Localization of intestinal intraepithelial T lymphocytes involves regulation of {alpha}Eß7 expression by transforming growth factor-ß

Ryuyo Suzuki1,2, Atsuhito Nakao1, Yutaka Kanamaru1,3, Ko Okumura1, Hideoki Ogawa1 and Chisei Ra1

1 Atopy (Allergy) Research Center, 2 Department of Pediatrics and 3 Department of Nephrology, Juntendo University School of Medicine, Tokyo 113-8421, Japan

Correspondence to: A. Nakao; Email: anakao{at}med.juntendo.ac.jp
Transmitting editor: M. Miyasaka


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of {alpha}Eß7 expression on T cells by transforming growth factor (TGF)-ß is thought to be important for intestinal intraepithelial T lymphocyte (IEL) entry into the epithelial compartment. However, there has been no in vivo evidence that up-regulation of {alpha}Eß7 expression on T cells by TGF-ß is critical for the selective localization of intestinal IEL in the epithelial area. We have recently established transgenic mice expressing Smad7 under the control of a distal lck promoter where TGF-ß/Smad signaling is specifically blocked in mature T cells. Here we showed that TGF-ß-mediated up-regulation of {alpha}Eß7 was impaired on T cells isolated from the Smad7 transgenic mice associated with reduced numbers of intestinal IEL when compared with that in wild-type littermates. These results indicated that failure to induce {alpha}Eß7 on T cells by TGF-ß resulted in reduced numbers of intestinal IEL, suggesting the importance of {alpha}Eß7 expression by TGF-ß in selective localization of intestinal IEL.

Keywords: mucosal immunity, transgenic mice, Smad7


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Intestinal intraepithelial T lymphocytes (IEL) represent one of the largest lymphoid populations in the body and are suggested to play an important role in mucosal immunity (1). Intestinal IEL show a characteristic surface profile which differs from T lymphocytes found in the lamina propria (LPL) and peripheral blood (PBL). In particular, >90% of intestinal IEL express high levels of the integrin {alpha}Eß7 (2). In culture, the surface profile of IEL changes and resembles that of PBL. Transforming growth factor (TGF)-ß restores the unique integrin profile of IEL by up-regulation of {alpha}Eß7 and is able to do the same on PBL (3,4).

The regulation of {alpha}Eß7 expression by TGF-ß is thought to be important for selective localization or retention of intestinal IEL in the intestinal epithelium (57). It has been postulated based on in vitro studies that lymphocytes entering the gastrointestinal tract from the peripheral blood do so via an interaction of {alpha}4ß7 on their surface with the mucosal-associated cell adhesion molecule (MadCam) on endothelial cells. Subsequent to migration, the {alpha}4 subunit is down-regulated and {alpha}E is up-regulated by TGF-ß in the microenvironment of the intestine. The {alpha}Eß7 integrin is suggested to interact with E-cadherin on the enterocyte surface, thereby mediating selective localization or retention of IEL in the epithelium. However, it remains obscure whether regulation of {alpha}Eß7 expression on T cells by TGF-ß is really critical for selective localization or retention of intestinal IEL in vivo.

TGF-ß is a multifunctional cytokine which has diverse effects on a variety of cell types (8). TGF-ß transduces signals from the receptors to the nucleus through the Smad family of proteins (9). We have recently established transgenic mice expressing Smad7, an intracellular antagonist of TGF-ß/Smad signaling (10), under the control of a distal lck promoter that directed high expression in peripheral T cells (11). Therefore, we were able to block the TGF-ß/Smad signaling pathway specifically in mature T cells. Peripheral T cells in the transgenic mice showed high expression of Smad7, which resulted in blockade of the Smad signaling pathway and resistance to TGF-ß action (12). The development of their immune system appeared to be normal and the mice survived into adulthood without any specific phenotype (12).

