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The Journal of Immunology, 1999, 163: 5843-5850.
Copyright © 1999 by The American Association of Immunologists

IL-15 Promotes Survival But Not Effector Function Differentiation of CD8+ TCR{alpha}ß+ Intestinal Intraepithelial Lymphocytes1

Yein-Gei Lai*, Vasily Gelfanov2,*, Valentina Gelfanova2,*, Liudmila Kulik*, Ching-Liang Chu*,{dagger}, Sheau-Wen Jeng* and Nan-Shih Liao3,*

* Institute of Molecular Biology, Academia Sinica, and {dagger} Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD8 single-positive cells, including CD8{alpha}{alpha}+ and CD8{alpha}ß+ subsets, constitute the majority of TCR{alpha}ß+ intestinal intraepithelial lymphocytes ({alpha}ß iIEL) in mice. CD8+ {alpha}ß iIEL show significantly weaker responses to TCR stimulation in the presence of exogenous IL-2 than do CD8+ T cells of the central immune system. IL-15 is a T cell growth factor likely expressed in the intestine mucosa. To understand the role of IL-15 in CD8+ {alpha}ß iIEL biology, we compared the effects of exogenous IL-15 and IL-2 on the survival and primary responses of the two CD8+ {alpha}ß iIEL subsets in vitro. In contrast to the death of ~60% of both CD8{alpha}{alpha}+ and CD8{alpha}ß+ iIEL cultured in IL-2 with or without TCR stimulation, IL-15 promoted survival of the CD8{alpha}{alpha}+ subset in the presence of TCR stimulation and promoted survival of both subsets in the absence of TCR stimulation. The higher proliferation level of TCR stimulated CD8{alpha}{alpha}+ {alpha}ß iIEL cultured in IL-15 compared with those cultured in IL-2 is likely due to IL-15’s prosurvival effects. In addition, unlike exogenous IL-2, exogenous IL-15 did not support the effector functions of either iIEL subsets, including IFN-{gamma} production, IL-4-induced Th2 cytokine production, and anti-TCR mAb-redirected cytotoxicity. These findings demonstrate that IL-15 and IL-2 are functionally distinct and suggest that IL-15 plays a unique role in the maintenance of the CD8+ {alpha}ß iIEL pool in the absence of Ag stimulation and in the survival and expansion of CD8{alpha}{alpha}+ {alpha}ß iIEL upon Ag stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The majority of TCR{alpha}ß+ intestinal intraepithelial lymphocytes ({alpha}ß iIEL)4 in mice are CD8 single positive. These CD8+ iIEL consist of CD8{alpha}{alpha}+ and CD8{alpha}ß+ subsets at a ratio ranging from 1:1 to 1:2 (1). These iIEL are different from T cells of the central immune system in their developmental pathway (2, 3, 4), their TCR complex composition (5), and in their activation requirements (6). Numerous studies have indicated that {alpha}ß iIEL are less responsive than peripheral {alpha}ß T cells to TCR stimulation in vitro (6, 7, 8, 9, 10). Analyses of purified CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL subsets revealed an ~100-fold and ~ 30-fold lower proliferation responder frequency, respectively, compared with CD8+ lymph node (LN) cells in response to TCR stimulation in the presence of exogenous IL-2 (6). Earlier studies on Thy1- iIEL, which are enriched for CD8{alpha}{alpha}+ cells, also demonstrated Thy1- iIEL’s poor cytokine response to TCR stimulation (7, 8). Consistently, purified CD8{alpha}{alpha}+ {alpha}ß iIEL produce little IFN-{gamma} and TNF when activated through TCR in the presence of exogenous IL-2 (6). Because activated CD8{alpha}{alpha}+ iIEL produce few cytokine(s) to support their own growth, exogenous IL-2 is often provided during experimentation. However, IL-2 may not be the only growth factor used by these cells in situ.

IL-15 is a T cell growth factor that belongs to the same four-helix bundle cytokine family as IL-2 (9, 10). Despite the lack of significant homology to IL-2, IL-15 binds to IL-2R ß and the common {gamma} ({gamma}c) chains and results in signal transduction (9, 11). One novel IL-15R {alpha}-chain binds by itself to IL-15 with high affinity (12, 13). Distinct from the T cell-restricted expression of IL-2, the IL-15 message is detected in various tissues and cell types (9). In the intestine, the IL-15 message has been detected in freshly isolated intestinal epithelial cells (IEC), in iIEL, and in lamina propria mononuclear cells (14, 15). A recent study demonstrated that Listeria monocytogenes infection in vitro and in vivo induced IL-15 production by IEC (16). As {alpha}ß iIEL express IL-15R{alpha}, IL-2Rß, and the {gamma}c chains (15), they likely use IL-15 in situ. To understand the role of IL-15 in iIEL biology, we compared the effects of exogenous IL-15 and IL-2 on the survival and activation of CD8+ {alpha}ß iIEL subsets and found that the cytokines’ effects are quite different. In the absence of TCR stimulation, after 36 h of culturing, most CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL died in the presence of IL-2 but survived in the presence of IL-15. IL-15 also promoted the survival of CD8{alpha}{alpha}+ but not CD8{alpha}ß+ {alpha}ß iIEL that had received TCR stimulation. Consistently, CD8{alpha}{alpha}+ but not CD8{alpha}ß+ {alpha}ß iIEL proliferated significantly better when activated in the presence of IL-15 rather than in the presence of IL-2. However, unlike IL-2, IL-15 did not support cytokine production nor TCR-triggered cytotoxicity of TCR-stimulated CD8+ {alpha}ß iIEL subsets. These findings suggest a unique role for IL-15 in the maintenance of the CD8+ {alpha}ß iIEL pool in the absence of Ag stimulation and in the survival and expansion of CD8{alpha}{alpha}+ {alpha}ß iIEL upon Ag stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

C57BL/6 mice purchased from National Cheng-Kung University (Tainan, Taiwan) were bred at the animal facility at the Institute of Molecular Biology, Academia Sinica under specific pathogen-free conditions. Eight- to 12-wk-old mice were used.

