8{alpha} mAbs. All the subsets were determined after gating on TCR{beta}+ cells. The percentage of CD8{alpha}{alpha} IELs was calculated by subtracting the percentage of TCR{alpha}{beta} CD4CD8{alpha}{alpha} (DP) IELs from the percentage of TCR{alpha}{beta} CD8{alpha}{alpha} obtained in the CD8{alpha} vs CD8{beta} staining. Statistical comparisons were conducted using Student’s two-tailed t test. *, p = 0.0000116; **, p = 0.01183; ***, p = 0.000513 when Tg lines are compared with WT mice. Indicated are results from small intestinal (B), large intestinal IEL (C), and splenocyte (D) of a representative line (Tg-07-6). TCR{alpha}{beta} CD4CD8{alpha}{alpha} DP IELs in the small intestine were increased (~70% of TCR{alpha}{beta} IELs) compared with those of WT mice (5–10% of TCR{alpha}{beta} IELs) (B). The frequency of DP IELs in the large intestine was not augmented in any Tg lines determined (C). The frequencies of CD4 and CD8 T cells in spleens of WT and Tg mice were not changed (D).

 
Because MICA expression affected the TCR{alpha}{beta} fraction of IEL, we next compared the changes in these cells between the small and large intestine of the T3b-MICA Tg mice. In Fig. 2B, representative flow cytometry analysis of the TCR V{beta}-gated IEL populations in the small intestines of WT mice and the Tg line (Tg-07-6) is illustrated; the population of DP IEL was markedly increased in all the Tg animals examined. In contrast, despite strong expression of MICA in the epithelium of the large intestine, no change in the colonic IEL population was seen (Fig. 2C). Splenic cells isolated from the Tg line had the same distribution of CD4 and CD8 T cell populations as that of WT mice (Fig. 2D). Thus, locally expressed MICA resulted in increased numbers of TCR{alpha}{beta} CD4CD8{alpha}{alpha} (DP) IELs selectively in the small intestine.

Maturity of the DP IELs by expression of other surface markers

DP IELs have been proposed to have a distinct origin and to use unique maturation pathways, unlike their thymus counterpart (CD8{beta} positive), which expresses low TCR levels and mediates the maturation of CD4 or CD8 T cells (26, 27). When the selectively propagated DP IELs in the T3b-MICA Tg mice were analyzed for the expression of other surface markers, the cells were shown to exclusively express CD69 and CD44, but not CD62L (data not shown) like those of normal C57BL/6 mice (Fig. 3A). When we analyzed the NKG2D expression in DP IELs isolated from Tg and C57BL/6 mice by RT-PCR, the expressions were detected in DP IELs from both mice, even though the expression levels were lower than those of CD8{alpha}{alpha} IELs (data not shown).



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  		FIGURE 3. Flow cytometric analysis of the DP IELs and {gamma}{delta} IELs of Tg mice for other surface molecules such as maturation markers. A, CD4CD8{alpha} (DP)-gated population of the Tg mice (right) exclusively expressed maturation surface markers CD69 and CD44 in comparison with that of C57BL/6 mice (left). B, TCR{gamma}{delta}-gated population of the Tg mice mainly expressed CD8{alpha}{alpha} (left) and CD69 and CD44 (right), showing the same pattern as that of C57BL/6 mice (data not shown).

 
In contrast, there was no change in {gamma}{delta} IEL repertoire in the Tg mice when compared with normal C57BL/6 mice in terms of usage of TCR V{gamma} and TCR V{delta} mRNA, as shown by RT-PCR analysis (data not shown). Moreover, when the phenotype of {gamma}{delta} IELs was examined with respect to CD4, CD8{alpha}, and CD8{beta}, the cells prominently expressed CD8{alpha}{alpha} homodimers, but not CD4 or CD8{alpha}{beta} subsets. The {gamma}{delta} IELs also expressed a high level of CD69 and CD44 (Fig. 3B). Thus, the pattern of cell surface markers indicates that the DP IEL and {gamma}{delta} IEL subpopulations have a characteristic of activated T cells (28) and, especially, that the propagated DP IELs are fully mature.

