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International Immunology 2007 19(10):1213-1221; doi:10.1093/intimm/dxm093
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

IL-21 synergizes with IL-7 to augment expansion and anti-tumor function of cytotoxic T cells

Shujuan Liu1, Gregory Lizée1, Yanyan Lou1, Chengwen Liu1, Willem W. Overwijk1, Gang Wang2,* and Patrick Hwu*

1 Department of Melanoma Medical Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA
2 Present address: U.S. Food and Drug Administration, Center for Biologics Evaluation and Research, 1401 Rockville Pike, HFM-675, Rockville, MD 20852, USA

Correspondence to: P. Hwu; E-mail: phwu{at}mdanderson.org


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
IL-21, a recently identified member of the common {gamma}-chain ({gamma}c) receptor cytokine family, has been shown to be an important regulator of both innate and adaptive immune responses. In this study, we investigated whether IL-21 could synergize with another {gamma}c cytokine, IL-7, to induce enhanced proliferation and effector function of tumor antigen-specific CD8+ T cells. Our results showed that IL-21 could significantly augment the IL-7-induced expansion of cytotoxic T cells, possibly by preventing the cytokine-induced down-regulation of the IL-7R{alpha} (CD127) on antigen-stimulated T cells. IL-21 also greatly enhanced the production of Th1 and inflammatory cytokines by activated T cells, including IFN-{gamma}, IL-2, tumor necrosis factor-{alpha}, granulocyte macrophage colony-stimulating factor, IL-1ß and IL-6. Finally, the addition of IL-21 to IL-7-cultured CTLs resulted in a considerably higher level of cytolytic activity, as measured by specific killing of tumor cells or antigen-pulsed target cells. These results suggest that IL-21 may play a cooperative role with IL-7 in modulating primary CD8+ T-cell responses and may have important implications for immunotherapy of cancer.

Keywords: cytotoxic T cell, IL-21, IL-7, immunotherapy, tumor immunology


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
CTLs can play a prominent role in the anti-tumor immune responses of both mice and humans (1–4). Many studies have demonstrated that {gamma}c receptor cytokines, including IL-2, IL-7 and IL-15, act at different stages of the immune response to promote the survival, proliferation and effector function of CD8+ T cells (5). IL-7 is critical for the homeostatic expansion of naive CD4+ and CD8+ T cells in immunodeficient hosts and can also mediate the survival of memory T cells (6, 7). Although IL-15 is the major cytokine known to stimulate the maintenance of memory CD8+ T cells, IL-7 can act in synergy with IL-15 to support their survival and expansion (8–10).

IL-21 is a more recently recognized member of the common {gamma}-chain ({gamma}c) receptor cytokine family. It is produced by activated CD4+ T cells, and its unique receptor (IL-21R{alpha}) is expressed in lymphoid tissues, in particular on NK cells, B cells, T cells, dendritic cells and macrophages (11–13). IL-21 enhances both the innate and adaptive components of tumor immunity, promoting the survival of tumor-bearing mice by potentiating the effector functions of NK and CD8+ T cells (14–22). IL-21 appears to modulate CD8+ T-cell responses by enhancing the homeostatic proliferation of CD8+ T cells in the presence of IL-15 and IL-7 (23). In addition, IL-21 can synergize with IL-15, IL-7 or IL-18 to induce augmented IFN-{gamma} production by CD8+ T cells upon anti-CD3 or allo-antigen stimulation (23–25).

Following the initiation of an immune response, CD4+ T cells secrete IL-21 along with a number of other cytokines, such that target immune cells are often exposed to several different {gamma}c cytokines simultaneously (26). The distinct yet overlapping functions of {gamma}c cytokine family members suggest that simultaneous signaling through multiple {gamma}c receptors may result in a range of biologically relevant outcomes. However, the functional role of IL-21 in the context of other {gamma}c cytokines known to regulate CD8+ T-cell function and survival during primary T-cell activation is not well understood.

