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The Journal of Immunology, 2005, 175: 755-762.
Copyright © 2005 by The American Association of Immunologists

IL-21 Sustains CD28 Expression on IL-15-Activated Human Naive CD8+ T Cells

Nuno L. Alves1,*,{dagger}, Fernando A. Arosa{dagger} and René A. W. van Lier*

* Department of Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands; and {dagger} Institute for Molecular and Cell Biology, Porto, Portugal


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human naive CD8+ T cells are able to respond in an Ag-independent manner to IL-7 and IL-15. Whereas IL-7 largely maintains CD8+ T cells in a naive phenotype, IL-15 drives these cells to an effector phenotype characterized, among other features, by down-regulation of the costimulatory molecule CD28. We evaluated the influence of the CD4+ Th cell-derived common {gamma}-chain cytokine IL-21 on cytokine-induced naive CD8+ T cell activation. Stimulation with IL-21 did not induce division and only slightly increased IL-15-induced proliferation of naive CD8+ T cells. Strikingly, however, IL-15-induced down-modulation of CD28 was completely prevented by IL-21 at the protein and transcriptional level. Subsequent stimulation via combined TCR/CD3 and CD28 triggering led to a markedly higher production of IL-2 and IFN-{gamma} in IL-15/IL-21-stimulated cells compared with IL-15-stimulated T cells. Our data show that IL-21 modulates the phenotype of naive CD8+ T cells that have undergone IL-15 induced homeostatic proliferation and preserves their responsiveness to CD28 ligands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunological protection to pathogens relies on the formation and maintenance of a diverse pool of T cells, each bearing a unique TCR. Following maturation in the thymus, naive T cells enter the periphery where they must survive for future encounters with Ag. Apart from the contribution of thymic emigrants, the number of mature T cells is tightly regulated through cell survival, proliferation, and apoptosis. This process, known as T cell homeostasis, is largely controlled by two cytokines, IL-7 and IL-15, belonging to the common {gamma} ({gamma}c)2 -chain-dependent cytokine family that also includes IL-2, IL-4, IL-9, and IL-21 (1, 2, 3).

For CD8+ T cells, it has been shown that the maintenance of the naive T cell population in the periphery is mainly dependent on IL-7 concurrently with signals from low affinity interactions between TCR and self-peptide-MHC (4, 5, 6). In addition, recent studies performed both in humans and mice suggest that IL-15 also contributes to the homeostasis of naive CD8+ T cells (7, 8, 9, 10, 11, 12). Although both cytokines induce proliferation in an Ag-independent manner, IL-15 drives naive CD8+ T cells to differentiate into effector-type cells, whereas IL-7 preserves their naive characteristics (11, 12, 13, 14).

IL-21 is a newly identified member of the {gamma}c-chain family produced by activated CD4+ T cells. Its effects are mediated through a class I cytokine family receptor, IL-21R{alpha}, which associates with the common {gamma} cytokine receptor chain for intracellular signaling transduction. The IL-21R{alpha} is widely expressed in lymphoid tissues and shows homology with the {beta}-chain of IL-2/15R{beta} (15, 16, 17). It has been demonstrated that IL-21 has pleiotropic effects, influencing both humoral and cell-mediated immune responses by regulating dendritic cell (DC), NK cell, B and T cell function (15, 18, 19, 20, 21).

IL-21 is a four helix-bundle cytokine structural related with IL-15 (15). It has been shown in mice that IL-21 inhibits IL-15-mediated expansion of NK cells and memory CD8+ T cells (22). IL-21 is produced specifically by CD4+ T cells following immune activation in an environmental context where IL-15 is likely being expressed (23, 24). Thus, is likely that T and NK cells are exposed to both cytokines simultaneously in a physiological setting. In this study, we analyze whether IL-21 modulates IL-15-induced homeostatic proliferation in human naive CD8+ T cells. We show that IL-21 moderately augments IL-15-driven expansion of naive CD8+ T cells. Noticeably, IL-21 prevents down-modulation of CD28 and enhances IFN-{gamma} production in naive CD8+ T cells that undergo IL-15-mediated proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and mAbs