Expansion of the DP IELs expressing V{beta}8.1, 8.2 repertoire

To further investigate the nature of the expanded DP IELs, we next characterized the cells’ usage of V{beta} chains. The total numbers of small intestinal IELs from WT and Tg mice (Tg-09-4 line) used for the V{beta} usage analysis were 1.6 x 107 ± 0.4 x 107 (n = 4, on average) and 1.9 x 107 ± 0.7 x 107 (n = 3, on average), respectively. The numbers of CD4 and DP IEL were 0.98 x 106 ± 0.32 x 106 and 0.62 x 106 ± 0.2 x 106 in WT mice, and 1.64 x 106 ± 0.64 x 106 and 5.13 x 106 ± 1.85 x 106 in T3b-MICA Tg mice. As illustrated in Fig. 4A, changes were detected in the numbers of CD4 and, especially, DP IELs in the small intestine of T3b-MICA Tg mice compared with those of WT mice; DP IELs harboring TCR V{beta}8.1, 8.2 were markedly expanded (3.74 x 106 ± 1.77 x 106, Tg mice; 0.78 x 105 ± 0.73 x 105, WT mice). The increased DP subset compared with that from WT mice was accounted for by this propagated repertoire. We also compared the CD4 and CD8 subsets for V{beta} repertoire usage in splenic cells from the Tg mouse line (Tg-07-6) that had the most plentiful DP IELs with these subsets in WT mice. In the absence of MICA, no propagation of the unique T cell clone having the V{beta}8.1, 8.2 chain was observed in the splenocytes of the T3b-MICA Tg mice (data not shown). These results are additional evidence that MICA mediated the clonal expansion of DP IELs expressing the TCR V{beta}8.1, 8.2 repertoire in a small intestine-restricted manner.



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  		FIGURE 4. A, Comparison of the frequency of different V{beta} chains expressed by DP T cells in SI-IEL of T3b-MICA Tg mice. Three WT mice and four mice of the Tg-09-4 line were explored in this flow cytometry analysis. IELs were stained with FITC anti-V{beta}x, PE anti-CD4, and APC CD8{alpha} mAbs. All the subsets were determined after gating on CD4 or CD4 plus CD8{alpha} IELs. The total number of lymphocytes in each population was calculated by multiplying the respective percentage of each population. The number of each subset or each V{beta} repertoire was calculated by multiplying the respective percentage of each repertoire by the total number of each subset. When splenocytes isolated from the mouse showing the greatest increase in DP IELs (4917 x 106) among four Tg mice were also analyzed by the same method, no significant differences were found between WT and Tg mice (data not shown). B, Immunoscope analysis of V{beta}8.2 clonotype of CD4 or CD8 T cells from splenocytes and CD4 or DP T cells from SI-IELs of WT or T3b-MICA Tg mice. Individual populations were purified by cell sorting using FACSVantage. cDNA from each FACS-sorted subset was subjected to PCR using V{beta}8.2- and C{beta}-specific primers, followed by a runoff with a nested fluorescent C{beta}-specific primer (23 ). The CDR3 size distribution was analyzed with the GeneScan software program. Arrows indicate expansions discussed in the text. The intensity of fluorescence is presented in arbitrary units as a function of CDR3 length in amino acids. The results shown are representative of five individual mice of T3b-MICA Tg (Tg-09-4 line) and WT mice.