Since cytotoxic T cells are capable of mediating substantial tumor regressions (2, 4), we explored whether the combination of IL-21 and IL-7 could potentiate the expansion and anti-tumor function of antigen-stimulated CD8+ T cells. To this end, we utilized CD8+ T cells derived from pmel-1 TCR transgenic mice, whose TCR recognizes an H-2Db-restricted epitope derived from gp100, a self/tumor antigen widely expressed by the majority of melanomas (27). Our results indicate that IL-21 can synergize with IL-7 to not only induce the expansion of antigen-activated CD8+ cells but also enhance their anti-tumor activity by significantly augmenting antigen-specific cytolysis of tumor cells and the production of Th1 and inflammatory cytokines. These results describe a novel synergy between two {gamma}c cytokines and may have implications for improving adoptive transfer-based regimens for the treatment of cancer.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Cells and cultures
Murine B16 melanoma cells and EL-4 lymphoma cells were cultured in RPMI 1640 complete medium (CM) supplemented with 10% fetal bovine serum, 50 U ml–1 penicillin, 50 µg ml–1 streptomycin, 2 mM L-glutamine and 50 µg ml–1 ß-mercaptoethanol (all from Invitrogen/Life Technologies, Inc., Rockville, MD, USA). Splenocytes (SPLs) were derived from pmel-1 TCR transgenic mice, whose TCR recognizes an H-2Db-restricted epitope corresponding to amino acids 25–33 of murine and human gp100, a self/tumor antigen widely expressed by a majority of melanomas. Approximately 90% of splenic CD3+ T cells in pmel-1 TCR mice are TCRVß13+ CD8+ T cells and demonstrate specificity for gp100 (27).

CTL stimulation
Single-cell suspensions of pooled spleens from pmel-1 mice were prepared by gently homogenizing the tissues and passing them through a 40-µm cell strainer. Erythrocytes were depleted with ACK lysis buffer (Biosource, Rockville, MD, USA). Cells were plated at 1 x 106 ml–1 in RPMI 1640 CM in six-well plates with 0.1 µg ml–1 hgp10025–33 peptide (KVPRNQDWL) and cultured for 2 days. Naive CD8+ cells showed a CD25–CD69–CD44low phenotype prior to peptide stimulation. After 2 days of stimulation, all CD8+ T cells showed an activated, CD25+CD69+CD44high phenotype (data not shown). Cells were then re-plated in 24-well plates at 4e5 cells per well in the presence of different doses of recombinant mIL-7 (R&D Systems, Minneapolis, MN, USA) and/or mIL-21 (R&D Systems) for further phenotypic and functional assays. Additional cytokines were added to the culture media whenever the cells were split.

Proliferation assay
Two days after peptide stimulation, T cells were washed and re-plated in 96-well flat-bottom plates at 2 x 104 cells per well in the presence of different doses of mIL-7 with or without titrated mIL-21. Cell proliferation was evaluated by adding [3H] thymidine (Amersham Biosciences, Piscataway, NJ, USA) to the cultures at 1 µCi per well for 16 h before harvesting and washing the cells. Incorporated [3H] thymidine was determined by liquid scintillation counting.

Flow cytometry
Cells were washed and re-suspended in FACS buffer (Dulbecco's phosphate-buffered saline containing 1% BSA), then incubated for 30 min at 4°C with the appropriate FITC-, PE- or APC-conjugated antibodies to the indicated cell-surface markers and then washed again with FACS buffer. For surface staining, the antibodies used were as follows: CD8–APC (53-6.7), CD132–PE (4G3), CD69–FITC (H1.2F3) (BD/Biosciences, San Diego, CA, USA) and CD127 (IL-7R{alpha})–PE (A7R34) (eBioscience, San Diego, CA, USA). For intracellular staining, the antibodies BCL2–PE (3F11) (BD/Biosciences), perforin–PE (JAW246) and granzyme B–FITC (16G6) (eBioscience) were used, according to the manufacturer's instructions. Apoptosis was assayed by flow cytometry using an Annexin V Apoptosis Detection kit from BD/PharMingen according to the manufacturer's instructions. Flow cytometric analysis was performed on a FACScan with CellQuest software (Becton-Dickinson, San Jose, CA, USA).