The mAbs PerCP-conjugated CD3, allophycocyanin-conjugated CD3, PerCP-conjugated CD8, allophycocyanin-conjugated CD27, PE-conjugated CD28, PE-conjugated CD62L, biotin-conjugated CD122, biotin-conjugated CD132, PE-conjugated anti-CCR7, PE-conjugated CD86, biotin-conjugated rat-anti-mouse IgM, allophycocyanin-conjugated streptavidin were purchased from BD Biosciences. Biotin-conjugated polyclonal anti-IL-15R{alpha} and anti-IL-21R{alpha} were obtained from R&D Systems. Isotype controls consisted of mouse IgG1, rat IgG2b, and goat IgG from BD Biosciences. Allophycocyanin-conjugated CD8, PE-conjugated CD45RA, and allophycocyanin-conjugated CD27 were obtained from Coulter. PE-conjugated anti-IL-7R{alpha} was purchased from Immunotech. PE-labeled CD80 mAb was kindly provided by Dr. M. de Boer (Pan Genetics, Utrecht, The Netherlands) (25). CTLA-4-IgG fusion protein was obtained from Bristol Myers Squibb. Intracellular detection of granzyme B (Sanquin) and perforin (BD Biosciences) was performed as described previously (12). Microbeads coated with human CD8 (CD8 microbeads) were purchased from Miltenyi Biotec, MACS. Cells were stained with indicated mAbs and were analyzed on a FACSCalibur (BD Biosciences). Cells were analyzed on live gating based on forward scatter/side scatter.

Cell preparation

Human PBMC from healthy donors and umbilical cord blood mononuclear cells were isolated by Ficoll-Isopaque density gradient centrifugation (Nycomed). Naive CD8+ T cells were purified from total umbilical cord blood mononuclear cells by positive selection using the MACS system, as described previously (12). The sample purity was assessed by FACS, with allophycocyanin-conjugated CD8 and PerCP-conjugated CD3 mAb (purity: >95% CD3+CD8+). The purity was >95%.

CFSE labeling

Purified naive CD8+ T cells were pelleted and resuspended in PBS at a final concentration of 5–10 x 106 cells/ml. Next, cells were labeled in 0.5 µM (final concentration) of CFSE (Molecular Probes Europe) in PBS for 10 min at 37°C. Cells were washed and subsequently resuspended in IMDM supplemented with L-Glutamine, 25 mM HEPES (BioWhittaker), containing 10% human pool serum (BioWhittaker), Streptomycin (100 ng/ml) (Invitrogen Life Technologies), Penicillin (10 U/ml) (Yamanouchi, Pharma) and 3.57 x 10–4% (v/v) 2-ME (Merck) (culture medium).

Cell culture

CFSE-labeled naive CD8+ T cell subsets were cultured for 7 days at 37°C in 5% CO2 atmosphere, in culture medium in the presence or absence of a range of concentrations (1.6–60 ng/ml) of IL-2 (Biotest), IL-7 (Strahtmann), IL-15 (R&D Systems) and IL-21 (ZymoGenetics). The precursor frequency (percentage of cells in the initial population that underwent one or more divisions after culture) was calculated as: [{Sigma}n ≥ 1(Pn/2n)]/[{Sigma}n ≥ 0(Pn/2n)], where n is the division number that cells have gone through and Pn is the number of cells in division n (26). Cells were analyzed on live gating based on forward scatter/side scatter.

RT-PCR

Total RNA was extracted from immediately ex vivo-isolated cells (resting) and cytokine-stimulated T cells using the GenElute Mammalian total RNA Miniprep kit (Sigma-Aldrich). Oligodeoxythymidine (oligo(dT))-primed cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals). From these cDNA pools specific targets, we amplified by PCR, using the sense and antisense CD28 primers 5'-CGCCCATGCTTGTAGCGTACG-3' and 5'-GATAGGCTGCGAAGTCGCGTG-3', respectively, and the sense and antisense GADPH primers 5'-GTGAAGGTCGGAGTCAACG-3' and 5'-TGAAGACGCCAGTGGACTC-3', respectively. Products were electrophoresed on 1% agarose gels.

Detection of IL-2 and IFN-{gamma} production

Human IL-2 and IFN-{gamma} were assayed in culture supernatants using an ELISA kit from Genzyme Diagnostic and Sanquin, respectively. Supernatants were collected 24 h following activation of resting cells or previously cytokine-stimulated human naive CD8+ T cells with plate-bound anti-CD3 mAb (0.3 µg/ml) (clone 16A9; Sanquin) in the presence or absence of soluble anti-CD28 mAb (5 µg/ml) (clone 15E8; Sanquin).