 
Clonality of DP IELs expressing TCR V{beta}8.2

Using immunoscope analysis associated with DNA sequencing of the CDR3, we next determined the clonality or redundancy of the TCR V{beta}8.1, 8.2 chain repertoire used by the DP IELs. By V{beta} chain-specific semiquantitative RT-PCR, we found that the expanded repertoire of DP IEL was preferentially associated with the V{beta}8.2 chain (data not shown). As shown in Fig. 4B, a typical Gaussian distribution of CDR3 lengths of V{beta}8.2-C{beta} PCR products was observed in the CD4 and CD8 splenocytes from both the WT and Tg mice. The CD4 IEL of the Tg mice had some oligoclonality for the V{beta}8.2 chain, compared with that of WT mice (Fig. 4A). The DP IEL of interest exhibited a bias of a single peak of CDR3 length for the V{beta}8.2 chain in the Tg line, but not in WT mice (Fig. 3B). This skewing suggested a clonal expansion of the V{beta}8.2-restricted DP IEL induced by MICA. We next analyzed the DNA sequence of the CDR3 region for each amplified V{beta}-J{beta} combination. As shown in Table II, the V{beta}-J{beta} pair had a tendency toward the V{beta}8.2-J{beta}2.7 or V{beta}8.2-J{beta}1.6 combinations, although this trend was not observed in all the Tg mice. Among the other T3b-MICA Tg mice examined, Tg-07-5-b mouse showed a V{beta}8.2-J{beta}2.7 combination containing a major CDR3 sequence (GDPDWEEQ) (8/11), and Tg-07-5-c showed a V{beta}8.2-J{beta}2.4 combination containing a major CDR3 sequence (SDWGGGQNTL) (6/8). Also, Tg-07-5-d, Tg-07-6-a, and Tg-09-4-d showed a V{beta}8.2-J{beta}2.5 (GEGLGGKDTQ) (4/5), a V{beta}8.2-J{beta}1.6 (GPGGRNSPL) (4/6), and a V{beta}8.2-J{beta}2.1 (SDWGNYAEQ) (4/5), respectively (data not shown). Furthermore, restricted motifs for the CDR3 region in the V{beta}8.2-J{beta}2.7 and V{beta}8.2-J{beta}1.6 combination were found to possess a sequence GDRQGFEQ in individual mice in two different lines (Tg-09-4-a and Tg-07-5-a) and SDRGHNSPL in two mice of Tg-09-4 (-b and -c), respectively, whereas no public CDR3 motif for V{beta}8.2 chain was detected in the DP among various WT mice. These data imply that MICA-induced clonal expansion of V{beta}8.2-harboring DP IEL developed a tendency to restrict limited sequences in the CDR3 region.


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  		Table II. V{beta}8.2-CDR3 sequence-J{beta} chain combination of CD4 and DP subset in SI-IELsa

 
Effect of MICA transgene against DSS-induced colitis

It was important to examine physiological and immunological contribution of transgenic MICA in vivo. The T3b-MICA Tg mice were subjected to the experimental disease-inducing protocol of DSS colitis. The onset of colitis in DSS-treated MICA Tg mice was substantially delayed when compared with that of DSS-treated non-Tg C57BL/6 mice. The DSS-treated MICA Tg mice lost significantly less body weight during the period of the observation when compared with DSS-treated non-Tg mice (Fig. 5A). The frequency of the physical change typical of those in DSS-treated C57BL/6 mice, i.e., hunched posture, anorectal prolapse, and diarrhea, was less observed in the DSS-treated MICA Tg mice. Histological analysis of colons, especially distal portion, from the DSS-treated MICA Tg mice revealed that the occurrence of ulcer, abscess, diminution of goblet cells, and disturbance of tissue architecture by infiltration of inflammatory neutrophils and mononuclear cells were reduced in the DSS-treated MICA Tg mice compared with DSS-treated C57BL/6 mice (Fig. 5B). Furthermore, recovery of the regeneration of epithelial layer and damaged tissue architecture was more rapidly observed in the DSS-treated MICA Tg mice compared with DSS-treated C57BL/6 mice. The average score in DSS-treated MICA Tg mice (e.g., proximal and distal colon; 4 and 1 on day 17, respectively) was less than that in DSS-treated C57BL/6 mice (proximal and distal colon; 8 and 12 on day 17, respectively). Thus, although MICA Tg mice developed mild acute-type colitis by administration of DSS, the severity of the colitis was markedly diminished compared with non-Tg mice.