Quantitative real-time (RT)-PCR for IL-7R{alpha} gene expression
T cells were stained with anti-CD8 and anti-IL-7R{alpha} mAbs. CD8+, CD8+CD127+ and CD8+CD127– T cells were sorted on a FACSAria (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) cell sorter. Total RNA from sorted cells was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. First-strand cDNA was reverse transcribed from total RNA using Superscript-III Reverse Transcriptase and random hexamers (Invitrogen). IL-7R{alpha} (Mm00434295_ml) and ß-actin (4352341E) primers and probes were ordered from ABI (Applied Biosystems). PCR for each primer/probe set was performed in triplicate using an ABI Fast Real-Time PCR System (ABI PRISM 7500 Sequence Detection System) according to the manufacturer's specifications. Relative gene expression was calculated in terms of the difference in threshold cycles (Ct) for target and reference ({Delta}Ct) (2E – {Delta}Ct).

Cytokine release assay
T cells were washed and re-plated at 1 x 105 cells per well in 96-well flat-bottom plates. EL-4 cells (1 x 106 ml–1) were pulsed with either 1 ng ml–1 hgp10025–33 peptide or an H-2Db-restricted epitope of the influenza nucleoprotein (NP) peptide (NP366–374, ASNENMETM) for 2 h at 37°C, and then washed twice. EL-4 cells (1 x 105 cells per well) and T cells were co-cultured for 18 h in a total volume of 200 µl. Supernatant was collected and analyzed for the production of Th1, Th2 and inflammatory cytokines using a Luminex 100 mouse cytokine ten-plex antibody bead kit, including IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-{gamma} and tumor necrosis factor-{alpha} (TNF-{alpha}) (Invitrogen/Biosource, Camarillo, CA, USA). IFN-{gamma} release was detected by ELISA (Pierce Endogen, Rockford, IL, USA). Specific cytokine production was calculated by subtracting the background cytokine release (from co-culture of T cells with NP366–374 pulsed EL-4 cells) from the cytokine production by co-cultured T cells and hgp10025–33 pulsed EL-4 cells.

Intracellular cytokine staining
T cells and hgp10025–33 or NP366–374 pulsed EL-4 cells were prepared as described above. T cells and peptide-pulsed EL-4 cells were co-cultured in 96-well round-bottom plates for 5 h at 37°C with 0.1 µl Golgi Plug (BD/PharMingen) in 100 µl CM each well. Cells were then harvested and stained for intracellular IFN-{gamma} (XMG1.2), TNF-{alpha} (MP6-XT22) or IL-2 (JES6-5H4) (BD/Biosciences) using a Cytofix/Cytoperm plus kit (BD/PharMingen) as per the manufacturer's instructions. Briefly, cells were stained with CD8–APC, washed with FACS buffer, then fixed and permeabilized with BD Cytofix/Cytoperm solution for 20 min at 4°C. Cells were then washed, incubated with intracellular cytokine mAbs for 30 min at 4°C and washed again with BD Perm/Wash solution. Flow cytometry was performed on a FACScan with CellQuest software.

Cytotoxicity assay
EL-4 cells were pulsed with 1 ng ml–1 hgp10025–33 or NP366–374 peptide for 2 h at 37°C, and then washed. Target cells (B16 or EL-4) were incubated at 37°C for 1 h with 100 µCi of 51Cr, washed and plated with effector cells at the indicated effector to target cell (E:T) ratios. After 4 h incubation at 37°C, supernatants were harvested and radioactivity was measured in a gamma counter. Maximum 51Cr release was determined by lysing target cells with 1 N HCl. Spontaneous release was determined as 51Cr released into the supernatant of targets incubated in the absence of effectors. Percent specific lysis was calculated according to the formula: (experimental release – spontaneous release)/(maximum release – spontaneous release) x 100.

Statistical analyses
Statistical comparisons were done using Student's t-tests. P values < 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
IL-21 synergizes with IL-7 to enhance proliferation of tumor antigen-activated T cells
In order to assess the capacity of IL-21 to enhance the proliferation and anti-tumor function of tumor antigen-specific T cells, we first stimulated pmel-1 TCR transgenic SPLs with hgp10025–33 peptide for 2 days, and then cultured the antigen-activated T cells with a range of doses of IL-21 alone or in combination with IL-7. Measurement of [3H] thymidine incorporation by these cells showed that IL-7 or IL-21 alone induced low levels of T-cell proliferation, while IL-21 could significantly augment proliferation of antigen-activated T cells when co-cultured with IL-7 in a dose-dependent manner (Fig. 1A).


