IL-21 Enhances and Sustains CD8+ T Cell Responses to Achieve Durable Tumor Immunity: Comparative Evaluation of IL-2, IL-15, and IL-21

Adrianna Moroz, Cheryl Eppolito, Qingsheng Li, Jianming Tao, Christopher H. Clegg and Protul A. Shrikant
J Immunol July 15, 2004, 173 (2) 900-909; DOI: https://doi.org/10.4049/jimmunol.173.2.900

Abstract

Cytokines that use the common receptor γ-chain for regulating CD8+ T cell responses to Ag include IL-2, IL-15, and the recently identified IL-21. The ability of these cytokines to regulate antitumor activity in mice has generated considerable interest in understanding their mode of action. In this study we compare the abilities of IL-2, IL-15, and IL-21 to stimulate immunity against tumors in a syngeneic thymoma model. Durable cures were only achieved in IL-21-treated mice. By monitoring both endogenous and adoptively transferred tumor Ag-specific CD8+ T cells, it was determined that IL-21 activities overlap with those of IL-2 and IL-15. Similar to IL-2, IL-21 enhanced Ag activation and clonal expansion. However, unlike IL-2 treatment, which induces activation-induced cell death, IL-21 sustained CD8+ T cell numbers long term as a result of increased survival, an effect often attributed to IL-15. These findings indicate that the mechanisms used by IL-21 to promote CD8+ T cell responses offer unique opportunities for its use in malignant diseases and infections.

CD8+ T cells play a prominent role in tumor immunity by recognizing and killing malignant cells that present tumor-associated Ags on MHC class I molecules. The orchestration of an effective CD8+ T cell response to tumor involves a number of cell types that help regulate a program of clonal expansion, differentiation, and subsequent memory formation or cell death. Ag-presenting dendritic cells regulate CD8+ T cell priming and promote proliferation (1), whereas CD4+ T cells, through an interaction with perhaps both APC and CD8+ cells, greatly influence the quantity and quality of the CD8+ T cell clonal expansion and memory generation (2, 3, 4). These and additional cell types use multiple signaling pathways to influence CTL development, including a number of cell surface receptors that mediate cell-cell contact (5, 6) and various cytokines that are secreted within the microenvironment (7, 8, 9).

One cytokine family that exerts overlapping and distinct regulation of CD8+ T cell responses to Ag uses the common γ-chain receptor subunit and includes IL-2, IL-7, and IL-15 (10, 11, 12, 13, 14, 15, 16, 17). Upon Ag activation, CD4+ T cells produce IL-2, which drives rapid clonal expansion of naive CD8+ T cells, leading to their subsequent differentiated state (7). However, IL-2 can also limit clonal expansion and the accumulation of Ag-specific effector cells by promoting activation-induced cell death (10, 18, 19). Unlike IL-2, IL-7 is not a potent mitogen, but functions as a survival factor that regulates naive T cell viability and promotes the maintenance of memory T cells (11, 20, 21). IL-15, another survival factor, exerts its effects primarily on memory CD8+ T cells (12, 22, 23). Moreover, IL-15 stimulates memory T cell proliferation rather than apoptosis (19, 22). Collectively, these cytokines influence several distinct phases of CD8+ T cell responses, and numerous studies have illustrated their effectiveness in augmenting antitumor immunity (10, 24, 25, 26, 27).

IL-21 is the most recent member of the IL-2 cytokine family to be identified (28, 29). Activated CD4+ T cells secrete IL-21, and the IL-21R is readily detectable on T cells (αβ, γδ, and NKT), B cells, NK cells, and monocyte-derived dendritic cells (28, 29, 30, 31). IL-21 costimulates lymphocyte proliferation, modulates gene expression, and displays attributes of both Th1 and Th2 cytokines in vitro (29, 32). Analysis of IL-21R-deficient mice indicates a role for this cytokine in regulating Ab production and humoral immunity (30, 33), and a variety of tumor challenge models using gene transfer methodologies indicate that IL-21 can influence both innate and cell-mediated immunity (31).

Considering the dominant role that CD4+ T cells and the IL-2 cytokine family play in CD8+ T cell-mediated tumor immunity, it seemed important to better characterize the relative mechanisms used by IL-2, IL-15, and IL-21 to influence CD8+ T cell responses to tumor Ag in vivo, as this information is likely to enable developing rational approach for improving their efficacy. In this study we present a comparative analysis of the abilities of IL-2, IL-15, and IL-21 to provide protection against a syngeneic mouse tumor challenge. Our results demonstrate that IL-21 has greater efficacy than IL-2 or IL-15 in stimulating tumor-free survival in the E.G7 thymoma model. This was due to activities that overlap with IL-2 and IL-15 and its superior ability to stimulate clonal expansion, differentiation, and survival of tumor-specific CD8+ T cells.

