Characterization of CD4+CD8
+ and CD4CD8
+ intestinal intraepithelial lymphocytes in rats
Katsuo Yamada1,2,
Yuki Kimura1,
Hitoshi Nishimura1,
Yasushi Namii1,3,
Mitsuya Murase2 and
Yasunobu Yoshikai1
1 Laboratory of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control,
2 Department of Thoracic Surgery, and
3 Second Department of Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan
Correspondence to:
Y. Yoshikai
 |
Abstract
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Intestinal intraepithelial lymphocytes (i-IEL) of aged rats comprise CD4+CD8
+ and CD4CD8
+ T cells expressing TCR
ß. In the present study, we compared characteristics between CD4+CD8
+ and CD4CD8
+ i-IEL, which were purified by a cell sorter from the i-IEL of 6-month-old Lewis rats. Most of the CD4+CD8
+ i-IEL were of the CD44high phenotype, while CD4CD8
+ i-IEL were CD44low. Vß usage in the CD4CD8
+ i-IEL was much diversified, while CD4+CD8
+ i-IEL showed a skewed Vß repertoire. The CD4+CD8
+ i-IEL but not the CD4CD8
+ i-IEL proliferated in response to syngeneic spleen cells, which was partially inhibited by addition of anti-MHC class I mAb. The CD4+CD8
+ i-IEL produced IFN-
and IL-2 but no IL-4 or transforming growth factor (TGF)-ß in response to syngeneic spleen cells, while CD4CD8
+ i-IEL produced abundant levels of TGF-ß but no IL-2, IFN-
or IL-4. CD4+CD8
+ i-IEL proliferated in response to exogenous IL-2 but not to IL-15, while CD4CD8
+ i-IEL could respond to IL-15 as well as IL-2. These results suggest that a significant fraction of CD4+CD8
+ i-IEL belongs to Th1-type T cells capable of responding to self-MHC class I, while CD4CD8
+ i-IEL are a unique population with a diversified Vß repertoire that respond to IL-15 in rats.
Keywords: cytokines, mucosa, rodent, T lymphocytes
 |
Introduction
|
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Intestinal intraepithelial lymphocytes (i-IEL) contain unique lymphoid populations such as TCR 
T cells and TCR
ß T cells expressing the CD8
homodimer. Approximately half of i-IEL consist of TCR
ß CD8+ T cells in mice and the rest are of the TCR 
T cell lineage (13). In contrast, the majority of rat and human i-IEL express TCR
ß, and only 10% represent TCR 
i-IEL (48). Unlike the TCR
ß CD8+ T cells in the periphery, which express exclusively the heterodimer (CD8
ß) form, TCR
ß CD8+ i-IEL contain those bearing the homodimer (CD8
) form (9,10). In addition, CD4+CD8
+ T cells are present in the i-IEL. We and others have previously reported that the CD4+CD8
+ population in the i-IEL increased with age in rats and mice, presumably by stimulation with intestinal microflora (7,8,1113). TCR
ß CD4CD8
+ i-IEL are thought to develop extrathymically, while TCR
ß CD4+CD8
+ i-IEL represent mature peripheral populations derived from thymus-dependent CD4+ lymphocytes (9).
Several lines of evidence suggest that i-IEL recognize endogenous antigens including tissue-specific peptides, TL antigen, CD1 and HSP70 (14,15). We and others reported that i-IEL of aged rats proliferated in response to irradiated syngeneic splenocytes (SPL) and the reactivity was enhanced by addition of bacterial antigens such as HSP70 (13,16). However, the specificity of each subset of i-IEL remains to be elucidated. Regarding functions, i-IEL exhibit non-MHC-restricted cytotoxicity mediated by perforin/serin esterase (17) and/or the FasFas ligand system (18). Furthermore, i-IEL produce a variety of cytokines including Th1-type cytokines, Th2-type cytokines and transforming growth factor (TGF)-ß, and show helper function for antibody synthesis (19). It can be speculated that a significant fraction of i-IEL can provide the first line of host defense against invasion of microbes through their cytotoxic activity against infected cells and helper function for IgA synthesis. Furthermore, i-IEL are thought to play important roles in homeostasis of intestinal epithelial cells through differentiation of epithelia (20). The growth factors for i-IEL are also varied because of their unique locations. IL-2 at least is involved in the proliferative response of activated i-IEL. However, i-IEL may also respond to other lymphokines such as IL-7 and CSF in vivo (2123). We have recently found that IL-15, sharing many properties of IL-2, can preferentially proliferate i-IEL, especially 
i-IEL in mice (24). i-IEL may use different cytokines as growth factors.