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  		FIGURE 5. The suppressive effect of transgenic MICA for the development of DSS-induced colitis. A, Body weight was measured daily. Data shown represent the mean ± SD of mice in each group (n = 5) (*, p < 0.05). B, Histological analysis of colons in DSS-treated MICA Tg mice. The severity of the colitis was also determined in each group of mice (n = 3) by using the histological disease-scoring system (25 ). The transgenic MICA ameliorated the severity of the colitis. Bars indicate 50 µm (proximal colon) and 100 µm (distal colon).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report the generation of a Tg model expressing human MICA selectively in mouse intestine. The T3b-MICA Tg mice possess some interesting properties. First, the mice expressed the MICA only in the intestinal tract (small intestinal villi and large intestinal tips). Second, the mice contained a greatly increased CD4CD8{alpha}{alpha} (DP) IEL in the small, but not the large intestine, and this population was present only in the IEL, not in lamina propria lymphocyte. Third, the increased DP subset was almost entirely biased toward the V{beta}8.2 chain repertoire, with some restricted CDR3 sequences. Based on these results, it is evident that the overexpression of intestinal MICA specifically promoted the development of clonally expanded DP IELs in the small intestine.

A major reason we chose to express intestinal MICA by the use of the T3b promoter was that both the T3b (a thymic leukemia Ag) and the MICA are nonclassical MHC class Ib molecules, and it has been reported that both of them are preferentially expressed in the intestinal epithelial cells (14, 19, 29). Thus, it could be expected that the T3b-driven MICA molecule expressed in the murine intestinal immune system would be represented and behave physiologically in the in vivo situation. In addition, our recent separate results showed that transgenic MICA is preferentially expressed in the basolateral side of the intestinal epithelial cells (data not shown), as previously reported (30). Thus, MICA and its derived mucosal T cells may be important components of the innate regulatory network that maintains immunologic homeostasis in the harsh environment of the intestinal tract. In this regard, T3b promoter-driven, transgenic MICA was able to attenuate the DSS-induced intestinal inflammation (Fig. 5), indicating the basolateral expression of MICA in intestinal epithelia be appropriate for the immunological surveillance against mucosal inflammation.

CD4CD8{alpha}{alpha} (DP) lymphocytes in mice are present mainly in the small intestine and, unlike thymic DP T cells, highly express CD3 on their surfaces (31). This interesting subset, harboring mutually exclusive coreceptors, is proposed to be involved in intestinal T cell maturation and development, and possibly to function in both innate and adaptive immunity (32). DP IELs have been shown capable of secreting Th2-type cytokines and providing help to B cells for the secretion of Igs (33). Also, TCR{alpha}{beta}-mediated signaling initiated cytotoxic function, but did not induce proliferation of the DP IELs (34), and murine CD4 IELs were found to give rise to DP IELs in an inflammatory bowel disease model (35). Recently, it was shown that CD4+CD8{alpha}{alpha} T cells in the intestinal epithelium were functioning as regulatory T cells for the prevention of inflammatory bowel disease in an IL-10-dependent fashion (36). Together with this report (37) and our present finding that T3b-MICA Tg mice are resistant to the development of DSS-induced colitis, an interesting scenario would be that a stress-associated nonclassical MHC class I molecule participates in the preferential induction of DP IELs with a regulatory function for the maintenance of host immune homeostasis from the development of the intestinal inflammation. Although the ontogeny, function, and precise reason for the propagation of DP IELs remain to be clarified, our unique T3b-MICA Tg mouse model most likely will be useful for this purpose.

There is evidence that DP IELs arise from thymus-derived CD4 T cells, which migrate into the epithelium and express CD8 (32); a report that the transfer of CD4 peripheral T cells into SCID micereconstituted DP IELs also supports this view (35). Furthermore, both CD4 and DP subsets of rat IELs reportedly showed oligoclonality and overlapping {beta}-chain repertoires, and the DP subset contains a considerably more restricted repertoire than do CD4+ IELs (37). Our data from immunoscope and CDR3 sequence analyses also reveal that DP IELs have more restricted oligoclonality than do CD4 IELs in T3b-MICA Tg mice (Fig. 3 and Table II). However, in addition to the possibility that DP IELs mature from CD4 IELs, it is possible that CD8{alpha}{alpha} IELs transform into DP IELs by acquisition of the CD4 coreceptor under the influence of MICA in the intestinal epithelium. As depicted in Fig. 2A, contrary to TCR{alpha}{beta} CD4 or CD8{alpha}{beta} population exhibiting no quantitative alteration, the percentage of CD8{alpha}{alpha} IELs was no more than 50% of those in WT mice, while CD4CD8{alpha}{alpha} (DP) IELs were concurrently increased in all Tg lines; perhaps this result reflects maturation of CD8{alpha}{alpha} IELs into CD4CD8{alpha}{alpha} IELs.