By using the Smad7 transgenic mice, we determined whether regulation of {alpha}Eß7 on T cells by TGF-ß was critical for selective localization or retention of intestinal IEL in vivo. We found that TGF-ß-mediated up-regulation of {alpha}Eß7 was impaired on T cells from Smad7 transgenic mice and that the number of intestinal IEL was reduced in the transgenic mice. These results suggested that regulation of {alpha}Eß7 expression on T cells by TGF-ß was involved in localization or retention of intestinal IEL.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Transgenic mice expressing Smad7 under control of a distal lck promoter were generated as previously described (12). Transgene positive founders were backcrossed to B6 mice to establish lines. All experiments used 8- to 10-week-old trans genic mice or the wild-type littermates, weighing 18–23 g, which were matched with sex.

Purification of T cells
Mouse CD4 T cells were purified from splenocytes harvested from the transgenic or wild-type mice by magnetic cell sorting using MACS anti-CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s recommendation. The purity of mouse CD4 T cells was confirmed by FACScan (Becton Dickinson, San Jose, CA) and was consistently >99%.

Stimulation of T cells
Purified mouse CD4 T cells (1 x 106 cells/ml) were stimulated with plate-bound anti-CD3 antibody (2C11) (10 µg/ml) (PharMingen, San Diego, CA) in the presence or absence of recombinant human TGF-ß1 (10 ng/ml) (R & D, Minneapolis, MN) and were cultured for 3 days in RPMI 1640 with 10% heat-inactivated FCS (Gibco, Grand Island, NY) in a humidified 5% CO2 at 37°C and followed by FACS analysis.

Flow cytometry
Mouse CD4 T cells suspended in PBS containing 0.1% NaN3 and 1% FCS were incubated on ice with FITC-labeled anti-{alpha}Eß7 (CD103) (2E7), phycoerythrin-labeled anti-CD4, anti-LFA-1, anti-ICAM-1, anti-VLA-4, anti-{alpha}4ß7 and anti-CD62L antibody (MEL14) (PharMingen). After washing, the cells were analyzed on a FACScan flow cytometer (Becton Dickinson) using CellQuest software (Becton Dickinson).

Immunofluorescence study
After removal of the small intestine, the samples were embedded in OCT compound and snap frozen in liquid nitrogen. Cryostat sections (4–8 µm thick) were blocked for 5 min with cold acetone and dried for 10 min. After washing the sections twice with PBS, they were incubated for 60 min at room temperature with FITC-labeled anti-CD3 antibody (C2C12), anti-{alpha}Eß7 antibody (2E7), anti-{alpha}ß TCR antibody and anti-{gamma}{delta} TCR antibody (PharMingen). For analysis of intestinal IEL number, the average number of cells stained with anti-CD3 antibody/100 villous epithelial cells in at least five randomly selected high power (x40) fields of the proximal jujunal tissue sections was determined with microscopic evaluation as previously described (13). For analysis of LPL number, a cell was counted as a LPL when it did not overlap the basement membrane and was contained within villi rather than crypts as previously described (13). The expression of Flag-tagged Smad7 transgene in intestinal IEL in Smad7 transgenic mice was assessed with immunohistochemical staining with optimally diluted biotinylated rabbit polyclonal anti-Flag antibody (Stratagene, La Jolla, CA) as previously described (14).

Isolation of IEL and LPL
IEL and LPL were isolated from Smad7 transgenic mice and the wild-type littermates by modified procedures as described previously (15). Briefly, each intestinal sample was removed and flushed with Ca2+/Mg2+-free HBSS, and all Peyer’s patches were removed. The intestines were then opened longitudinally and cut laterally into small pieces. Each segment was incubated in Ca2+/Mg2+-free HBSS containing 0.1 mM EDTA and stirred for 30 min twice to remove epithelial cells. Cell suspensions were filtered through nylon mesh and then centrifuged. The cell pellet, consisting of IEL and epithelial cells, was suspended in RPMI1640 medium with 10% FCS. The remaining fragments were then transferred to flasks containing RPMI 1640 with 90 U/ml of collagenase and stirred gently for 60 min in a 37°C water bath. Cell suspensions, containing LPL, were filtered through nylon mesh and then centrifuged. IEL and LPL were purified using a 45–70% discontinuous Percoll gradient. After the isolation, the number of viable IEL and LPL was counted by Trypan blue dye exclusion. The isolated IEL from Smad7 transgenic mice or the wild-type littermates were subjected to FACscan using anti-CD3, CD4, CD8, {alpha}Eß7, {alpha}ßTCR and {gamma}{delta}TCR antibodies as described earlier.

