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The Journal of Immunology, 2007, 178: 7640-7648.
Copyright © 2007 by The American Association of Immunologists, Inc.
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IL-21 Promotes Differentiation of Naive CD8 T Cells to a Unique Effector Phenotype1

Kerry A. Casey and Matthew F. Mescher2

Center for Immunology, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-21, the most recently described member of the common {gamma}-chain cytokine family, is produced by activated CD4 T cells, whereas CD8 T cells express the IL-21 receptor. To investigate a possible role for IL-21 in the priming of naive CD8 T cells, we examined responses of highly purified naive OT-I CD8 T cells to artificial APCs displaying Ag and B7-1 on their surface. We found that IL-21 enhanced OT-I clonal expansion and supported development of cytotoxic effector function. High levels of IL-2 did not support development of effector functions, but IL-2 was required for optimal responses in the presence of IL-21. IL-12 and IFN-{alpha} have previously been shown to support naive CD8 T cell differentiation and acquisition of effector functions through a STAT4-dependent mechanism. Here, we show that IL-21 does not require STAT4 to stimulate development of cytolytic activity. Furthermore, IL-21 fails to induce IFN-{gamma} or IL-4 production and can partially block IL-12 induction of IFN-{gamma} production. CD8 T cells that differentiate in response to IL-21 have a distinct surface marker expression pattern and are characterized as CD44high, PD-1low, CD25low, CD134low, and CD137low. Thus, IL-21 can provide a signal required by naive CD8 T cells to differentiate in response to Ag and costimulation, and the resulting effector cells represent a unique effector phenotype with highly effective cytolytic activity, but deficient capacity to secrete IFN-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Engagement of the TCR and CD28 on naive CD8 T cells stimulates the cells to enter the cell cycle and undergo several rounds of division. However, an additional signal is required to initiate the differentiation program leading to acquisition of effector functions and establishment of a responsive memory population (1, 2, 3). In the absence of this additional signal, clonal expansion is compromised due to poor survival of the expanding cells, the cells do not acquire cytolytic activity or the ability to produce IFN-{gamma}, and long-term the remaining cells are tolerant. The signal needed for differentiation and establishment of memory can be provided by either IL-12 or IFN-{alpha} (4, 5), both of which are produced by activated dendritic cells (DC)3 (6, 7, 8, 9). Thus, DCs activated by CD4 T cells or by TLR ligands can provide all of the signals needed to fully activate naive CD8 T cells: Ag; costimulation; and IL-12 or IFN-{alpha}.

In addition to the indirect influence of DC conditioning, CD4 T cells can also directly influence CD8 T cell responses through the secretion of cytokines. Activated CD4 T cells are the major producers of IL-2, a cytokine important to CD8 T cells for the relief of split anergy (10, 11), the maintenance of effector populations (12, 13), and the promotion of antitumor immunity (14, 15). Although the effects of IL-2 on CD8 T cells are diverse, they are limited to the promotion of proliferation and/or survival and do not include the induction of de novo effector functions. There is some evidence, however, that CD4 T cells may secrete a factor capable of stimulating naive CD8 T cells to develop effector functions. Supernatant from activated CD4 T cell cultures was shown to be sufficient to rescue CD4-dependent CD8 T cell cytolytic activity after removal of the CD4 cells (16). The authors concluded that the soluble factor responsible for providing direct help to the CD8 T cells was secreted by the activated CD4 T cells. None of the lymphokines found in the culture supernatant could directly account for this effect on CD8 T cell cytolytic activity, leaving open the possibility that an unidentified lymphokine was responsible.

Members of the common {gamma}-chain ({gamma}c) cytokine family, including IL-2, IL-7, and IL-15, have important roles in regulating CD8 T cell response. IL-21, a newly identified member of the {gamma}c family, has recently also been shown to positively influence CD8 T cell responses (17). Like other {gamma}c cytokine family receptors, the IL-21 receptor is expressed by a variety of cell types including resting and activated B, T, NK, and DCs (18), whereas activated CD4 T cells are the only cell type found to express IL-21 (17). Initially, IL-21 was reported to induce proliferation of bulk T cells upon stimulation with anti-CD3 mAb by enhancing the effects of IL-2, IL-15, and, to a lesser extent, IL-7 (17). IL-21 has also been shown to act in synergy with IL-18 and IL-15 in the induction of IFN-{gamma} production by human bulk T and NK cells (19). In addition, administration of IL-21 in three separate tumor models proved to have protective and/or therapeutic benefits (20, 21, 22). Mediation of these antitumor responses correlated strongly with the activation and clonal expansion of CD8 T cells, and in one case the antitumor effect was ablated by Ab depletion of CD8 T cells (22). This suggested that the IL-21 was acting through a CD8 T cell-dependent mechanism. Most recently, IL-21 has been reported to act synergistically with IL-15 to directly promote Ag-independent clonal expansion of CD44high and CD44low CD8 T cells (23). The same authors also observed a marked defect in Ag-specific CD8 T cell cytolytic activity after response to viral challenge in IL-21R–/– mice.

Although IL-21 clearly has effects on CD8 T cell responses, the basis for these effects is not clear, nor is it clear whether IL-21 is acting on the CD8 T cells or a different cell type, such as APC. The observations reported thus far raised the possibility that IL-21 might directly provide a signal to the naive CD8 T cell that acts in conjunction with TCR and CD28 signals to support proliferation, survival, and/or differentiation to acquire effector functions. To examine this possibility, we stimulated highly purified naive CD8 T cells from OT-I TCR-transgenic mice with artificial APC (aAPC) that provided Ag and costimulation and found that IL-21 could enhance clonal expansion and support development of cytolytic function. The resulting OT-I effector cells, however, were unable to produce IFN-{gamma} or IL-4 and displayed a distinct cell surface phenotype characterized as CD44high, CD25low, CD134low, CD137low, and PD-1low. Thus, IL-21 can act directly on naive CD8 T cells to support clonal expansion and differentiation to a unique effector phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, cell lines, and reagents