Materials and Methods

Tumor challenge, cytokine treatment, and cell depletions

Mice were maintained under specific pathogen-free conditions at the DLAR facilities of Roswell Park Cancer Institute. All experimental procedures were performed in compliance with protocols approved by the institutional animal care and use committee of the institute. Female C56BL/6 (Thy1.2) mice (The Jackson Laboratory, Bar Harbor, ME), 6–8 wk old, were used for all tumor experiments. Syngeneic E.G7 thymoma cells expressing chick OVA (34) were injected at a concentration of 3 × 106 cells/0.5 ml of PBS into the peritoneum of mice (considered day 0) and monitored thereafter for morbidity. Alternatively, E.G7 cells (3–5 × 106 cells) were injected s.c. on the flanks of mice, and tumor growth was monitored after day 6. Recombinant murine IL-2 and IL-15 were purchased from R&D Systems (Minneapolis, MN). Recombinant mouse IL-21 was produced using a baculovirus expression system. Protein was purified from conditioned medium by sequential ion exchange and size exclusion chromatography. Murine IL-21 was stored at a concentration of 1.9 mg/ml in PBS (pH 6.0) at −80°C. The endotoxin level of this working solution was determined to be <0.92 endotoxin unit/ml. Cytokines were injected i.p. in 100-μl volumes containing PBS. The doses and days of delivery relative to tumor challenge varied in the experiments as indicated. The Abs used to deplete specific cell types in mice (American Type Culture Collection, Manassas, VA) were clones GK1.5 (CD4+), PK136 (NK1.1), and 2.43 (CD8+). These were administered i.p. at 0.3–0.5 mg/day every fourth day. The depletion efficiency was routinely monitored by flow cytometric evaluation of tail vein bleeds.

OT-I cell adoptive transfers

CD8+ OT-I T cells express a transgenic TCR specific for the chick OVA peptide SIINFEKL in the context of H-2Kb. These cells were isolated from OT-I.PL Thy1.1+ transgenic mice as previously described (25, 26). Briefly, lymph nodes (LN)3 from 6- to 8-wk-old transgenic mice were homogenized and subjected to RBC lysis and adherence depletion. After purification by adherence depletion and negative selection, 3 × 106 cells CD8+ T cells were injected through the tail vein of mice in a volume of 500 μl of PBS. Mice were challenged 1 day later with tumor. At varying times thereafter, animals were killed, and cells were isolated from the spleen, draining LN (periaortic, mesenteric, axillary, and brachial), distal LN (cervical), and peritoneal cavity. OT-I cells were quantitated by flow cytometry using anti-Thy 1.1-PE and anti-CD8α-CyChrome mAbs. For some experiments, the OT-I suspension was labeled with the intracellular fluorescent dye CFSE-DA (Molecular Probes, Eugene, OR) before adoptive transfer (35). These cells were analyzed for the number of cell divisions at various times after tumor challenge. The purity of the cells used for these adoptive transfers was always >98% CD8+ cells, with <1% CD4+ cells.

Flow cytometry

Cells harvested from tissues were incubated with Fc Block (anti-CD16/32) for 15 min and then reacted with Abs specific for several cell surface proteins, including anti-Thy1.1, anti-Thy1.2, anti-CD8α, and anti-CD69 (BD Pharmingen, San Diego, CA). MHC class I tetramers folded with SIINFEKL were obtained from the National Institutes of Health tetramer facility (Bethesda, MD) and were used as recommended and previously described (35). PE-conjugated annexin V was purchased from Caltag Laboratories (Burlingame, CA). After reacting with Abs, the cells were washed twice in HBSS containing 0.01% NaN3 and 1% FCS, fixed with 1% paraformaldehyde, and stored at 4°C in the dark before acquisition. All flow cytometric evaluations were performed on FACScan or FACSCalibur flow cytometer. After gating on forward and side scatter parameters, at least 10,000 gated events were routinely acquired and analyzed using CellQuest software (BD Biosciences, Mountain View, CA). To determine T cell proliferation, CFSE-DA fluorescence was evaluated in a minimum of 5000 CD8+/Thy1.1+ gated T cells.

Measuring T cell proliferation by BrdU incorporation

E.G7 tumor-bearing mice transferred with OT-I T cells were injected i.v. with 200 μl of BrdU (0.4 mg/ml; Sigma-Aldrich, St. Louis, MO) on day 3 or 26 post-tumor challenge. The animals were killed at the indicated time points, and the draining LN cells were stained for CD8 and Thy1.1, then ethanol-permeabilized, fixed, and reacted with anti-BrdU Abs. A minimum of 5000 OT-I T cells were gated and analyzed by flow cytometry.

Cytotoxic assay

The effector cell population consisted of CD8+ T cells that were purified from tumor-challenged animals by harvesting the draining nodes and/or the spleen and were enriched by negative selection using CD8 Cellect columns (Cedarlane Laboratories, Hornsby, Canada) and/or magnetic bead separation (Miltenyi Biotech, Auburn, CA). EL-4 thymoma cells pulsed with 10 nM SIINFEKL peptide and labeled with 100 μCi of Na251Cr for 60 min at 37°C in RPMI 1640 medium containing 10% FCS were used as targets after repeated washing. Cytotoxicity was measured by incubating varying ratios of effector to target cells for 6 h in 96-well plates at 37°C for 6 h. Supernatants from each culture well (100 μl) were measured for radioactivity in a Micro-β counter (Wallac, Turku, Finland). The maximum 51Cr release was determined by detergent lysis (Nonidet P-40) of the labeled target cells. Spontaneous 51Cr release was always <10% of the maximum release. The percentage of specific 51Cr release was calculated by the following formula: % specific lysis = (experimental − spontaneous × 100/maximal − spontaneous).

