IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8⁺ T Cells

Abstract

IL-15 and IL-2 possess similar properties, including the ability to induce T cell proliferation. However, whereas IL-2 can promote apoptosis and limit CD8⁺ memory T cell survival and proliferation, IL-15 helps maintain a memory CD8⁺ T cell population and can inhibit apoptosis. We sought to determine whether IL-15 could enhance the in vivo function of tumor/self-reactive CD8⁺ T cells by using a T cell receptor transgenic mouse (pmel-1) whose CD8⁺ T cells recognize an epitope derived from the self/melanoma antigen gp100. By removing endogenous IL-15 by using tumor-bearing IL-15 knockout hosts or supplementing IL-15 by means of exogenous administration, as a component of culture media or as a transgene expressed by adoptively transferred T cells, we demonstrate that IL-15 can improve the in vivo antitumor activity of adoptively transferred CD8⁺ T cells. These results provide several avenues for improving adoptive immunotherapy of cancer in patients.

Immunotherapies employing IL-2 as a T cell growth/activation factor have been used extensively for the treatment of patients with metastatic melanoma and renal cell carcinoma (1). Despite clinical successes, IL-2 possesses qualities that may preclude it from being the optimal T cell growth/activation factor for use in immunotherapy. IL-2 plays a pivotal role in activation-induced cell death of T cells (2–4) and inhibits memory CD8⁺ T cell proliferation and survival (5, 6). Also, high-dose IL-2 can cause severe, dose-limiting toxicities in patients (7).

IL-15 and IL-2 possess similar properties. Both cytokines bind to and signal through a common, intermediate affinity receptor complex composed of β (CD122) and γ_c receptor (CD132) subunits; thus, IL-2 and IL-15 can share similar in vitro activities (8, 9). Each cytokine, however, interacts with a unique, ligand-specific α chain receptor (10). Despite similarities, it is now clear that IL-2 and IL-15 can play very different, and at times oppositional, roles in T cell biology. IL-2 can promote T cell activation and proliferation, and signaling through the IL-2 receptor (IL-2R) complex may, however, trigger the elimination and/or suppression of activated lymphocytes (3, 4, 11). By contrast, signaling through the IL-15R complex is necessary for the development of elements of the innate immune system, and it contributes to the maintenance of memory CD8⁺ T cells (12, 13).

Although significant in vitro data supporting the role of IL-15 as a T cell growth factor have accumulated (14), comparatively few published reports have assessed the direct function of IL-15 on CD8⁺ T cells in vivo. This point is especially critical because it has been demonstrated that components of the IL-2R and IL-15R complexes are differentially regulated in vitro compared with in vivo (15). Further, there have been very few accounts of IL-15 use in immunotherapy (16–18) and, to our knowledge, no reports of IL-15 use in an antitumor therapeutic model involving adoptive transfer of CD8⁺ T cells.

Recently, we reported that an immunotherapy regimen that combined adoptive transfer of CD8⁺ T cells reactive against the tumor/self antigen (Ag) gp100, Ag-specific vaccination by using an altered peptide ligand, and administration of exogenous IL-2 could reproducibly cause significant regression of large, established s.c. B16 melanoma in mice (19). Using this model, we sought to determine whether IL-15 could enhance the in vivo function of tumor/self-reactive CD8⁺ T cells. By removing endogenous IL-15 by using tumor-bearing IL-15 knockout hosts or supplementing IL-15 by means of exogenous administration, as a component of culture media, or as a transgene expressed by adoptively transferred T cells, we demonstrate that IL-15 can improve the in vivo antitumor activity of adoptively transferred CD8⁺ T cells.

Materials and Methods

Mice and Tumor Lines. pmel-1 T cell receptor transgenic (Tg) (19) mice were crossed with human IL-15 Tg (20) and C57BL/6-TgN (ACTbE-GFP) mice (The Jackson Laboratory) to derive pmel-IL-15 and pmel-GFP double Tg mice, respectively. Expression of transgenes was confirmed by PCR analysis for the pmel-α and β T cell receptor chains, quantification of serum human IL-15 by ELISA (R & D Systems), and UV fluorescence for GFP. B16 (H-2^b), a spontaneous gp100⁺ murine melanoma, was maintained in culture media (19). The gp100^– tumor lines EL-4 (American Type Culture Collection) and methylcholanthrene 205 (MCA-205; National Cancer Institute Tumor Repository, Bethesda) were maintained in culture media and used as irrelevant H-2^b targets.