OT-I mice having a transgenic TCR specific for H-2Kb and OVA257–264 were a gift from Dr. F. Carbone (University of Melbourne, Melbourne, Australia), and breeding colonies were maintained under specific pathogen-free conditions at the University of Minnesota (Minneapolis, MN). BALB/c and STAT4–/– mice were purchased from The Jackson Laboratory. 129S6 and STAT1–/– mice were purchased from Taconic Farms. Experiments were performed in compliance with relevant laws and institutional guidelines and with the approval of the Institutional Animal Care and Use Committee at the University of Minnesota. E.G7 tumor cells (EL-4 thymoma transfected with OVA) were used as targets in the 51Cr release cytotoxicity assay, and EL-4 cells were used as controls for specificity. 2C11 hybridoma cells (24) that produce the anti-CD3e mAb were used for the redirected lysis assay, and EL-4 cells were used as controls. Directly conjugated fluorescent anti-human granzyme B mAb and the corresponding mouse IgG1 isotype were purchased from Caltag Laboratories. All other directly conjugated fluorescent Abs were purchased from BD Pharmingen or eBioscience. The medium used for all cell cultures was RPMI 1640 supplemented with 10% FCS, 4 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin and streptomycin, 10 mM HEPES, and 5 mM 2-ME (RP-10).

Naive CD8 T cell purification

Inguinal, axillary, brachial, cervical, and mesenteric lymph nodes were collected, pooled, homogenized, and enriched for CD8+CD44low naive cells using a negative selection protocol for MACS (Miltenyi Biotec). Briefly, cells were coated with FITC-labeled Abs specific for CD4, B220, I-Ab, and CD44. Cells were subsequently coated with Anti-FITC magnetic MicroBeads and passed over a separation column coupled to the MACS magnet. Flowthrough cells were >95% CD8+ and <0.5% CD44high.

Preparation of aAPC

Methods for immobilizing MHC Ags and costimulatory ligands on 5-µm-diameter latex microspheres have been previously described in detail (25). Briefly, microspheres were coated with Dimer X H-2Kb:Ig fusion protein (BD Pharmingen) using 2.5 µg of Dimer X H-2Kb:Ig/107 latex microspheres. Peptide was loaded onto the H-2Kb by incubating the coated microspheres with 0.2 µM (unless otherwise indicated) OVA257–264 for 2 h at 37°C, followed by extensive washing to remove free peptide. B7-1 in the form of a recombinant mouse B7-1/Fc chimera (R&D Systems) was coimmobilized using 0.15 µg/107 microspheres. Immobilization of proteins was verified by staining with fluorescent Abs and flow cytometric analysis. Microspheres prepared in this manner, referred to as Ag/B7 aAPC, were used for stimulation of OT-I.

Purified anti-mouse Vb5.1,5.2 TCR mAb (BD Pharmingen) at 2.5 mg/107microspheres and B7-1/Fc chimera at 0.56 µg/107 microspheres were coimmobilized onto 5-µm-diameter latex microspheres during a 30-min incubation period on a rotator at 4°C. A parallel set of microspheres was prepared using purified anti-mouse Vb8 TCR mAb (BD Pharmingen) at 2.5 µg/107microspheres and B7-1/Fc chimera at 0.56 µg/107 microspheres. Both types of microspheres were then blocked with an equal volume of 1% BSA for 30 min on a rotator at 4°C, washed, and resuspended. Vb5.1,5.2/B7-1 and Vb8/B7-1 microspheres were then mixed at a 1:1 ratio. Microspheres prepared in this manner, referred to as anti-TCR/B7 aAPC, were used for in vitro stimulation of BALB/c, STAT4–/–, 129S6, and STAT1–/– polyclonal naive CD8 T cell populations.

In vitro clonal expansion and cytotoxicity assays

Purified CD8 T cells (5 x 104) and 2 x 105 Ag/B7-1-coated microspheres were placed in flat-bottom microtiter wells in 200 ml of RP-10. Where indicated, cultures were supplemented with the following cytokines: murine IL-21 at 100 ng/ml unless otherwise indicated (R&D Systems); human rIL-2 at 2.5 U/ml (1 µg = 1.3 x 104 WHO units; TECIN; National Cancer Institute Biological Resources Branch, Frederick, MD); murine rIL-12 at 2 U/ml (Genetics Institute); murine IFN-{alpha} at 1000 U/ml (PBL Biomedical Laboratories). Where indicated, the following blocking Abs were added to cultures: culture supernatant from the PC61.5.3 hybridoma (nonstimulatory anti-IL-2R mAb) was used at a dilution of 1/10; neutralizing anti-mouse IL-21 Ab at 10 µl/ml (R&D Systems); neutralizing sheep anti-serum to IL-12 at 3.5 µg/ml (Genetics Institute); and neutralizing anti-mouse IFN-{alpha} Ab at 2000 neutralization units/ml (PBL Biomedical Laboratories). Cell recovery was determined after 3.5 days of culture and is expressed as the average number of cells recovered per well with six replicates per condition. Cytolytic activity of OT-I cells was determined in a standard 4-h 51Cr release assay using E.G7 cells as targets with EL-4 cells included as a control for specificity; triplicate wells of each E:T ratio were assayed. Cytotoxicity of BALB/c, STAT4–/–, 129S6, and STAT1–/– cells was determined using a redirected lysis assay (24) with the use of 2C11 hybridoma targets with EL-4 cells to control for specificity.