The cytokine-producing capacity was assessed after PMA (1 ng/ml) and ionomycin (1 µM/ml) stimulation in the presence of brefeldin A (1 µM; all from Sigma-Aldrich) for 4 h. Cells were treated with FACS Lysing Solution and FACS Permeabilizing Solution (BD Biosciences), followed by staining with FITC-labeled anti-IFN-{gamma}, PE-labeled anti-IL-2, and a control mAb ({gamma}-1) (all from BD Biosciences).

Redirected cytotoxicity assay

The cytotoxic activity of IL-15 and IL-15/IL-21 stimulated naive CD8+ T cells was determined in a 4-h 51Cr release assay using FcR {gamma}-bearing P815 cells as targets. Target cells were labeled with 200 µCi (7.4 MBq) of 51Cr (Amersham) in 100 µl of FCS (FCS) for 1 h at 37°C. After three washes, target cells were plated in 96-well round-bottom plates at 5000 cells per well. CD8+ T cells were mixed with target cells at E:T ratios of 20:1, 10:1, 5:1, and 1:1, in the presence or absence of 5 µg/ml anti-CD3 mAbs (clone CD3.4/1; Sanquin). After 4 h of incubation, supernatants were harvested and counted in a gamma counter. Specific lysis was calculated according to the following formula: percent-specific release = [(experimental release – spontaneous release)/(maximum release – spontaneous release)] x 100.

Statistical analysis

The two-tailed Mann-Whitney U test was used for analysis of differences between groups. Values of p <0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Distinct responsiveness of naive CD8+ T cells to IL-7, IL-15, and IL-21

IL-15 and IL-7 are two key cytokines involved in the Ag-independent proliferation of human naive CD8+ T cells (10, 11, 12, 13). A previous study showed that the CD4+ T cell-derived cytokine IL-21 inhibits the IL-15-mediated proliferation of mouse memory CD8+ T cells (22). We considered examining IL-21 effects on cytokine-driven expansion of human naive CD8+ T cells. We started by analyzing the responsiveness of adult total CD8+ T cells to the {gamma}c-dependent cytokines, IL-2, IL-7, IL-15, and IL-21. The proliferative response was measured by monitoring cell division of CFSE-labeled PBMCs cultured for 7 days in the presence of graded concentrations of cytokines. As depicted in Fig. 1A, IL-21 did not induce Ag-independent division of CD8+ T cells at any of the concentrations tested. In contrast, IL-7 and IL-15 were able to induce a clear response in CD8+ T cells, IL-7 being a more potent mitogenic stimulus. CD8+ T cells were unresponsive to IL-2 although a clear response was observed at the higher concentration in CD4+ T cells (divided cells underwent six to seven divisions, precursor frequency 4–10%) (data not shown). We next examined whether IL-21 influenced IL-15-mediated expansion of CD8+ T cells, as reported previously (22). PBMCs were labeled with CFSE and cultured with an optimal concentration of IL-15 (10 ng/ml) alone or in presence of a range concentration of IL-21. In contrast with the report by Kasaian et al. (22), IL-21 enhanced the IL-15-dependent expansion of total CD8+ T cells in a dose-dependent manner (Fig. 1, B and C). Having established that IL-21 on its own did not induce expansion but increased IL-15-mediated expansion of total adult CD8+ T cells, we determined the effect of IL-21 on cytokine-driven expansion of human naive CD8+ T cells. The proliferative response was measured by monitoring cell division of CFSE-labeled cord blood-derived naive CD8+ T cells cultured in the presence of IL-7, IL-15, and IL-21 alone or combining two cytokines. IL-21 did not induce Ag-independent proliferation of naive CD8+ T cells. Moreover, IL-21 had no effect on T cell survival as a similar reduction in T cell cellularity was observed in cells cultured in medium (Fig. 2). In contrast, IL-7 stimulated the majority of naive CD8+ T cells to enter cell cycle, with 86% of responding cells. IL-15 revealed a less potent stimulus compared with IL-7, with 40% of the cells initiating cell cycling. However, IL-15-responding cells underwent more rounds of cell divisions than those cultured in IL-7. IL-7-mediated proliferation was not influenced by IL-21. In contrast, IL-21 moderately increased IL-15-mediated expansion with a slight augmentation in the precursor frequency and in the cell yield (Fig. 2). Overall, our data show that CD8+ T cells display distinct responsiveness to IL-7 and IL-15. Moreover, IL-21 slightly affects the Ag-independent expansion of human naive CD8+ T cells induced by IL-15.