Abs

Pure Ab used included anti-CD8 (clone 3.155) (17), anti-TCRß (clone H57.597) (18), anti-mouse IL-2 (clone S4B6), and anti-IFN-{gamma} mAb (clone R4-6A2), and goat anti-mouse IL-15R{alpha} (C-19) polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA). Conjugated Ab included anti-CD4-FITC (clone RM-4-5; PharMingen, San Diego, CA), anti-CD8{alpha}-PE or -biotin (clone 53-6.7; Caltag Laboratories, San Francisco, CA) (19), anti-CD8ß-FITC or -biotin (clone 53-5.8) (20), anti-TCR{delta}-FITC (clone GL3) (21), anti-IL2R{alpha}-PE (clone PC 61.5.3; Caltag Laboratories), anti-IL2Rß-FITC (clone TM-ß1; PharMingen), anti-{gamma}c-biotin (clone 4G3; PharMingen), and donkey-anti-goat IgG FITC (Jackson ImmunoResearch Laboratories, West Grove, PA).

Preparation of CD8+ iIEL subsets and LN cells

Total iIEL were isolated as previously described (22). Briefly, iIEL were dissociated from small intestine pieces in Ca2+- and Mg2+-free Hank’s balanced salt solution (Life Technologies, Grand Island, NY) containing 1 mM DTT and 1 mM EDTA, and enriched by filtration through a nylon wool column and by centrifugation in a discontinuous Percoll gradient (44%/67%). Total LN cells and iIEL were enriched for CD8+ cells by positive panning on plates precoated with anti-CD8 mAb (clone 3.155). The CD8-enriched LN cells were stained with anti-CD4-FITC and anti-CD8{alpha}-PE and then sorted for CD8+ cells by using FACStarPlus (Becton Dickinson, Mountain View, CA). CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL subsets were purified by three-color sorting after staining the CD8-enriched iIEL with anti-CD4-FITC, anti-TCR{delta}-FITC, anti-CD8{alpha}-PE, and anti-CD8ß-biotin mAb, and then followed with streptavidin (SA)-allophycocyanin (APC) (Caltag Laboratories). The purity of each CD8+ subset was routinely above 98%. No contamination of TCR{gamma}{delta}+ cells was detected in CD8{alpha}{alpha}+ TCR{alpha}ß+ iIEL cultures stimulated for 11 days. Panning on anti-CD8 mAb and staining with anti-CD8{alpha} and anti-CD8ß mAb did not activate nor inactivate iIEL or LN cells (data not shown).

Analysis of IL-15R and IL-2R expression

CD8+ LN cells were enriched by positive panning of total LN cells on an anti-CD8 mAb-coated plate. CD8+ {alpha}ß iIEL were enriched by positive panning of total iIEL on an anti-CD8 mAb-coated plate and then by negative panning on an anti-TCR{delta} mAb-coated plate. Expression of IL-15R and IL-2R by CD8+ LN cells and {alpha}ß iIEL subsets were examined by three-color FACS analysis before and after activation by immobilized anti-TCRß mAb in the presence of recombinant human (rh) IL-15 (200 ng/ml) or recombinant murine (rm) IL-2 (20 ng/ml). CD8{alpha}ß+ LN cells and iIEL were distinguished by staining with anti-CD8ß mAb. CD8{alpha}{alpha}+ {alpha}ß iIEL were distinguished by gating out the CD8{alpha}+, CD4-, CD8ß-, and TCR{delta}- population from the CD8-enriched iIEL after staining with respective mAb. IL-2R {alpha}- and ß-chains and the {gamma}c chain were detected by cell-surface staining with each specific mAb in combination with each of the three iIEL subsets’ mAb. Cells stained with each subset’s mAb alone served as negative controls. IL-15R{alpha} was detected by intracellular staining with a goat anti-mouse IL-15R{alpha} Ab and then with donkey-anti-goat Ig FITC of permeabilized cells that had previously been fixed in paraformaldehyde after staining with each subset’s mAb. Goat Ig instead of goat anti-mouse IL-15R{alpha} Ab was used to prepare negative controls.