Intracellular cytokine staining for IFN-{gamma}, granzyme B, and IL-4

Cells from in vitro cultures were harvested at 24, 48, or 72 h as indicated. For IFN-{gamma} and granzyme B staining, where restimulation was indicated, 1 µM OVA257–264 was added for 1 h followed by the addition of 0.6 µl/ml monensin-containing GolgiStop (BD Pharmingen) for an additional 3–4 h. For IL-4 staining (or IL-4 + IFN-{gamma}), where restimulation was indicated, 1 µM OVA257–264 was added together with brefeldin A-containing GolgiPlug (BD Pharmingen) for a period of 4 h. Cells were washed, fixed in Cytofix buffer (BD Pharmingen) for 15 min at 4°C, and permeabilized in saponin-containing Perm/Wash buffer (BD Pharmingen) before staining with allophycocyanin-conjugated Ab to IFN-{gamma}, PE-conjugated Ab to IL-4, PE-conjugated Ab to granzyme B, and/or PE-conjugated Ab to CD8 for 30 min at 4°C. Cells were then washed twice and analyzed by flow cytometry.

ELISA for IFN-{gamma} production

Cells from in vitro cultures were harvested at 90 h and replated at a density of 1.5 x 105 cells/ml. Supernatants were collected from replated cultures at 24 h. The concentration of IFN-{gamma} in the supernatants was determined using a Mouse IFN-{gamma} ELISA Ready-SET-Go! kit (eBioscience) according to the protocol provided with the exception that standards and samples were diluted in culture medium instead of assay diluent.

Real-time PCR

Total RNA was isolated from 5 x 106 MACS-purified naive CD8 T cells or cells cultured for 72 h in the presence of Ag/B7 aAPC with or without IL-12 and/or IL-21 using the RNeasy Mini Kit and on-column DNase digestion (Qiagen) according to the manufacturer’s protocol. First-strand cDNA was synthesized using the Reverse-iT 1st Strand Synthesis Kit (ABgene) with anchored oligodeoxythymidylate and random decamers. IFN-{gamma} and HPRT relative expression levels were quantified with iQ SYBR Green I Master Mix (Bio-Rad) using mouse-specific primers (Quantitect Primers; Qiagen). Duplicate PCR reactions were performed in an iQ4 Real-Time PCR Detection System (Bio-Rad) by denaturing at 95°C for 4.5 min, followed by 45 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 40 s. To confirm amplicon specificity, the PCR products were subjected to melting curve analysis (tm) at 95°C for 1 min and 55°C for 1 min, and then ramping from 55 to 95°C over 13 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-21 enhances clonal expansion and supports development of cytotoxicity during naive CD8 T cell activation

Naive CD8 T cells were purified from OT-I TCR-transgenic mice specific for H-2Kb/OVA257–264 (26) to yield a cell population of >95% CD8+ and >99% CD44low. For stimulation, aAPCs were prepared by immobilizing H-2Kb and B7-1 on latex microspheres and pulsing with OVA257–264 (Ag/B7 aAPC). Artificial APC prepared in this manner can fully activate CD44high memory CD8 T cells from OT-I mice, but activation of CD44low naive cells requires an additional signal that can be provided by IL-12 (4) or IFN-{alpha} (5). At low Ag concentrations, addition of IL-12 or IFN-{alpha} can strongly enhance clonal expansion in response to the aAPC, and this was also the case for IL-21. Naive OT-I cells stimulated for 3.5 days with aAPC pulsed with 2 nM OVA257–264 underwent minimal clonal expansion, whereas addition of IL-21 increased cell recovery >5-fold, similar to the increased expansion that results from addition of IL-12 to the cultures (Fig. 1a). The addition of both cytokines, IL-21 and IL-12, did not synergistically enhance clonal expansion. IL-21 also enhanced clonal expansion when the aAPC were made using 10 and 100 nM concentrations of OVA peptide (data not shown).


Figure 1
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  		FIGURE 1. IL-21 promotes CD8 T cell clonal expansion and provides a necessary signal for development of cytolytic activity. a, Naive OT-I T cells were stimulated with Ag/B7 aAPC (loaded with 2 nM OVA257–264). Where indicated, IL-21 was added at 100 ng/ml and IL-12 was added at 2 U/ml. Clonal expansion on day 3.5 is expressed as average number of OT-I cells recovered per well. Error bars display SD (n = 6), and results are representative of three independent experiments. b, Naive OT-I cells were stimulated with Ag/B7 aAPC (loaded with 0.2 µM OVA257–264) and IL-21 as indicated at 0, 2, 10, or 100 ng/ml. To control for Ag specificity, Ag/B7 aAPC lacking peptide were used to stimulate the naive OT-I cells at the highest concentration of IL-21. Cells were harvested on day 3, and cytolytic activity was assessed in a 4-h 51Cr release assay. For all conditions, nonspecific cytotoxicity was assessed in parallel using EL4 targets; in all cases, lysis was ≤5%. Results are representative of two independent experiments.

 
To test whether IL-21 can stimulate development of cytolytic function, naive cells were stimulated with aAPC made using 200 nM OVA peptide. This results in substantial clonal expansion in the absence of cytokine, providing sufficient numbers of cells to examine in cytolytic assays. Despite clonal expansion, cells stimulated in the absence of cytokine fail to develop significant cytolytic activity by day 3 (Fig. 1b). However, addition of IL-21 supported the development of highly effective cytolytic activity in a dose-dependent manner, and this was dependent on Ag/B7-1 stimulation, given that addition of IL-21 alone resulted in no cytolytic activity (Fig. 1b). B7-1 was not required for development of IL-21-dependent cytolytic activity if IL-2 was present in the cultures (data not shown).

Optimal IL-21 induction of CD8 T cell cytolytic activity requires IL-2, but not IL-12 or IFN-{alpha}

Because IL-21 is structurally similar to IL-2 and both cytokines have been shown to positively regulate CD8 T cell clonal expansion (4, 17) and because relatively high concentrations of IL-21 were required to elicit optimal effects on CD8 T cell cytolytic activity, we tested whether similarly high concentrations of IL-2 would promote development of cytolytic activity. Purified naive OT-I were cultured for 3 days with Ag/B7 aAPC with or without IL-2 at concentrations up to 100 ng/ml. At all concentrations tested, IL-2 failed to stimulate cytolytic activity above background levels even though clonal expansion was maintained at levels equal to those obtained when IL-21 was present (Fig. 2a). Thus, although both IL-2 and IL-21 signal through a {gamma}c cytokine receptor, IL-21 is uniquely capable of inducing CD8 T cell differentiation.


