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The Journal of Immunology, 2007, 178: 757-764.
Copyright © 2007 by The American Association of Immunologists, Inc.

Enforced Expression of Bcl-2 Partially Restores Cell Numbers but Not Functions of TCR{gamma}{delta} Intestinal Intraepithelial T Lymphocytes in IL-15-Deficient Mice1

Kenji Nakazato*,{dagger}, Hisakata Yamada*, Toshiki Yajima*,{dagger}, Yoshiko Kagimoto*, Hiroyuki Kuwano{dagger} and Yasunobu Yoshikai2,*

* Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; and {dagger} First Department of Surgery, Gunma University School of Medicine, Maebashi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-15 knockout (KO) mice have severely reduced numbers of TCR{gamma}{delta} intestinal intraepithelial T lymphocytes (i-IEL), suggesting requirements of IL-15 signaling in the development or maintenance of i-IEL. To determine an involvement of survival signals via Bcl-2 in IL-15-mediated homeostasis of TCR{gamma}{delta} i-IEL, we introduced a bcl-2 transgene into IL-15 KO mice. In situ apoptosis of TCR{gamma}{delta} i-IEL was decreased in Bcl-2 transgenic (Tg) x IL-15 KO mice compared with IL-15 KO mice. The enforced expression of Bcl-2 partially restored the numbers of TCR{gamma}{delta} i-IEL in IL-15 KO mice. However, effector functions of TCR{gamma}{delta} i-IEL, including cytokine production and cytotoxic activity, were not recovered in Bcl-2 Tg x IL-15 KO mice. Importantly, TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice expressed a reduced level of eomesodermin, a transcription factor critical for effector functions of NK cells and CD8+ T cells. Similar to the case of TCR{gamma}{delta} i-IEL, enforced expression of Bcl-2 restored the numbers but not the functions of NK cells in IL-15 KO mice. These results suggest that Bcl-2-mediated survival signal is involved in the IL-15-mediated homeostasis of TCR{gamma}{delta} i-IEL and NK cells, but other signals from IL-15 are critical for inducing transcription factors, such as eomesodermin for their effector functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-15 uses beta-chain of IL-2R and common cytokine receptor common {gamma}-chain (c{gamma})3 for signal transduction and thus shares many properties of IL-2 despite having no sequence homology with IL-2 (1, 2, 3, 4, 5). In contrast to IL-2, which is produced mainly by activated T cells, IL-15 is produced by wide variety of cells, including the placenta, skeletal muscle, kidney, as well as intestinal epithelium (6, 7). Recent study has shown that IL-15 is trans-presented by unique IL-15R{alpha} on IL-15-producing cells (8). Mice lacking IL-15R{alpha}, IL-2/15Rbeta, or IL-15 lack TCR{gamma}{delta} skin intraepithelial T lymphocytes (s-IEL), but have severely reduced but appreciable numbers of NK cells, NKT cells, TCR{gamma}{delta} intestinal-IEL (i-IEL), and memory phenotype CD8+ T cells (9, 10, 11, 12, 13, 14). Therefore, while IL-15-mediated signals are indispensable for the development of TCR{gamma}{delta} skin IEL, it has potential roles in the homeostasis of TCR{gamma}{delta} i-IEL, NK cells, NKT cells, and memory phenotype CD8+ T cells.

Homeostasis of peripheral lymphocytes is regulated by proliferation and apoptosis. It has been shown that IL-15 is a potent inhibitor of apoptotic pathways in lymphocytes, inducing antiapoptotic molecules such as Bcl-2 and Bcl-xL (15, 16, 17, 18). Previous studies have shown that IL-15 is critical for the maintenance of NK cells by up-regulating expression levels of Bcl-2 (18, 19, 20). We also reported that IL-15 promoted survival of mouse TCR{gamma}{delta} i-IEL and memory CD8+ T cells through up-regulation of Bcl-2 expression (21, 22). Involvement of Bcl-2-mediated signals in the maintenance of lymphocytes in vivo has been addressed more directly by introducing bcl-2 transgenes (23). Minagawa et al. (24) reported that enforced expression of Bcl-2 restored the number of NK cells but not TCR{gamma}{delta} i-IEL in IL-2/15Rbeta knockout (KO) mice. Thereafter, it was suggested that the decreased number of TCR{gamma}{delta} i-IEL in IL-2/IL-15Rbeta KO mice might be resulted primarily from impaired development or proliferation of TCR{gamma}{delta} i-IEL (24). Consistent with this, we found that IL-15 induced proliferation of TCR{gamma}{delta} i-IEL (21). However, IL-2 has also been shown to participate in the development of TCR{gamma}{delta} i-IEL (25, 26), leaving a possibility that a lack of IL-2-mediated signaling is also involved in the mechanism of reduced numbers of TCR{gamma}{delta} i-IEL in IL-2/15Rbeta KO mice.