Proliferation assay

Purified CD8+ LN cells and CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL were activated in a 96-well half-area plate precoated with anti-TCRß mAb (0.1 µg/well) in tissue culture medium containing rhIL-2 (Hoffmann-La Roche, Nutley, NJ), rmIL-2 (R & D Systems, Minneapolis, MN), or rhIL-15 (R&D Systems) for the indicated periods of time. The tissue culture medium used in all experiments was RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 2 mM L-glutamine, 20 mM HEPES, 2000 U/L penicillin/streptomycin, 5 x 10-5 M 2-ME, and 10% FCS (Life Technologies). Cell proliferation was measured by [3H]TdR incorporation by pulsing the cells with 1 µCi/well of [3H]TdR (Amersham, Buckinghamshire, U.K.) for 12 h before harvesting. All samples were set up in triplicate. Recombinant hIL-2 and rhIL-15 were used at 50 U/ml and 200 ng/ml, respectively, for both iIEL and LN cells unless otherwise indicated, whereas rmIL-2 was used at 20 ng/ml for iIEL and 5 ng/ml for LN cells. These concentrations of rIL-2 and rIL-15 were found to support maximal proliferation of the corresponding cell types (data not shown).

Frequency analysis

Frequency assay was designed based on the principle of limiting dilution (23). Wells of Terasaki plates (Nunc, Roskilde, Denmark) were coated with 10 µl of anti-TCRß mAb (20 µg/ml) overnight at 4°C. After removal of unbound mAb, purified CD8+ LN cells and {alpha}ß iIEL subsets were cultured in these coated wells in 10 µl of tissue culture medium containing rhIL-2 (50 U/ml) or IL-15 (200 ng/ml). Four different numbers of cells per well were chosen for each cell type, and 30 wells were set up for each culture condition. Cell morphology was examined under microscope on day 8. Wells containing live blast cells, as characterized by enlarged cell size and intact and smooth cell margins, were scored positive for proliferation. Wells that did not contain live blast cells were scored as negative wells. The validity of the scoring method was ensured by determining that the plot of percentage negative wells against the number of cells per well showed Poison distribution (plot not shown). The fraction of negative wells (F0) was used to calculate the proliferation responder frequency, which equals µ/N where µ equals -lnF0 and N represents numbers of cells in each well. For negative controls, each type of cell in a quantity equal to the highest number of cells per well used in each experiment was cultured in either IL-2 or in IL-15 without TCR stimulation. No positive well was found among the negative controls.

Apoptosis assay

Cells were washed and stained with Annexin-V-FLUOS (Boehringer Mannheim, Mannheim, Germany) following the manufacturer’s instructions, and then analyzed immediately by using FACSCalibur (Becton Dickinson).

ELISA

Cytokine contents in supernatants collected from stimulated iIEL and LN cell cultures were determined by ELISA using a purified unconjugated capture mAb and a biotinylated detecting mAb (24, 25). The mAb pairs were purchased from PharMingen, including R4-6A2 and XMG1.2-biotin for IFN-{gamma}, BVD4-1D11 and BVD6-24G2-biotin for IL-4, TRFK5 and TRFK4-biotin for IL-5, and JES5-2A5 and SXC-1-biotin for IL-10. The sensitivity of the ELISA assays was 39 pg/ml for IFN-{gamma}, 8 pg/ml for IL-4, 16 pg/ml for IL-5, and 39 pg/ml for IL-10.

Cytotoxicity assay

Effector cells were harvested and centrifuged through Ficoll to remove dead cells. Target cells P815 were labeled with Na51Cr (100 µCi/106 cells/100 µl RPMI 1640–10% FCS) (Amersham) at 37°C for 1.5 h, washed, and then mixed (2000 cells/well) with effector cells at indicated ratios in 200 µl RPMI 1640–5% FCS/well of a 96-well V-bottom plate in the presence of 3 µg/ml of anti-TCR-ß mAb (clone H57.597). After incubation for 4 h at 37°C, cells were pelleted by centrifugation, and 100 µl of supernatant was collected from each well and counted for radioactivity by a gamma counter. Percent of specific lysis was calculated as described (26).

Statistics

Model I ANVOA (27) was used to determine the statistical significance of differences in proliferation responder frequency (Table IGo) and in cytokine production (see Figs. 5Go and 6Go) among cells receiving different cytokine treatments. For experiments involving three different cytokine treatments (IL-2, IL-15, and IL-15 plus anti-IL-2 mAb), the significance between each two treatment groups was confirmed by analysis of data pairs using the Welsch step-up procedure (28).


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  		Table I. Frequency analysis of the proliferation respondersa

 


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  		FIGURE 5. IL-15 is less supportive than IL-2 for IFN-{gamma} production by TCR-stimulated CD8{alpha}ß+ iIEL and LN cells. Sorted CD8{alpha}ß+ iIEL (105 cells/well) and LN cells (104 cells/well) were activated with immobilized anti-TCRß mAb in the presence of rmIL-2, rhIL-15, or rhIL-15 plus anti-mIL-2 mAb (15 µg/ml) for 7 days. Cells were transferred to new wells without Ab coating to rest for 1 day, and then restimulated at 106 cells per well for 24 and 48 h under conditions similar to primary activation. IFN-{gamma} content in the primary supernatants ({square}) and 24-h () and 48-h () secondary supernatants were determined by ELISA. A presents the data from one of three independent experiments. B tabulates the values of p for each cell type obtained from Model I ANOVA of the three experiments. The amount of anti-mIL-2 mAb used was enough to block proliferation of sorted CD8+ LN cells activated with immobilized anti-TCRß mAb and 20 ng/ml of exogenous mIL-2 (data not shown).