An intriguing question is what is the driving force responsible for human MICA accelerating a bias to clonal selection of DP IELs and their propagation? Some interesting possibilities can be entertained. First, the transgenic MICA molecules could be recognized by an NKG2D-like or -related receptor for MICA, expressed on the DP IELs. In this regard, in a separate study, we are assessing the reactivity of the NKG2D tetramer with intestinal epithelial cells (IECs) isolated from the small intestine of the T3b- MICA Tg mice by flow cytometric analysis and immunohistochemical analysis. We have found that IECs isolated from the MICA Tg, but not WT, mice bind strongly to the NKG2D tetramer, and an intense signal is present in the basolateral portions of IECs (data not shown). In contrast, splenocytes isolated from both T3b- MICA Tg and WT mice did not react with the NKG2D tetramer. These results suggest that ectopically expressed MICA molecules in the Tg mice can interact specifically with NKG2D molecules on neighboring IELs in a physicochemical fashion. Second, CD8{alpha}{alpha} coreceptors on DP IEL could directly bind to MICA independent of TCR specificity, similar to the reported interaction between CD8{alpha}{alpha} and the thymic leukemia Ag, which is another nonclassical MHC class I molecule expressed on intestinal epithelial cells, modulating T cell responses (38). And the lack of expansion of {gamma}{delta} IELs in the small intestine of the T3b-MICA Tg mice might be due to the species (rodents vs humans), aging (adults vs neonates), and/or tissue (small vs large intestine) differences. In this regard, our unpublished data demonstrated that MICA could positively regulate the development of {gamma}{delta} IELs in the neonatal stage of the T3b-driven MICA Tg mice and in the early phase of bone marrow-chimeric irradiated MICA-Tg/RAG-2-deficient mice.

In our Tg mice, MICA was designed to be expressed in both the small and large intestine, and indeed both sites harbored many MICA proteins. However, the prominent change in IELs was only restricted to the small intestine. One possible explanation for this observation might be differences in the commensal microflora. In the colon, the symbiotic microbes might block the ability of MICA to increase DP IELs by protecting the colonic epithelium and intraepithelial T cells from stress-induced inflammation. Alternatively, the different effects on IELs might reflect qualitative and quantitative differences in the IEL subsets residing in the small and large intestines; large intestinal IELs contain more CD4 T cells with a lower frequency of DP T cells than do small intestinal IELs (39, 40). Collectively, we emphasize that our T3b-MICA Tg mice are a unique in vivo model that can be used to elucidate the biological role of the stress-inducible nonclassical MHC molecules for the regulation of gastrointestinal immune surveillance and homeostasis.


   Acknowledgments
 
We thank Dr. Toshiro Suzuki at Japan SLC for his help in generating T3b-MICA Tg mice, Dr. Olga Naidenko (Washington University, St. Louis, MO) for kind provision with NKG2D tetramer, Dr. Masami Nozaki at Central Instrumentation Laboratory of The Research Institute for Microbial Diseases in Osaka University for his help in DNA sequence analysis, and Dr. William Brown (University of Colorado School of Medicine, Denver, CO) for his critical reading of the manuscript.


   Footnotes
 
1 This study was performed through Special Coordination Funds (to I.T.) for Promoting Science and Technology and Grant-in-Aid (to I.T.) for Scientific Research on Priority Areas from the Ministry of Education, Cultures, Sports, Science, and Technology, the Japanese Government. This work was also supported by grants from Core Research for Engineering, Science, and Technology of Japan Science and Technology; the Ministry of Education, Science, Sports, and Culture; and the Ministry of Health and Welfare in Japan. Back

2 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. E-mail address: kiyono{at}ims.u-tokyo.ac.jp Back

3 Abbreviations used in this paper: IEL, intraepithelial lymphocyte; APC, allophycocyanin; CDR3, complementarity-determining region 3; DP, double positive; DSS, dextran sodium sulfate; IEC, intestinal epithelial cell; MICA, MHC class I-related chain A; Tg, transgenic; WT, wild type. Back

Received for publication March 14, 2003. Accepted for publication August 12, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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