To directly address the importance of survival signals via Bcl-2 in the IL-15- mediated homeostasis of TCR{gamma}{delta} i-IEL, we crossed IL-15 KO mice with human Bcl-2 Tg mice. We found that enforced expression of Bcl-2 partially restored the number, but not functions of TCR{gamma}{delta} i-IEL in IL-15 KO mice. Furthermore, the recovered {gamma}{delta} i-IEL developed in Bcl-2 Tg x IL-15 KO mice expressed a reduced level of eomesdermin, transcription factor shown to be critical for effector function of NK and CD8+ T cells (27, 28). These results indicate nonredundant roles of IL-15 in the development of functional TCR{gamma}{delta} i-IEL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6-background IL-15 KO mice were purchased from Taconic Farms. Eµ-bcl-2-25 Tg mice, which express human Bcl-2 under the control of the 5' IgH enhancer (Eµ) in T cells, were described previously (29). Mice heterozygous for the human bcl-2 transgene were used as Bcl-2 Tg mice. Age- and sex-matched C57BL/6 mice obtained from Japan SLC were used as control mice. All mice were kept in specific pathogen-free conditions in our laboratory and were used at 6–8 wk of age.

Cell preparation

I-IEL, s-IEL, splenocytes, liver lymphocytes, bone marrow cells, and PBMC were prepared as described previously (30, 31, 32). The number of viable cells was counted by staining of trypan blue or tulk’s solution. To examine the survival of TCR{gamma}{delta} i-IEL, i-IEL were cultured without stimulation for 24 h at 37°C under 5% CO2 in 96-well flat-bottom plates (Falcon; BD Biosciences) in a volume of 0.2 ml of RPMI 1640 containing 10% FBS. At various time points, cells were harvested, and the numbers of surviving TCR{gamma}{delta} i-IEL were calculated after the flow cytometric analysis. In some experiments, TCR{gamma}{delta} i-IEL were positively separated by MACS (Miltenyi Biotec) using FITC-conjugated anti-TCR{gamma}{delta} mAb with anti-FITC microbeads. The purity of TCR{gamma}{delta} i-IEL was >90% as assessed by flow cytometry.

Flow cytometric analysis and mAbs

For flow cytometric analysis, isolated cells were preincubated with an Fc{gamma}R-blocking mAb (CD16/32; 2.4G2) for 15 min at 4°C. Then the cells were incubated with saturating amounts of FITC-, PE-, Cy-Chrome-, and biotin-conjugated mAbs for 30 min at 4°C. The following mAbs were used: FITC-conjugated anti-CD3e (145-2C11), anti-CD8{alpha} (53-6.7), anti-TCRbeta (H57-597), and anti-TCR{gamma}{delta} (GL-3) mAbs; PE-conjugated anti-TCRbeta (H57-597), anti-TCR{gamma}{delta} (GL-3), anti-NK1.1 (PK136), anti-CD122 (5H4), anti-CD132 (4G3), anti-CD25 (PC61.5), anti-CD127 (A7R34), anti-NKG2D (CX5), anti-Ly49 (14B11), and anti-Thy1.2 (53-2.1) mAbs; CyChrome-conjugated anti-CD3{epsilon} (145-2C11) and anti-TCRbeta (H57-597) mAbs; biotin-conjugated anti-TCR{gamma}{delta} (UC7–13D5), anti-CD8beta (Ly-3.2), and anti-DX-5 (DX-5) mAbs. To detect biotin-conjugated mAb, cells were stained with CyChrome- or APC-conjugated streptavidin. The stained cells were run on a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed with CellQuest software (BD Biosciences). To detect of mouse and human Bcl-2, intracellular staining was performed using the Cytofix/Cytoperm kit (BD Pharmingen) and the following mAbs: FITC-conjugated anti-human Bcl-2 (DakoCytomation) or hamster anti-mouse Bcl-2 (3F11) (BD Pharmingen). Apoptosis of TCR{gamma}{delta} i-IEL was determined by staining with Annexin VFITC conjugate according to the manufacturer’s instructions.