 


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  		FIGURE 6. IL-15 did not support IL-4-directed Th2 cytokine production by TCR-stimulated CD8+ {alpha}ß iIEL subsets and LN cells. Sorted CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL and CD8+ LN cells were activated with immobilized anti-TCRß (1 µg/well of 24-well plate) and anti-CD28 (8 µg/well) mAb together with rmIL-4 (400 U/ml) and anti-IFN-{gamma} mAb (10 µg/ml) in the presence of IL-2, IL-15, or IL-15 plus IL-2-neutralizing mAb (15 µg/ml) for 8 days. Each culture was fed with 0.1 ml of fresh medium containing the respective cytokine and mAb every 2 days. After resting for 1 day in noncoated wells, cells were washed and restimulated under the same conditions as in the primary activation except that IL-4 and anti-IFN-{gamma} mAb were not included. Supernatants collected 24 and 48 h after restimulation were analyzed for IL-4, IL-5, and IL-10 contents by ELISA. A shows the data from one of four independent experiments. B tabulates the value of p for each cell type calculated by Model I ANOVA of the data of the four independent experiments.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IL-15R and IL-2R

Expression of IL-2R and IL-15R by CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL was analyzed before and after TCR stimulation in the presence of IL-2 or IL-15 (Fig. 1Go). CD8+ LN cells treated in the same manner were also analyzed for comparison purposes. Before TCR stimulation (0 h), all three subsets expressed low levels of IL-2R ß and {gamma}c chains and no detectable level of IL-2R{alpha}. IL-15R{alpha} was also expressed by all three subsets before stimulation. TCR stimulation induced IL-2R{alpha} expression by all CD8{alpha}ß+ {alpha}ß iIEL and LN cells, but only by a fraction of CD8{alpha}{alpha}+ {alpha}ß iIEL. Higher IL-2R{alpha} expression was found in LN cells stimulated in IL-2 than in those stimulated in IL-15 for 4 h. This difference subsided after 18 h of TCR stimulation. The level of IL-2R{alpha} expression was similar between iIEL stimulated either in IL-2 or IL-15. TCR stimulation also enhanced expression of IL-15R{alpha} and {gamma}c by all three subsets. The level of IL-2R{alpha}, IL-15R{alpha}, and {gamma}c increased with the length of stimulation time. The IL-2Rß staining was too faint to analyze the change in expression level, although the same amount of anti-IL-2Rß mAb stained activated {gamma}{delta} iIEL to an extent such that the positive peak was clearly distinguishable from the negative peak (data not shown). Because both CD8+ {alpha}ß iIEL subsets express IL-15R{alpha} but not IL-2R{alpha} before TCR stimulation, they may use IL-15 more readily than IL-2. The IL-2R{alpha}- subpopulation of TCR-stimulated CD8{alpha}{alpha}+ {alpha}ß iIEL may also use IL-15 more readily than IL-2.



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  		FIGURE 1. Expression of IL-2R and IL-15R by CD8+ {alpha}ß iIEL and LN cells. LN and iIEL enriched for CD8+ and CD8+ TCR{alpha}ß+ cells, respectively, were prepared as described in Materials and Methods. Expression of IL-2R {alpha}-, ß-, and {gamma}-chains and the IL-15R{alpha} by cells before and after TCR stimulation in the presence of IL-2 or IL-15 were determined. Cell activation and staining conditions are described in Materials and Methods. The filled peaks represent staining with the cytokine receptor-specific Ab, while the open peaks represent negative controls. Intracellular staining of anti-IL-15R{alpha} polyclonal Ab was found to localize in the cytoplasmic membrane by confocal microscopy (data not shown).

 
Differential effects of IL-15 and IL-2 on the proliferation of TCR-stimulated CD8+ {alpha}ß iIEL

To examine whether IL-15 affects iIEL growth, the proliferation kinetics of CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL subsets in exogenous IL-15 or IL-2 with or without TCR stimulation were determined. CD8+ LN cells treated in the same manner were included as positive controls. As shown in Fig. 2Go (left), all three types of cells proliferated, to various extents, in response to TCR stimulation in the presence of IL-15. The CD8{alpha}{alpha}+ {alpha}ß iIEL proliferated significantly better in IL-15 than in IL-2 at all time points examined. The CD8{alpha}ß+ iIEL and LN cells showed a similar level of proliferation in IL-2 and in IL-15 except at late time points, when higher proliferation in IL-15 than in IL-2 was observed. The growth-supporting effect of IL-15 was not mediated through induction of endogenous IL-2, because the addition of IL-2 neutralizing mAb to the IL-15 groups did not reduce the cellular yield (data not shown). In the absence of TCR stimulation (Fig. 2Go, right), little proliferation was observed in either iIEL subset cultured in IL-2 or in IL-15. In contrast, in the absence of TCR stimulation, CD8+ LN cells proliferated in IL-15 but not in IL-2 and exhibited a delayed kinetics compared with CD8+ LN cells that received TCR stimulation. The proliferation of unstimulated CD8+ LN cells in IL-15 likely reflects the response of memory cells (29).



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  		FIGURE 2. Differential effects of IL-15 and IL-2 on proliferation of CD8+ {alpha}ß iIEL. Sorted CD8{alpha}{alpha}+ {alpha}ß iIEL (square), CD8{alpha}ß+ {alpha}ß iIEL (triangle), and CD8+ LN cells (circle) at 104 cells/100 µl/well were cultured in rhIL-2 (open symbols) or in IL-15 (filled symbols) in wells with or without immobilized anti-TCRß mAb for the indicated periods of time. Fresh medium (25 µl) containing the respective cytokine was added into each well at 96 and 216 h. Cell proliferation was determined by [3H]TdR incorporation as described in Materials and Methods. Similar results were obtained from three independent experiments.