Induction of oral tolerance
Transgenic mice or wild-type littermates (8 weeks old) were fed with 1 mg of ovalbumin (OVA) (Sigma, St Louis, MO) dissolved in 0.2 ml of PBS or PBS alone 3 times every 24 h by gastric intubation with an 18-gauge stainless steel animal feeding needle. Two weeks after the final feeding, the mice were immunized i.p. twice at 2-week intervals with 1 µg of OVA in 4 mg of aluminum hydroxide.

Proliferation assay
Proliferation assay was performed as previously described (12).

The spleen was removed from the mice 10 days after the second immunization of OVA and a single-cell suspension of spleen cells was prepared. Splenocytes were then cultured in triplicate in the absence or presence of OVA (100 µg/ml) in a 96-well microtiter plate (2 x 105 cells/well) at 37°C in 5% CO2 for 72 h and 1 µCi of [3H]thymidine (Amersham, Little Chalfont, UK) was added to the culture for the last 12 h. Thymidine incorporation was assessed by harvesting cells with a PHD cell harvester (Cambridge Technologies, Cambridge, MA).

Data analysis
Data are summarized as mean ± SD. The statistical analysis of the results was performed by the amount of variance using Fisher’s least significant difference test for multiple comparisons. P < 0.05 was considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of {alpha}Eß7 expression by TGF-ß is abrogated on splenic T cells from Smad7 transgenic mice
Induction of {alpha}Eß7 expression by TGF-ß is thought to be important for selective localization or retention of intestinal IEL in the intestinal epithelium (6). However, it remains obscure whether up-regulation of {alpha}Eß7 expression on T cells by TGF-ß is critical for selective localization or retention of intestinal IEL in vivo. We hypothesized that T cells from the transgenic mice expressing Smad7 under control of a distal lck promoter (T cell-specific promoter) should be resistant to TGF-ß action and up-regulation of {alpha}Eß7 by TGF-ß might be impaired. If this was the case, we thought that we could determine whether regulation of {alpha}Eß7 expression on T cells by TGF-ß was critical for selective localization or retention of intestinal IEL in vivo by examining the number of intestinal IEL in the transgenic mice.

As shown in Fig. 1(A), up-regulation of {alpha}Eß7 by TGF-ß was impaired on T cells isolated from Smad7 transgenic mice. Purified CD4+ T cells from Smad7 transgenic mice and the wild-type littermates were stimulated with plate-bound anti-CD3 antibody in the absence or presence of recombinant TGF-ß1 (10 ng/ml). Three days after the culture, {alpha}Eß7 expression on CD4+ T cells was evaluated by FACScan. TGF-ß up-regulated {alpha}Eß7 expression on splenic T cells obtained from wild-type mice as previously described (4), but not from Smad7 transgenic mice.



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Fig. 1. FACScan analysis of splenic CD4+ T cells. (A) Expression of {alpha}Eß7 on CD4 T cells from Smad7 transgenic mice (TG) and the wild-type littermates (WT). Purified mouse CD4 T cells were then stimulated with plate-bound anti-CD3 antibody (10 µg/ml) in the presence (open profile) or absence (closed profile) of recombinant human TGF-ß1 (10 ng/ml) and were cultured for 3 days. Mouse CD4 T cells were then stained with FITC-labeled anti-{alpha}Eß7 antibody and the expression was assessed by FACS analysis. (B) Basal expression levels of LFA-1, VLA-4, ICAM-1 and {alpha}4ß7 on CD4 T cells from Smad7 transgenic mice and the wild-type littermates. Splenic CD4 T cells were purified from the transgenic mice and the wild-type littermates as described above. Expression of LFA-1, VLA-4, ICAM-1 and {alpha}4ß7 on CD4 T cells was assessed by FACS analysis using phycoerythrin-labeled anti-LFA-1, VLA-4, ICAM-1 and {alpha}Eß7 antibodies (open profiles). Closed profiles represent negative staining. The results are from one representative experiment. Similar results were obtained from other two experiments.