RT-PCR and Southern blot analysis

Total RNA was extracted by the acid-guanidinium-phenol-chloroform method and was primed with 20 pmol of random primer in 20-µl reaction mixtures for reverse transcription. For determination of the V{gamma}/{delta} repertoire, synthesized cDNA was amplified by PCR using specific 3' primers for C{gamma} (5'-CTTATGGAGGATTTGTTTCAGC-3') or C{delta} (5'-CGAATTCCACAATCTTCTTG-3') and specific 5' primers for V{gamma}1/2 (5'-ACACAGCTATACATTGGTAC-3'), V{gamma}2 (5'-CGGCAAAAAACAAATCAACAG-3'), V{gamma}4 (5'-TGTCCTTGCAACCCCTACCC-3'), V{gamma}5 (5'-TGTGCACTGGTACCAACTGA-3'), V{gamma}6 (5'-GGAATTCAAAAGAAAACATTGTCT-3'), V{gamma}7 (5'-AAGCTAGAGGGGTCCTCTGC-3'), V{delta}1 (5'-ATTCAGAAGGCAACAATGAAAG-3'), V{delta}2 (5'-AGTTCCCTGCAGATCCAAGC-3'), V{delta}3 (5'-TTCCTGGCTATTGCCTCTGAC-3'), V{delta}4 (5'-CCGCTTCTCTGTGAACTTCC-3'), V{delta}5 (5'-CAGATCCTTCCAGTTCATCC-3'), V{delta}6 (5'-TCAAGTCCATCAGCCTTGTC-3'), V{delta}7 (5'-CGCAGAGCTGCAGTGTAACT-3'), and V{delta}8 (5'-AAGGAAGATGGACGATTCAC-3'). Southern blots of the V{gamma} or V{delta} PCR products were hybridized with 32P-labeled C{gamma}2 (MNG6) probe or J{delta}1 (5'-TTGGTTCCACAGTCACTTGG-3') or J{delta}2 (5'-CTCCACAAAGAGCTCTATGCCCA-3') oligonucleotide probe. The radioactivity of each band of PCR product was analyzed with a Fujix BAS2000 Bio-image analyzer (Fuji). For examination of the eomesdermin/T-bet expression, cDNA synthesized from the mRNA of purified TCR{gamma}{delta} i-IEL was amplified using 10 pmol of each primer specific for murine beta-actin, eomesodermin or T-bet. The specific primers were as follows: beta-actin sense, 5'-GGAATCCTGTGGCATCCATGAAAC-3'; antisense, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'; eomesodermin sense, 5'-TGAATGAACCTTCCAAGACTCAGA-3'; antisense, 5'-TGTCAACACTTTGCCTCAAGCC-3'; T-bet sense, 5'-CCTGCAGTGCTTCTAACACACAC-3'; antisense, 5'-GAACACAGTTATGAAGCGGAG-3'.

Cytokine ELISA

The MACS-purified {gamma}{delta} T cells (5 x 104/well) were incubated in the anti-CD3 mAb-coated plates for 48 h. IFN-{gamma} levels in the culture supernatants were determined by ELISA (Genzyme) according to the manufacturer’s instructions.

Cytotoxic assay

TCR{gamma}{delta} i-IEL were incubated with 103 51Cr-labeled UC-7 hybridomas expressing anti-TCR{gamma}{delta} mAb at varying E:T ratios for 4 h. NK activities of spleen cells from mice pretreated i.p. with 150 µg of poly(I:C) 24 h previously were assessed by a standard 4-h 51Cr release assay using an NK-sensitive YAC-I cell line. The spleen cells were incubated at the indicated E:T ratios with 104 51Cr-labeled YAC-1 cells in 96-well round-bottom plates. Cytotoxicity was calculated as follows: (experimental release – spontaneous release)/(total release – spontaneous release) x 100.

Statistical analysis

The statistical significance of the data was determined by Student’s t test. The value of p < 0.05 was taken as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Enforced expression of Bcl-2 partially restores the numbers of TCR{gamma}{delta} i-IEL

It has been shown that IL-15 plays a critical role in the development of TCR{gamma}{delta} i-IEL and TCR{gamma}{delta} s-IEL (9, 12). Consistent with previous studies, TCR{gamma}{delta} skin IEL almost completely vanished in IL-15 KO mice and TCR{gamma}{delta} i-IEL were selectively reduced in IL-15 KO mice (Fig. 1, A–C). As we have found that IL-15 promoted survival of mouse TCR{gamma}{delta} i-IEL through up-regulation of Bcl-2 molecules in vitro (21), we examined intracellular expression of Bcl-2 in TCR{gamma}{delta} i-IEL in IL-15 KO mice. Compared with TCR{gamma}{delta} i-IEL in the control C57BL/6 mice, those in IL-15 KO mice showed severely reduced expression of Bcl-2 (Fig. 1D), suggesting an impaired survival of TCR{gamma}{delta} i-IEL in IL-15 KO mice in vivo. To directly examine an involvement of the reduced expression of Bcl-2 in the decreased number of TCR{gamma}{delta} i-IEL in IL-15 KO mice, we introduced a human bcl-2 transgene driven by Eµ promoter into IL-15 KO mice. Expression of exogenous human Bcl-2 in TCR{gamma}{delta} i-IEL was detected in both Bcl-2 Tg mice and Bcl-2 Tg x IL-15 KO mice, although the expression level was slightly higher in Bcl-2 Tg mice (Fig. 1D). There were no differences in the expression levels of endogenous mouse Bcl-2 between IL-15 KO and Bcl-2 Tg x IL-15 KO mice. The percentage of TCR{gamma}{delta} cells in i-IEL of Bcl-2 Tg x IL-15 KO mice was comparable to that of the control WT mice (Fig. 1B). Absolute number of TCR{gamma}{delta} i-IEL in IL-15 KO was also significantly increased by the enforced expression of Bcl-2 (p < 0.05; Fig. 1C). Although the increase of TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice was seen in both CD8{alpha}{alpha} and CD8{alpha}beta TCR{gamma}{delta} cells, statistically significant increase in the number was detected only in CD8{alpha}{alpha} TCR{gamma}{delta} cells (p < 0.01). It is of note that enforced expression of human Bcl-2 did not influence the number of TCR{gamma}{delta} i-IEL in WT C57BL/6 mice, suggesting endogenous Bcl-2, which is induced by basal levels of IL-15, is sufficient for the maintenance of TCR{gamma}{delta} i-IEL.