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  		FIGURE 1. Distinct responsiveness of human CD8+ T cells to cytokine stimulation. CFSE-labeled PBMCs were cultured in the presence of variable concentrations of cytokines for 7 days, followed by FACS analysis. CD8+ T cells were gated on CD3+CD8+ T cells. Precursor frequencies following cytokine stimulation: the percentage of cells in the initial population that had undergone one or more rounds of division after cytokine stimulation was calculated as described in He et al. (26 ). Data depicted are the mean ± SD from three experiments performed. A, Response curve for IL-2, IL-7, IL-15, and IL-21. B, CFSE-labeled PBMCs were cultured in presence of IL-15 (10 ng/ml) alone or adding variable concentrations of IL-21 as indicated (nanograms per milliliter). C, Data are from a representative experiment shown in B.

 


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  		FIGURE 2. Different responsiveness of human naive CD8+ T cells to cytokine stimulation. CFSE-labeled naive CD8+ T cells were left unstimulated or stimulated for 7 days with IL-7 (10 ng/ml), IL-15 (10 ng/ml), and IL-21 (25 ng/ml) either alone or by combining two cytokines. A, Precursor frequencies following cytokine stimulation. Data depicted are the mean ± SD from four experiments performed. B, Cell division was measured by flow cytometry at the time indicated. Data are from a representative experiment of four performed. C, Naive CD8+ T cell numbers following stimulation. Cultures were started with ~1 x 106 cells. Data depicted are the mean ± SD from seven experiments performed.

 
IL-15 enhances the expression of IL-21R{alpha} and {gamma}c chain in naive CD8+ T cells

IL-7 and IL-21 bind to a dimeric receptor constituted by the {gamma}c chain (CD132) and their respective high-affinity {alpha}-chains, i.e., IL-7R{alpha} (CD127) and IL-21R{alpha}. IL-15 binds to a trimeric receptor composed of the {gamma}c chain, the IL-2/IL-15 {beta}-chain (CD122), and the unique IL-15R {alpha}-chain (2, 27). It has been shown that the IL-15R {alpha}-chain is dispensable on CD8+ T cells for IL-15-mediated proliferation (28). The expression of the cytokine receptors on resting and cytokine-stimulated naive CD8+ T cells was analyzed, to examine a possible basis for the different responsiveness of naive CD8+ T cells to the aforementioned cytokines (Fig. 3). IL-7R{alpha} was highly expressed when examined directly ex vivo. Its expression was completely down-regulated in the presence of IL-7, alone or combined with other cytokines. Stimulation with IL-15 or IL-21 separately or together also induced a decrease on IL-7R{alpha} expression although to a lower extent. As we reported previously (12), IL-15R{alpha} was barely expressed in naive T cells and completely down-regulated in cultures where IL-15 is present. IL-7 or the combination of IL-7 and IL-21 (not shown) induced the up-regulation of IL-15R{alpha} on naive CD8+ T cells. The IL-2/IL-15{beta} and the {gamma}c chain were visibly up-regulated upon stimulation with most cytokines, although the expression of the {beta}-chain was hardly affected by IL-21. The expression of IL-21R{alpha} was low on freshly isolated naive CD8+ T cells. Although IL-7 had no significant effect on its expression level, IL-21R{alpha} expression was sharply up-regulated following stimulation with IL-15, IL-21, and the combination of both. Taken together, our data show that responsiveness of naive CD8+ T cells to IL-7, IL-15, and IL-21 correlates with the levels of expression of their receptors found on cells analyzed ex vivo. Particularly, our results suggest that IL-15 might affect the responsiveness of naive CD8+ T cells to IL-21 through the up-regulation of IL-21R{alpha}.



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  		FIGURE 3. Regulation of cytokine receptors on human naive CD8+ T cells following cytokine stimulation. CFSE-labeled naive CD8+ T cells were stimulated for 7 days with IL-7 (10 ng/ml), IL-15 (10 ng/ml), and IL-21 (25 ng/ml) either alone or by combining two cytokines. After 7 days the cells were stained for IL-7R {alpha}-chain (CD127), IL-15R {alpha}-chain, IL-2/15R {beta}-chain (CD122), IL-21R {alpha}-chain and {gamma}c chain (CD132), followed by FACS analysis. Dashed line indicates control staining; thin line, ex vivo naive CD8+ T cells; and bold line, indicated cytokine-stimulated cells. Data are from one representative experiment of three performed.