Figure 1
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  		Fig. 1. IL-21 synergizes with IL-7 to promote enhanced proliferation, but not survival, of activated CD8+ T cells. SPLs from pmel-1 transgenic mice were stimulated in vitro with hgp10025–33 peptide (100 ng ml–1) for 2 days, followed by addition of IL-7 alone, IL-21 alone or a combination of both cytokines for an additional 48 h. (A) Proliferation of day-4 T cells cultured in the presence of indicated concentrations of IL-7 and/or IL-21 was evaluated using a [3H] thymidine incorporation assay. c.p.m., counts per minute. (B) Apoptosis and bcl-2 expression of day-4 CD8+ T cells cultured in IL-7 and/or IL-21 was evaluated by Annexin V or bcl-2 staining and flow cytometry. Dotted line histograms indicate no added cytokines; solid histograms indicate addition of IL-7 (10 ng ml–1), IL-21 (10 ng ml–1) or both cytokines together (10 ng ml–1 of each). Numbers indicate perecentages of Annexin V-Negative cells. Data shown are representative of three independent experiments.

 
We next evaluated the viability of the T cells cultured under the different cytokine conditions. As shown by Annexin V staining, the majority of tumor antigen-activated pmel-1 CD8+ T cells underwent apoptosis 4 days after peptide stimulation when cultured in the absence of {gamma}c cytokines. As expected, known T-cell survival {gamma}c cytokine IL-7 rescued the cells from undergoing activation-induced apoptotic cell death (Fig. 1B). However, IL-21 alone was unable to rescue these cells from apoptosis, and had no obvious influence on the anti-apoptotic effects of IL-7 (Fig. 1B). Staining of these cells for expression of the anti-apoptotic molecule bcl-2 further revealed that IL-21 was unable to up-regulate bcl-2 expression as compared with the cells cultured with IL-7 (Fig. 1B). These data indicate that IL-21 acts in synergy with IL-7 by increasing T-cell proliferation, while IL-7 appears to mediate cell survival. IL-21 also demonstrated a similar synergy with IL-15, but not IL-2, in the expansion of antigen-activated CD8+ T cells (data not shown). Because IL-21 alone was unable to rescue activated CD8+ T cells from apoptosis, we could not obtain an adequate number of viable T cells from this experimental group to perform further experiments.

IL-21 prevents {gamma}c cytokine-induced IL-7R{alpha} down-regulation on activated CD8+ T cells
Having determined that IL-21 synergized with IL-7 to enhance T-cell proliferation but not decrease apoptosis, we next sought to determine the mechanism by which IL-21 mediated increased T-cell growth. Both IL-7 and IL-21 bind to dimeric receptors consisting of the {gamma}c chain (CD132) and their respective high-affinity {alpha}-chains, IL-7R{alpha} (CD127) and IL-21R{alpha} (5, 12). It has been previously shown that IL-7R{alpha} is specifically down-regulated in response to IL-7 and the other pro-survival {gamma}c cytokines IL-2, IL-4, IL-6 and IL-15 (28) and that IL-7R{alpha} expression on activated effector CD8+ T cells is a marker for the sub-population that is capable of differentiating into long-lived memory cells (29). In this study, we evaluated the expression of IL-7R{alpha} before and after antigen stimulation of pmel-1 T cells, in the presence or absence of {gamma}c cytokines, alone or in combination.

As expected, IL-7R{alpha} was highly expressed on naive CD8+ T cells when examined directly ex vivo, and its expression was down-regulated after antigen stimulation and culture in IL-7 (data not shown). Surprisingly, IL-21 was capable of inhibiting the IL-7-induced down-regulation of IL-7R{alpha} in a dose-dependent manner (Fig. 2A). At higher, saturating doses of IL-7 (≥10 ng ml–1), however, addition of IL-21 was less effective at maintaining expression of IL-7R{alpha} on the activated CD8+ T cells (Fig. 2B). In contrast to IL-7R{alpha}, {gamma}c chain (CD132) expression was only marginally affected by the addition of IL-21 to IL-7-cultured T cells (Fig. 2A and B). To further examine the mechanism leading to sustained IL-7R{alpha} expression, we performed quantitative real-time (RT)-PCR on T cells cultured in IL-7 plus IL-21 versus IL-7 alone. As shown in Fig. 2(C), T cells exposed to IL-21 showed a significantly higher level of IL-7R{alpha} transcript expression, indicating that IL-21 acts to enhance IL-7R{alpha} gene transcription or to increase mRNA stability. The specific maintenance of IL-7R{alpha} expression on activated T cells cultured in IL-21 may suggest a possible mechanism by which IL-21 synergizes with IL-7 to enhance T-cell proliferation.