Figure 1
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  		FIGURE 1. Enforced expression of Bcl-2 restored the number of TCR{gamma}{delta} i-IEL in IL-15 KO mice. A, IEL from the skin of WT, Bcl-2 Tg, IL-15 KO, or Bcl-2 Tg x IL-15 KO mice were analyzed for the presence of Thy1.2+ TCR{gamma}{delta}+ cells by a flow cytometer. B, Flow cytometric analysis of i-IEL from WT, Bcl-2 Tg, IL-15 KO, and Bcl-2 Tg x IL-15 KO mice. Analysis gate was set as indicated. Five mice for each group were analyzed independently, and representative profiles are shown in the figures. C, Absolute number of TCR{gamma}{delta}, CD8{alpha}{alpha}TCR{gamma}{delta}, and CD8{alpha}betaTCR{gamma}{delta} cells in i-IEL was calculated by multiplying total cells by the percentage of each cell population. Values are means ± SD for five mice in each group. *, p < 0.05; **, p < 0.01. D, Expression of Bcl-2 in TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice. Freshly isolated i-IEL from WT, Bcl-2 Tg, IL-15 KO, or Bcl-2 Tg x IL-15 KO mice were surface stained with anti-TCR{gamma}{delta} mAb and then subjected to intracellular staining for mouse Bcl-2 or human Bcl-2. Data are representative of three independent experiments.

 
Apoptosis of TCR{gamma}{delta} i-IEL in vitro culture

We examined the percentage of apoptotic cells in freshly isolated TCR{gamma}{delta} i-IEL and the number of TCR{gamma}{delta} i-IEL after in vitro culture without any stimulation. As shown in Fig. 2A, freshly isolated TCR{gamma}{delta} i-IEL in IL-15 KO mice contained high percentage of apoptotic cells. Enforced expression of human Bcl-2 in TCR{gamma}{delta} i-IEL in IL-15 KO mice decreased the percentage of apoptotic cells. The number of TCR{gamma}{delta} i-IEL from IL-15 KO mice decreased to less 20% by 24 h, whereas the number of TCR{gamma}{delta} i-IEL from Bcl-2 Tg x IL15 KO mice decreased to only 80% after in vitro culture (Fig. 2B). These results clearly demonstrated that the decreased expression of Bcl-2, which might be resulted in increase of apoptotic cells, was involved in the mechanism of the decreased number of TCR{gamma}{delta} i-IEL in IL-15 KO mice.


Figure 2
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  		FIGURE 2. A, Annexin V staining of TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice. Freshly isolated i-IEL from IL-15 KO or Bcl-2 Tg x IL-15 KO mice were stained with anti-TCR{gamma}{delta} mAb and annexin V. B, In vitro culture of TCR{gamma}{delta} i-IEL without any stimulation. At various time points, the survival rate was calculated by dividing the numbers of surviving TCR{gamma}{delta} i-IEL by the numbers of preculture total TCR{gamma}{delta} i-IEL. Data are representative of three independent experiments.

 
V{gamma} and V{delta} gene expression by the rescued TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice

{gamma}{delta} T cells in specific tissue locations use distinct TCR V{gamma} regions. It has been shown that TCR{gamma}{delta} i-IEL mainly use V{gamma}7, V{gamma}1, and V{gamma}2 (33, 34, 35). To examine TCR repertoire of the TCR{gamma}{delta} i-IEL restored by enforced expression of Bcl-2 in an IL-15-deficient environment, we compared the V gene expressions of TCR{gamma}{delta} i-IEL of Bcl-2 Tg, IL-15 KO, Bcl-2 Tg x IL-15 KO and control WT mice. Total RNA was extracted from i-IEL from small intestine, and the V gene expressions were analyzed by RT-PCR. As have been reported, the TCR{gamma}{delta} i-IEL from control mice expressed V{gamma} 1/2, 2, and 7 (Fig. 3). In contrast, the few TCR{gamma}{delta} i-IEL that did develop in IL-15 KO mice were predominately V{gamma}1 and 2, whereas expression of V{gamma}7 was hardly detected. In contrast, the rescued TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice were virtually indistinguishable from those in control mice. Therefore, the increase in the TCR{gamma}{delta} i-IEL population in Bcl-2 Tg x IL-15 KO mice resulted from a selective increase of i-IEL that express the V{gamma}7. This also suggests that IL-15 induced up-regulation of Bcl-2 is specifically involved in the maintenance of TCR{gamma}{delta} i-IEL expressing V{gamma}7.