Figure 2
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  		FIGURE 2. High levels of IL-2 do not support CD8 T cell development of cytolytic activity, but IL-2 is required for optimal IL-21-mediated gain of function. a, Naive OT-I cells were stimulated with Ag/B7 aAPC and IL-2 at the indicated concentrations, or IL-21 at 100 ng/ml for a positive control. b, Naive OT-I cells were stimulated for 3 days with Ag/B7 aAPC with and without IL-21 or with IL-21 + anti (a-)-CD25 (PC61.5.3)-blocking Ab. Cells were harvested on day 3, and cytolytic activity was assessed in a 4-h 51Cr release assay. For all conditions, nonspecific cytotoxicity was assessed in parallel using EL4 targets; in all cases, lysis was ≤6%. Results are representative of two independent experiments.

 
It is clear that IL-2 cannot substitute for IL-21 to support development of cytolytic activity (Fig. 2a); however, synergistic interactions have been reported for IL-21 and other IL-2 family members (19, 23). Although we do not add exogenous IL-2 to the cultures, CD8 T cells are capable of transient autocrine IL-2 production upon activation (10, 27, 28). To test whether IL-2 was cooperating with IL-21 in development of cytolytic activity, we blocked the ability of the cells to respond to IL-2 by addition of an anti-IL-2R{alpha} (CD25) mAb. Naive OT-I cells were cultured with Ag/B7 aAPC in the presence or absence of IL-21 and anti-CD25 Ab. By day 3, cells given IL-21 were effective cytolytic mediators compared with cells cultured without cytokine. In contrast, cytolytic activity was significantly impaired when the IL-2R{alpha} chain was blocked (Fig. 2b). Therefore, IL-21 cooperates with CD8 T cell-derived IL-2 to elicit maximal induction of cytolytic activity.

IL-12 and IFN-{alpha} can support development of cytolytic activity by naive CD8 T cells (4, 5). To rule out the possibility that one of these cytokines might be contributing to the responses seen here, perhaps through IL-21 stimulation of a small number of contaminating cells in the naive T cell population, we tested the ability of each cytokine to elicit cytolytic activity in the presence of neutralizing Abs specific for the other two cytokines. In all cases, the only neutralizing Ab capable of blocking development of function was the Ab specific for the supplemented cytokine; only the anti-IL-21 Ab blocked the response to IL-21 (Fig. 3). Thus, each cytokine is capable of supporting development of cytolytic activity independently.


Figure 3
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  		FIGURE 3. IL-21, IL-12, and IFN-{alpha} (IFNa) independently deliver the necessary signal for development of cytolytic activity. Naive OT-I cells were stimulated with Ag/B7 aAPC and IL-12 (a), IL-21 (b), or IFN-{alpha} (c). In each panel, the cytokine used to stimulate the OT-l cells was also incubated with its specific neutralizing or a mixture of the neutralizing Abs specific for the other two cytokines. Cells were harvested on day 3, and cytolytic activity was assessed in a 4-h 51Cr release assay. For all conditions, nonspecific cytotoxicity was assessed in parallel using EL4 targets; in all cases, lysis was ≤5%. Results are representative of two independent experiments.

 
IL-21 signaling requirements for the initiation of differentiation are distinct from those of IL-12 and IFN-{alpha}

Development of CD8 T cell cytolytic activity in response to IL-12 and IFN-{alpha} is STAT4-dependent (5). Because IL-21 has been shown to activate STAT4 (19), we tested whether the IL-21-mediated acquisition of cytolytic activity was also dependent on STAT4. Naive CD8 T cells were purified from BALB/c and STAT4–/– mice. Because these mice are not TCR transgenics, cells were stimulated with aAPC coated with Abs for the Vb5 and Vb8 TCR chains along with recombinant B7-1 protein (anti-TCR/B7 aAPC) to provide polyclonal stimulation. Cytolytic activity was determined on day 4, the peak of the polyclonal response, in a redirected lysis assay using 2C11 hybridoma targets bearing the anti-CD3 mAb on their surface (24). IL-21 induced comparable cytolytic activity in both BALB/c and STAT4-deficient CD8 T cells, whereas, as expected, STAT4–/– cells failed to respond to IL-12 (Fig. 4, a and b). Thus, in contrast to IL-12 and IFN-{alpha}, IL-21 induction of cytolytic function is not STAT4 dependent.


Figure 4
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  		FIGURE 4. IL-21 does not signal via a STAT4-dependent or STAT1-dependent pathway to mediated CD8 T cell development of cytolytic activity. Naive CD8 T cells purified from BALB/c (a), STAT4–/– (b), 129S6 (c), or STAT1–/– (d) mice were stimulated with anti-TCR/B7 aAPC and cytokines IL-21, IL-12, or IFN-{alpha} as indicated. To allow for maximal expansion of the polyclonal population, cells were harvested on day 4, and cytolytic activity was assessed via a redirected lysis assay using 2C11 hybridoma targets to redirect the killing to the anti-CD3-bearing targets. For all conditions, nonspecific cytotoxicity was assessed in parallel using EL4 targets; and in all cases lysis was ≤7%. Results are representative of two independent experiments.

 
IL-21 is also a strong inducer of STAT1 activation (19, 29), and mice deficient in STAT1 have demonstrated defective tumor rejection, suggesting a failure to develop an effective CD8 T cell response (30). A direct role for STAT1 in the up-regulation of cytolytic effector function is also suggested by the IL-6-induced binding of STAT1a to a functional STAT-binding element in the human perforin promoter (31, 32). To test whether IL-21-mediated enhancement of cytolytic activity required STAT1, naive CD8 T cells were purified from 129S6 and STAT1–/– mice. Polyclonal stimulation using anti-TCR/B7 aAPC and redirected lysis assays were conducted as above. The STAT1-deficient CD8 T cells did not exhibit any defect in cytotoxicity compared with the wild-type controls when the required signal was provided by IL-21 or IL-12 (Fig. 4, c and d). In contrast, IFN-{alpha} induced development of cytotoxicity was significantly enhanced in STAT1-deficient CD8 T cells compared with their wild-type counterparts. A similar enhancement of clonal expansion in response to Ag and IFN-beta in STAT1–/– T cells has been reported (33). Thus, STAT1 is not required for IL-21-mediated development of cytolytic activity, and it may limit the response to IFN-{alpha}.