 
IL-15-induced CD28 down-regulation is prevented by IL-21

In contrast to IL-7, IL-15 induces a primed phenotype in naive CD8+ T cells (11, 12, 13). Recent studies in mice have shown that IL-21 modulates phenotypic changes in NK cells and IL-15-expanded CD8+ T cells (19, 22). In light of the up-regulation of IL-21R{alpha} following IL-15-activation, we next examined whether IL-21 affected the differentiation process of naive CD8+ T cells mediated by IL-15.

Freshly isolated naive CD8+ T cells were CD27+, CD28+, CCR7+, CD45RA+, CD62L+, and IL-7R{alpha}+ (12) (data not shown). As corroborated in Fig. 4, cells expanded in the presence of IL-7 maintain their naive phenotype. In contrast, IL-15-stimulated cells differentiated into effector-type cells, characterized by down-regulation of CD28, CCR7, CD62L, and CD45RA (12). Naive CD8+ T cells exposed to IL-21 or the combination of IL-7 and IL-21 (not shown) did not change their phenotype, apart from a small increase in CD28 expression. As observed in cells cultured in medium, the expression of CCR7 was slightly reduced in cells cultured with IL-21. The combination of IL-7 and IL-15 induced a similar effector phenotype to the one observed in IL-15-stimulated cells. For the majority of the cell surface markers analyzed, naive CD8+ T cells cultured with IL-15 plus IL-21 exhibited a nearly identical phenotype as IL-15-activated cells. However, IL-15-induced down-regulation of CD28 was prevented by IL-21. Additionally, IL-21 also affected the CD62L down-regulation observed in IL-15-stimulated cells, although at a lower extent compared with the effect on CD28 expression. All together, our data indicate that IL-21 modulates the phenotype of naive CD8+ T cells undergoing IL-15-driven proliferation.



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  		FIGURE 4. Phenotypic properties of human naive CD8+ T cells following cytokine stimulation. CFSE-labeled naive CD8+ T cells were stimulated for 7 days with IL-7 (10 ng/ml), IL-15 (10 ng/ml), and IL-21 (25 ng/ml) either alone or by combining two cytokines. After 7 days the cells were stained for the indicated cell surface markers, followed by FACS analysis. Data are from one representative experiment of three performed.

 
Having determined that the down-regulation of CD28 in IL-15-stimulated cells could be prevented by culture with IL-21, we examined whether this effect was both dose-dependent and a specific property of IL-21. Naive CD8+ T cells were cultured in the presence of IL-15 alone or by adding IL-21 in different doses. In parallel we analyzed the effect of IL-2 and IL-7. As shown in Fig. 5, neither IL-2 nor IL-7 could prevent the IL-15-induced down-regulation of CD28, although a moderate increase in expression of CD28 was observed at higher doses. In contrast to the previous {gamma}c cytokines, IL-21 prevented CD28 down-regulation already at a very low concentration. Further, at higher concentrations, CD28 expression was enhanced. Thus, our data indicate that among the {gamma}c cytokines tested, IL-21 specifically maintains CD28 expression in IL-15-activated human naive CD8+ T cells.



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  		FIGURE 5. IL-21 sustains CD28 expression on IL-15-activated human naive CD8+ T cells in a dose-dependent manner. Naive CD8+ T cells were cultured in presence of IL-15 (10 ng/ml) alone or by adding variable concentrations of IL-2, IL-7, and IL-21 as indicated. After 7 days the cells were stained for CD28, followed by FACS analysis. The numbers indicate the mean fluorescent intensity (MFI) for CD28. Thin line, ex vivo naive CD8+ T cells (italic number indicates MFI); and bold line, cytokine-stimulated cells. Data are from a representative experiment of two performed.

 
IL-21 prevents IL-15-induced CD28 down-regulation at the transcriptional level

Because IL-21 sustained CD28 expression in IL-15-activated cells we next analyzed whether this effect was associated with the transcriptional control of the CD28 gene, and/or occurred through direct cell-cell contact effect mediated via its ligands, CD80 and CD86. Analyses of the mRNA levels revealed that CD28 was down-regulated in naive CD8+ T cells stimulated with IL-15 or IL-7 plus IL-15. Remarkably, IL-15-induced CD28 down-regulation was blocked in presence of IL-21 (Fig. 6A).