TUNEL assay

TUNEL was performed using the ApoAlert DNA fragmentation assay kit (BD Clontech, San Diego, CA). In brief, CD8+ T cells were negatively selected by column purification and subjected to anti-Thy1.1 (PE) staining before fixation with 95% ethanol and paraformaldehyde. The fixed cells were reacted with Tdt reaction buffer and Tdt/biotin-dUTP reagent for 1 h at 37°C. The cells were washed extensively, and FITC-conjugated streptavidin was added for 30 min at room temperature. The cells were analyzed by flow cytometry, where a minimum 10,000 OT-I T cells were evaluated for each experimental condition.

Results

IL-21 demonstrates potent antitumor activity in mice relative to IL-2 and IL-15

To study the antitumor activity of IL-2, IL-15, and IL-21, we compared their abilities to promote the survival of mice challenged with E.G7 thymoma. C57BL/6 mice were injected with 3 × 106 syngeneic E.G7 thymoma cells (day 0) and then treated with optimal doses of IL-2 (2000 IU/day) (10), IL-15 (5 μg/day), and IL-21 (20 μg/day) on days 2, 4, 6, 8, 10, and 12. The optimal dose for use was established in survival studies involving thorough titration of each cytokine (data not shown). As indicated in Fig. 1⇓, IL-2, IL-15, and IL-21 enhanced overall survival relative to the PBS-treated control group, but to varying degrees. Low dose IL-2 (Fig. 1⇓A) had only a marginal effect by extending overall survival from day 40 to day 60, whereas this benefit was lost after treatment with high dose IL-2. IL-15 (Fig. 1⇓B) performed better than IL-2 at the two injected doses, with overall survival being extended to day 80 (5 μg/dose) and day 90 (50 μg/dose). IL-21 administration, in contrast to IL-2 and IL-15, resulted in a near doubling of the 50% survival time relative to the control, but, more importantly, 20–30% of the mice survived disease-free for >120 days (Fig. 1⇓C). We conclude that IL-21 is more potent than IL-2 or IL-15 in promoting survival in E.G7 thymoma-challenged animals.

FIGURE 1.
FIGURE 1.

IL-21 enhances the survival of thymoma-bearing mice. Groups of 10 C57BL/6 animals challenged i.p. with 3 × 106 E.G7 tumor cells on day 0 received either vehicle control (PBS) or the indicated doses of cytokine on days 2, 4, 6, 8, 10, and 12. Mice were then monitored for survival. A, IL-2 (2,000 IU/day (1 μg/day) and 20,000 μU/day (10 μg/day)); B, IL-15 (5 and 50 μg/day); C, IL-21 (20 and 100 μg/day). D, To optimize the delivery of cytokines relative to tumor challenge, mice received six doses of IL-21 (20 or 100 μg/day) on alternating days between days −4 and 6 (pre), days 2 and 12 (early), or days 12 and 22 (late). The average percentage of animals surviving on day 120 is plotted. The SEM derived from three independent experiments is shown.

IL-2’s ability to regulate CD8+ T cell responses is dependent on the timing of cytokine administration (10, 48). To determine when, relative to the time of tumor challenge, IL-21 produces optimal protection, we injected E.G7 tumor-bearing mice with IL-21 on 6 alternating days beginning with prechallenge delivery (days −4, −2, 0, 2, 4, and 6), early challenge delivery (days 2, 4, 6, 8, 10, and 12), or late challenge delivery (days 12, 14, 16, 18, 20, and 22). Only 5% or less of the animals receiving the predelivery schedule survived >120 days, whereas 25–30% of the animals receiving the early delivery schedule survived long term (Fig. 1⇑D). Surprisingly, a near-equivalent percent survival was also achieved in the late delivery groups. These results indicate that treating mice with IL-21 before or shortly after tumor challenge is not optimal for long term survival. Rather, IL-21 provision augments protective mechanisms several days and even weeks after tumor challenge.

IL-21 administration generates CD8+ T cell-mediated antitumor immunity

To identify the immune cell type(s) that may mediate IL-21’s antitumor effect, we injected IL-21 into groups of tumor-bearing mice that were selectively depleted of CD4+ T cells, CD8+ T cells, NK and/or NK T cells. As indicated in Fig. 2⇓, the IL-21 benefit was completely lost in mice depleted of CD8+ T cells, whereas no significant differences were observed between CD4+ T cell-depleted mice and nondepleted mice in terms of either 50% survival or long term survival (>100 days). This indicates that CD8+ T cells are an essential requirement for the antitumor activity of IL-21, whereas CD4+ T cells are dispensable for the CD8+-mediated protection. Interestingly, the depletion of NK 1.1+ cells (i.e., NK and NKT cells) reduced the 50% survival time from day 70 to day 35, but a similar frequency of long term survivors was maintained. Thus, NK and/or NKT cells may be involved during the initial stages of this antitumor response. In separate experiments, IL-21 treatment produced the same survival benefit in mature B cell-deficient uMT mice as in control animals, thus eliminating the possibility that IL-21 mediated antitumor activity via B cells (data not shown).