IL-21 does not induce production of IFN-{gamma}

Cytolytic activity and the ability to produce IFN-{gamma} upon restimulation with Ag are characteristic effector functions of most activated CD8 T cell populations. IL-12 and IFN-{alpha} promote CD8 T cell acquisition of both of these effector functions by a STAT4-dependent mechanism (5, 34). There are conflicting reports concerning the effect of IL-21 on IFN-{gamma} production by CD8 T cells and other cell types (19, 23, 35, 36). To examine the effect of IL-21 on naive CD8 T cell acquisition of IFN-{gamma}-secreting capacity, purified naive OT-I cells were stimulated with Ag/B7 aAPC alone or with IL-12 or IL-21 added to the wells on day 0. Cells were harvested on days 1, 2, and 3 and stained for intracellular IFN-{gamma}. Without cytokine, OT-I cells did not acquire the ability to produce IFN-{gamma}. As expected, IL-12 elicited substantial populations of IFN-{gamma}-producing cells on days 1, 2, and 3. IL-21, in contrast, failed to produce a significant population of IFN-{gamma}-producing cells on any of the days, even when day 3 cells were restimulated with additional peptide before intracellular staining (Fig. 5). When cells stimulated for 3 days were tested for cytolytic activity, comparable target cell lysis was found whether stimulation was with IL-21 or IL-12 (data not shown). The IL-21-stimulated cells were not completely devoid of the capacity to make cytokines, given that day 3 cells produced TNF-{alpha} upon restimulation to an extent similar to that of IL-12-stimulated cells (data not shown). Thus, IL-21 supports development of effective cytolytic function but fails to promote naive CD8 T cell acquisition of IFN-{gamma} production capacity.


Figure 5
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  		FIGURE 5. IL-21 does not support CD8 T cell development of IFN-{gamma} (IFN-g) production capacity. Naive OT-I cells were stimulated with Ag/B7 aAPC without cytokine, with IL-12, or with IL-21 as indicated. Cells were harvested on day (D) 1, day 2, or day 3 and restimulated (restim) where indicated. IFN-{gamma} production was assessed via intracellular cytokine staining. Results are representative of three independent experiments.

 
The inability of IL-21 to induce IFN-{gamma} production raised the possibility that it might also inhibit the IFN-{gamma} response to IL-12. To examine this, purified naive OT-I cells were stimulated with Ag/B7 aAPC and IL-12, IL-21, or the combination. Cells stimulated in the presence of IL-12 beginning at the initiation of the cultures expressed IFN-{gamma} on day 2 and strongly up-regulated IFN-{gamma} production in response to peptide restimulation on day 3, whereas cells stimulated with IL-21 produced little IFN-{gamma} at any time (Fig. 6, a–c). The same results were obtained when cytokine addition was delayed until 16 h after the cultures were initiated. To examine potential inhibitory effects of IL-21 on IL-12-induced IFN-{gamma} production, IL-12 was added at 0 h and IL-21 at 16 h, or vice versa. IFN-{gamma} production on day 2 in response to IL-12 was substantially inhibited by IL-21, and the inhibition was greater when IL-21 was present first (Fig. 6a). The inhibitory effect of IL-21 was still significant though less pronounced, when IFN-{gamma} production in response to restimulation with additional peptide was examined on day 3 (Fig. 6c). The IFN-{gamma} mean fluorescence intensity for cells stimulated with just IL-12 was 448, and this was reduced to 244 and 235 when IL-21 was added at either 0 h or 16 h, respectively.


Figure 6
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  		FIGURE 6. IL-21 partially inhibits IL-12 mediated induction of IFN-{gamma} production, but fully up-regulates granzyme B levels. Naive OT-I cells were stimulated with Ag/B7 aAPC. IL-21 or IL-12 was added at the indicated time points. Cells were harvested on day 2 or day 3 and restimulated where indicated. IFN-{gamma} (IFN-g) production (a–c) or granzyme B production (d–f) was assessed via intracellular cytokine staining. c, The mean fluorescent index (MFI) for each population has been annotated. g, Naive OT-1 cells were stimulated with Ag/B7 aAPC (loaded with 20 nM OVA257–264) with and without IL-12 or IL-21 added at 0 h as indicated. Cells were harvested at 90 h and replated at 1.5 x 105 cells/ml with and without 1 µM OVA257–264). IFN-{gamma} production was assessed via ELISA on supernatants collected 24 h postreplating. Error bars represent SD of the mean for triplicate samples. a–f, Data are representative of three or more independent experiments except for conditions (iv + v) which were repeated twice. g, Results are representative of three independent experiments.

 
When granzyme B expression was examined for cells stimulated with IL-12, IL-21, or the combination, induction of expression was very similar for all conditions (Fig. 6, d–f). For the experiment shown in Fig. 6, a–f, cells stimulated in the absence of cytokine had no cytolytic activity, whereas potent and comparable cytolytic activity was obtained for all of the populations stimulated with IL-12, IL-21, or the combination (data not shown).