Figure 2
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  		Fig. 2. IL-21 sustains the expression of IL-7R{alpha} on TCR-stimulated CD8+ T cells. Pmel-1 SPLs were stimulated in vitro with 100 ng ml–1 hgp10025–33 peptide for 2 days, and then cultured with IL-7, with or without IL-21, for 4 additional days. (A) Day-6 T cells were stained with anti-CD8 and anti-IL-7R{alpha} (CD127) or anti-{gamma}c (CD132) mAbs and analyzed by flow cytometry. Histograms show gated, propidium iodide (PI)-negative, CD8-positive T cells cultured in IL-7 (2 ng ml–1) and indicated concentrations of IL-21. (B) Summary of flow cytometric analysis depicting relative IL-7R{alpha} and {gamma}c chain expression on day-6 T cells following culture in IL-7, with or without addition of IL-21. Data depict the average MFIs as measured in three independent experiments. (C) Quantitative RT-PCR analysis of IL-7R{alpha} transcript expression in CD8+ T cells cultured in IL-7 alone, IL-7 plus IL-21, and sorted CD8+CD127+ and CD8+CD127– T cells cultured in IL-7 plus IL-21. Quantitative RT-PCR was performed after the synthesis of first-strand cDNA according to the manufacturer's instructions. For these experiments, ß-actin was used as a control gene. *P < 0.05.

 
IL-21 sustains CD69 expression on tumor antigen-activated CD8+ T cells
CD69 is an early and transient T-cell activation marker that is up-regulated shortly after TCR stimulation. Six days following antigen stimulation with hgp10025–33 peptide, CD8+ pmel-1 T cells cultured in IL-7 alone showed significant CD69 down-regulation (Fig. 3). Higher doses of IL-7 (>10 ng ml–1) were capable of sustaining CD69 expression in a maximum of ~50% of the T cells; however, addition of IL-21 led to sustained CD69 expression on a significantly higher proportion of cultured CD8+ T cells (80–95%, Fig. 3). IL-21 also increased CD69 expression on cells cultured with IL-15 (data not shown). These results suggest that IL-21 may prolong the activation status of a subset of antigen- and cytokine-stimulated T cells. Addition of IL-21 to IL-7-cultured CD8+ T cells had no discernable effect on the expression of other T-cell phenotypic markers examined, including CD25, CD62L, CD27 and CD44 (data not shown).


Figure 3
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  		Fig. 3. IL-21 sustains the expression of CD69 on TCR-stimulated CD8+ T cells. Pmel-1 SPLs were stimulated in vitro with 100 ng ml–1 hgp10025–33 peptide for 2 days, and then cultured with IL-7 (2 or 10 ng ml–1), with or without indicated concentrations of IL-21, for 4 additional days. Day-6 T cells were stained with anti-CD8 and anti-CD69 mAbs, and then analyzed by flow cytometry. Histograms show only gated, propidium iodide (PI)-negative, CD8-positive T cells. Dotted line histograms indicate isotype control staining and solid lines depict CD69 staining. Numbers indicate of CD69-positive cells. Data shown are representative of three independent experiments.

