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MyD88-Dependent Signaling for IL-15 Production Plays an Important Role in Maintenance of CD8 TCR and TCR Intestinal Intraepithelial Lymphocytes¹

Qingsheng Yu^*, Ce Tang^*, Sun Xun^*, Toshiki Yajima^*, Kiyoshi Takeda and Yasunobu Yoshikai^2,*

^* Division of Host Defense and Division of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

	Abstract

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Interaction between commensal bacteria and intestinal epithelial cells (i-ECs) via TLRs is important for intestinal homeostasis. In this study, we found that the numbers of CD8 TCR and TCR intestinal intraepithelial lymphocytes (i-IELs) were significantly decreased in MyD88-deficient (–/–) mice. The expression of IL-15 by i-ECs was severely reduced in MyD88^–/– mice. Introduction of IL-15 transgene into MyD88^–/– mice (MyD88^–/– IL-15 transgenic mice) partly restored the numbers of CD8 TCR and TCR i-IELs. The i-IEL in irradiated wild-type (WT) mice transferred with MyD88^–/– bone marrow (BM) cells had the same proportions of i-IEL as WT mice, whereas those in irradiated MyD88^–/– mice transferred with WT BM cells showed significantly reduced proportions of CD8 TCR and TCR i-IELs, as was similar to the proportions found in MyD88^–/– mice. However, irradiated MyD88^–/– IL-15 transgenic mice transferred with WT BM cells had increased numbers of CD8 TCR and TCR subsets in the i-IEL. These results suggest that parenchymal cells such as i-ECs contribute to the maintenance of CD8 TCR and i-IELs at least partly via MyD88-dependent IL-15 production.

	Introduction

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Intestinal intraepithelial lymphocytes (i-IELs),³ which are located at the basolateral surfaces of intestinal epithelial cells (i-ECs), comprise unique T cell populations that include CD4^–/CD8⁺ T cells expressing TCR or TCR and exhibit non-MHC-restricted cytotoxicity (1, 2, 3). The interaction of i-ECs and i-IELs through E-cadherin/integrin _E7 is important for the homing and maintenance of i-IELs (4). We previously showed that i-IELs recognized and eliminated effete i-ECs for homeostatic regulation of intestinal epithelia (5, 6). It has been reported that i-IELs, especially TCR i-IELs, play an important role in the regulation of generation and differentiation of i-ECs at crypts (7, 8). Taken together, results of previous studies suggest that mutual interaction of i-IELs and i-ECs is important for homeostasis of intestinal epithelia.

IL-15 is a cytokine that resembles IL-2 in its biological activity, stimulating macrophages (M), NK cells, TCR T cells, and B cells to proliferate, secrete cytokines, exhibit increased cytotoxicities, and produce Ab (9, 10, 11, 12). IL-15 was found to be produced only by limited populations of cells, such as activated monocyte/M and epithelial cells, but not by activated T cells (13, 14). We previously reported that CD8 i-IELs proliferate preferentially in response to exogenous IL-15 (15). It has been shown that mice deficient in the IL-15 or IL-15R gene had reduced numbers of CD8 and/or TCR i-IELs (16, 17). Taken together, results of these studies suggest that IL-15 is involved in the development and proliferation of CD8⁺ T cells in i-IELs.

It is widely accepted that intestinal microflorae play an important role in the maintenance of homeostasis of the intestinal microenvironment by inhibiting colonization by many pathogens and stimulating the growth of beneficial microorganisms. It has been reported recently that intestinal microflorae play an important role in maintaining i-ECs via TLRs (18). TLRs are a group of pattern recognition receptors that cooperate in recognizing a series of pathogens by binding to the pathogen-associated molecular patterns (19). After binding to their ligands, most of TLRs induce a series of intracellular signal transductions through MyD88, an adaptor protein for transcriptional activation of cytokine genes (20, 21). We and others previously reported that TLR signaling played important roles in activation of IL-15 transcription in LPS-stimulated M and virus-infected cell lines (22, 23). Thus, it is hypothesized that TLR signaling for IL-15 production via microflorae is involved in interaction of i-EC and i-IEL for maintaining homeostasis of the intestinal microenvironment.