In the present study, we compared the characteristics of CD4+CD8
+ i-IEL with CD4CD8
+ i-IEL in aged rats. A significant fraction of CD4+CD8
+ i-IEL showed a Th1 response against self-MHC class I, while CD4CD8
+ i-IEL displayed a diversified Vß repertoire and produced TGF-ß. The implications of these findings for the different roles of these i-IEL subsets in mucosal immunity are discussed.
 |
Methods
|
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Animals
Male Lewis/Crj (Lewis) rats were purchased from Japan Charles River (Yokohama, Japan) and bred under conventional conditions. Six- to 8-month-old rats were used for the experiments. Four or five rats were used for each experiment. Rats were housed in compliance with the guidelines established by the Canadian Council on Animal Care.
Cell preparation
A previously published method was used for isolation of i-IEL with some modifications (25). Briefly, the small intestine was surgically removed, flushed with 20 ml of PBS and inverted. The intestine was washed with PBS and cut into four segments. The segments of intestine were stirred in medium 199 (Gibco, Grand Island, NY) supplemented with 20% Nu serum (Collaborative Research, Bedford, MA) and 1 mM dithioerythritol (Sigma, St Louis, MO) at room temperature for 30 min. The debris was removed and the remaining cell suspension was centrifuged. The supernatant was aspirated and the cell pellet resuspended in 7 ml of 45% Percoll (Sigma) and then layered on 5 ml of a 66.7% Percoll solution. The gradient was centrifuged (600 g) at 20°C for 20 min. i-IEL harvested from the gradient interface were washed twice with HBSS. For each experiment, 522x106 i-IEL were isolated from one rat. In some experiments, thymi were isolated and the thymocytes released by the application of gentle pressure.
Antibodies
FITC-conjugated anti-CD44 mAb (OX-49), FITC-conjugated mouse IgG2a mAb (G155-178), biotin-conjugated anti-CD3 mAb (G4.18), biotin-conjugated anti-CD8ß mAb (341), anti-class I mAb (OX-18) (26) and anti-RT1D mAb (OX-17) were purchased from PharMingen (San Diego, CA). FITC-conjugated anti-CD8
mAb (OX-8), FITC-conjugated anti-CD8ß mAb (341.6), phycoerythrin (PE)-conjugated anti-CD8
mAb (OX-8) and PE- or biotin-conjugated anti-CD4 mAb (W3/25) were purchased from Serotec (Oxford, UK). Anti-class I mAb (R4-8B1) were purchased from Seikagaku (Tokyo, Japan). Anti-nitrophenol mAb (H1-6-2) was obtained from the supernatants of H1-6-2 hybridoma cells as described previously (27). Anti-RT1D mAb, anti-class I mAb and anti-nitrophenol mAb were dialyzed to remove sodium azide before culture. The concentrations of all dialyzed mAb were determined by the Lowry method.
Flow cytometric analysis
For three-color FACS analysis, cells were stained with FITC- or PE-conjugated anti-CD8
mAb, FITC- or biotin-conjugated CD8ß, FITC-conjugated anti-CD44 mAb, FITC-conjugated mouse IgG2a, PE- or biotin-conjugated anti-CD4 mAb, and biotin-conjugated anti-CD3 mAb followed by streptavidinRed670. StreptavidinRed670 was purchased from Gibco/BRL (Gaithersburg, MD). The stained cells were analyzed by a FACS flow cytometer (Becton Dickinson, Oxnard, MA).
Cell sorting
Cell populations were stained for cell sorting. Positive selection by cell sorting was performed on an Epics Elite ESP (Coulter, Miami, FL). The purity of them was 99.3% as assessed by FACS analysis. Contamination of intestinal epithelial cells was <0.5% as assessed by forward and side scatter profile.