FIGURE 2.
FIGURE 2.

IL-21 promotes CD8+ T cell-mediated protection against thymoma. Relative to the day of E.G7 tumor challenge (day 0), groups of 10 animals were either left intact or treated between days −4 and 40 with specific mAbs to deplete them of CD4+ T cells, NK1.1+ lymphocytes, or CD8+ T cells (see Materials and Methods). All animals received 20 μg of IL-21 on days 4, 6, 8, 10, and 12. The percent survival is plotted. A representative of three independent experiments is shown. The dotted line indicates 50% survival.

IL-21 augments CD8+ T cell responses to promote durable tumor immunity

To test IL-21’s ability to stimulate durable immunity via CD8+-dependent control, we rechallenged animals that survived >100 days after pre-, early, and late deliveries of IL-21 (see Fig. 1⇑D) with an s.c. injection of E.G7 tumor cells. As shown in Fig. 3⇓A, tumor growth was severely restricted in the early and late delivery groups, 100% of which survived >100 days (Fig. 3⇓B). However, this effect was lost in a subset of the early delivery group that was depleted of CD8+ T cells before tumor rechallenge. These data clearly demonstrate that IL-21 administration generates a durable CD8+ T cell-mediated immunity to E.G7 thymoma. The partial survival achieved in the predelivery group re-emphasizes the previous observation (Fig. 1⇑D) that the timing of IL-21 administration, relative to tumor challenge, is critical for achieving the maximum benefit. In separate experiments, mice that survived E.G7 thymoma after IL-21 treatment failed to reject parental EL4 tumor cells (data not shown). This result identifies OVA as a critical Ag for the maintenance of durable immunity against E.G7.

FIGURE 3.
FIGURE 3.

Establishment of CD8+ T cell memory in IL-21-treated mice. Groups of 30 C57BL/6 mice were challenged with E.G7 cells (3 × 106). Animals were then treated with 20 μg of IL-21 every other day beginning on days −4 to 6 (pre), days 2–12 (early), or days 12–22 (late). Mice (five per group) that were tumor free for 100 days were rechallenged s.c. with 5 × 106 E.G7 tumor cells; the rechallenge was considered day 0. One cohort of surviving mice from the early delivery group was depleted of CD8+ T cells (see Materials and Methods) and then rechallenged s.c. with E.G7 tumor. A, The diameter of E.G7 tumor (millimeters) measured over time. B, The percentage of mice that survived tumor rechallenge for 120 days. Naive animals (n = 5) served as the nonimmunized control for E.G7 tumor rechallenge. EL-4 tumor challenge served as the Ag control (data not shown). The results shown are representative of two independent experiments.

To investigate how IL-21 stimulates CD8+ T cell responses to tumor relative to IL-2 and IL-15, we next evaluated the frequencies of tumor-specific CD8+ T cells in cytokine-treated mice using MHC class I tetramers complexed with an OVA-specific peptide (H-2Kb and SIINFEKL) (35). The frequency of CD8+ T cells positive for tetramer binding was noticeably increased 15 days after tumor challenge; the highest numbers were in the IL-2 group, followed by IL-21-treated animals (Fig. 4⇓). By day 45, the percent tetramer+ CD8+ T cells had dropped precipitously in IL-2-treated mice, whereas this frequency increased significantly in the IL-21 group. For IL-15-treated animals, the frequency of tetramer-positive cells remained unchanged beyond day 15. These results demonstrate that IL-21 is more effective than IL-2 or IL-15 in achieving an expanded frequency of tumor-specific CD8+T cells that affords long term protection against thymoma.

FIGURE 4.
FIGURE 4.

IL-21 promotes endogenous CD8+ T cell responses to tumor Ag. Naive C57BL/6 animals were challenged with E.G7 tumor (3 × 106) on day 0 and treated on days 2, 4, 6, 8, 10, and 12 with vehicle only (PBS), IL-2 (2000 IU/day), IL-15 (20 μg/day), or IL-21 (20 μg/day). The CD8+ T cells derived from draining LN at indicated time points were analyzed for H-2Kb/SIINFEKL tetramer reactivity by flow cytometry. A, The percentage of CD8+ T cells that are tetramer positive is plotted. The error bars represent the SEM from four independent experiments with three animals each. B, CD8+ T cells stained with H-2Kb/SIINFEKL on day 45 post-tumor challenge. A representative histogram from four independent experiments is shown. The marker indicating tetramer positivity was established after staining with H-2Kb tetramers bearing irrelevant Ag.

To evaluate whether IL-21 can promote cytolytic ability of the tumor-bearing hosts, we tested the lytic activity of CD8+ lymphocytes purified from cytokine-treated, tumor-bearing mice against syngeneic thymoma pulsed with the nominal antigenic peptide SIINFEKL. As indicated in Fig. 5⇓, CTL activity was detected in cells recovered on day 10 post-tumor challenge, with the IL-2- and IL-21-treated groups sharing the highest activity relative to the PBS-treated group (Fig. 5⇓A). A similar level of lysis was achieved by cells derived from the IL-21-treated mice on day 30, but this capacity was lost in the corresponding cells isolated from the IL-2-treated group (Fig. 5⇓B). In contrast, IL-15 treatment resulted in only a marginal benefit in terms of cytolytic activity on day 10, but it was sustained until day 30. Thus, relative to IL-2 and IL-15, IL-21 may promote tumor free survival and durable immunity by increasing the frequency of tumor Ag-specific CD8+ T cells and their cumulative cytolytic activity within the hosts after tumor challenge. This effect with IL-21 was OVA-specific, because non-Ag-pulsed EL-4 target cells were not killed, thus confirming the previous observation that the durable immunity induced by IL-21 was restricted to E.G7 tumor.