 
IL-21 synergizes with IL-7 to enhance Th1 and inflammatory cytokine production by activated CD8+ T cells
Since IL-21 acted in concert with IL-7 to increase proliferation and sustain the activation phenotype of T cells following antigen stimulation, we next examined whether IL-21 could modulate the effector function of tumor antigen-specific CD8+ T cells. Thus, seven days following the initiation of pmel-1 T-cell cultures, we evaluated the release of Th1, Th2 and inflammatory cytokines in the supernatants of T cells cultured in IL-7 alone or IL-7 in combination with IL-21, following antigen-specific re-stimulation. As shown in Fig. 4, inclusion of IL-21 increased by >10-fold the production of Th1 (IL-2 and IFN-{gamma}) and inflammatory (IL-1ß, IL-6, GM-CSF and TNF-{alpha}) cytokines when combined with low doses of IL-7 (<2 ng ml–1). Even in the presence of high doses (>10 ng ml–1) of IL-7, addition of IL-21 to the cultures increased T-cell cytokine production by 2- to 4-fold. In contrast, IL-21 showed only a marginal effect on IL-10 production by CD8+ T cells (Fig. 4B). Antigen-stimulated CD8+ T cells showed little production of IL-4, IL-5 or IL-12 in all cultures tested (data not shown). Similar cytokine release profiles were observed at 14 days following the initiation of T-cell cultures (data not shown). Intracellular cytokine staining of antigen-stimulated d14-cultured CD8+ T cells showed that IL-21 not only increased the number of T cells producing IL-2 and IFN-{gamma} but also increased the amount of TNF-{alpha} production on a per cell basis, as indicated by comparative mean fluorescence intensities (MFIs) of TNF-{alpha}-producing cells (MFI 420 versus MFI 622, Fig. 5). Taken together, these data suggest that IL-21 is capable of synergizing with IL-7 to significantly enhance Th1 and inflammatory cytokine production during a tumor antigen-specific T-cell response.


Figure 4
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  		Fig. 4. IL-21 strongly enhances the secretion of Th1 and inflammatory cytokines by TCR-stimulated CD8+ T cells. Pmel-1 SPLs were stimulated in vitro with 100 ng ml–1 hgp10025–33 peptide for 2 days, and then cultured with IL-7 (2 or 10 ng ml–1), with or without IL-21 (10 ng ml–1). At day 7 or 14, EL-4 cells were pulsed with hgp10025–33 peptide (1 ng ml–1) and used to re-stimulate T cells at a responder to target ratio of 1:1 at 37°C for 18 h. (A) Culture supernatants were collected and analyzed for IL-2 secretion by Luminex. (B) Day-7 T-cell culture supernatants were collected and analyzed for the secretion of Th1, Th2 or inflammatory cytokines using Luminex or ELISA. For all cytokines tested, the differences in secretion between cells cultured with IL-7 alone versus IL-7 plus IL-21 were statistically significant (P < 0.05).

 

Figure 5
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  		Fig. 5. IL-21 augments the production of Th1 and inflammatory cytokines by tumor antigen-specific CD8+ T cells. Pmel-1 SPLs were stimulated in vitro with 100 ng ml–1 hgp10025–33 peptide for 2 days, and then cultured with IL-7 (10 ng ml–1), with or without IL-21 (10 ng ml–1) for 12 additional days. EL-4 cells were pulsed with hgp10025–33 or NP366–374 peptide, washed and incubated with day-14 T cells at a responder to target ratio of 1:1 for 5 h. T cells were then permeabilized and stained for intracellular IFN-{gamma}, IL-2 and TNF-{alpha}. Numbers indicate the percentage of cytokine-producing CD8+ T cells. Data shown are representative of three experiments.

 
IL-21 synergizes with IL-7 to enhance cytotoxicity of tumor antigen-specific CD8+ cells
Melanoma-specific CD8+ T cells cultured in IL-7 alone, or IL-7 in combination with IL-21, were next compared for their respective abilities with lyse peptide-pulsed and bona fide tumor targets. As measured using a 4-h 51Cr release cytotoxicity assay, T cells cultured in IL-7 alone had a very low, nearly undetectable capacity for killing gp100-expressing B16 murine melanoma cells; however, exposure of the T cells to IL-21 in culture increased cytotoxicity to levels well above detection at 20:1 or higher E:T ratios (Fig. 6A). Since B16 melanoma cells are known to express very low levels of MHC Class I molecules, we also tested the cytolytic capacity of the cultured T cells using peptide-pulsed EL-4 cells as targets. In these experiments, T cells cultured in IL-7 and IL-21 demonstrated dramatically higher specific cytotoxicity (>20-fold) compared with T cells cultured in IL-7 alone (Fig. 6B). This increased cytolytic capacity of T cells could not be explained by a difference in perforin or granzyme B expression, as IL-21 did not significantly affect expression of either marker (Fig. 6C). Taken together, these results suggest that IL-21 can synergize with IL-7 to strongly enhance the cytotoxic, anti-tumor function of melanoma-specific CD8+ T cells in a manner that appears to be independent of perforin and granzyme B.