 
To understand the differences between the proliferation response of CD8{alpha}ß+ and CD8{alpha}{alpha}+ {alpha}ß iIEL subsets (Fig. 2Go), the proliferation responder frequency was determined (Table IGo). CD8{alpha}ß+ and CD8{alpha}{alpha}+ {alpha}ß iIEL stimulated in the presence of exogenous IL-2 showed ~35- and 150-fold lower responder frequency, respectively, than did identically treated CD8+ LN cells. The low responder frequency of iIEL likely contributed to their delayed proliferation kinetics compared with CD8+ LN cells (Fig. 2Go). Furthermore, a 4-fold higher responder frequency for CD8{alpha}{alpha}+ iIEL and a 2-fold lower responder frequency for CD8{alpha}ß+ iIEL were observed in cells stimulated in IL-15 compared with those stimulated in IL-2. This observation is consistent with the higher proliferation of CD8{alpha}{alpha}+ but not CD8{alpha}ß+ iIEL stimulated in IL-15 compared with the same iIEL stimulated in IL-2 up to day 9 of culturing (Fig. 2Go, left). The elevated proliferation of CD8{alpha}ß+ {alpha}ß iIEL stimulated in IL-15 on day 11 might reflect the continuous proliferation of the responders that started at lower numbers than the CD8{alpha}{alpha}+/IL-15 responders, whose proliferation plateaued on day 9 probably due to cytokine and/or nutrient consumption. These results demonstrate that IL-15 is a growth factor for both CD8+ {alpha}ß iIEL subsets in response to TCR stimulation and that CD8{alpha}{alpha}+ {alpha}ß iIEL proliferated better when activated in the presence of IL-15 than in the presence of IL-2.

Differential effects of IL-15 and IL-2 on the survival of CD8+ {alpha}ß iIEL

Although CD8{alpha}{alpha}+ {alpha}ß iIEL stimulated in IL-15 and CD8{alpha}ß+ iIEL stimulated in IL-2 showed similar responder frequencies (Table IGo), the level of [3H]TdR incorporation was significantly higher in the former group, suggesting that IL-15 may affect aspect(s) of cell biology other than proliferation. We hence examined the effects of IL-15 vs IL-2 on the death/survival of these cells. CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL were cultured in IL-2 or IL-15 in wells with or without immobilized anti-TCRß mAb and monitored for cell death at various time points up to 36 h, at which point little increase in cell numbers occurred due to proliferation (data not shown). CD8+ LN cells were treated in identical manners for comparison purposes. As shown in Fig. 3Go, <20% of CD8+ LN cells died under all four culture conditions, while up to 60% of CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL died when cultured in the presence of IL-2. In contrast to IL-2, IL-15 protected the CD8{alpha}{alpha}+ {alpha}ß iIEL from apoptosis regardless of TCR stimulation, but only protected the CD8{alpha}ß+ iIEL in the absence of TCR stimulation. These results are consistent with the higher proliferation and responder frequency of CD8{alpha}{alpha}+ iIEL stimulated in IL-15 compared with those stimulated in IL-2, and with the higher proliferation of CD8{alpha}{alpha}+ {alpha}ß iIEL stimulated in IL-15 compared with CD8{alpha}ß+ iIEL stimulated in either cytokine (Fig. 2Go). Together, these findings demonstrate the different roles of IL-15 vs the roles of IL-2 in the growth and survival of CD8+ {alpha}ß iIEL subsets and the preferential survival-promoting effect of IL-15 on TCR-stimulated CD8{alpha}{alpha}+ {alpha}ß iIEL.



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  		FIGURE 3. Differential effects of IL-15 and IL-2 on the death/survival of CD8+ {alpha}ß iIEL. Sorted CD8{alpha}{alpha}+ {alpha}ß iIEL at 4.1 x 104/100 µl/well, CD8{alpha}ß+ {alpha}ß iIEL at 3.75 x 104/100 µl/well, and CD8+ LN cells at 5 x 104/100 µl/well were cultured in rmIL-2 (triangle) or in IL-15 (circle) in wells precoated with (filled symbols) or without (open symbols) anti-TCRß mAb for the indicated periods of time. Cell death was determined by annexin V staining. Similar results were obtained from four independent experiments.

 
TCR-stimulated CD8{alpha}{alpha}+ {alpha}ß iIEL produce little IFN-{gamma}

To understand the role of IL-15 in the effector functions of CD8+ {alpha}ß iIEL, IL-15’s effects on cytokine responses were first examined. IFN-{gamma} is a major cytokine produced by CD8+ T cells of the central immune system in response to TCR stimulation and serves as a key effector and regulator for cell-mediated immune responses. However, CD8{alpha}{alpha}+ {alpha}ß iIEL stimulated in the presence of IL-2 produce little IFN-{gamma} (6, 7). Because IL-15 supports better growth of CD8{alpha}{alpha}+ {alpha}ß iIEL compared with IL-2, IL-15’s effect on IFN-{gamma} production by these cells was determined. CD8+ LN cells treated in identical manners were included as positive controls. As shown in Fig. 4Go, IL-15 and IL-2 supported proliferation of CD8{alpha}{alpha}+ {alpha}ß iIEL and CD8+ LN cells in response to TCR stimulation in a dose-dependent manner. Significant amounts of IFN-{gamma} were produced by LN cells stimulated in either cytokine, and the level of IFN-{gamma} production correlated with the level of proliferation. In contrast, CD8{alpha}{alpha}+ {alpha}ß iIEL stimulated in either IL-2 or IL-15 produced little IFN-{gamma} (0.7–1.6 ng/ml) regardless of their proliferation status.