RT-PCR and Southern blotting for expressions of TCR Vß and cytokine genes
Whole i-IEL were pooled from five rats for one experiment and then separated onto each T cell population by the cell sorter. Approximately 510x106 cells of each T cell population were recovered and then analyzed for Vß and cytokine gene expression. mRNA was extracted from the sorted T cells and thymocytes by using a QuickPrep Micro mRNA purification kit (Pharmacia Biotech, Milwaukee, WC). cDNAs were synthesized with 1 µg of each mRNA in a total volume of 20 µl containing 10 mM TrisHCl, 8 mM MgCl2, 50 mM KCl, 0.01% gelatin, 500 mM each dNTP, 1 mM DTT and 1.5 µg of random primers (BRL, Gaithersburg, MD). The mixture was added with 1 µl of 200 U SuperScript II reverse transcriptase (Gibco/BRL) and then incubated at 37°C for 60 min. The reaction was stopped at 95°C for 5 min and then quenched at 4°C. Products were diluted to a final volume of 100 µl. An aliquot of the synthesized first-strand cDNA was amplified by means of PCR using 40 pmol of each primer with 2.5 U of AmpliTaq (Takara Shuzo, Kyoto, Japan) in a total volume of 100 µl reaction buffer consisting of 10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin and 200 µM dNTP. PCR cycles were run for 1 min at 94°C, followed by 2 min at 60°C and 2 min at 72°C with 2230 cycles. Aliquots of reactions were taken for each sample after different numbers of cycles to ensure that reactions were not saturated, thus allowing quantitative estimations to be performed. Five microliters of PCR products were electrophoresed on a 1.8% agarose gel, and visualized with ethidium bromide and transferred to Gene Screen Plus (New England Nuclear, Boston, MA). Southern blot analyses were performed with cytokine-specific oligonucleotide probes, which were labeled with [
-32P]ATP (Amersham Life Science, Amersham, UK) using the Megalabel 5' labeling kit (Takara Shuzo), according to the manufacturer's instructions. After hybridization of 18 h at 60°C in 1% SDS, 1 M NaCl, 10% dextran sulfate and 100 µg/ml heat-denatured salmon sperm DNA, the filters were washed in 2xSSC (0.3 M NaCl, 0.03 M sodium citrate) and 1% SDS, and exposed to X-ray film at room temperature with intensifying screens. Radioactivities were assessed using a Fujix BAS2000 Bio-image analyzer (Fuji Photo Film, Tokyo, Japan).
To ensure that equivalent amounts of cDNA were used in each reaction, PCR was also performed for ß-actin and Cß from each sample and the cDNA adjusted to equivalent levels. All primers and probes were designed from published sequences. The used primer sequences were as follows; ß-actin sense: 5'-AGAAGAGCTATGAGCTGCCTGACG-3'; antisense: 5'-CTTCTGCATCCTGTCAGCCTACG-3', which produce a 236 bp product. Cß sense: 5'-CGGTGACTC- CACCCAAGGTC-3'; antisense: 5'-GCCTCCGCACTGATGTTCTG-3', which produce a 350 bp product. IFN-
sense: 5'-ACACTCATTGAAAGCCTAGAAAGTCTG-3'; antisense: 5'-ATTCTTCTTATTGGCACACTCTCTACC-3', which produce a 432 bp product. IL-2 sense: 5'-AACAGCGCACCCACTTCAA-3'; antisense: 5'-TTGAGATGATGCTTTGACA-3', which amplify a 400 bp fragment. IL-4 sense: 5'-ACCTTGCTGTCACCC- TGTTCTGC-3'; antisense: 5'-ACACTCATTGAAAGCCTAGAAAGTCTG-3', which produce a 352 bp product. TGF-ß1 sense: 5'-CTTTAGGAAGGACCTGGGTT-3'; antisense: 5'-CAGGAGCGCACAATCATGTT-3', which produce a 257 bp product. The oligonucleotide probes were as follows; ß-actin: 5'-CTATCGGCAATGAGCGGTTC-3'; Cß: 5'-CTGGTGGGTGAATGGAGGAG-3'; IFN-
: 5'-CTCCTTTTCCGCTTCCTTAG-3'; IL-2: 5'-CACTGAAGATGTTTC-3'; IL-4: 5'-ATGCACCGAGATGTTTGTACC-3'; TGF-ß1: 5'-ACCTTGCTGTACTGTGTGTC-3'. With the exception of TGF-ß1, primers span one or more introns (e.g. three introns for IL-2) to distinguish amplicons of cDNA from those of genomic DNA. In the case of TGF-ß1, it is necessary to prepare RNA free of contaminating genomic DNA. We confirmed no contamination of genomic DNA by PCR amplification without reverse transcriptase for each sample. To determine Vß usage, 2 µl of cDNA products was transferred to individual tubes that contained one of the Vß oligonucleotide primers (28), Cß-1 primer, and the components described above. PCR cycles were run for 1 min at 94°C, followed by 1.5 min at 55°C and 2 min at 72°C with 2630 cycles. Electrophoresis and Southern blotting of PCR products were carried out as described above. The used oligonucleotide probe was designed based on the known sequence of the rat Cß region (at the 5' side from the Cß-1 primer) using the OLIGO computer program (National Biosciences, Plymouth, MN): Cß-P, 5'-CGGTGACTCCACCCAAGGTC-3'.