FIGURE 5.
FIGURE 5.

IL-21 stimulates persistent tumor-specific CTL activity in mice. Animals were challenged with Ag-specific E.G7 tumor or control EL-4 on day 0 and then treated with vehicle alone, IL-2 (2000 IU/day), IL-15 (20 μg/day), or IL-21 (20 μg/day) for various times. LN and spleens were harvested on the indicated days, and purified CD8+ T cells were tested for CTL activity in a standard 6-h chromium release assay (see Materials and Methods). A, Lytic activity of cells isolated from on day 10 after exposure to cytokines on days 2, 4, 6, and 8. B, Lytic activity of cells isolated on day 30 after treatment with cytokines on days 2, 4, 6, 8, 10, and 12. Lytic activity of cells isolated from EL-4-challenged mice (No Ag) receiving IL-21 served as an in vivo control for Ag. Although EL-4 cells pulsed with an irrelevant peptide served as an additional control for Ag-specific target cell lysis, no lytic activity was demonstrated (data not shown). A representative dataset from four independent experiments is shown, with error bars indicating the SD.

IL-21 enhances proliferation of OT-I CD8+ T cells after adoptive transfer into tumor-bearing mice

Adoptively transferred naive OT-I cells have been effectively used to evaluate the effects of cytokines on E.G7-specific CD8+ T cell responses (25, 26, 36, 37, 38, 39). Within 3–4 days after E.G7 tumor challenge, naive OT-I cells are activated to migrate into the peritoneal cavity (PC), where they undergo clonal expansion, and develop effector functions that control tumor growth for the next 2–3 days (25). This process is short-lived, however, and a more sustained antitumor response requires the presence of IL-2 and/or additional activities associated with CD4+ Th cells (26).

Given that IL-21, in contrast to IL-2 and IL-15, generated sustained endogenous CTL responses, we monitored the impact of the cytokines on adoptively transferred naive OT-I T cells in E.G7 tumor-challenged mice. Within 4 days of tumor challenge, OT-I cell numbers had increased in the PC of both PBS-treated and cytokine-treated mice, with IL-2 having the dominant effect (Fig. 6⇓A). Thereafter, OT-I cell numbers decreased dramatically in the IL-2- and PBS-treated mice, whereas their numbers remained elevated in the IL-21- and IL-15-treated groups. At the delivered doses, IL-21 was more potent than IL-15 in sustaining the OT-I response to tumor. Treatment with these three cytokines caused a delay in tumor cell growth, but only in the IL-21 group was tumor progression prevented (Fig. 6⇓B). We conclude that, similar to its effects on endogenous Ag-specific CD8+ T cells, IL-21 is more potent than IL-2 or IL-15 in stimulating a long term expansion of OT-I cells after tumor challenge.

FIGURE 6.
FIGURE 6.

IL-21-mediated regulation of OT-I T cell response in tumor-bearing mice. C57BL/6 congenic animals were adoptively transferred with naive (3 × 106) OT-I T cells and challenged i.p. with 3 × 106 E.G7 tumor on day 0. Mice were injected with vehicle alone (PBS), IL-2 (2000 IU/day), IL-15 (5 μg/day), or IL-21 (20 μg/day) on days 4, 6, 8, 10, and 12 post-tumor challenge. Cells were isolated from the draining LN and the peritoneal cavity on the indicated days and subjected to flow cytometric evaluations. Cell numbers were calculated as previously described (25 ). A, The numbers of OT-I cells measured in draining LN. B, The number of E.G7 tumor cells measured in the peritoneal cavity. Two animals were analyzed independently from each experimental treatment group. The values plotted are derived from three independent experiments, and the error bars represent the SEMs. +, Time points at which 100% fatality was noted.

The initial stages of Ag activation of T cells can be monitored by CD69 expression. Approximately 40–60% of the OT-I cells isolated from LN of E.G7 tumor-bearing mice were CD69+ after 4 days (Fig. 7⇓A). However, ∼90% of OT-I T cells were CD69+ at a similar time point after treatment with either IL-2 or IL-21, indicating that these cytokines promote Ag responsiveness in tumor Ag-specific T cells. To demonstrate that these activated T cells subsequently initiate DNA synthesis, we pulsed tumor-bearing mice with BrdU between days 3 and 5 and then measured the percentage of BrdU+ OT-I T cells (Fig. 7⇓B). Significantly more OT-I cells were BrdU+ in the cytokine-treated mice than in the control animals, and IL-21 was nearly equivalent to IL-2. As independent measures of cell proliferation, we monitored OT-I clonal expansion (Fig. 7⇓C) and CFSE dilution (Fig. 7⇓D) on day 10 post-tumor challenge. Both IL-2 and IL-21 enhanced the numbers of OT-I cells in LN and PC, relative to vehicle-treated mice. Moreover, based on the CFSE dilution experiment, the vast majority of OT-I cells isolated from mice treated with each cytokine had undergone at least five rounds of cell division in the PC and three rounds in the LN. Collectively, these results indicate that IL-21, like IL-2, can significantly increase the Ag-specific T cell proliferation that results in increased clonal expansion.