 
Additional FACS analyses were performed to determine whether the expression levels of other adhesion molecules were altered on T cells from Smad7 transgenic mice.

As shown in Fig. 1(B), basal expression levels of LFA-1, VLA-4, ICAM-1 and {alpha}Eß7 on splenic T cells were not significantly different between the transgenic mice and the wild-type littermates. We also found that TGF-ß did not significantly alter the basal expression levels of LFA-1, VLA-4 and ICAM-1 on splenic T cells (data not shown). {alpha}Eß7 expression was slightly enhanced by TGF-ß on splenic T cells from wild-type mice, but not from Smad7 transgenic mice (data not shown). These findings indicated that regulation of {alpha}Eß7 expression by TGF-ß was abrogated on splenic T cells obtained from Smad7 transgenic mice, probably due to the blockade of TGF-ß/Smad signaling pathway by overexpression of Smad7 in T cells.

The number of intestinal IEL is reduced in Smad7 transgenic mice
Based on the findings that up-regulation of {alpha}Eß7 expression by TGF-ß was completely abrogated on splenic T cells from Smad7 transgenic mice in vitro, we next examined the number of intestinal IEL in Smad7 transgenic mice and the wild-type littermates by immunofluorescence with anti-CD3 antibody.

We found that the number of CD3+ T cells located in the epithelial area of proximal jujunal tissue was reduced by 46% in Smad7 transgenic mice when compared with the wild-type littermates (Fig. 2A and B). Staining with anti-{alpha}Eß7 antibody showed that the number of {alpha}Eß7+ cells in intestinal epithelium was reduced in Smad7 transgenic mice, although there were some significant {alpha}Eß7+ cells present in the transgenic mice (Fig. 2A and B). In addition, the number of {alpha}ß+ and {gamma}{delta}+ T cells in the intestinal epithelium was equally reduced in Smad7 transgenic mice (Fig. 2A and C). These findings indicated the number of intestinal IEL was reduced in the transgenic mice when compared with the wild-type littermates, and intestinal localization of both subsets of {alpha}ß and {gamma}{delta} IEL were equally affected in Smad7 transgenic mice.



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Fig. 2. Immunofluorescence study of intestinal mucosa. (A) Immunofluorescence staining of IEL in the intestinal mucosa. The small intestines (the proximal jujunal tissue) sections from Smad7 transgenic mice (TG) or the wild-type littermates (WT) were stained with FITC-labeled anti-CD3 antibody, anti-{alpha}Eß7 antibody, anti-{alpha}ß TCR antibody and anti-{gamma}{delta} TCR antibody. (B) The number of intestinal IEL in Smad7 transgenic mice and the wild-type littermates. Proximal jejunal tissue sections prepared from Smad7 transgenic mice and the wild-type littermates as described above were stained with FITC-labeled anti-CD3 antibody (closed columns) or anti-{alpha}Eß7 antibody (open columns). The average number of cells stained with anti-CD3 antibody or anti-{alpha}Eß7 antibody/100 villous epithelial cells in five randomly selected high power (x40) fields of the proximal jujunal tissue sections was determined with microscopic evaluation as described in Methods. Data are mean ± SD for five randomly selected sections. The results are from one representative experiment. Similar results were obtained from other two experiments. *P < 0.05. (C) The number of {alpha}ß and {gamma}{delta} TCR+ cells in the intestinal epithelium of Smad7 transgenic mice and the wild-type littermates. Proximal jejunal tissue sections prepared were stained with FITC-labeled anti-{alpha}ß or {gamma}{delta} TCR antibody (PharMingen) and the number of stained cells/100 villus epithelial cells was determined by microscopic evaluation as described above. Data are mean ± SD as described in (B). *P < 0.05.