In the present study, we found that the CD8 TCR and TCR i-IELs were selectively decreased in MyD88-deficient (–/–) mice, accompanied with impaired IL-15 expression by i-ECs. Introduction of IL-15 transgene into MyD88^–/– mice was able to restore the numbers of CD8 TCR and TCR i-IELs, albeit partly. The experiments with bone marrow (BM) chimeras revealed that radioresistant parenchymal cells played an important role in MyD88-dependent maintenance of the i-IELs. We concluded that MyD88-dependent signaling was involved in interaction of i-EC and i-IEL for maintaining homeostasis of the intestinal microenvironment at least partly via IL-15 production.

	Materials and Methods

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Animals

MyD88^–/– and IL-15 transgenic (Tg) mice with C57BL/6 background were prepared as described previously (24, 25). MyD88^–/–/IL-15Tg mice were prepared by mating MyD88^–/– and IL-15Tg mice and backcrossing them for three generations. Typing of MyD88^–/– mice was performed by PCR with the primers described previously. C57BL/6NCrj mice used as wild-type (WT) controls were purchased from Charles River Laboratories. C57BL/6 Ly-5.1-congenic WT mice were obtained from The Jackson Laboratory. All of the mice were fed in a sterile, isolated room and used in experiments at 8–10 wk of age. The animal experiments were approved by our institutional review committee according to a notice of the Prime Minister’s Office of Japan (no. 6 of March 27, 1980) for the care and use of laboratory animals.

Preparation of i-IELs and i-ECs

Mice were sacrificed according to the relevant rules for animal usage. Peyer’s patches and contents were removed from the small intestines of the mice, and small intestines were cut into pieces of <5 mm in length and stirred at 37°C for 30 min in medium 199 (Invitrogen Life Technologies) containing 10% FBS (Sigma-Aldrich). After stirring, the cells were passed through gauze to remove debris and coarse pieces and were centrifuged through a 25–40–75% discontinuous Percoll (Pharmacia) gradient that had been adjusted by 10x PBS at 940 x g for 20 min at 20°C in Multipurpose Refrigerated Centrifuge (Tomy). I-ECs and i-IELs are obtained at the interfaces of 25–40 and 40–75%, respectively. Cells were suspended in HBSS. A total of 10 µl of cell suspension was mixed with 90 µl of Turk’s dye. A total of 10 µl of cell-Turks dye mixture was loaded onto a set of cell-counting plates, and the living cells were counted under a common optical microscope. The cells in each square were counted, and the total number of living cells was calculated by adjusting the number of cells in each square to the volume defined by the square and the whole volume of the cell suspension.

FACS analysis

Cells intended for FACS analysis were washed twice with FACS flow and suspended in 100 µl of FACS flow. Equal volumes of 2.4G2 (specific inhibitor for FcR) were added to each sample and incubated on ice for 15 min. Cells of each sample were washed twice with FACS flow and suspended in 100 µl of FACS flow. Cells of each sample were stained with fluorescent dye- or biotin-conjugated Abs (PE-, FITC-, CyChrome (PE-Cy5)-, and biotin-conjugated anti-CD3, -CD4, -CD8, -CD8, -TCR, -TCR, -CD45.1, -CD45.2 Abs (eBioscience)), which were finally diluted 200 times. To detect the cells specifically bound by biotin-conjugated Abs, the cells were incubated with allophycocyanin-conjugated streptavidin (eBioscience) after incubating the cells with biotin-conjugated Abs. All incubations were performed at 4°C for 30 min. The cells for each sample were washed by FACS flow twice after the incubation and suspended in 500 µl of FACS flow. The samples were analyzed by FACSCalibur and interpreted by CellQuest software (BD Biosciences). Living lymphocytes were selected by gating on forward and side scattering. CD3⁺, CD45.1⁺, CD45.2⁺, TCR, and TCR cells were selected by gating on histograms.

ELISA

Anti-CD3 mAb (clone number 145-2C11) was immobilized onto the bottom of each well of 96-well tissue culture plates (Falcon; BD Discovery Labware) overnight at 4°C at concentration of 50 µg/ml. MyD88^–/– and WT i-IELs were cultured at concentration of 5 x 10⁵ cells/well for 48 h at 37°C with 5% CO₂. Sandwich enzyme immunoassay was performed with the ELISA kits for IL-4, IL-10, and IFN- (R&D Systems). Recombinant mouse IL-4, IL-10, and IFN- were used as standard. The OD of each well was immediately read by Multiskan JX (ThermoLabsystems). The standard curve was drawn, and the concentration of each kind of cytokine was calculated according to the standard curve.