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  		FIGURE 6. Modulation of CD28 by IL-21 both on the RNA and protein level in IL-15-activated naive CD8+ T cells. CFSE-labeled naive CD8+ T cells were stimulated for 7 days with IL-7 (10 ng/ml), IL-15 (10 ng/ml), and IL-21 (25 ng/ml) either alone or by combining two cytokines. A, After 7 days the cells were harvested, total RNA was immediately isolated and subjected to RT-PCR analysis for CD28 transcripts; ex vivo-isolated cells (resting). Data are from one representative experiment of three performed. B, After 7 days the cells were stained for CD80, followed by FACS analysis. C, Naive CD8+ T cells were stimulated for 7 days with IL-15 (10 ng/ml) ± CTLA-4-IG construct, IL-15 (10 ng/ml) + IL-21 (25 ng/ml) or IL-21. Cells were stained for the indicated cell surface markers, followed by FACS analysis. i, Upper panel, resting (gray), IL-15-stimulated (thin line), IL-15+CTLA-Ig (bold line). ii, Lower panel, resting (gray), IL-15+IL-21-stimulated (thin line), IL-21 (bold line). D, Total RNA was immediately isolated and subjected to RT-PCR analysis for CD28 transcripts. Data are one representative experiment of two performed.

 
We next examined the expression of CD80 and CD86 on naive CD8+ T cells following cytokine stimulation. The expression of CD86 was not detectable in any condition (data not shown). As depicted in Fig. 6B, culture with IL-15 or the combination of IL-15 plus IL-7 induced a clear up-regulation of CD80 on proliferating cells. Strikingly, culture of naive CD8+ T cells with the combination of IL-15 and IL-21 prevented IL-15-induced CD80 up-regulation. This observation might indicate that the IL-15-induced down-regulation of CD28 was also due to the cell-cell contact effect via interaction between CD28 and CD80 (29). To test this, we blocked CD28 interaction with its ligands by addition of the CTLA4-IgG fusion protein to IL-15-stimulated cells. As depicted in Fig. 6C, under this condition, CD28 expression was reduced to the same extent as on IL-15-stimulated cells cultured without the construct. Analyses of the mRNA showed that the reduction of CD28 was not influenced by blocking interaction with its ligand (Fig. 6D). Hence, our data suggest that IL-21 precludes IL-15-induced down-modulation of CD28 at transcriptional level via a mechanism independent of receptor-ligand interaction.

IL-21 augments IL-2 and IFN-{gamma} production in IL-15-activated naive CD8+ T cells

Upon interaction with its ligands, CD80 and CD86, CD28 transduces activating signals that coupled with TCR signaling promotes entry into cell cycle, survival, and IL-2 production (30). Thus, the expression level of CD28 is of particular importance for the outcome of T cell activation. We next assessed the functionality of the CD28 molecule by measuring the levels of IL-2 following combined TCR/CD3 and CD28 stimulation of previously cytokine-activated cells (Fig. 7A, upper panel). The values obtained following different stimulations were normalized to the level of IL-2 produced following TCR activation of freshly isolated naive cells. In concordance with CD28 expression, the combined triggering of TCR/CD3 and CD28 gave a markedly higher production of IL-2 in IL-15/IL-21-stimulated T cells compared with those stimulated with IL-15 alone. The production of IL-2 was not detectable following combined TCR/CD28 stimulation on IL-21-activated cells (data not shown). This observation could be explained by the low "fitness" of IL-21-stimulated cells because they neither proliferated nor had an advantage in survival (as shown in Fig. 2).



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  		FIGURE 7. IL-21 increases IL-2 and IFN-{gamma} production in IL-15-activated naive CD8+ T cells following combined TCR/CD3 and CD28 triggering. Naive CD8+ T cells were stimulated for 7 days with IL-15 (10 ng/ml) alone or in combination with IL-21 (25 ng/ml). A, Ex vivo-isolated cells (resting) or cytokine-stimulated cells were then harvested and subsequently activated for 24 h with anti-CD3 mAb (0.3 µg/ml) ± anti-CD28 mAb (5 µg/ml). (i) IL-2 and (ii) IFN-{gamma} content in the supernatants following stimulation was assessed by ELISA. The values obtained for different conditions were normalized to the level obtained following TCR activation of naive CD8+ T cells immediately isolated ex vivo (resting). Anti-CD3 activation ({square}), anti-CD3 + anti-CD28 ({blacksquare}). B, After 7 days, cytokine production was assessed by intracellular cytokine staining 4 h after restimulation with PMA/ionomycin and brefeldin A in the presence (lower panel) or absence (upper panel) of anti-CD28 mAb (5 µg/ml) and (C) after 7 days, cells were stained intracellularly for perforin and granzyme B followed by FACS analysis. Dotted line indicates control staining; thin line, day 0; and bold line, stimulated cells.