Cell culture
Triplicate cultures of the enriched cells (1x105 cells/ml) were incubated with or without 10 times the number of 33 Gy-irradiated normal syngenic SPL in the presence or absence of cytokines or mAb in 96-well flat-bottom plates (Falcon, Becton Dickinson, Lincoln Park, NJ) for proliferation assay. RPMI 1640 complete culture medium contained 10% FBS (CSL, Victoria, Australia), 100 U/ml penicillin, 100 mg/ml streptomycin and 10 mM HEPES. Optimal doses of cytokines or mAb were added to the culture as follows: 100 ng/ml rIL-2 derived from human (Takeda Chemicals, Osaka Japan), 100 ng/ml rIL-15 derived from human (Genezyme, Cambridge, MA), 10 µg/ml R4-8B1 mAb, 10 µg/ml OX-18 mAb, 10 µg/ml OX-17 mAb and 10 µg/ml isotype-matched anti-nitrophenol mAb as a control. The amounts of mAb were confirmed to be sufficient to block
ß T cell responses (29). Triplicate cultures were incubated at 37°C in 5% CO2 for 72 h. For the last 18 h, cells were pulse-labeled with [3H]thymidine (1 µCi/well; Amersham) before harvesting on glass-fiber filters. Incorporation of [3H]thymidine was determined by a liquid scintillation counter.
Assay for cytokines
Culture supernatants were harvested after 24 or 72 h incubation. IL-2, IFN-
and active forms of TGF-ß1 activities in the culture supernatants were measured by ELISA using a rat IL-2 ELISA kit (Cosmo Bio), a rat IFN-
ELISA Kit (Gibco/BRL) and a TGF-ß1, human, ELISA system (Amersham) respectively.
Statistical analysis
Data were analyzed by using Student's t-test. Values of P < 0.05 were considered statistically significant. All experiments were performed at least twice.
 |
Results
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Cell surface phenotype of i-IEL in aged rats
i-IEL are composed of a majority of CD8
+ cells and a few CD4CD8 and CD4+ cells in young Lewis rats (78 weeks of age), while CD4+CD8
+ cells were hardly detected in i-IEL at this stage (8). Consistent with our previous reports (8,13), the number of CD4+CD8
+ i-IEL increased to a considerable level in aged Lewis rats (6 months old) (Fig. 1A
). To further characterize the CD8
+ i-IEL, flow cytometric analysis for the expression of CD8ß and CD44 was carried out on CD4CD8
+ and CD4+CD8
+ i-IEL. CD4CD8
+ i-IEL contained ~50.4±12.4% CD8ß+ i-IEL (n=5), while no CD4+CD8
+ i-IEL expressed CD8ß, suggesting that CD4+CD8+ i-IEL have exclusively the CD8
homodimer. Typical results from five rats are shown in Fig. 1
(B). Only a few CD44high cells (20.0±8.7%) were detected in CD4CD8
+ i-IEL comprising approximately equal numbers of CD8
+ and CD8
ß+ cells, while most of the CD8
ß+ i-IEL were CD44high (80.3±9.6%), indicating that CD4CD8
+ i-IEL are of the CD44low phenotype. On the other hand, a majority of CD4+CD8
+ i-IEL expressed CD44high (86.1±5.1%) (n=5) (Fig. 1C
). These results suggest that most of the CD4+CD8
+ and CD4CD8
ß+ i-IEL may be in an activated state in situ.