Figure 6
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  		Fig. 6. IL-21 enhances the cytotoxic activity of tumor antigen-specific CD8+ T cells. Pmel-1 SPLs were stimulated in vitro with hgp10025–33 peptide (100 ng ml–1) for 2 days, and then cultured with IL-7 (10 ng ml–1), with or without IL-21 (10 ng ml–1) for 6 additional days. Cultured T cells were then analyzed for cytotoxicity by incubating for 4 h with 51Cr-labeled (A) B16 melanoma cells or (B) hgp10025–33 or NP366–374 peptide-pulsed EL-4 thymoma target cells. Supernatants were harvested and analyzed for radioactivity using a gamma counter. (C) CD8+ T cells were evaluated for granzyme B and perforin expression by intracellular staining and flow cytometry. Shadowed histograms indicate isotype control staining; solid gray histograms indicate T cells cultured in IL-7 (10 ng ml–1) and dotted line histograms indicate T cells cultured in IL-7 plus IL-21 (10 ng ml–1 of each). Data shown are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Over the past two decades, much evidence has accumulated to support the notion that the immune system, particularly CTLs, can mediate substantial tumor regressions in both mouse tumor models and cancer patients (2, 4). Thus, a number of current immunotherapeutic approaches involve active immunization to boost levels of circulating, tumor antigen-specific T lymphocytes (30–32). Alternatively, peripheral blood-derived or tumor-infiltrating T lymphocytes may be expanded in culture and infused back into patients, an approach that has met with some clinical success in advanced melanoma patients (33, 34). In the context of adoptive cell transfer, the {gamma}c cytokine IL-2 has historically been widely used to successfully expand T cells in vitro, and IL-15 is now being considered as an alternative due to its capacity to promote memory responses (35, 36). However, much still remains unknown about how activation and expansion in different {gamma}c cytokines, alone or in combination, can affect the resulting anti-tumor function of CD8+ T cells.

In this study, we specifically investigated the role of the {gamma}c cytokine IL-21 on the proliferation and effector function of tumor antigen-activated CD8+ T cells. It has been previously demonstrated that IL-21 can promote the expansion of T cells stimulated with anti-CD3 or allo-antigens (13, 37). Consistent with this, we found that IL-21 had a marked synergistic effect with IL-7 on the proliferation of tumor antigen-specific CD8+ T cells following in vitro stimulation with tumor-associated peptides. However, in contrast to {gamma}c cytokines IL-2, IL-4, IL-7 and IL-15, that have been demonstrated to support T cell proliferation and T-cell survival (5, 38), IL-21 alone could not support the survival of activated T cells.

IL-21 has previously been demonstrated to enhance IFN-{gamma} production by CD8+ T cells grown in IL-7, IL-15 or IL-18 (23–25). We have confirmed these studies and extended them by showing that IL-21 could enhance the function of TCR-stimulated CD8+ T cells grown in IL-7, not only by significantly augmenting the production of Th1 cytokines IFN-{gamma} and IL-2 but also by inflammatory cytokines TNF-{alpha}, GM-CSF, IL-1ß and IL-6. Interestingly, the production of IL-10 was only slightly enhanced, suggesting a potential skewing by IL-21 away from a Th2, and toward a Th1, cytokine release profile. IL-21 has been reported to stimulate IFN-{gamma} gene expression in activated T cells and enhances the transcription of other genes involved in Th1 immune responses, including IL-12Rß2, IL-18 receptor components and the transcription factor T-bet (39).