FIGURE 7.
FIGURE 7.

IL-21 enhances OT-I cell activation and expansion induced by tumor. Naive CD8+ OT-I cells (3 × 106) were adoptively transferred into congenic recipient mice challenged i.p. with tumor and injected with vehicle alone (PBS), IL-2 (2000 IU/day), or IL-21 (20 μg/day) at various intervals. OT-I cells from the PC or draining LN were analyzed for activation and proliferation in the following manner. A, CD69 expression on LN cells. Tumor-bearing mice were injected with cytokine on days 0 and 2. Draining LN cells were isolated on day 4. The overlay histogram is a measure of relative CD69 expression on gated OT-I cells (representative of five independent experiments). OT-I cells isolated from mice injected with EL-4 cells (no Ag) served as a negative control. B, BrdU incorporation. Mice were treated with cytokines on days 0 and 2 after tumor challenge and injected with BrdU on day 3, and LN cells gated for CD8+/Thy1.1+ were analyzed on day 5 for BrdU staining. The mean percentage of BrdU+ OT-I cells obtained from four independent experiments is shown. The error bars represent the SDs. C, Clonal expansion. Before adoptive transfer, naive OT-I T cells were labeled with CFSE-DA. Animals were treated with cytokine on days 2, 4, 6, and 8 after tumor challenge. The percentage of CD8+/Thy1.1+ cells from total lymphocytes on day 10 was determined and is indicated in each dot plot. A representative of five independent experiments is shown. D, CFSE dilution. The gated OT-I T cells were assessed for their CFSE-DA staining by flow cytometry. The percentage of CD8+/Thy1.1+ cells that had undergone more than three rounds of cell division (LN) or more than five rounds of cell division (PC) is indicated. A representative of five independent experiments is shown.

IL-21 stimulates OT-I cell survival in tumor-bearing mice

Mice treated with IL-21, but not IL-2, maintained relatively high numbers of OT-I cells in LN for several weeks and even months after tumor challenge (see Fig. 6⇑). To determine whether this might occur through sustained proliferation, we monitored OT-I cells for BrdU incorporation between days 28 and 30 (Fig. 8⇓A), and in parallel studies, we analyzed their CSFE dilution profile to assay the number of cell divisions completed by OT-I T cells (Fig. 8⇓B). Surprisingly, the proliferative rate in the IL-21 -treated group was relatively slow compared with that in the IL-2-treated group (∼10% BrdU+ vs ∼25% BrdU+, respectively). Moreover, CSFE dilution analysis suggested that although ∼80% of CFSE+ OT-I cells in IL-21-treated mice had undergone more than three rounds of cell divisions, there were ∼10% OT-I T cells that remained in an undivided state 30 days post-tumor challenge. In contrast, in the IL-2-treated mice, ∼25% of the CFSE+ OT-I T cells in IL-2-treated animals had undergone more than three rounds of cell divisions, and nondividing cells were undetectable. These results indicate that IL-21, unlike IL-2, promotes the survival of both dividing and nondividing CD8+ T cells.

FIGURE 8.
FIGURE 8.

IL-21 sustains OT-I cell numbers in tumor-bearing mice. Naive CD8+ OT-I cells (3 × 106) were adoptively transferred into congenic recipient mice challenged i.p. with tumor and injected with vehicle alone (PBS), IL-2 (2000 IU/day), or IL-21 (20 μg/day) on days 4, 6, 8, 10, and 12. OT-I cells from the PC or draining LN were then analyzed in the following manner. A, BrdU incorporation. Mice were injected i.v. with BrdU on day 28, and LN cells were analyzed by flow cytometry on day 30. The mean percentage of BrdU+ OT-I cells obtained from four independent experiments is shown. The error bars represent the SDs. B, Clonal expansion. The percentages of CD8+/Thy1.1+ cells in LN and PC were measured on day 30. A representative of five independent experiments is shown. C, CFSE dilution. The percentage of gated CD8+/Thy1.1+ cells that had undergone more than three rounds of cell division as determined by CFSE staining is indicated. A representative of five independent experiments is shown.

To test the extent of OT-I T cell apoptosis induction in IL-2- and IL-21-treated animals, we assayed their level of annexin V binding and DNA breaks by TUNEL staining at 20 days after tumor challenge. As shown in Fig. 9⇓A, only a small percentage of OT-I T cells isolated from IL-21-treated mice had initiated programmed cell death relative to PBS- and IL-2-treated mice. These results demonstrate that IL-21 protects Ag-activated OT-I T cells from apoptosis, and the sustained clonal expansion achieved by IL-21 treatment is due in part to a reduction in programmed cell death. This property undoubtedly contributes to IL-21’s ability to enhance CD8+ memory and achieve durable antitumor immunity.