In Vitro Activation, Cytokine Release, and Cytolytic Assays. pmel-1 splenocytes were isolated as described (19) and cultured in the presence of 1 μM human gp100_25–33 (hgp100_25–33) and culture media containing 10 ng/ml recombinant human IL-2 (rhIL-2; Chirion) or 10 ng/ml rhIL-15 (PeproTech, Rocky Hill, NJ). IL-2 and IL-15 cultured cells (henceforth described as pmel^IL2 and pmel^IL15, respectively) were used for adoptive cell transfer, fluorescence-activated cell sorting (FACS) analysis, recognition, or cytolytic assays on d 6–8. Cytokine release assays were performed as described (19). When naïve cells were used, pmel-1 splenocytes were depleted of non-CD8⁺ T cells by using a magnetic cell sorting negative-selection column (Miltenyi Biotec, Auburn, CA). Supernatants were analyzed by murine Lincoplex (Linco Research Immunoassay, St. Charles, MO) or murine IFN-γ, murine tumor necrosis factor α (TNF-α), murine IL-10 (Endogen, Cambridge, MA), and murine IL-2 (R & D Systems) ELISA kits. For cytolytic assays, MCA-205 targets were pulsed with 100 μCi (1 Ci = 37 GBq) ⁵¹Cr (Amersham Biosciences) and 1 μM peptide for 2 h at 37°C and washed. We plated 1 × 10³ labeled cells per well with effector cells at indicated concentrations in a 96-well, U-bottomed plate for 4 h at 37°C. The amount of ⁵¹Cr released was determined by γ-counting, and the percentage of specific lysis was calculated from triplicate samples by using the following formula: [(experimental cpm – spontaneous cpm)/(maximal cpm – spontaneous cpm)] × 100.

RNA Preparation and Real-Time RT-PCR. Naïve, CD8⁺-enriched pmel-1 splenocytes, pmel^IL2, or pmel^IL15 cells were plated at 2 × 10⁶ cells per well in a 24-well plate and then stimulated for 6 h with 2 × 10⁶ cells per well of EL-4 targets pulsed with 1 × 10^–7 M hgp100_25–33 peptide in a final volume of 2 ml of culture media. RNA was isolated by using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. DNase I (Qiagen) treatment was used to remove residual DNA. We made 1 μg of cDNA by reverse transcription. The levels of IL-2 and 18S rRNA mRNAs were evaluated by real-time PCR using commercially available IL-2 probes and primers (Applied Biosystems) and a Prism 7900HT (Applied Biosystems). We calculated the ratio of the 18S cyclic threshold (C_T) of highest unknown to the C_T of a specific sample. The C_T for a sample was multiplied by this ratio, and a relative IL-2 message per 18S message was determined.

Adoptive Cell Transfer, Vaccination, and Cytokine Administration. Female C57BL/6 (The Jackson Laboratory), IL-15^–/– (Taconic Farms), and IL-2^–/– (The Jackson Laboratory) mice at 6–12 wk of age were injected s.c. with 2–5 × 10⁵ B16F10 melanoma cells. Mice (n = 5 for all groups) were treated 12–14 d later with i.v. adoptive transfer of pmel-1 or pmel-IL-15 splenocytes or in vitro activated splenocytes, as indicated. In some experiments, lymphopenia was induced by sublethal irradiation (500 cGy) of tumor-bearing mice on the day of treatment. Where specified, mice were vaccinated with 2 × 10⁷ plaque-forming units of a previously described recombinant fowlpox virus encoding hgp100 (rFPhgp100; Therion Biologics, Cambridge, MA) (21). Indicated doses of rhIL-2 or rhIL-15 were administered by i.p. injection twice daily for a total of six doses. Tumors were measured in a blinded fashion by using calipers. The products of the perpendicular diameters are presented as mean ± SEM. Animal survival was analyzed by using Kaplan–Meier statistics.