The Vß repertoire of CD4CD8
+ and CD4+CD8
+ i-IEL
We addressed the TCR Vß usages of CD4CD8
+ and CD4+CD8
+ i-IEL by RT-PCR and Southern blotting. Each subset of i-IEL in aged rats (n=5) was purified to >99.3% with an Epics Elite ESP of Coulter cell sorting system and mRNA was extracted as described in Methods. As shown in Fig. 2
, the Vß repertoire of CD4CD8
+ i-IEL was much diversified, while the CD4+CD8
+ i-IEL used limited Vß segments. CD4CD8
ß+ i-IEL also displayed an oligoclonal Vß repertoire. We obtained the same tendency in three individual experiments, using pooled i-IEL populations. Thymocytes consisting mainly of CD4+CD8
ß+ T cells showed a diversified Vß repertoire. These results suggest that CD4+CD8
+ i-IEL may be limited antigen-driven and that CD4CD8
+ i-IEL may be composed of a large number of T cells randomly rearranging TCR genes.
Involvement of MHC class I molecules in the proliferative response of CD4+CD8
+ i-IEL to syngeneic SPL
We have previously reported that CD4+CD8
+ i-IEL of aged rats proliferate in response to syngeneic SPL (8,13). Consistent with this finding, purified CD4+CD8
+ i-IEL proliferated significantly in response to irradiated syngeneic SPL, but purified CD4CD8
+ or CD4CD8
ß+ i-IEL did not respond to the syngeneic SPL (Fig. 3
, data not shown). To investigate whether MHC molecules are involved in the response of CD4+CD8
+ i-IEL, we examined the effects of addition of anti-MHC class I or class II mAb on the proliferation of CD4+CD8
+ i-IEL in response to the syngeneic SPL. The addition of anti-class I mAb (R4-8B1 and OX-18) significantly inhibited the proliferative response of CD4+CD8
+ i-IEL to irradiated syngeneic SPL (P<0.05), while the addition of anti-class II mAb (OX-17) did not affect the response against the irradiated syngeneic SPL (Fig. 3
). These results indicate that MHC class I molecules are at least partially responsible for the proliferation of CD4+CD8
+ i-IEL in response to syngeneic SPL.

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Fig. 3. The effect of mAb to MHC class I and class II molecules on the proliferative response of i-IEL in mixed culture with irradiated syngeneic SPL. Proliferation was assayed by [3H]thymidine uptake after 3 day incubation of primary cultures in the presence or absence of anti-class I (R4-8B1 and OX-18) (10 µg/ml), anti-RT1D (OX-17) (10 µg/ml) or isotype IgG (10 µg/ml) mAb. Results are expressed as the mean ± SD of culture triplicates. Data are representative of three independent experiments using pooled cells from five aged rats. *P < 0.05, **P < 0.01.
|
|
Cytokine expression of CD4CD8
+ and CD4+CD8
+ i-IEL
To gain insight into differences in functions between CD4CD8
+ and CD4+CD8
+ i-IEL, we analyzed gene expression of IL-2, IFN-
, IL-4 and TGF-ß1 of each population in the freshly isolated i-IEL pooled from five aged rats. The amount of cDNA was adjusted by PCR and the Southern hybridization analysis of serially diluted cDNA with ß-actin primers as described in Methods, and expression of mRNA specific for these cytokines was analyzed by RT-PCR. As shown in Fig. 4
, the CD4+CD8
+ population purified from the freshly isolated i-IEL expressed mRNA specific for IL-2 and IFN-
but no mRNA specific for IL-4 or TGF-ß1, suggesting that these cells are Th1-type cells. On the other hand, the CD4CD8
+ i-IEL expressed mRNA specific for TGF-ß1 but little mRNA specific for Th1- or Th2-type cytokines. We confirmed that the IL-4 internal probe could hybridize to the PCR product derived from concanavalin A-stimulated spleen cells using the IL-4-specific primers (data not shown).