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  		FIGURE 4. Activated CD8{alpha}{alpha}+ {alpha}ß iIEL produce little IFN-{gamma}. Sorted CD8{alpha}{alpha}+ {alpha}ß iIEL ({triangleup}) and CD8+ LN cells ({circ}) at 2 x 104 cells/well were activated with plate-bound anti-TCRß mAb in the presence of titrating amounts of rhIL-2 for 132 h or rhIL-15 for 72 h. Cell proliferation was measured by [3H]TdR incorporation (solid line). Supernatants collected immediately before pulsing were analyzed for IFN-{gamma} content (dashed line) by ELISA. The low IFN-{gamma} production by CD8{alpha}{alpha}+ iIEL activated in either IL-2 or IL-15 was observed in seven independent experiments.

 
IL-15 is less supportive than IL-2 for IFN-{gamma} production by TCR-stimulated CD8{alpha}ß+ iIEL and LN cells

The effects of IL-15 and IL-2 on IFN-{gamma} production by CD8{alpha}ß+ iIEL and LN cells were also compared (Fig. 5Go). Cells were stimulated with immobilized anti-TCRß mAb in the presence of IL-2, IL-15, or IL-15 plus anti-IL-2 mAb for 7 days and then restimulated under identical conditions for 24 and 48 h. The anti-IL-2 mAb was used to neutralize endogenous IL-2 and did not inhibit cell proliferation (data not shown). Fig. 5GoA indicates the amounts of IFN-{gamma} detected in the primary and secondary supernatants collected from one of three independent experiments. IFN-{gamma} production was higher in both CD8{alpha}ß+ iIEL and LN cells stimulated in IL-2 compared with those stimulated in IL-15 or in IL-15 plus anti-IL-2 mAb. Except for the LN cells in primary activation, the differences in IFN-{gamma} production among cells receiving different cytokine treatments were significant as indicated by the values of p obtained from Model I ANOVA of data generated from all three experiments (Fig. 5GoB). The significance of the differences in IFN-{gamma} production between cells stimulated in IL-2 vs IL-15 and between cells stimulated in IL-2 vs IL-15/anti-anti-IL-2 mAb were confirmed using the Welsch step-up procedure. These results indicate that IL-15 is less supportive than IL-2 for IFN-{gamma} production by CD8{alpha}ß+ iIEL and LN cells in response to TCR stimulation.

IL-15 did not support IL-4-directed Th2 cytokine production by TCR-stimulated CD8+{alpha}ß iIEL subsets

The effect of IL-15 on IL-4-directed Th2 cytokine production by CD8+ {alpha}ß iIEL subsets and by CD8+ LN cells was next examined. Cells were induced for Th2 differentiation by IL-4 and anti-IFN-{gamma} mAb treatment (30) during primary activation in the presence of IL-2, IL-15, or IL-15 plus IL-2-neutralizing mAb and then restimulated for 24 and 48 h under the same conditions as in primary activation but without IL-4 and anti-IFN-{gamma} mAb (Fig. 6Go). Fig. 6GoA indicates the amounts of IL-4, IL-5, and IL-10 detected in the secondary supernatants collected from one of four independent experiments. Both CD8+ {alpha}ß iIEL subsets stimulated in IL-2 produced higher amounts of Th2 cytokines than when stimulated in IL-15. CD8+ LN cells stimulated in either IL-15 or IL-2 produced similar levels of cytokines. Cytokine production by the LN cells stimulated in IL-15 can be attributed to the effect of endogenous IL-2 as shown by the inhibitory effect of IL-2 neutralizing mAb on cytokine production. The differences in cytokine production among all cells receiving different cytokine treatments were significant as indicated by the values of p for each cell type obtained by Model I ANOVA of data from four independent experiments (Fig. 6GoB). Analysis of data pairs by Welsch step-up procedure indicated significant differences in Th2 cytokine production between iIEL stimulated in IL-2 and in IL-15 and between iIEL stimulated in IL-2 and in IL-15 plus anti-IL-2 mAb. For LN cells, a significant difference was found between cells stimulated in IL-2 and in IL-15 plus anti-IL-2 mAb and between cells stimulated in IL-15 and in IL-15 plus anti-anti-IL-2 mAb. These results indicate that, unlike IL-2, IL-15 does not support IL-4-directed Th2 differentiation of CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL and CD8+ LN cells.