Enumeration of Adoptively Transferred Cells. Enumeration of adoptively transferred pmel-GFP cells in blood was performed by bleeding mice (n = 3) by tail vein daily into heparin-containing microcentrifuge tubes (Marsh Biomedical Products, Rochester, NY). Samples were analyzed by the National Institutes of Health Clinical Laboratory (Bethesda) for the absolute lymphocyte count and by FACS analysis for CD8 and GFP expression by cells. The absolute number of pmel-GFP cells per μl of blood was calculated by multiplying the absolute lymphocyte count by the ratio CD8⁺GFP⁺/lymphocyte gate.

Flow Cytometry. Cells were labeled with mAbs (BD Biosciences, Pharmingen) against Vβ13-FITC (MR12–3), CD25-phycoerythrin (PE) (PC61), CD44-PE (IM7), CD62L-PE (MEL-14), CD69-PE (H1.2F3), CD122-PE (TM-β1), and CD8α-APC (53–6.7). To detect CCR7 expression, a CCL19-Fc fusion protein (a gift from Jason Cyster, University of California, San Francisco) was used in combination with a PE-conjugated anti-human Fc mAb. Propidium iodide-stained cells were excluded from analysis. Samples were analyzed by using a FACSCalibur flow cytometer and cellquest software (BD Biosciences).

Results

Exogenous IL-15 Enhances the in Vivo Antitumor Activity of Tumor-Reactive CD8⁺ T Cells. IL-2 has been shown to enhance the antitumor activity of adoptively transferred immune cells in patients with cancer (1). Further, the exogenous administration of IL-2 was found to be strictly necessary yet individually insufficient to obtain significant and durable regression of established s.c. B16 tumors in the pmel-1 model (19). We sought to determine whether the exogenous administration of IL-15, a T cell growth/activation factor with functions distinct from that of IL-2 (14), could replace the administration of IL-2 in an adoptive immunotherapy treatment regimen. To produce a treatment window, we administered a dose of cells (1–3 × 10⁶) that was determined to be suboptimal (data not shown). Pmel^IL2 cells were adoptively transferred into sublethally irradiated C57BL/6 (WT) mice bearing 14-d established s.c. B16 tumors. In addition to cells, rFPhgp100 vaccine was administered alone or in combination with titrated doses (12–108 μg per dose) of either rhIL-2 or rhIL-15. Adoptive transfer of cells plus vaccination alone did not significantly delay tumor growth compared with untreated controls (Fig. 1A). When cells and vaccination were combined with the exogenous administration of either rhIL-15 or rhIL-2, a pronounced delay in tumor growth was observed. There was no statistical difference observed in tumor size (Fig. 1) or animal survival (data not shown) between treatment groups that received rhIL-2 or rhIL-15 at any of the cytokine dose levels tested. Similar results were observed in four independent experiments using both irradiated and nonirradiated tumor-bearing mice. Thus, we found that the exogenous administration of IL-15 could significantly enhance the in vivo antitumor activity of adoptively transferred, tumor/self-reactive CD8⁺ T cells.

Fig. 1.

Exogenous IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8⁺ T cells. WT mice bearing 13-d established s.c. B16 tumors were sublethally irradiated and left untreated as controls (⋄) or received adoptive transfer of 1 × 10⁶ pmel^IL2 cells and rFPhgp100 vaccination (▴). In addition to cells and vaccine, indicated groups also received exogenous administration of either rhIL-2 (○) or rhIL-15 (▪) at doses of 12 μg (A) 36 μg (B), or 108 μg (C). Data shown are representative of four independent experiments.

Endogenous IL-15 Enhances Tumor/Self-Reactive CD8⁺ T Cell Function in Vivo. Despite reported differences in the in vivo biologic functions of IL-2 and IL-15, we determined that the exogenous provision of either cytokine produced similar antitumor effects when combined with adoptively transferred, tumor-reactive CD8⁺ T cells and vaccination. Because we administered potentially saturating doses of IL-2 and IL-15, we next asked whether physiologic levels of host-derived IL-15 could influence the function of adoptively transferred CD8⁺ T cells. Therefore, we assessed the efficacy of the tripartite treatment regimen of pmel^IL2 cells, rFPhgp100 vaccination, and exogenous IL-2 (36 μg per dose) in tumor-bearing IL-15^–/– hosts. Tumor-bearing IL-2^–/– hosts were used as a control for the absence of a γ_c-signaling cytokine. Tumor growth was similar in untreated, sublethally irradiated WT, IL-15^–/–, and IL-2^–/– mice (Fig. 2). However, we found that the long-term treatment effect of the combined immunotherapy regimen was diminished in IL-15^–/– hosts relative to WT controls (Fig. 2A). Although tumor growth was initially suppressed in both treatment groups, tumor growth in the IL-15^–/– group diverged from that of the WT group 2–3 wk after tumor treatment. By contrast, treatment in IL-2^–/– hosts was comparable with that of WT controls (Fig. 2B). Similar results were observed in two independent experiments. Thus, physiologic levels of host-derived IL-15, but not IL-2, contributed to a sustained antitumor treatment response.