RT-PCR

Total RNA was extracted from i-ECs using TRIzol (Invitrogen Life Technologies). The first-strand cDNA was synthesized from 4 µg of RNA using 5x first-strand buffer, DTT, random primer, and SuperScript II RNase H-Reverse Transcriptase from Invitrogen Life Technologies and dNTP and RNase inhibitor from Toyobo Biochemicals for Life Science in a 20-µl system. The synthesized cDNA (2 µl) was amplified using PCR kit (Takara Bio) in a 50-µl system with primers described previously. The specific primers were as follows: -actin sense, 5'-TTCTGCATCCTGTCAGCAAT-3', and antisense, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'; primers for IL-15: sense from exon 1, 5'-GGAAGGCTGAGTTCCACATC-3', and antisense from exon 5, 5'-AGGGAGACCTACACTGACAC-3'; sense from exon 3, 5'-GTTCTGGATGGATGGATGGCAGCT-3', and antisense from exon 7, 5'-CTGTTTGCAAGGTAGAGCACG-3' (26).

Generation of BM chimeras

BM hemopoietic cells were harvested from femurs and tibias of Ly-5.2⁺MyD88^–/– and Ly-5.1⁺WT mice by lavaging the cavity of the bones with HBSS. The BM cells from Ly-5.2⁺MyD88^–/– mice were transferred to Ly-5.1⁺WT mice. BM cells from Ly-5.1⁺WT mice were transferred to Ly-5.2⁺MyD88^–/– mice or Ly-5.2⁺MyD88^–/– IL-15Tg mice. Recipients were irradiated with 1100 rad (11 Gy) of -ray in a single dose 3 h before the transfer with 5 x 10⁶ BM cells through the tail vein. i-IELs derived from the donor in the BM chimeras were analyzed 8 wk after the BM transfer. i-IELs were gated by CD45.1 (Ly-5.1) or CD45.2 (Ly-5.2) according to their expression on the donor of the BM transfer by histogram to distinguish the donor-derived i-IELs from the host-derived i-IELs.

Statistical analysis

Student’s t test was used to determine the statistically significant differences for cell counts between experimental groups. A value of p < 0.05 was taken as existence of statistically significant difference between experimental groups.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD8/TCR and TCR i-IELs were decreased in naive MyD88^–/– mice

Differences between i-IEL subpopulations in naive MyD88^–/– mice and WT mice were analyzed by FACS. Typical results are presented in Fig. 1A, and the mean number of each i-IEL subpopulation from five mice is summarized in Fig. 1B. Proportions of CD8/CD3⁺ and TCR/CD3⁺ i-IELs were markedly decreased in MyD88^–/– mice (Fig. 1A). MyD88^–/– mice had significantly decreased numbers of CD8/CD3⁺ and TCR/CD3⁺ i-IELs (p < 0.01; Fig. 1B). There were no significant differences in the numbers of CD8/CD3⁺ or TCR/CD3⁺ i-IELs between MyD88^–/– and WT mice, although their proportions relatively were increased in MyD88^–/– mice (Fig. 1, A and B). Nearly half of the TCR⁺ i-IELs expressed CD8 in WT mice, while the proportion of CD8 subpopulation in TCR⁺ i-IELs was decreased in MyD88^–/– mice (Fig. 1C). In contrast, most of TCR⁺ i-IELs expressed CD8 WT mice, and the proportion of CD8 subpopulation was only slightly decreased in TCR⁺ i-IELs (Fig. 1C). Thus, CD8⁺ subpopulation was selectively reduced in TCR⁺ i-IELs in MyD88^–/– mice (p < 0.05; Fig. 1D), while TCR⁺ i-IELs were decreased in MyD88^–/– mice regardless of CD8 (p < 0.05) or (p < 0.01; Fig. 1D).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Analysis of i-IEL phenotypes of WT and MyD88–/– mice. A, Expression of CD8 and CD8 chains or TCR and TCR on CD3+ i-IEL. Cells were stained with anti-CD3, anti-CD8, and CD8 or TCR and TCR mAbs and positively gated by CD3. B, Absolute numbers of i-IEL subsets obtained from MyD88–/– mice. The absolute number of each subset was calculated by multiplying total number of i-IEL by the percentage of each subset. The data are shown as the mean of four mice ± SD. Significant differences compared with the value for WT mice are shown: **, p < 0.01. C, Expression of CD8 and CD8 chains on TCR or TCR i-IEL. Cells were stained with anti-TCR and anti-TCR, anti-CD8 and anti-CD8 mAbs, and positively gated on TCR or TCR. D, Absolute numbers of i-IEL subsets obtained from MyD88–/– mice. The absolute number of each subset was calculated by multiplying total number of i-IEL by the percentage of each subset. The data are shown as the mean of four mice ± SD. Significant differences compared with the value for WT mice are shown: *, p < 0.05; **, p < 0.01.