FIGURE 9.
FIGURE 9.

IL-21 limits programmed cell death of OT-I cells in tumor-bearing mice. CD8+ OT-I cells (3 × 106) were adoptively transferred into congenic recipient mice challenged i.p. with tumor and injected with vehicle alone (PBS), IL-2 (2,000 or 20,000 IU/day), or IL-21 (20 μg/day) on days 2, 4, 6, 8, 10, and 12. OT-I cells from the draining LN on day 20 were assayed by flow cytometry for the indicated markers. A, Annexin V; the marker indicating annexin V+ cells was established using naive OT-I T cells as a negative control. A representative of four independent experiments is shown. B, TUNEL; cells were stained by DNA terminal nick end labeling and subjected to flow cytometry evaluation (see Materials and Methods). The mean percentage of positive cells and the SD obtained from four independent experiments are shown.

Discussion

In this report we present data on the comparative effects of IL-2, IL-15, and IL-21 as a monotherapy for thymoma in a syngeneic murine model system. Similar to our previous studies (26), low dose IL-2 administration (2,000 IU/day/mouse) prolonged survival relative to PBS controls, but failed to induce tumor-free survival (Fig. 1⇑). However, the beneficial effect achieved with IL-2 was eliminated at the higher dose (20,000 IU/day/mouse) (10, 14). IL-15 administration (0.25 mg/kg) produced a slight improvement in survival over IL-2, but all animals eventually died. Increasing the dose of IL-15 10-fold (2.5 mg/kg) failed to improve this outcome. This finding was rather surprising given IL-15’s ability to enhance effector/memory generation in CD8+ T cells (22). In contrast to IL-2 and IL-15, IL-21 treatment (1–5 mg/kg) increased the 50% survival by almost 2-fold, and more importantly, ∼30% of the animals survived tumor free for >150 days. Interestingly, IL-21 delivery at either early (days 4–12) or late (days 12–22) time points produced similar numbers of long term survivors (Fig. 1⇑D), whereas pretreatment produced suboptimal benefits by promoting only 2–5% long term survivors, suggesting that IL-21 promotes adaptive immunity by promoting the expansion and/or restoration of induced tumor-specific T cell responses, but only marginally augments immune surveillance or affects Ag priming of naive T cell responses in this thymoma model.

Several lines of evidence indicate that IL-21 stimulates CD8+ T cell immunity to E.G7 thymoma. First, depleting CD8+ T cells in mice before and during IL-21 treatment abrogated the antitumor effect (Fig. 2⇑). Eliminating CD4+ T cells had no deleterious effect relative to controls. Similarly, NK1.1 cell depletions failed to diminish the frequency of long term survivors, although the reduction in days to reach 50% survival implicates NK cell responses during the early phases of IL-21-dependent tumor control (see below). Second, the increased survival of IL-21-treated mice correlated with a substantial increase in the frequency of both tumor-specific (OVA) CD8+ T cell numbers and effector cell differentiation. This enhancement consistently exceeded that achieved with IL-2 or IL-15 treatment (Figs. 4⇑ and 5⇑). Third, IL-21’s induction of memory formation and durable immunity, as measured by rejection of s.c. tumors in rechallenge experiments, was abolished in mice after CD8+ T cell depletion (Fig. 3⇑).

The mechanism(s) by which IL-21 stimulates CD8+ T cell-mediated antitumor immunity relative to IL-2 and IL-15 was further investigated by monitoring adoptively transferred OT-I T cells responding to E.G7 challenge. IL-21 clearly enhanced tumor Ag-activated OT-I cell responses, as measured by parameters of early T cell activation (CD69 expression), DNA synthesis (BrdU incorporation), extent of cell division (CSFE dilution), and the accumulation of clonotypic CD8+ T cells (Figs. 6⇑ and 7⇑). In our comparative studies, IL-21 was not as effective as IL-2 during this early induction phase, whereas IL-15 had little impact on the expansion occurring in response to E.G7 tumor challenge. A similar hierarchy in potency among IL-2, IL-21, and IL-15 was also observed in the numbers of endogenous OVA-specific CD8+ T cells (tetramer specific) and the adoptively transferred OT-I T cells (Fig. 4⇑). However, the kinetics of the adoptively transferred OT-I T cell response were accelerated relative to those of the endogenously generated OVA-specific response, perhaps due to the larger pool of high avidity transgenic T cells or the inability of the tetramer to detect earlier initiation of the clonal expansion due to rather small frequency or lower avidity. The overall conclusion that IL-21 effectively costimulates Ag-specific T cell expansion by promoting activation/proliferation is supported by a variety of in vitro studies (28, 30, 32, 40, 41).