To better quantify the combined effect of having a smaller population of cells secreting IFN-{gamma} at a lower average mean fluorescence intensity per cell, we performed a similar experiment adding IL-12, IL-21, or both cytokines at 0 h. Cells were harvested washed and replated at a normalized cell density after 4 days in culture, and supernatants were harvested on day 5 for an IFN-{gamma} ELISA. Similar to the results obtained using intracellular staining, supernatants from IL-12-stimulated cells contained nearly 10-fold more IFN-{gamma} than supernatants from cells stimulated without cytokine or cells stimulated with IL-21 (Fig. 6g). Supernatants from cells stimulated simultaneously with IL-12 and IL-21 contained only one-third the amount of IFN-{gamma} as supernatants from IL-12-stimulated cells. Thus, IL-21 fails to stimulate significant IFN-{gamma} production and can partially inhibit IFN-{gamma} production in response to IL-12, while inducing comparable granzyme B expression and generation of cytolytic activity.

To determine whether the IL-21-mediated inhibition of IFN-{gamma} production was due to regulation at the mRNA level, real-time PCR and flow cytometric analysis were used to compare levels of IFN-{gamma} mRNA and protein in naive OT-1 stimulated with aAPC and IL-12 with and without IL-21. Supplementation with IL-21 inhibited ~90% of the IFN-{gamma} protein and 80% of the IFN-{gamma} mRNA induced by IL-12 (Fig. 7b). Although restimulation with additional peptide greatly increased IFN-{gamma} mRNA levels in all conditions tested (Fig. 7a), it could only partially restore IFN-{gamma} mRNA and protein levels in IL-21 inhibited cells (Fig. 7b). Thus, IL-21-mediated inhibition of IL-12-induced IFN-{gamma} occurs at least partially at the mRNA level.


Figure 7
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  		FIGURE 7. IL-21 inhibits IL-12-induced up-regulation of IFN-{gamma} (IFNg) mRNA levels. Naive OT-I cells were stimulated with Ag/B7 aAPC with or without IL-12 and/or IL-21 as indicated. Cells were harvested at 72 h and incubated with and without 1 µM OVA257–264 for an additional 4 h. Real-time PCR was used to quantify the fold increase of IFN-{gamma} mRNA levels over naive controls, whereas the percent of OT-1 cells positive for IFN-{gamma} protein was assessed by intracellular staining. a, Fold increase in cells stimulated with Ag/B7 aAPC and the indicated cytokines versus mRNA expression in naive cells. Results are shown for one representative experiment. Error bars show the range of duplicate samples. b, Percent IL-21-mediated inhibition of IL-12-induced IFN-{gamma} protein and mRNA expression. The percent inhibition was calculated as [(IL-12 response – IL-12 + 21 response)/(IL-12 response)] x 100. Values are the mean ± SD for three independent experiments. Restim, Restimulated.

 
IL-21 does not induce production of IL-4

Dutton and coworkers (37, 38) showed that CD8 T cells, including OT-1 T cells, could be skewed to a Th1-like or Th2-like phenotype when cultured in the presence of either IL-12 or IL-4. The Tc2 population resulting from stimulation in the presence of IL-4 had cytolytic activity and produced IL-4 and IL-5, but not IFN-{gamma}. To determine whether IL-21 produces CD8 effectors of the Tc2 phenotype, purified naive OT-I cells were stimulated with Ag/B7 aAPC with and without IL-12 or IL-21. Cells were harvested on day 3, restimulated, and stained for intracellular IL-4 and IFN-{gamma}. As expected, cells stimulated with IL-12 induced a robust IFN-{gamma} response whereas cells stimulated with IL-21 did not. Neither cytokine induced significant IL-4 production (Fig. 8). IL-21 also failed to induce any significant IL-5 production (data not shown). Commercially available fixed, polarized CD4 cells served as positive controls for the cytokine staining. Mick 1 cells demonstrated characteristic Th1 skewing toward IFN-{gamma} production, whereas Mick 2 cells demonstrated characteristic Th2 skewing toward IL-4 production (Fig. 8). Thus, IL-21 does not result in generation of CD8 effector cells having a Tc2 phenotype.


Figure 8
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  		FIGURE 8. IL-21 does not support CD8 T cell development of IL-4 production capacity. Naive OT-I cells were stimulated with Ag/B7 aAPC. IL-21 or IL-12 was added at the indicated time points. Cells were harvested on day 3 and restimulated. IL-4 and IFN-{gamma} production were assessed via intracellular cytokine staining. Mick 1 and Mick 2 control cells were also stained with IL-4 and IFN-{gamma}.

 
IL-21 promotes naive CD8 T cell acquisition of a distinct surface molecule expression pattern

Effector cells that have differentiated in response to IL-21 are quite different from previously described CD8 T cell subtypes with regard to effector function regulation and signaling. To determine whether IL-21-differentiated cells might also have a distinct set of surface molecules, the surface phenotype of day 3 effector cells generated with aAPC and IL-21 was analyzed by flow cytometry. Cells stimulated with aAPC alone served as controls for the effects of Ag and costimulation, whereas cells stimulated with aAPC and IL-12 were included to represent the more classical CD8 effector phenotype. Synchronous CD44 up-regulation above naive control levels confirmed uniform activation by day 3 for all effector groups (Fig. 9a). Uniform expression of CD122 (IL-2Rbeta) on all effector groups served as an additional control for cell size, ensuring that populations could be appropriately compared (Fig. 9b). We found that, in contrast to cells stimulated in the absence of cytokine or with IL-12, IL-21-differentiated effector CD8 T cells were CD25 (IL-2R{alpha})low, CD134 (OX-40)low, CD137 (4-1BB)high, and CD279 (PD-1)low (Fig. 9b) Therefore, IL-21-differentiated effector CD8 T cells can be distinguished from their more classical counterparts based on surface phenotype.