IL-15 did not support cytotoxicity of TCR-stimulated CD8+{alpha}ß iIEL

Freshly isolated CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL are able to mediate perforin-based cytotoxicity (31, 32). Activation through TCR somehow reduces the cytotoxicity of CD8{alpha}{alpha}+ iIEL but augments the cytotoxicity of CD8{alpha}ß+ iIEL (32). Because IL-15 enhances the cytotoxicity of NK cells (33, 34) and promotes the survival of activated CD8{alpha}{alpha}+ {alpha}ß iIEL, IL-15’s effect on the cytotoxicity of activated CD8+ {alpha}ß iIEL subsets was determined. CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL were stimulated with immobilized anti-TCRß mAb in the presence of IL-2 or IL-15 for 8 days and then analyzed for anti-TCR mAb-redirected lysis against P815 targets cells (Fig. 7Go). When stimulated in the presence of IL-2, CD8{alpha}ß+ iIEL were highly cytotoxic, while CD8{alpha}{alpha}+ iIEL displayed a very low level of cytotoxicity as was previously reported (32). When activated in the presence of IL-15, the cytotoxicity of the CD8{alpha}ß+ iIEL decreased ~9-fold compared with cells harvested from the IL-2 culture, while no cytotoxicity was induced in the CD8{alpha}{alpha}+ iIEL. These results indicate that IL-15 does not support cytotoxicity of either CD8+ {alpha}ß iIEL subsets.



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  		FIGURE 7. IL-15 did not promote cytotoxicity of TCR-stimulated CD8+ {alpha}ß iIEL subsets. Sorted CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL were activated by immobilized anti-TCRß mAb in the presence of IL-2 (triangle) or IL-15 (circle) for 8 days and then tested for their ability to mediate anti-TCRß mAb-redirected lysis of P815 cells (solid symbols) as described in Materials and Methods. Open symbols represent the control group without addition of anti-TCRß mAb. Similar results were obtained from two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-15 and IL-2 share many biological activities. However, the wide tissue distribution and the novel receptor {alpha}-chain of IL-15 suggest IL-15’s unique function. In the present study, we examined the effects of IL-15 vs IL-2 on the responses of CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL to TCR stimulation, because IL-15 is likely used by iIEL in situ. We found that both IL-15 and IL-2 supported the growth of CD8+ {alpha}ß iIEL subsets upon primary activation in vitro. However, CD8{alpha}{alpha}+ {alpha}ß iIEL proliferate significantly better in IL-15 than in IL-2 (Fig. 2Go). The effect of IL-15 on activated CD8{alpha}{alpha}+ {alpha}ß iIEL was exerted at the levels of proliferation responder frequency (Table IGo) and cell survival (Fig. 3Go), which likely contributed to the better proliferation of CD8{alpha}{alpha}+ {alpha}ß iIEL in IL-15 than in IL-2. The observation that IL-15 is a better growth factor than IL-2 for CD8{alpha}{alpha}+ {alpha}ß iIEL is noteworthy, because these cells produce little IL-2 and have been known for their poor proliferation response to TCR stimulation under conditions optimal for the growth of peripheral T cells in vitro (6).

IL-15 and IL-2 have distinct roles in the survival and death of CD8+ {alpha}ß iIEL. IL-2 is the prototype T cell growth and survival factor. Accumulating evidence indicates that IL-2 also promotes apoptosis of T cells that have been repeatedly stimulated through TCR, a phenomenon known as activation-induced cell death (AICD) (35, 36, 37). In this study, a large fraction of CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL underwent apoptosis when cultured in IL-2 with or without TCR stimulation, which is in great contrast to the low level of cell death observed in CD8+ LN cells cultured under similar conditions (Fig. 3Go). One ready explanation for the death of freshly isolated iIEL is that these iIEL had started the AICD process before isolation, and thus were susceptible to the death-promoting activity of IL-2 in vitro without further TCR stimulation. However, freshly isolated CD8+ {alpha}ß iIEL expressed no detectable level of surface IL-2R{alpha}, which was induced by TCR cross-linking within 4 h (Fig. 1Go), making the ready explanation less convincing. Another possibility is that CD8+ iIEL are intrinsically different from CD8+ cells of the central immune system in such a way that they are insensitive to the survival-promoting activity of IL-2; therefore, culturing in IL-2 was insufficient for the survival of isolated iIEL.

Both freshly isolated CD8+ {alpha}ß iIEL subsets survived well in IL-15 (Fig. 3Go). However, when cells received TCR stimulation, the survival-promoting effect of IL-15 was only observed on the CD8{alpha}{alpha}+ but not on the CD8{alpha}ß+ subset. The ability of IL-15 to prevent passive cell death, such as under the condition of cytokine withdrawal, and TCR-triggered cell death has been demonstrated in human and murine peripheral T cells (38, 39, 40, 41) as well as in murine {gamma}{delta} iIEL, which are enriched for CD8{alpha}{alpha}+ cells (15, 42). In contrast, we found that IL-15 does not protect murine CD4+ and CD8+ LN cells from AICD (our unpublished observations). Together, these findings suggest that IL-15 preferentially prevents the TCR-triggered death of CD8{alpha}{alpha}+ gut T cells. The preferential effects of IL-15 on the growth and survival of activated CD8{alpha}{alpha}+ iIEL is consistent with the observation in rats orally infected with Listeria monocytogenes, in which the number of CD8{alpha}{alpha}+ but not CD8{alpha}ß+ iIEL increased significantly (16).