To further examine the cytokine synthesis by CD4CD8
+ or CD4+CD8
+ i-IEL at the protein level, we measured the IL-2, IFN-
and TGF-ß1 levels in culture supernatants of i-IEL incubated with 33 Gy-irradiated normal syngeneic SPL by ELISA. CD4+CD8
+ i-IEL incubated with irradiated SPL secreted a large amount of IL-2 and IFN-
but not TGF-ß1 (Fig. 5
), which was consistent with the results obtained by RT-PCR analysis (Fig. 4
). On the other hand, CD4CD8
+ i-IEL produced TGF-ß1 spontaneously even without irradiated SPL but not IL-2 or IFN-
in the culture supernatants (Fig. 5
).
Responsiveness of CD4CD8
+ and CD4+CD8
+ i-IEL to IL-2 or IL-15
Our RT-PCR data suggested that IL-2 was produced by CD4+CD8
+ i-IEL but not by CD4CD8
+ i-IEL. We have recently found that IL-15 preferentially proliferate 
i-IEL expressing CD8
+ in mice (24). Therefore, we next compared the proliferative response to IL-2 or IL-15 between CD4CD8
+ and CD4+CD8
+ i-IEL. rIL-2 induced the proliferation of both CD4CD8
+ and CD4+CD8
+ i-IEL but rIL-15 only induced CD4CD8
+ i-IEL proliferation (Fig. 6
). These results suggest that CD4CD8
+ i-IEL can use IL-15 for a growth factor, while CD4+CD8
+ i-IEL exclusively use IL-2 for it.
 |
Discussion
|
---|
In this report, we showed that CD4+CD8
+ i-IEL in aged rats displayed a skewed TCR Vß repertoire and a significant fraction of this subset proliferated to produce Th1-type cytokines in response to self-MHC class I. On the other hand, CD4CD8
+ i-IEL bearing a diversified Vß repertoire neither respond to syngeneic stimulator cells nor express mRNA specific for Th1- or Th2-type cytokines. These results suggest that CD4+CD8
+ i-IEL may have different specificities and functions from CD4CD8
+ i-IEL in mucosal immunity.
The oligoclonal repertoire of TCR
ß CD8+ i-IEL has been reported in the small intestine of human and mouse (30,31). Reimann and Rudolphi reported that CD4+CD8+ i-IEL in mice displayed a limited diversity of the TCR Vß repertoire (32). Consistent with this finding in mice, our results revealed that CD4+CD8
+ i-IEL in rats expressed a skewed Vß repertoire and responded to self-MHC class I molecules. Mature human and rat CD4+ T cells can be induced to express CD8 in vitro under certain conditions (33,34). The gut epithelial environment plays both attractive and inductive roles in the appearance of unique i-IEL co-expressing CD4 and homodimeric 
CD8+ (9). It has been reported that CD4+ CD8
+ T cells appeared in the epithelial layer of the small intestine of scid mice after transfer of CD4+ T cells from lymph nodes (32). Taking into account all the results, it appears that mature CD4+ T cells induce CD8
expression under intestinal circumstances and in turn have a reactivity against self-MHC class I. On the other hand, CD4CD8
+ i-IEL have a relatively diversified Vß repertoire and no ability to proliferate in response to syngeneic SPL. The presence of RAG mRNA in CD3CD4CD8
+ i-IEL is compatible with the hypothesis of TCR gene rearrangements occurring in the gut (9). Furthermore, positive and negative selections are not evident in CD4CD8
+ i-IEL (14,15). We speculate that CD4CD8
+ i-IEL are derived from a large number of precursors randomly rearranging TCR genes in the intestine. Regnault et al. have reported that both CD8
and CD8
ß i-IEL in mouse exhibited limited diversities (35). They speculate that the repertoire of the CD8
i-IEL clones is established from a small number of precursors and the bacterial flora is not responsible for the oligoclonality of i-IEL T cell clones, but is involved in the clonal expansion (35). The repertoire varied among genetically identical individual mice maintained in similar environmental conditions. Therefore, the generality of our finding and its possible implications await analysis of T cell clones from i-IEL of more genetically identical rats under similar environmental and germ-free conditions.