Figure 3
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  		FIGURE 3. V{gamma} or V{delta} usage by TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice. Total RNA extracted from i-IEL of WT, Bcl-2 Tg, IL-15 KO, or Bcl-2 Tg x IL-15 KO mice was reverse-transcribed into cDNA and amplified by PCR using primers for C{gamma} or C{delta} and various V{gamma} or V{delta} segments, respectively. The Southern blot of {gamma} and {delta} PCR products was hybridized with MNG6 cDNA containing the C{gamma}2 (A) and J{delta}1 or J{delta} 2 (B), respectively. Data are representative of three independent experiments.

 
Expression of surface molecules on TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice

We next compared the expression levels of CD25, CD122, CD127, and CD132 on i-IEL from control WT, Bcl-2 Tg, IL-15 KO and Bcl-2 Tg x IL-15 KO mice (Fig. 4). There were no differences in the expression levels of CD132 and CD25 on TCR{gamma}{delta} i-IEL in these four groups of mice. However, TCR{gamma}{delta} i-IEL in IL-15 KO mice or Bcl-2 Tg x IL-15 KO mice showed decreased expression of CD122 compared with WT or Bcl-2 Tg mice. In contrast the expression level of CD127 was increased in TCR{gamma}{delta} IEL in IL-15 KO mice or Bcl-2 Tg x IL-15 KO mice compared with WT or Bcl-2 Tg mice. Thus, although an enforced expression of Bcl-2 restores the number of TCR{gamma}{delta} i-IEL with normal TCR repertoire in IL-15 KO mice, it is not sufficient to generate phenotypically equivalent TCR{gamma}{delta} i-IEL.


Figure 4
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  		FIGURE 4. Expression of surface molecules on i-IEL in Bcl-2 Tg x IL-15 KO mice. Surface expression of CD122, CD132, CD25, or CD127 on TCR{gamma}{delta} i-IEL of WT, Bcl-2 Tg, IL-15 KO, or Bcl-2 Tg x IL-15 KO mice was examined by a flow cytometry. Staining with an isotype control Ab was overlaid on each histogram as a dotted line. The analysis gate was set on TCR{gamma}{delta}+ cells. Data are representative of three independent experiments.

 
IL-15 is indispensable for the functional maturation of TCR {gamma}{delta} i-IEL

The differential expression patterns of surface markers suggest TCR{gamma}{delta} i-IEL in IL-15 KO mice are also functionally different from those in WT mice. Therefore, we next compared IFN-{gamma} production by the TCR{gamma}{delta} i-IEL in response to stimulation with anti-CD3 mAb. As shown in Fig. 5, purified TCR{gamma}{delta} i-IEL from WT mice produced substantial amount of IFN-{gamma}, whereas those from IL-15 KO mice as well as Bcl-2 Tg x IL-15 KO mice produced only small amount of IFN-{gamma} (p < 0.01). The defect in IFN-{gamma} production by TCR{gamma}{delta} i-IEL from Bcl-2 Tg x IL-15 KO mice was not due to the overexpression of human Bcl-2, as TCR{gamma}{delta} i-IEL in Bcl-2 Tg mice produced even higher levels of IFN-{gamma} than those in WT mice. We also compared redirectional CTL activities of the TCR{gamma}{delta} i-IEL. Similar to the case of IFN-{gamma} production, TCR{gamma}{delta} i-IEL in IL-15 KO mice or Bcl-2 Tg x IL-15 KO mice showed significantly lowered CTL activity to the target cells expressing anti-TCR{gamma}{delta} mAb compared with those in WT mice or Bcl-2 Tg mice (p < 0.01; Fig. 5). The observed defect in anti-CD3 mAb-induced IFN-{gamma} production or CTL activity of TCR{gamma}{delta} i-IEL in IL-15 KO mice or Bcl-2 Tg x IL-15 KO mice was not due to a decreased expression of CD3 molecules, as expression levels of CD3 were comparable between these four groups of mice (Fig. 5).


Figure 5
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  		FIGURE 5. Effector functions of the rescued TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice. CD3 expression level on TCR{gamma}{delta} i-IEL from WT, Bcl-2 Tg, IL-15 KO, or Bcl-2 Tg x IL-15 KO mice was examined by a flow cytometry. TCR{gamma}{delta} i-IEL were positively enriched using auto MACS. The sorted TCR{gamma}{delta} i-IEL (5 x 104 cells/well) were cultured with immobilized anti-CD3 mAb (20 µg/ml) for 48 h at 37°C. Amount of IFN-{gamma} in the culture supernatants was determined by ELISA. To examine redirected cytotoxicity, TCR{gamma}{delta} i-IEL were incubated with 103 51Cr-labeled hybridomas secreting anti-TCR{gamma}{delta} (UC-7) mAb for 4 h, at varying E:T ratios. Data are representative of three independent experiments and are shown as the mean of triplicates ± SD. Statistically significant differences between Bcl-2 Tg x IL-15 KO or IL-15 KO and WT or Bcl-2 Tg mice are shown (*, p < 0.01). N.D., Not detectable.