IL-15 and IL-2 also affect the effector functions of CD8+ {alpha}ß iIEL differently. Analysis of IFN-{gamma} production, IL-4-induced Th2 cytokine productivity, and anti-TCR Ab-redirected cytotoxicity of activated CD8+ {alpha}ß iIEL subsets demonstrated that IL-15, in contrast to IL-2, was inefficient in supporting these effector function ( Figs. 4–7GoGoGoGo). These results are consistent with earlier observations that IL-15, compared with IL-2, induced weaker IFN-{gamma} production by human NK cells stimulated with tumor cells (43) and weaker cytotoxicity of murine peritoneal lymphocytes primed with alloantigens in vivo (44). IL-15 also did not support IL-4-primed IL-4 production by naive CD4+ transgenic TCR+ cells stimulated with the cognate Ag (45). However, supportive effects of IL-15 on T cell effector function have also been reported. IL-15 and IL-2 induced similar levels of IFN-{gamma} production by naive CD4+ LN cells upon primary activation (45), and IL-15 was more potent than IL-2 in induction of IFN-{gamma} production and cytotoxicity of total iIEL (46). The reported supportive effect of IL-15 might have been due to the presence of endogenous IL-2 in these experiments, because IL-2 is produced by CD4+ LN cells and by CD4+ and CD8{alpha}ß+ iIEL in response to TCR stimulation.

The observed different functions of IL-15 and IL-2 suggest that IL-15 and IL-2 trigger different signals within the cells. As IL-2 and IL-15 share two of the three receptor chains, ß and {gamma}c, the causes of different signaling might occur in two ways, either independently or in combination. One cause may occur during the interaction between the cytokine and the ß/{gamma}c receptor. Both IL-15 and IL-2 bind to the ß/{gamma}c chains with intermediate affinity. However, their binding can be qualitatively different as suggested by the observation that the {gamma}c chains were coimmunoprecipitated by anti-IL2Rß Ab in the presence of IL-2 but not in the presence of IL-15 (11, 13). The other cause may be due to differences in the IL-2R{alpha} and IL-15R{alpha}-chains, which could result in either different interactions between each {alpha}-chain and the ß/{gamma}c chains or in a novel function of the IL-15R{alpha}-chain in addition to its high affinity for IL-15 (47).

IL-15 is likely present in the intestine epithelium during normal as well as disease conditions (14, 15, 16). That IL-15 supports the growth and survival of iIEL but not their effector function as observed in this study appears to be a feasible and beneficial design for the animal, because IL-15 and IL-2 are expressed by different cells under different conditions. A likely scenario is as follows. In the absence of pathogen stimuli, some IL-15 may be produced by non-T cells, such as IEC, which maintains the survival of CD8+ {alpha}ß and {gamma}{delta} iIEL without inducing their effector functions. In response to pathogen invasion, IEC likely produce large amounts of IL-15 in a few hours (16), which may serve as a chemoattractant to recruit iIEL into the infection site (48). IL-15 may also be critical for the initial growth of CD8{alpha}{alpha}+ {alpha}ß and {gamma}{delta} iIEL reactive to the invading pathogens, because these cells are poor IL-2 producers. At the same time, IL-15 may protect the CD8{alpha}{alpha}+ iIEL from death induced by TCR stimulation. For CD8{alpha}ß+ and possibly CD4+ {alpha}ß iIEL activated by Ags presented by the infected IEC, IL-15 may support their proliferation and enhance their IL-2 responsiveness (49). These activated cells produce IL-2, which then promote the growth and effector function differentiation of all iIEL subpopulations. The appearance of IL-2 may also render the CD8{alpha}{alpha}+ iIEL under the regulation of AICD as do CD8{alpha}ß+ iIEL and peripheral T cells (42).

In summary, the present study demonstrates distinct effects of IL-15 vs IL-2 on CD8 {alpha}ß iIEL. IL-15 promoted the survival of both freshly isolated CD8{alpha}{alpha}+ and CD8{alpha}ß+ {alpha}ß iIEL subsets, but only protected the formal subset from TCR-triggered cell death. Consistently, TCR-stimulated CD8{alpha}{alpha}+ but not CD8{alpha}ß+ {alpha}ß iIEL proliferated significantly better in the presence of IL-15 than in the presence of IL-2. In contrast to IL-2, IL-15 did not support effector functions of CD8+ {alpha}ß iIEL subsets, including IFN-{gamma} production, IL-4-directed Th2-type cytokine production and TCR-triggered cytotoxicity. The observed unique functions of IL-15 suggest its role in maintaining the CD8+ {alpha}ß iIEL pool in the absence of Ag stimulation and maintaining the survival and expansion of CD8{alpha}{alpha}+ {alpha}ß iIEL upon Ag stimulation.


   Acknowledgments
 
We thank Yah-Min Lin for cell sorting and Douglas Platt for editing the manuscript.


   Footnotes
 
1 This work was supported by a grant (National Science Council 86-2316-B-001-019) and a postdoctoral fellowship for V. Gelfanov from the National Science Council, Taipei, Taiwan and by a grant (86-5202401224-4) from Academia Sinica, Taipei, Taiwan. Back

2 Current address: Indiana University School of Medicine, 1044 West Walnut Street, Indianapolis, IN 46202. Back

3 Address correspondence and reprint requests to Dr. Nan-Shih Liao, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan. E-mail address: Back

4 Abbreviations: iIEL, intestinal intraepithelial lymphocytes; LN, lymph node; IEC, intestinal epithelial cells; SA, streptavidin; APC, allophycocyanin; {gamma}c, common {gamma}; rh, recombinant human; rm, recombinant murine; AICD, activation-induced cell death. Back

Received for publication February 1, 1999. Accepted for publication September 14, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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