 
To further confirm the immunofluorescence data, we examined the number of intestinal IEL in Smad7 transgenic mice and the wild-type littermates by carrying out isolation of IEL from the transgenic mice. The number of IEL, but not LPL, isolated from the intestine of Smad7 transgenic mice was significantly reduced when compared with the wild-type littermates (Fig. 3A). FACS analyses of isolated IEL showed that proportions of subsets of CD4, CD8, and {alpha}ß and {gamma}{delta} IEL in Smad7 transgenic mice did not appear to be different from those in the wild-type littermates (Fig. 3B). We also found that most IEL isolated from the wild-type mice were {alpha}Eß7+ cells, whereas about half of IEL isolated from the transgenic mice were {alpha}Eß7+ (Fig. 3B). These findings were consistent with the earlier results obtained by immunofluorescence study.



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Fig. 3. The number and surface expression profile of IEL. (A) The number of intestinal IEL and LPL in Smad7 transgenic mice and the wild-type littermates. IEL and LPL were isolated from Smad7 transgenic mice and the wild-type littermates by modified procedures as described in Methods. After the isolation, the number of viable IEL and LPL was counted by Trypan blue dye exclusion. Data are mean ± SD for three mice. (B) Surface expression profile on isolated IEL. The isolated IEL from Smad7 transgenic mice or the wild-type littermates as described above were subjected to FACscan using anti-CD3, CD4, CD8, {alpha}Eß7, {alpha}ßTCR and {gamma}{delta}TCR antibodies. Bold lines represent staining with the indicated antibodies.

 
In addition, expression of Smad7 transgene in Smad7 transgenic mice, but not in the wild-type littermates, was confirmed by immunohistochemical staining with anti-Flag antibody (Fig. 4), because we used a plasmid construct expressing Flag-tagged mouse Smad7 (10) under the control of a distal lck promoter for generation of the transgenic mice (12).



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Fig. 4. Expression of Smad7 transgene in IEL of Smad7 transgenic mice. Intestinal mucosa sections derived from Smad7 transgenic mice (left panel) or the wild-type littermates (right panel) were stained with optimally diluted biotinylated rabbit polyclonal anti-Flag antibody.

 
Oral tolerance is induced in Smad7 transgenic mice
Subsets of intestinal IEL are suggested to be involved in induction of oral tolerance (16). Since we found that the number of intestinal IEL was reduced in both {alpha}ß and {gamma}{delta} subsets of IEL in Smad7 transgenic mice, we determined whether oral tolerance was induced in Smad7 transgenic mice to the same degree as that seen in the wild-type littermates. As shown in Fig. 5, in vitro OVA-induced T cell proliferation was significantly decreased in sensitized wild-type mice after oral administration of OVA. Significant inhibition of T cell proliferation was also observed after oral administration of OVA in sensitized Smad7 transgenic mice (Fig. 5). There was little T cell proliferation in the absence of OVA in sensitized wild-type and Smad7 transgenic mice (data not shown). Interestingly, OVA-induced T cell proliferation was ~3 times higher in sensitized Smad7 transgenic mice than that in sensitized wild-type mice, suggesting that endogenous TGF-ß might be involved in the suppression of antigen-induced T cell proliferation. These findings indicated that oral tolerance was inducible in Smad7 transgenic mice to the same extent as that observed in wild-type mice.



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Fig. 5. Effect of oral administration of antigen on antigen-induced T cell proliferation in Smad7 transgenic mice. Mice were fed with PBS (open bars) or OVA (closed bars), and immunized with OVA and alum at 2-week intervals. At 10 days after the second immunization, splenocytes from Smad7 transgenic (TG) and the wild-type littermates (WT) were stimulated with OVA. Aliquots of cells were pulsed with [3H]thymidine, and the incorporated counts (in c.p.m.) were determined. Shown are the mean ± SD of [3H]thymidine corporation from triplicate cultures. The results are from one representative experiment. Similar results were obtained from two other experiments. *P < 0.05, significantly different from the mean value of the corresponding control response (PBS-fed mice).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we showed that TGF-ß-mediated up-regulation of {alpha}Eß7 was impaired on T cells isolated from Smad7 transgenic mice and that the number of intestinal IEL was reduced in the transgenic mice by 46% when compared with the wild-type littermates (Figs 1–3). These results suggested that regulation of {alpha}Eß7 expression on T cells by TGF-ß was partially involved in selective localization or retention of intestinal IEL.