To investigate the qualitative difference of i-IEL in MyD88^–/– mice from those in WT mice, we next examined IFN-, IL-4, and IL-10 production in the supernatant of i-IEL cultured with immobilized anti-CD3 mAb by ELISA (Fig. 2). The concentrations of IL-10, IFN-, and IL-4 in the supernatant of cultured i-IEL of MyD88^–/– mice were significantly increased as compared with WT mice (p < 0.05). Thus, these results suggest that i-IELs in MyD88^–/– mice are comprised of larger numbers of i-IELs capable of producing higher levels of cytokines.

View larger version (8K): [in this window] [in a new window]   FIGURE 2. ELISA analysis of production of IL-4, IL-10, and IFN- by MyD88–/– i-IEL. Whole i-IEL (1 x 105) was cultured for 72 h with CD3 mAb, and the culture supernatants were collected. The concentration of IFN-, IL-10, or IL-4 in the culture supernatants was determined by ELISA. The data are representative of three separate examinations using pooled cells from three mice and are shown as the mean of triplicate determinations ± SD. Significant differences compared with the value for WT mice are shown: *, p < 0.05; **, p < 0.01; ****, p < 0.005.

Introduction of IL-15 transgene can recover the number of CD8 TCR and TCR i-IEL subpopulations in MyD88^–/– mice

We and others have previously reported that IL-15 plays an important role in the development of CD8 i-IEL and TCR i-IEL (15, 16, 17). Furthermore, TLR signaling is important for transcriptional activation of IL-15 gene (22, 23). These findings raise the possibility that impairment of IL-15 production by radioresistant parenchymal cells such as i-EC may be responsible for decreased number of CD8 TCR and TCR i-IEL in MyD88^–/– mice. To address this issue, we examined the expression of IL-15 mRNA by i-ECs of MyD88^–/– mice using RT-PCR. We used two pairs of primers for IL-15: one specific for exons 3 and 7 and another specific for exons 1 and 5 (Fig. 3). The expression of IL-15 gene by i-ECs of MyD88^–/– mice was severely reduced in both experiments using two kinds of primers (Fig. 3). These results strongly suggest that MyD88-dependent signaling is important for transcriptional activation of IL-15 gene in i-EC and consequently for development and/or maintenance of CD8 TCR and TCR i-IEL.

View larger version (64K): [in this window] [in a new window]   FIGURE 3. Expression of IL-15 mRNA by i-ECs from MyD88–/– and MyD88–/– IL-15Tg mice. IL-15 mRNA was reversely transcribed into first-strand cDNA. The first-strand cDNA was amplified using primers from exons 3 and 7 of IL-15 or using primers from exons 1 and 5 of IL-15. Two kinds of mRNA were shown. One was normal transcript comprising all exons. The other was alternatively spliced transcript that lacked exon 2, thereby 120 bp smaller than the normal transcript.

We then analyzed the phenotype of i-IEL subpopulations in MyD88^–/– IL-15Tg mice. As shown in Fig. 4, the proportions of CD8 TCR and TCR i-IELs were significantly increased in MyD88^–/– IL-15Tg mice compared with MyD88^–/– mice (p < 0.05 for TCR and p < 0.01 for CD8; Fig. 4, A and B). Thus, introduction of exogenous IL-15 by Tg manipulation was able to restore the CD8 TCR and TCR i-IELs in MyD88^–/– mice.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Analysis of i-IEL phenotypes of MyD88–/– mice and MyD88–/– IL-15Tg mice. A, Expression of CD8 and CD8 chains on CD3+ i-IEL. Cells were stained with anti-CD3, anti-CD8, and anti-CD8 mAbs and positively gated by CD3. Expression of TCR or TCR i-IEL on CD3+ i-IEL. Cells were stained with anti-CD3, anti-TCR, and anti-TCR mAbs, and positively gated by CD3. B, Absolute numbers of i-IEL subsets obtained from MyD88–/– mice and MyD88–/– IL-15Tg mice. The absolute number of each subset was calculated by multiplying total number of i-IEL by the percentage of each subset. The data are shown as the mean of four mice ± SD. Significant differences compared with the value for WT mice are shown: **, p < 0.01. Significant differences compared with the value for MyD88–/– mice are shown: ***, p < 0.05; ****, p < 0.01.