 
We have previously shown that IL-15-activated naive CD8+ T cells acquire the ability to produce IFN-{gamma}, express key cytotoxic molecules such as perforin and granzyme B, and acquire cytotoxic potential (12). It has been reported that in murine T cells IL-21 increases IL-15-induced IFN-{gamma} production (22, 31). To further address the IL-21 effects on the functionality of IL-15-activated naive CD8+ T cells, we measured the production of IFN-{gamma} following combined TCR/CD3 and CD28 stimulation (Fig. 7A, lower panel). We found that IL-21 significantly augmented IFN-{gamma} production of IL-15-activated T cells, independently of CD28 signaling. Moreover, in concordance with the results obtained with culture supernatants following combined TCR/CD28 stimulation, an increased number of IL-2 and IFN-{gamma}-producing cells was observed in IL-15/IL-21-activated cells following PMA/ionophore/CD28 stimulation (Fig. 7B). Further, activation with IL-15 plus IL-21 augmented IL-2 and IFN-{gamma} production on a per-cell basis (Fig. 7B). Analyses on the expression of key cytotoxic effector molecules revealed that IL-21 had no effect on IL-15-induced up-regulation of perforin and granzyme B (Fig. 7C). In agreement with this finding we found no differences when IL-15 and IL-15/IL-21-activated cells were compared in redirected cytotoxicity assays (data not shown).

Our data show that IL-21 preserves CD28 responsiveness of naive CD8+ T cells that have undergone IL-15-induced proliferation


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies have shown that IL-21, a recently identified cytokine produced by activated CD4+ Th cells, affects both humoral and cell-mediated immune responses (23). IL-21 promotes the full maturation of NK cells and enhances effector function of NK and memory CD8+ T cells (19, 21, 22, 31, 32, 33). Interestingly, clear interspecies differences exist with respect to the biological effect of IL-21. In particular, in contrast to what has been observed in humans, IL-15-mediated expansion of murine NK cells is inhibited by IL-21 (15, 22). A similar inhibitory effect was seen on the proliferation of IL-15-activated murine memory CD8+ T cells (22). Herein, we examined the effects of IL-21 on IL-15-induced expansion and differentiation of human naive CD8+ T cells. We demonstrated that IL-21 slightly increased IL-15-mediated expansion. However, IL-21 prevented IL-15-induced CD28 down-regulation with a concomitant maintenance of CD28 functionality. Moreover, IL-21 augmented the IL-15-induced IFN-{gamma} production in naive CD8+ T cells.

IL-21 has been shown to enhance the proliferation of Ag-driven naive and memory T cells (22, 34, 35). However, in the absence of TCR activation, IL-21 is not able to induce proliferation, in contrast to other {gamma}c chain signaling cytokines such as IL-2, IL-7, and IL-15 (10, 12, 13, 15). Consistently, we observed that IL-21 had no effect on the division of human naive CD8+ T cells. We found no inhibitory effect of IL-21 on IL-15-driven expansion of naive CD8+ T cells (Fig. 2) whereas others have reported that IL-15-induced proliferation of murine memory cells is lowered in the presence of IL-21 (22). A possible explanation to reconcile both observations is that effects of IL-21 might depend on the activation or differentiation state of the T cell (23). Alternatively, discrepancies could result from differences between human and mouse. However, in concordance with our data, a recent article by Zeng et al. (33) has shown that IL-21 synergies with IL-15 promoting the expansion of both naive and memory murine CD8+ T cells. As suggested in the latter report, differences in the concentration of IL-21 used in the course of the experiments by Kasaian et al. (22) may explain the discrepancies observed.