A particularly striking feature of IL-21’s enhancement of antitumor immunity is the prolonged elevation of CD8+ T cells that can occur in mice for many days and even weeks after cytokine administration. This was apparent for both adoptively transferred OT-I cells (Fig. 6⇑) and the endogenously generated OVA-specific CD8+ T cells (Fig. 4⇑). Using both BrdU incorporation as well as CSFE dilution (Fig. 8⇑), the relatively large clonal pools of CD8+ T cells were not produced by IL-21 due to its ability to sustain the enhanced rate of proliferation that typifies the effects observed in the induction phase. Rather, IL-21 appeared to promote the CD8+ T cell clonal pool by augmenting survival by tempering their proliferation, as judged by the relatively low rate of cell division and a significantly reduced frequency of apoptosis (Fig. 9⇑). This effect of IL-21 differed considerably from that in IL-2-treated animals, where the rate of CD8+ T cell proliferation was sustained late into the response and was associated with the increased appearance of apoptotic cells that accompanied a dramatic decline in the number of OVA-specific CD8+ T cells and OT-I cells (Figs. 4⇑ and 6⇑), but was similar to IL-15 in maintaining induced CD8+ T cell responses. Thus, IL-21 contrasts with IL-2 with regard to clonal contraction observed after multiple injections (10), but shares features of IL-15 in promoting the survival of previously activated, Ag-specific CD8+ T cells.

How IL-21 regulates a long term effect on these Ag-driven CD8+ T cell populations remains to be determined, although some clues exist. For instance, IL-21 induces STAT3 phosphorylation, a regulator of several antiapoptotic pathways (42), in both human and mouse primary lymphocytes and cell lines (43). Consistent with this observation, IL-21 is nearly equivalent to IL-15 in regulating the survival of Ag-activated OT-I cells in vitro, but, in contrast to IL-15, these cells acquire a central memory phenotype, as defined by their expression of Ly6C, CD44, CCR7, and CD62L (our unpublished observations). Similarly, human Vγ9/Vδ2 T cells grown in the presence of IL-21 also differentiate into putative central memory CD45RO+ T cells that maintained expression of CD62L, CD81, and CCR7 (44). Although these observations argue that IL-21 regulates the survival of Ag-activated CD8+ T cells directly, it could also have an indirect mode of action by synergizing with other regulatory proteins implicated in CD8+ T cell survival, such as 4-1BB, IL-7, or IL-15, which are available in vivo (39, 45).

In addition to enhancing the long term accumulation of endogenous OVA-specific CD8+ T cells, IL-21 treatment sustained the cytolytic activity within tumor-bearing hosts (Fig. 5⇑). Although, the data presented in this study do not demonstrate enhanced ability of IL-21 to promote CTL differentiation, emerging evidence suggests that IL-21 is a potent inducer of CTL activity (28, 30). For instance, OT-I cells cultured in IL-21 show enhanced killing on a per cell basis relative to IL-2 exposure, similar to the results obtained from OVA-specific CD8+ T cells isolated from E.G7 challenged mice (our unpublished observations). In parallel studies, IL-21 readily induces the expression of granzyme B, perforin, and IFN-γ in CD8+ T cells as well as NK cells (30, 31). Based on these observations, we conclude that the immunity induced in IL-21-treated mice includes the augmented differentiation of CD8+ T cells into CTLs that effectively kill E.G7 thymoma. This interpretation is further supported by several studies that have documented an enhancement of CTL-mediated responses by IL-21 against lymphoma and colon carcinoma (24, 46) along with the recent report demonstrating that transgenic IL-21 expression by tumor cells cures B16 melanoma, primarily through a CD8 T cell and perforin-dependent pathway (31). Not surprisingly, given its impact on innate immunity (29), IL-21 can also stimulate NK cell-mediated antitumor responses (24, 31, 46, 47). When combined, these results identify IL-21 as a potent regulator of cell-mediated immunity.

In this study, we have delineated the mechanisms by which IL-21, a product of CD4+ Th cells, stimulates CD8+ T cell immunity to thymoma and provided a conceptual framework for its ability to regulate adaptive immune responses in other tumor models. Similar to IL-2, IL-21 has the capacity to enhance Ag activation and clonal expansion. Distinct from IL-2, however, these Ag-expanded cell populations do not undergo activation-induced cell death in the presence of IL-21, but, rather, they survive while maintaining effector activity. In this regard, IL-21 functions more like IL-15 than IL-2. Thus, the unique ability to behave in certain respects like both IL-2 and IL-15 endows IL-21 with critical attributes for achieving durable immunity. In lieu of the critical role played by CD4+ T cells in regulating CD8+ T cell differentiation (9), it becomes increasingly important to determine the basis by which their interaction promote beneficial responses to infectious disease and cancer. The mechanistic understanding achieved in our studies may enable a more rational approach for the use of cytokines such as IL-21 in the clinical setting.

Acknowledgments

We are grateful to Drs. M. Mescher and P. Sivakumar for their critical review and comments.

Footnotes

  • ↵1 This work was supported by Zymogenetics, Alliance Foundation-Roswell Park Cancer Institute, and the National Institutes of Health. P.A.S. was a Special Fellow of the Leukemia and Lymphoma Society of America.

  • ↵2 Address correspondence and reprint requests to Dr. Protul A. Shrikant, Department of Immunology, 322 CCC Elm and Carlton Streets, Roswell Park Cancer Institute, Buffalo, NY 14263. E-mail address: protul.shrikant{at}roswellpark.org

  • ↵3 Abbreviations used in this paper: LN, lymph node; PC, peritoneal cavity.

  • Received February 17, 2004.
  • Accepted May 5, 2004.

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