 
IL-15 is indispensable for the induction of eomesodermin in TCR {gamma}{delta} i-IEL

As shown in the above experiments, enforced expression of Bcl-2 only restored the numbers but not functions of TCR{gamma}{delta} i-IEL in IL-15 KO mice, suggesting the presence of IL-15-dependent signaling events other than inducing Bcl-2. Thereafter, we examined expression of T-bet and eomesodermin, transcription factors critical for effector functions, and homeostasis of NK and CD8+ T cells (28). As shown in Fig. 6, TCR{gamma}{delta} i-IEL from WT mice or Bcl-2 Tg mice express significant levels of both T-bet and eomesodermin. However, expression of T-bet and eomesodermin was reduced in TCR{gamma}{delta} i-IEL from Bcl-2 Tg x IL-15 KO mice. Because we were not able to obtain TCR{gamma}{delta} i-IEL from IL-15 KO mice with enough purity for RT-PCR analysis, this group was excluded in this experiment. Nevertheless, these results suggest that induction of T-bet and eomesodermin is one of the IL-15-induced events critical for the acquisition of effector functions of TCR{gamma}{delta} i-IEL, independent of Bcl-2 induction.


Figure 6
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  		FIGURE 6. Expression of mRNA of transcription factors in i-IEL. TCR{gamma}{delta}+ cells were purified from i-IEL of WT, Bcl-2 Tg, or Bcl-2 Tg x IL-15 KO mice. The purified TCR{gamma}{delta} i-IEL were subjected to RT-PCR analysis for eomesodermin and T-bet. beta-Actin was used as an internal control. The relative intensity of Bcl-2 Tg or Bcl-2 Tg x IL-15 KO to WT mice were calculated by density meter.

 
Enforced expression of Bcl-2 restores the numbers of NK cells but not NKT cells in IL-15 KO mice

IL-15 has also been shown to play critical roles in the development of NK cells and NKT cells. Therefore, we also examined the effects of enforced expression of Bcl-2 on the development of these lymphocytes in the absence of IL-15. As shown in Fig. 7A, there were reduced but substantial number of NK1.1+CD3– cells or NK1.1+CD3+ cells in the spleen, liver, bone marrow, and PBMC of IL-15 KO mice. Enforced expression of Bcl-2 in IL-15 KO mice significantly increased the absolute number of NK1.1+CD3– cells but not of NK1.1+CD3+ cells (p < 0.05; Fig. 7B). Thus IL-15-induced up-regulation of Bcl-2 might be differently involved in the maintenance of NK cells and NKT cells.


Figure 7
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  		FIGURE 7. Enforced expression of Bcl-2 restored the number of NK cells but not NK-T cells in IL-15 KO mice. A, Splenocytes, liver mononuclear cells, bone marrow cells, and PBMC from WT, Bcl-2 Tg, IL-15 KO, or Bcl-2 Tg x IL-15 KO mice were subjected to flow cytometric analysis for the expression of CD3 and NK1.1. Representative data are shown in the figures. B, Absolute number of NK1.1+CD3– and NK1.1+CD3+ cells in the spleen was calculated. Data are shown as means ± SD for five mice in each group. Statistically significant difference between IL-15 KO and Bcl-2 Tg x IL-15 KO mice are shown (*, p < 0.05).

 
Impaired functions of the restored NK cells in Bcl-2 Tg x IL-15 KO mice

We next examined the level of maturation of the rescued NK cells in Bcl-2 Tg x IL-15 KO mice in the spleen. Several cell surface markers, such as DX-5, CD122, NKG2D, and Ly-49, have been proposed as indicators of mature NK cells. As shown in Fig. 8A, NK cells from Bcl-2 Tg x IL-15 KO and IL-15 KO mice have profoundly reduced level of DX5, a marker shown to be up-regulated during NK development and reduced levels of NKG2D and Ly49, markers shown to be activating and inhibitory receptor. Surprisingly, NK cells, defined as NK1.1+CD3– cells, from Bcl-2 Tg x IL-15 KO and IL-15 KO mice expressed lower level of CD122, which is usually expressed before expression of NK1.1 during NK cell maturation. Therefore, the rescued NK cells in Bcl-2 Tg x IL-15 KO mice were phenotypically distinguishable from mature NK cells in control mice.


Figure 8
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  		FIGURE 8. Expression of surface molecules and cytotoxic function of NK cells in Bcl-2 Tg x IL-15 KO mice. A, Expression of DX5, CD122, NKG2D, and Ly49 on NK1.1+CD3– cells in WT, Bcl-2 Tg, IL-15 KO, and Bcl-2 Tg x IL-15 KO mice was examined by a flow cytometry. Staining with an isotype control Ab was overlaid on each histogram as a dotted line. B, Splenocytes from WT, Bcl-2 Tg, IL-15 KO, and Bcl-2 Tg x IL-15 KO mice pretreated with poly(I:C) 24 h previously were examined for cytotoxic activity against NK-sensitive YAC-I cell line. Data are representative of two independent experiments and expressed as the means of triplicate ± SD. Statistically significant differences between Bcl-2 Tg x IL-15 KO or IL-15 KO and WT or Bcl-2 Tg mice are shown (*, p < 0.01).