Previous studies have shown that IL-21 regulates phenotypic changes in the later stage of NK cell maturation (15, 19). Yet, despite the numerous reports on the role of IL-21 on the function of T cells (21, 22, 31, 32, 33, 35, 36), a modulatory effect of IL-21 on the cell surface phenotype of human T cells has not been ascertained hitherto. Our results demonstrated that on naive CD8+ T cells, IL-21 prevented both the IL-15-induced down-regulation of CD28 and the up-regulation of its ligand, CD80 (Figs. 4, 5, and 6B). It has been reported that IL-21 inhibits DC maturation, blocking the LPS-induced up-regulation of the CD28 ligands, CD80 and CD86 (18), suggestive of a similar mechanism between T cells and DCs. Strikingly, IL-15-induced CD28 down-regulation was still observed, both at the transcriptional and protein level, when the interaction of CD28 with its ligands was blocked (Fig. 6, C and D). Thus, we would favor the hypothesis that IL-15 suppresses the transcription of the CD28 gene independent of receptor-ligand interaction. Several observations have shown that CD28 expression can be down-modulated by cytokines such as IL-15 and TNF-{alpha} on CD8+ and CD4+ T cells, respectively (12, 14, 37). TNF-{alpha} represses the transcriptional activity of the CD28 promoter by inhibiting the binding of nuclear complexes that recognize two regulatory sequence motifs, site {alpha} and {beta}, within the CD28 promoter (37). It would be interesting to investigate whether IL-15-induced CD28 down-regulation occurs through a similar mechanism. It has been shown that IL-4 prevents the loss of CD28 expression in long-term TCR-activated CD8+ T cells (38). IL-4 and IL-21 belong to the {gamma}c-chain-dependent cytokine family and bind to overlapping epitopes on the {gamma}c chain (39). Indeed, we found that, in contrast to IL-2 and IL-7 (Fig. 5), IL-15-induced down-regulation of CD28 was not only blocked by IL-21, but also by IL-4 (our unpublished data).

IL-21 has been reported to be produced by activated CD4+ T cells, predominantly in Th2 cells. Moreover, it has been shown that IL-21 inhibits the production of IFN-{gamma} in naive Th cell precursors (40). In contrast to what has been reported for CD4+ T cells, IL-21 augments both IFN-{gamma} production and cytolytic function of CD8+ T cells (21, 22, 31, 36). We have previously shown that IL-15 induces IFN-{gamma} production in naive CD8+ T cells upon secondary stimulation (12). In concordance with previous studies (22, 31, 33), our results showed that addition of IL-21 enhanced IL-15-induced IFN-{gamma} production in T cells. A recent paper showed that granzyme B is preferentially induced in murine naive CD8+ T cells following short-time activation with IL-15 and IL-21 (33). Differently, in our experiments IL-21 had little effect on other functional properties induced by IL-15, such as the expression of perforin and granzyme B (Fig. 7). Rather, our results suggest that IL-21 contributes to enhance particular IL-15-induced effector cytokines in naive CD8+ T cells. Importantly, IL-21 preserves the function of CD28, a costimulatory molecule of paramount importance for the establishment of an effective T cell-mediated immune response. IL-21 has been primarily described as a T cell costimulatory cytokine enhancing TCR-mediated proliferation and function of CD8+ T cells (15, 22, 22, 33). We would consider an analysis of the influence of IL-21 on the regulation of CD28 during Ag-dependent immune responses of great interest.

It is relevant to note that the biological effects induced by IL-15 and IL-21 may depend on their concurrent availability within the lymphoid microenvironment. Although they are produced by distinct cell types, parallel mechanisms might regulate the level of both cytokines. IL-15 is produced by activated monocytes/macrophages, DCs, and epithelium mainly under stress conditions (e.g., infection) whereas IL-21 is produced specifically by CD4+ T cells following immune activation (23, 24). Thus, we would envision that both cytokines can be present simultaneously in sites of infection.

In summary, we demonstrated that IL-21, a CD4+ Th-derived cytokine, exerts specific immunoregulatory properties in naive CD8+ T cells that have undergone IL-15-driven homeostatic proliferation. Importantly, IL-21 preserves the ability of naive CD8+ T cells to respond to costimulatory ligands on future encounters with activated APCs expressing the cognate Ag. In addition, IL-21 can potentiate specific effector T cell functions, such as IFN-{gamma} production, induced by IL-15. Hence, IL-21 may serve as an immunomodulatory cytokine in IL-15-mediated activation of human naive CD8+ T cells.


   Acknowledgments
 
We thank Drs. Eric Eldering, Ester M. M. van Leeuwen, and Robert H. Hoek for critical reading of the manuscript. We thank Si-La Yong for all technical support.


   Disclosures
Top
 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 Address correspondence and reprint requests to Dr. Nuno L. Alves, Laboratory for Experimental Immunology, Academic Medical Center, G1-133, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail address: N.M.LagesAlves{at}amc.uva.nl Back

2 Abbreviations used in this paper: {gamma}c, common {gamma}; DC, dendritic cell. Back

Received for publication February 8, 2005. Accepted for publication May 6, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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