We have previously shown that the transgenic mice expressing Smad7 selectively in mature T cells survive into adulthood without any spontaneous phenotype (12). The development of thymic and splenic T cells and B cells appeared to be normal in the transgenic mice. No pathological findings were observed in the intestine or other organs in the transgenic mice. In addition, basal expression levels of adhesion molecules such as LFA-1, ICAM-1, VLA-4, {alpha}4ß7 and CD62L in the transgenic mice were not different from the wild-type littermates (Fig. 1B and data not shown). We thus reasoned that usage of the Smad7 transgenic mice would be appropriate to analyze homeostasis of the mucosal immune system in the absence of experimentally induced infection or inflammation.

We found that up-regulation of {alpha}Eß7 by TGF-ß was completely impaired on T cells isolated from Smad7 transgenic mice in vitro (Fig. 1A). However, the reduction of CD3+ T cells in the intestinal epithelium was partial and significant numbers of CD3+ T cells were still present in the intestinal epithelium in Smad7 transgenic mice (Figs 2 and 3). Recently, Schon et al. reported that the number of intestinal IEL was reduced in {alpha}E (CD104)-deficient mice (13). Consistent with our results, they found that the number of intestinal IEL was diminished, but not completely lost, and significant numbers of intestinal IEL were present in {alpha}E-deficient mice. Some studies suggested that {alpha}Eß7 was not essential for the constitution of IEL (4,17) and ß2 integrin may be also involved in the localization of IEL (18). Taken together with our current findings, not only the regulation of {alpha}Eß7 by TGF-ß but also other molecular regulation mechanisms including ß2 integrins could be involved by which intestinal IEL localize in the epithelial area in vivo.

Interestingly, there were some {alpha}Eß7+ cells present in the epithelium of Smad7 transgenic mice (Figs 2 and 3). These findings suggested that there was redundancy in the regulation of {alpha}Eß7 expression on T cells and stimuli other than TGF-ß might be involved in the regulation of {alpha}Eß7 in vivo. Alternatively, it is possible that we did not achieve complete blockade of the Smad signaling pathway in vivo and ‘leakage’ of Smad7 occurred in Smad7 transgenic mice, which might lead to {alpha}Eß7 expression on some intestinal IEL in Smad7 transgenic mice. This issue should be investigated in future studies.

Subsets of intestinal IEL are suggested to play a role in induction of oral tolerance (16). Although we found that the number of intestinal IEL was reduced in Smad7 transgenic mice, oral tolerance was inducible in Smad7 transgenic mice to the same degree as that seen in the wild-type littermates. These findings indicated that reduced numbers of intestinal IEL in Smad7 transgenic mice did not affect induction of oral tolerance. It is possible that residual intestinal IEL present in Smad7 transgenic mice are sufficient for induction of oral tolerance. Since intestinal IEL are suggested to have various functions (19), we cannot exclude the possibility that other in vivo functions of intestinal IEL are affected in Smad7 transgenic mice.

In summary, we showed that TGF-ß-mediated up-regulation of {alpha}Eß7 was impaired on T cells isolated from Smad7 transgenic mice and that the number of intestinal IEL was reduced in the transgenic mice when compared with the wild-type littermates. We have thus provided evidence that regulation of {alpha}Eß7 expression by TGF-ß on T cells is involved in selective localization or retention of intestinal IEL in vivo. These findings demonstrate an important role of {alpha}Eß7 regulation by TGF-ß in modulating the homeostasis of T lymphocyte number in the intestine.


    Acknowledgements
 
We thank S. Miike, M. Hatano, T. Tokuhisa and I. Iwamoto for generation of Smad7 transgenic mice, K. Maeda, H. Ushio, T. Tokura, S. Nagata and H. Ohtsuka for discussion and technical assistance, Y. Yamashiro for support, and E. Kawasaki and M. Matsumoto for secretarial assistance. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture, Japan, and the Uehara Memorial Foundation for Biomedical Science (to A. N.).


    Abbreviations
 
PBL—peripheral blood lymphocyte

IEL—intraepithelial T lymphocyte

LPL—lamina propria T lymphocyte

OVA—ovalbumin

TGF—transforming growth factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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