 
We lastly examined the function of NK cells developed in Bcl-2 Tg x IL-15 KO mice. Splenocytes from poly(I:C)-injected WT mice or Bcl-2 Tg mice exhibited strong NK activity against NK-sensitive target cells, whereas splenocytes from poly(I:C)-injected IL-15 KO mice or Bcl-2 Tg x IL-15 KO mice exhibited undetectable NK activity (Fig. 8B). Thus, these results indicate that IL-15-dependent signaling events other than inducing Bcl-2 is required for the development of functional NK cells, similar to the case of TCR{gamma}{delta} i-IEL.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
As TCR{gamma}{delta} i-IEL are severely decreased in IL-2/IL-15Rbeta KO mice (9), IL-15R{alpha} KO mice (10), or IL-15 KO mice (11), an importance of IL-15 mediated signaling in the development of TCR{gamma}{delta} i-IEL has been suggested, although the mechanism for their decrease is not well defined. In the present study, we showed that enforced expression of Bcl-2 partially restored the numbers of TCR{gamma}{delta} i-IEL in IL-15 KO mice, clearly indicating an involvement of IL-15 in survival of TCR{gamma}{delta} i-IEL through up-regulation of Bcl-2 expression. This is not simply due to a decreased apoptosis of the cells that undergo apoptosis even in normal IL-15 sufficient conditions, because enforced expression of Bcl-2 in WT mice did not affect the cell number of TCR{gamma}{delta} i-IEL.

Minagawa et al. (24) previously reported that enforced expression of Bcl-2 restored the number of NK cells but not TCR{gamma}{delta} i-IEL in IL-2/15Rbeta KO mice. They concluded that additional signals other than survival signals from IL-2/15R were important for the development or maintenance of TCR{gamma}{delta} i-IEL. In this regard, it is of note that the number of TCR{gamma}{delta} i-IEL is also decreased in IL-2 KO mice (26). Taken together with our results, it is speculated that defects of both IL-2 and IL-15 mediated signaling are involved in the decreased number of TCR{gamma}{delta} i-IEL in IL-2/15Rbeta KO mice. The primary role of IL-15 is up-regulation of Bcl-2 and prevention of apoptosis, whereas IL-2 has additional role for the optimal development of TCR{gamma}{delta} i-IEL. It is also possible that another unknown IL-2/15Rbeta-using cytokine plays a role in the homeostasis of TCR{gamma}{delta} i-IEL. In addition, there is a possible difference between these studies in the expression of transgenic Bcl-2. In the Bcl-2 Tg mice used in our study, transgenic bcl-2 was expressed under control of Igh E{alpha} enhancer (29), while Minagawa et al. (24) used Bcl-2 Tg mice in which transgenic human bcl-2 was driven by H-2 K promoter. Therefore, we cannot completely exclude the possibility that differential regulation of the expression of Bcl-2 results in difference in the number of restored TCR{gamma}{delta} i-IEL. We found the absolute number of TCR{gamma}{delta} i-IEL restored in Bcl-2 Tg x IL-15 KO mice did not reach the normal level (Fig. 1). The homeostasis of TCR{gamma}{delta} i-IEL is thought to depend on balance of cell death and cell division. IL-15 plays important roles not only in protection of memory CD8+ T cells from apoptosis via induction of Bcl-2 but also in homeostatic proliferation via activation of c-Myc (36). This may explain why enforced expression of Bcl-2 could not completely restore the number of TCR{gamma}{delta} i-IEL.

We found that the rescued TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice were nonfunctional as assessed by cytokine production and cytotoxic activity. This indicates that IL-15 is involved in functional maturation of TCR{gamma}{delta} i-IEL in addition to their survival. It is notable that the TCR{gamma}{delta} i-IEL in Bcl-2 Tg x IL-15 KO mice expressed a reduced level of T-box transcriptional factors, eomesodermin and T-bet. Eomesodermin is expressed by activated CD8+ T cells and NK cells and regulates expressions of genes encoding IFN-{gamma} and cytolytic molecules (27, 28). T-bet has a critical function in the differentiation and function of Th1 cells through regulating the expression of IFN-{gamma} and IL-12Rbeta2 (37, 38). T-bet also contributes to the expression of IFN-{gamma}, perforin, and granzyme B in activated CD8+ T cells and NK cells (39, 40). Therefore, the reduced expression of eomesodermin and T-bet in TCR{gamma}{delta} i-IEL developed in the absence of IL-15 might be, at least in part, related to their impaired effector functions. Recently, eomesodermin has been reported to be necessary and sufficient for the expression of CD122 that consequently confers cellular responsiveness to IL-15 (28). Interestingly, we found that the expression level of CD122 on the TCR{gamma}{delta} i-IEL was reduced in Bcl-2 Tg x IL-15 KO mice as well as IL-15 KO mice. Therefore, it is likely that expression of eomesodermin depends on IL-15-mediated signaling, which in turn up-regulates CD122 expression and further promotes maintenance of functional TCR{gamma}{delta} i-IEL by IL-15. Alternatively, IFN-{gamma} produced by TCR{gamma}{delta} i-IEL themselves may regulate gene expression of eomesodermin in an autocrine manner. Further investigation is required to clarify the signaling events downstream of IL-15, which control the function of TCR{gamma}{delta} i-IEL.