Our present results revealed that the proliferative response of purified CD4+CD8
+ i-IEL to syngeneic spleen cells was partially inhibited by addition of anti-MHC class I mAb. These results raise a possibility that at least some of the CD4+CD8
+ i-IEL respond to self-MHC class I molecules. MHC class I molecules, composed of an
chain associated with ß2-microglobulin, are responsible for peptide presentation to T cells. The fact that the CD4+CD8
+ i-IEL proliferated in response to irradiated syngeneic SPL excludes the possibility that this subset recognizes tissue-specific peptides in the context of MHC class I molecules. Nakamura et al. have reported that the reactivity of i-IEL to syngeneic SPL is enhanced by bacterial antigens such as purified protein derivative, HSP70 or HSP60 (16). It is possible that CD4+CD8
+ i-IEL recognize such conserved peptides in the context of MHC class I molecules. Sydora et al. have demonstrated that the number of CD4+CD8+ i-IEL is decreased in ß2-microglobulin deficient mice but not in TAP-deficient mice (36). This suggests that CD4+CD8+ i-IEL recognize TAP-independent peptides in the context of self-MHC class I molecules. However, our results with anti-MHC class I mAb do not necessarily imply that presentation of antigen by class I MHC has been inhibited. Further experiments are required to determine the specificity of CD4+CD8+ i-IEL.
Th1 cells uniquely secrete IL-2, IFN-
and tumor necrosis factor-
for induction of cell-mediated immunity characterized by macrophage activation and cytotoxic T lymphocyte induction, whereas Th2 cells uniquely secrete IL-4, IL-5 and IL-6 for providing help for antibody production (37,38). Th2 type cytokines not only induce B cell development but also inhibit activities of macrophage and NK cells by shutting down the cytokine synthesis of Th1 cells such as IFN-
. On the other hand, IFN-
derived from Th1 cells inhibit proliferation of Th2. Thus Th1 and Th2 cells are mutually regulated (3741). Recently, a unique subpopulation of Th cells termed Th3 cells was reported to play an important role in immune regulation, especially oral tolerance, through TGF-ß production (42,43). Our present study indicated that unique CD4+CD8
+ i-IEL in aged rats are Th1-type cells, suggesting that this subset plays an important role in the first line of defense against pathogens and/or effete self cells through evoking inflammation. On the other hand, CD4CD8
+ i-IEL expressed TGF-ß1, which is a multi-functional cytokine regulating growth and differentiation of various cell types and directing class switch to IgA (44). This subset may play a role in immunoregulation in mucosal immunity.
Despite a lack of structural homology with IL-2, IL-15 utilizes the ß and
subunits of IL-2R which are the essential signal transducing components of the IL-2R system (4547). The activities of IL-2 and IL-15 include stimulation of T, B and NK cell proliferation, cytotoxic T lymphocyte and NK cell activation, and augmenting antibody secretion by B cells (4547). We have reported that TCR 
T cells appearing during salmonellosis in mice can proliferate in response to IL-15 (48). Furthermore, we have recently found that the TCR 
i-IEL in mice utilize IL-15 preferentially as a growth factor (24). Although IL-2 are generated exclusively by activated T cells, IL-15 are generated by various tissues including intestinal epithelial cells (48,49). We speculate that unique CD4CD8
+i-IEL use IL-15 for their expansion in situ in the small intestine because they cannot produce IL-2, while CD4+CD8
+i-IEL may expand in the autocrine pathway via IL-2 production.
In conclusion, CD4+CD8
+i-IEL in aged rats expressed a skewed Vß repertoire and CD44high, and a significant fraction of this subset proliferated to produce IL-2 and IFN-
in response to self-MHC clss I molecules. On the other hand, CD4CD8
+i-IEL showed a diversified Vß repertoire, CD44low of phenotype, and TGF-ß production. Thus, CD4+CD8
+ and CD4CD8
+i-IEL may have different specificities and play different roles in mucosal immunity.
 |
Acknowledgments
|
---|
We thank Takeda Chemicals (Osaka, Japan) for providing human rIL-2 and Mr Y. Yamakawa for technical assistance in the sorting of lymphocytes. This work was supported in part by grants from the Ministry of Education, Welfare, and Culture, and Grant-in-Aid for COE Research.
 |
Abbreviations
|
---|
i-IEL | intestinal intraepithelial lymphocytes |
PE | phycoerythrin |
SPL | splenocytes |
TGF | transforming growth factor |
 |
Notes
|
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Transmitting editor: T. Hünig
Received 14 May 1998,
accepted 21 September 1998.
 |
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