TLRs are expressed not only by radio-susceptible hemopoietic cells such as M/dendritic cells, but also by radioresistant parenchymal cells such as epithelial cells (27). To determine whether hemopoietic or parenchymal cells are responsible for the development, differentiation, and maintenance of CD8/TCR and TCR i-IELs in MyD88^–/– mice, we transferred BM cells for Ly-5.1⁺WT mice and Ly-5.2⁺MyD88^–/– mice into lethally irradiated Ly-5.2⁺MyD88^–/– mice or Ly-5.2⁺MyD88^–/– IL-15Tg mice and Ly-5.1⁺WT mice, respectively. As a result, we generated three types of BM chimera mice: the first type had parenchymal cells with functional MyD88 and hemopoietic cells deficient in MyD88 (Ly-5.2⁺MyD88^–/– mice as donors and Ly-5.1⁺WT mice as recipients); the second type had parenchymal cells deficient in MyD88 and hemopoietic cells with functional MyD88 (Ly-5.1⁺WT mice as BM donor and Ly-5.2⁺MyD88^–/– mice as recipients); and the third type had parenchymal cells deficient in MyD88, but carrying IL-15 transgene and hemopoietic cells with functional MyD88 (Ly-5.1⁺WT mice as BM donor and Ly-5.2⁺MyD88^–/– IL-15Tg mice as recipients). Consistent with results obtained in our previous studies (28), hemopoietic cells were almost completely replaced by donor-derived hemopoietic cells in i-IELs by 8 wk after BM cell transfer in both types of BM chimera mice (data not shown). We analyzed the phenotypes of donor-derived CD8/CD3⁺, CD8/CD3⁺, TCR/CD3⁺, and TCR/CD3⁺ i-IELs of BM chimera mice at 8 wk after BM transfer (Fig. 5). The Ly-5.2⁺MyD88^–/– donor-derived i-IELs in BM chimera mice with Ly-5.1⁺WT parenchymal cells had the same proportions of CD8/CD3⁺ and TCR/CD3⁺ i-IELs as WT mice (Fig. 5). The Ly-5.1⁺WT donor-derived i-IELs in BM chimera mice with Ly-5.2⁺MyD88^–/– parenchymal cells showed significantly reduced proportions of CD8/CD3⁺ i-IEL (p < 0.05) and TCR/CD3⁺ i-IELs (p < 0.01), as was similar to the proportions found in MyD88^–/– mice, whereas the Ly-5.1⁺WT donor-derived i-IELs in BM chimera mice with Ly-5.2⁺MyD88^–/– IL-15Tg parenchymal cells showed significantly increased numbers of CD8/CD3⁺ i-IEL (p < 0.05) and TCR/CD3⁺ i-IELs (p < 0.05). These results suggest that the changes in the proportions of CD8 TCR and TCR i-IELs in MyD88^–/– mice essentially depend on the deficiency of MyD88 in radioresistant parenchymal cells rather than on hemopoietic cells, and that introduction of exogenous IL-15 in the parenchymal cells by Tg manipulation was able to restore the CD8 TCR and TCR i-IELs.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Analysis of phenotypes of the donor-derived CD3+ i-IELs in BM chimera mice. Ly-5.1+WT mice and Ly-5.2+MyD88–/– or Ly-5.2+MyD88–/– IL-15Tg mice transferred with Ly-5.2+MyD88–/– BM cells and Ly-5.1+WT BM cells after 1100 rad of gamma ray irradiation, respectively. i-IELs derived from the donor in the BM chimeras were analyzed 8 wk after the BM transfer. i-IEL were stained with anti-CD3, anti-CD8, or anti-TCR, anti-CD8, or anti-TCR, and anti-Ly-5.1 or anti-Ly-5.2 mAbs and gated on CD3 and Ly-5.1- or Ly-5.2-positive cells. The data are shown as the mean of four mice ± SD. Significant differences compared with the value for irradiated MyD88–/– mice transferred with WT BM cells are shown: *, p < 0.05.