Similar to TCR{gamma}{delta} i-IEL, the number of NK cells was restored in IL-15 KO mice with enforced expression of Bcl-2, although these NK cells lack cytotoxic functions. Therefore, Bcl-2-mediated signals induced by IL-15 appear to be critical for the homeostasis of TCR{gamma}{delta} i-IEL and NK cells, but additional signals are necessary for acquisition of effector function by these lymphocytes. We have found that the rescued NK cells that developed in Bcl-2 Tg x IL-15 KO mice are nonlytic and are phenotypically different from mature splenic NK1.1+ cells, and they resemble immature NK1.1+ cells in neonatal and fetal mice. Although IL-15 induces a critical survival signals in the maintenance of NK cells, IL-15 may be involved in different stages of the NK cell development. The rescued NK cells also showed no cytotoxic activity in IL-2/15Rbeta KO mice (24). It has been shown that cell surface expression of most Ly-49 receptor is not detectable on immature NK cells and requires several weeks after birth to achieve adult expression profiles (41). The Ly-49 homodimers and CD94-NKG2 heterodimers are inhibitory or activation NK cell receptors expressed on overlapping subsets of mature NK cells (42). A recent study (43) showed that IL-15 was required for Ly-49 expression on NK cells. In this study, we found that the expression of Ly-49 and NKG2D was reduced on NK cells in Bcl-2 Tg x IL-15 KO mice. Therefore, IL-15-mediated signals might be involved in the expression of activating Ly-49 and NKG2 receptors by NK cells to recognize target cells.

In contrast to TCR{gamma}{delta} i-IEL, we found that enforced expression of Bcl-2 did not restore TCR{gamma}{delta} s-IEL in IL-15 KO mice. It was shown that the V{gamma}5/V{delta}1 TCR transgene completely rescued V{gamma}5+ cells in the fetal thymus and the skin of IL-7R{alpha} KO mice, whereas the same transgene failed to restore V{gamma}5+ cells in the skin of IL-2/15Rbeta KO mice (44). These results indicate that, while IL-7 is required for the expression of V{gamma}5/V{delta}1 TCR, IL-15 plays essential roles in either expansion or survival of TCR{gamma}{delta} skin IEL. However, recent studies showed that enforced expression of Bcl-2 did not restore TCR{gamma}{delta} IEL in the skin of not only IL-2/15Rbeta KO mice but also V{gamma}5/V{delta}1 TCR-transgenic IL-2/15Rbeta KO mice (44), suggesting that TCR{gamma}{delta} s-IEL most likely receive proliferation and survival signals from IL-15 independently of Bcl-2. In support of this, it was shown that exogenous addition of IL-15 to organ culture of fetal skin induced proliferation of V{gamma}5 s-IEL (45). Similar to the case with TCR{gamma}{delta} s-IEL, enforced expression of Bcl-2 did not restore the number of NKT cells in IL-15 KO mice. As BrdU incorporation studies have shown that peripheral NKT cells are slowly dividing in an IL-15-dependent manner (46), the primary role of IL-15 in the homeostasis of these cells may be to provide proliferation signals rather than survival signals.

Taken together, these results suggest that IL-15-mediated signaling is differentially required for the development, proliferation, survival, or functions of lymphocytes depending on cell types. It is of importance to clarify the difference in the signaling events induced by IL-15 in different lymphocyte populations.


   Acknowledgments
 
We thank Yohko Kobayashi and Kazue Kaneda for their excellent technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 by Funds for Japan Science and Technology, Grant-in-Aid for Scientific Research on Priority Areas and Young Scientists (B), Japan Society for Promotion of Science, and grants from the Japanese Ministry of Education, Science, and Culture (to Y.Y.), Yakult Bioscience Foundation (to Y.Y.), and Uehara Memorial Foundation (to Y.Y.). Back

2 Address correspondence and reprint requests to Dr. Yasunobu Yoshikai, Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail address: yoshikai{at}bioreg.kyushu-u.ac.jp Back

3 Abbreviations used in this paper: c{gamma}, common {gamma} chain; i-IEL, intestinal intraepithelial T lymphocyte; s-IEL, skin intraepithelial T lymphocyte; KO, knockout; Tg, transgenic. Back

Received for publication August 17, 2006. Accepted for publication November 7, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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