Figure 9
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  		FIGURE 9. IL-21- and IL-12-differentiated CD8 T cells display distinct surface molecule expression patterns. Naive OT-I cells were stimulated with Ag/B7 aAPC without cytokine, with IL-12, or with IL-21 as indicated. Surface molecule expression after 72 h was determined via flow cytometric analysis. a, Uniform activation of cells for each culture condition is confirmed with CD44 staining. Naive control is shown for comparison. b, Expression of the indicated cell surface proteins. Naive controls overlap with isotype controls and are not included.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous work strongly suggested that IL-21 had the capacity to modulate CD8 T cell responses. However, the experimental systems utilized in these investigations did not permit resolution of which cell type(s) the IL-21 was acting on, or precisely what effect the IL-21 had on that cell type. Using highly purified cell populations and stable, defined sources of Ag and costimulation, we have shown that IL-21 supports the Ag-dependent clonal expansion and effector function acquisition of naive CD8 T cells. This puts IL-21 into a small class of proinflammatory cytokines capable of acting directly on naive CD8 T cells to provide the additional signal required to initiate their differentiation into effector cells. We have designated this signal as signal 3, because it is required in conjunction with signals 1 and 2 (Ag and costimulation) to generate effector function and avoid long-term tolerance.

IL-21 induces a unique CD8 T cell differentiation program in comparison with the other signal 3 cytokines, IL-12 and IFN-{alpha}, as demonstrated by the distinct surface phenotype of IL-21-stimulated cells (Fig. 9), and the inability of these cells to produce IFN-{gamma}. Of the three known third-signal cytokines, IL-21 is the only one belonging to the {gamma}c cytokine family. Although all of the third-signal cytokines activate STAT4 (19, 34, 39), IL-21 is uniquely capable of inducing cytolytic activity in the absence of STAT4 (Fig. 4). This suggests that a STAT4-independent pathway is sufficient to support naive CD8 T cell acquisition of cytolytic activity and that among the known third-signal cytokines, this pathway is exclusive to IL-21. It is unlikely that this alternate pathway is STAT1 mediated, because all-third signal cytokines activate STAT1 (19, 29, 40, 41), but none requires it for the induction of cytolytic activity (Fig. 4). In fact, IFN-{alpha}-induced cytotoxicity is actually enhanced in STAT1-deficient CD8 T cells, suggesting that STAT1 might play an inhibitory role in the induction of cytotoxicity. This is consistent with a recently published report by Tanabe et al. (33) demonstrating a strong mitogenic effect of INF-{alpha}beta on STAT1-deficient but not wild-type T cells. However, Liang et al. (42) have shown that STAT1 is required for IFN-{alpha}-mediated NK cell cytotoxicity, and Morishima et al. (43) have shown that IL-27-mediated CD8 T cell up-regulation of granzyme B and perforin is also dependent on STAT1. Thus, STAT1 appears to play a complex role in the regulation of cytotoxic activity.

Perhaps mechanistically related to its distinct signaling pathway, IL-21 is the only third-signal cytokine that fails to support development of effector type IFN-{gamma} production (Fig. 5). Furthermore, IL-21 partially inhibits an otherwise strong IFN-{gamma} response induced by IL-12, and this inhibition is seen at the level of protein (Fig. 6) and mRNA (Fig. 7) expression. Suto et al. (44) have recently shown that IL-21 inhibits IFN-{gamma} production by CD4 T cells through the repression of eomesodermin. Whether this is involved in IL-21-mediated inhibition of IFN-{gamma} production by CD8 T cells remains to be determined. IL-21-mediated inhibition of IFN-{gamma} production by CD8 T cells is weakest for day 3-restimulated samples, and treatment with both IL-21 and IL-12 results in equivalent IFN-{gamma} production regardless of the order of administration. This suggests that IL-21 can mediate its inhibitory effect well into the effector differentiation program. In summary, the IL-21 third signal is mechanistically and effectively distinct from the type of third signal provided by IL-12 or IFN-{alpha}, with respect to both the signaling pathway used and the functional outcome.

Of perhaps greatest significance, these experiments describe the generation of a unique CD8 effector subtype capable of mediating highly effective cytolytic activity, but deficient in IFN-{gamma} production and not belonging to the previously described Tc2 subset (37). There have been previous reports of dissociation of cytolytic function and cytokine secretion by CD8 T cells. Effector CD8 T cells (45) and cloned CTL lines (10) have cytolytic activity but are unable to produce IL-2 upon restimulation. Snyder et al. have reported the generation of CD8 effector cells capable of secreting IFN-{gamma}, but defective for cytolytic activity (46); however, their individual cell-based assay was not designed to detect effectors of the opposite phenotype. Two groups have independently described CD8 T cell populations capable of cytolytic function, but not IFN-{gamma} secretion (47, 48). However, because both of these groups were working with human T cell clones derived from peripheral blood lymphocytes, it is not possible to directly compare their findings with naive CD8 T cell activation and subsequent gain of effector function. Additionally, both of these groups assayed IFN-{gamma} production by ELISA, which does not permit resolution of cytokine secretion capacity on an individual cell basis. Our work demonstrating the concurrent production of high levels of granzyme B by nearly all cells of a highly cytolytic effector population that is uniformly unable to produce IFN-{gamma}, provides the strongest evidence to date for the uncoupling of cytotoxicity and IFN-{gamma} secretion within a single CD8 T cell. Consistent with the results described here, Ma et al. (21) have described clearance of an IL-21-secreting tumor by a CD8- and NK cell-dependent mechanism requiring perforin but not IFN-{gamma}. Furthermore, we have observed an E.G7 tumor-infiltrating, Ag-specific population of granzyme B high, IFN-{gamma} negative CD8 effector cells (J. M. Curtsinger, M. Y. Gerner, D. C. Lins, and M. F. Mescher, unpublished data). Together, these findings carry important implications for the common presumption of CD8 effector cell cytolytic functionality based solely on the assessment of IFN-{gamma} production.