In addition to increased cytokine production, we have also shown that tumor antigen-specific CD8+ T cells grown in the presence of IL-7 and IL-21 demonstrate superior cytolytic ability compared with T cells cultured in IL-7 alone. This enhanced anti-tumor function is reminiscent of published in vivo studies showing superior CTL responses and long-term tumor control in mice receiving IL-21, compared with IL-15, or IL-2 alone (17). It is also consistent with reports of impaired cytotoxic T cell responses from IL-21 receptor-deficient mice (23, 37). Although our previous study showed that the anti-tumor effects of IL-21 treatment could be attributed to enhanced NK cell function in vivo (15), the present results suggest that in the context of IL-7, IL-21 may also enhance anti-tumor immunity through the augmentation of CTL function.

While the effects of IL-21 on CD8+ T cells are now starting to be appreciated, elucidation of the mechanisms behind this observed functional enhancement will require further study. We observed that activated T cells expanded in the presence of IL-21 showed sustained expression of two markers, CD69 and IL-7R{alpha}, that may have functional implications for improved T-cell activity. CD69 is a type II integral membrane protein with a C-type lectin-binding domain and a member of the NK cell gene complex family of cell-surface receptors. It is rapidly induced on the surface of activated lymphocytes following stimulation and its expression is closely correlated with various immunological functions (40). The acquisition of CD69 expression after antigen stimulation directly correlates with the functional maturation of CD8+ T lymphocytes, in both IFN-{gamma} production and specific cytotoxicity (41). Because much is unknown about CD69 function, it is unclear whether prolongation of CD69 expression on activated IL-21-cultured cells is simply a surrogate marker of increased T-cell function or whether it actively contributes to enhanced CTL activity.

Our study also showed that addition of IL-21 sustained IL-7R{alpha} expression on T cells cultured in IL-7 by augmenting IL-7R{alpha} mRNA transcript levels. Presumably, sustained expression of IL-7R{alpha} may result in prolonged responsiveness to IL-7, which could explain at least part of the observed synergy between IL-7 and IL-21. Intriguingly, IL-7R{alpha} has recently garnered much interest as a marker of T cells that have the potential to transition to memory cells. It has been reported that at the peak of primary CD8+ T cell responses, a small subset of effector cells with elevated expression of IL-7R{alpha} preferentially survive and differentiate into long-lived memory cells capable of generating protective recall responses (29). Conversely, lack of IL-7R{alpha} expression during acute viral infection has been shown to stunt memory CD8+ T cell generation because of excessive effector cell death (6). In an adoptive-transfer setting, IL-21 was shown to be more potent than either IL-2 or IL-15 at sustaining the persistence of tumor antigen-specific CTLs (17).

Other studies have shown that IL-21 and IL-15 synergize to generate enhanced anti-tumor immunity and induce long-lived protection from challenge with syngeneic tumor cells (23, 42). Recent data indicate that different signaling pathways are activated by IL-21 and IL-15, which may provide a molecular basis for their synergy (23, 43). Thus, it is possible that activation of different pathways may also explain the synergy of IL-7 and IL-21, as reported here.

Since IL-21 is produced by activated CD4+ T cells in a context where other {gamma}c cytokines like IL-2, IL-7 or IL-15 are simultaneously being expressed, it is likely that effector CD8+ T cells are exposed to combinations of these cytokines in vivo. It is thus possible that IL-21 in this setting contributes to the critical transition from effector to memory cells. We are currently examining whether IL-21-mediated maintenance of IL-7R{alpha} expression on antigen-activated CD8+ T cells is important for the generation of tumor-specific memory CD8+ T cells in vivo following anti-tumor vaccination.

Taken together, our in vitro data show that IL-21 can effectively synergize with IL-7 to significantly increase the proliferation, cytokine secretion and cytotoxic activity of TCR-stimulated, tumor antigen-specific CD8+ T cells. These results shed light on the role of IL-21 during a primary CD8+ T-cell response and may have important applications for the generation of optimal anti-tumor CTL responses in cancer patients.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
National Institutes of Health (R21 CA111547 [GenBank] -02).


   Abbreviations
 
Ct, threshold cycle
CM, complete medium
{gamma}c, common {gamma}-chain
E:T, effector to target cell
GM-CSF, granulocyte macrophage colony-stimulating factor
NP, nucleoprotein
RT, real time
SPL, splenocyte
TNF-{alpha}, tumor necrosis factor-{alpha}

   Notes
 
* These authors contributed equally to this study. Back

Transmitting editor: C. Paige

Received 22 February 2007, accepted 19 July 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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