	Discussion

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Murine i-IELs consist of approximately equal amounts of TCR and TCR i-IELs and unique populations bearing CD8 homodimeric -chains as well as those bearing CD8 heterodimeric - and -chains (32, 33). i-IELs produce a variety of cytokines, including Th1-type cytokines, Th2-type cytokines (34), and immunosuppressive cytokines such as TGF- and IL-10 (35). i-IELs are thought to play important roles in the helper function for local IgA response and homeostasis of i-ECs through production of cytokines such as TGF- (36). i-IELs also exhibit non-MHC-restricted cytotoxicity via serine esterase-dependent and Fas/Fas ligand-dependent mechanisms that provide surveillance against infected cells, premalignant cells, and effete cells (5, 6). TCR^–/– mice showed impaired development of villi (7, 37), suggesting that TCR i-IELs play an important role in homeostasis of i-EC differentiation. It has been reported that a significant fraction of i-IELs such as TCR i-IELs and CD8 i-IELs is thought to down-regulate excessive inflammation caused by infection and autoimmunity (8, 38, 39). In the present study, we found that whole i-IEL in MyD88^–/– mice produced the larger amounts of IFN- upon TCR triggering than those in WT mice. It is most likely that the increased cytokine production simply reflects the relatively increased number of CD8 TCR i-IEL in MyD88^–/– mice. However, it is also possible that MyD88-dependent i-IEL populations may suppress the function of CD8 TCR i-IEL upon TCR triggering via their suppressive activities.

Rakoff-Nahoum et al. (18) have reported recently that MyD88^–/– mice showed a defect in steady state intestinal epithelial homeostasis, resulting in high susceptibility to intestinal injury induced by dextran disulfide sodium. These results suggest that MyD88-dependent TLR signaling in i-ECs plays an important role in the homeostasis of i-ECs. They also reported increase in count of proliferating i-ECs in MyD88^–/– mice and reduced levels of cytoprotective cytokines such as IL-6 and KC/CXCL1 and cytoprotective protein, heat shock protein, produced by i-ECs in MyD88^–/– mice. They speculated that the direct stimulation of i-ECs by intestinal microflorae via MyD88-dependnet TLR signaling induces cytoprotective cytokines and proteins, including IL-6 and heat shock protein, which result in steady state intestinal epithelial homeostasis. We previously reported that CD8^–/– mice showed high susceptibility to 5-fluorouracil-induced intestinal injury (40), suggesting that CD8 i-IELs are important for intestinal homeostasis. Therefore, we suggest that impairment of development, differentiation, and maintenance of CD8 and TCR i-IELs in MyD88^–/– mice accounts at least partially for the defect in steady state intestinal epithelial homeostasis and susceptibility to intestinal injury.

In summary, we found that MyD88-dependent signaling is important for the development and maintenance of CD8 TCR and TCR i-IELs in mice. Experiments with BM chimera mice demonstrated that MyD88-dependent signaling in radioresistant host parenchymal cells is important in keeping the number of the i-IEL populations. The expression level of IL-15 was greatly reduced in i-ECs of MyD88^–/– mice, and introduction of IL-15 transgene in MyD88^–/– mice restored the numbers of CD8 TCR and TCR i-IELs in MyD88^–/– mice. These results suggest that MyD88-dependent signaling for IL-15 production from interaction between commensal bacteria and i-ECs via TLRs plays an important role in maintenance of the number of CD8/TCR and TCR i-IELs.

	Acknowledgments

 
We express our gratitude to Y. Kobayashi and K. Kaneda for their excellent technical assistance.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas, Japan Society for the Promotion of Science, and grants from the Japanese Ministry of Education, Science, and Culture (to Y.Y.), and Uehara Memorial Foundation (to Y.Y.).

² Address correspondence and reprint requests to Dr. Yasunobu Yoshikai, Division of Host Defense, Center for Prevention of Infectious Disease, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan. E-mail address: yoshikai{at}bioreg.kyushu-u.ac.jp

³ Abbreviations used in this paper: i-IEL, intestinal intraepithelial lymphocyte; BM, bone marrow; i-EC, intestinal epithelial cell; M, macrophage; Tg, transgenic; WT, wild type.

Received for publication September 12, 2005. Accepted for publication March 7, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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