Although we have clearly demonstrated that IL-21 fails to induce CD8 T cells to make IFN-{gamma} and can even inhibit IL-12-mediated IFN-{gamma} production, it is important that we are examining only the direct effect of IL-21 on naive CD8 T cells responding in an Ag-specific manner. Zeng et al. (23) have shown that IL-21 can synergize with IL-15 to increase the total number of IFN-{gamma}+ CD8 T cells obtained after culture of splenocytes for 7 days with IL-15, IL-21, or IL-15 and IL-21 in the absence of Ag followed by stimulation with anti-CD3 and anti-CD28 for 0, 1, 2, or 4 h. Because there are no IFN-{gamma}+ cells in their IL-21 cultures and there is only a minor increase in the percent IFN-{gamma}+ cells in cultures containing IL-15 and IL-21 as compared with IL-21 alone, it is possible that the majority of the increase in absolute number of IFN-{gamma}+ cells in the cultures stimulated with IL-15 and IL-21 is due to IL-21-mediated increased proliferation and/or survival of the IFN-{gamma}+ cells induced by the IL-15. Alternatively, cells cultured with IL-21 alone in the absence of Ag had a predominantly naive (CD44low) phenotype, whereas the cells cultured with IL-15 alone had a predominantly memory (CD44high) phenotype (23). It is possible that IL-21 promotes IFN-{gamma} production by the memory phenotype cells obtained in the IL-15 cultures but is unable to elicit a similar effect on naive cells that were not exposed to IL-15. The same authors also demonstrate a reduction in the percentage of IFN-{gamma}+ CD8 T cells obtained after immunization of IL-21R–/– mice vs wild-type mice with recombinant vaccinia virus followed by a week-long Ag restimulation of bulk splenocytes harvested 5 days after infection. Here the authors are looking at an Ag-dependent primary CD8 response, and it is possible that in this in vivo system IL-21 does synergize with other cytokines to promote IFN-{gamma} production. This is especially of interest given that IL-12 has been shown to be required for CTL responses to vaccinia, and thus it would appear that in this in vivo system IL-21 is not inhibiting IL-12-induced Ag-dependent IFN-{gamma} production because the IL-21R–/– have fewer IFN-{gamma}+ CD8s. However, it is unclear what the mechanism for this effect would be, given that the IL-21R–/– mice were directly immunized with the virus; thus, all the cells and not just the CD8 T cells lacked an intact IL-21-signaling pathway. Therefore, the direct effect of IL-21 on CD8 T cells in the context of in vivo priming remains to be elucidated.

We have described here a direct role in naive CD8 T cell activation for a cytokine made exclusively by activated CD4 T cells, raising the question as to whether IL-21 secretion may be a novel mechanism for CD4 help. If IL-21 is capable of providing help for CD8 T cell responses, there is more than one way that this could occur. It is possible that IL-21 could be a contributing factor in CD4 help during the primary activation of naive CD8 T cells. Our data would suggest that IL-21 is certainly capable of mediating substantial effects during this crucial period in the life of a CD8 T cell. However, an activated CD4 T cell capable of making IL-21 would also likely be competent to activate or condition a resting DC. DCs activated via ligation of their CD40 molecules, as is thought to occur during CD4-mediated conditioning, have been shown to produce both IL-12 and IFN-{alpha} (49, 50, 51) and provision of any one of the signal 3 cytokines is sufficient to support differentiation of naive CD8 T cells. Even though CD4 help at this stage is thought to be mechanistically dependent on CD40/CD40L interactions, implying that DC-derived cytokines IL-12 and IFN-{alpha} may be important during this stage, it is also possible that the CD40/CD40L interaction is necessary to trigger CD4 T cell secretion of IL-21. One group has reported that IL-21 negatively impacts the T cell-priming capacity of DCs (52, 53). However, this work was done using bone marrow-derived DCs which are not always representative of resident lymph node or splenic DC subsets; furthermore, delayed-type hypersensitivity responses were used as the readout for T cell activation and therefore are not necessarily indicative of CD8 T cell influence. When this group examined the direct influence of IL-21 on the DC, they found that DCs treated with IL-21 failed to up-regulate costimulatory molecules. However, in the presence of a signal three cytokine, basal levels of costimulation appear to be sufficient to support full activation of naive CD8 T cells (K. A. Casey and M. F. Mescher, unpublished data). They also observed that IL-21 treatment of DCs resulted in increased uptake of Ag which was interpreted as reversion to an immature phenotype. In the presence of third-signal and basal levels of costimulatory molecules, increased capacity for Ag uptake could be highly beneficial to CD8 T cell responses.

IL-21 might also contribute to CD4 help by acting to support the maintenance phase of the CD8 T cell response. Because IL-21 is a member of the {gamma}c cytokine family, there is a distinct likelihood that it mediates some effect, at least collaboratively, on CD8 T cells during this stage given that similar roles have already been described for IL-2, IL-7, and IL-15 (54). In support of this idea, {gamma}c-chain-deficient mice show a much more severe CD8 T cell defect than any of the individual cytokine knockouts. Furthermore, Zeng et al. (23) have previously described a collaborative role for IL-21 and IL-15 in regulating Ag-independent proliferation of CD8 T cell populations. Alternatively or additionally, it is possible, given our data, that IL-21 production by activated CD4 T cells could play a role in down-modulating IFN-{gamma} secretion by CD8 T cells at the site of secondary challenge with Ag. Evidence exists to indicate that both Th1 and Th2 cells can produce IL-21 (35, 55, 56, 57). Because activated CD4 Th1 cells are already secreting copious quantities of IFN-{gamma}, this may provide a mechanism for the avoidance of cytokine storm repercussions. In contrast, TH2 CD4 cells could utilize IL-21 secretion to prevent Th1 from polarizing IFN-{gamma} production by CD8 T cells or other cell populations. Thus, IL-21 could influence CD8 T cells at the effector/memory stage by a variety of mechanisms. Elucidation of the in vivo roles for IL-21 in CD8 T cell responses will require further investigation.


   Acknowledgments
 
We thank Drs. Dan Mueller and Timothy Starr for critical reading of the manuscript, Pujya Agarwal for helpful suggestions, and Kendra Hyland for 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 National Institutes of Health Grants CA82596 and A134824. Back

2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, Box 334 Mayo, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail address: mesch001{at}umn.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; aAPC, artificial APC; {gamma}c, common {gamma}-chain. Back

Received for publication November 1, 2005. Accepted for publication March 7, 2007.


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 Materials and Methods
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 Discussion
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