Fig. 2.

Endogenous IL-15, but not IL-2, enhances the in vivo antitumor activity of adoptively transferred CD8⁺ T cells. WT, IL-15^–/–, and IL-2^–/– mice bearing 14-d s.c. B16 tumors received sublethal irradiation and were left untreated as controls or given a triple regimen of 6 × 10⁶ pmel^IL2 cells, rFPhgp100 vaccine, and exogenous IL-2 (36 μg per dose). Tumors grew similarly in untreated WT (⋄), IL-15^–/– (□), and IL-2^–/– (▵) controls. (A) The treatment effect of the combined immunotherapy regimen is impaired in IL-15^–/– hosts (▪) relative to WT controls (○). (B) Treatment in IL-2^–/– hosts (▴) is similar to that of WT controls (•). Data shown are representative of two independent experiments.

Phenotypic Polarization of Cells Cultured in IL-15 Vs. Cells Cultured in IL-2. Previously, Weninger et al. (22) demonstrated that Agprimed splenocytes from P14 T cell receptor Tg mice (whose CD8⁺ T cells recognize the LCMV Ag gp33) differentially polarized to either an effector memory (T_EM)- or central memory (T_CM)-like phenotype when cultured in the presence of IL-2 or IL-15, respectively (22). To determine whether a similar phenotypic polarization is observed in CD8⁺ T cells reactive against a tumor/self Ag, rather than a foreign viral Ag, we cultured pmel-1 splenocytes primed with 1 μM of hgp100_25–33 in either 10 ng/ml rhIL-2 (pmel^IL2) or 10 ng/ml rhIL-15 (pmel^IL15). Fresh pmel-1 splenocytes expressed surface markers characteristic of naïve cells; specifically, CD8⁺ pmel-1 T cells (identified by Vβ13 expression) stained negatively for the activation markers CD25 and CD69, were CD44 intermediate, and expressed the lymph node homing markers CD62L and CCR7 (Fig. 3). Both pmel^IL2 and pmel^IL15 cells were uniformly CD44^hi 7 d after peptide stimulation, indicating that the cells had become Ag-experienced. Further, cells from both culture conditions also stained positively for CD25, although expression on pmel^IL15 cells was lower than expression on pmel^IL2 cells. Importantly, pmel^IL15 cells maintained positive staining for CD62L and CCR7, whereas pmel^IL2 cells had down-regulated expression for both lymph node homing markers relative to naïve controls. In addition, expression of the early activation Ag CD69 was generally positive for pmel^IL2 and generally negative for pmel^IL15 cells. These phenotypic data demonstrated that naïve, tumor/self-reactive CD8⁺ T cells cultured in the presence of IL-15 polarized to a T_CM-like phenotype, whereas cells cultured in IL-2 assumed a T_EM-like phenotype.

Fig. 3.

pmel-1 splenocytes differentially polarize either to a T_EM-orT_CM-like population when cultured in IL-2 or IL-15, respectively. Splenocytes from a pmel-1 Tg mouse were primed with 1 μM hgp100_25–33 peptide and cultured in culture media containing 10 ng/ml rhIL-2 or 10 ng/ml rhIL-15. On d 7, FACS analysis for surface expression of the activation markers CD25, CD44, and CD69 and the lymph node homing markers CD62L and CCR7 was performed. Naïve pmel-1 cells were obtained from splenocytes of an age-matched littermate. Results from a representative experiment are shown after gating on propidium iodide-negative and CD8⁺ T cells.

Functional Polarization of Cells Cultured in IL-15 Vs. Cells Cultured in IL-2. We next asked whether these phenotypic differences correlated with functional changes in vitro and in vivo. To compare cytokine release profiles, naïve CD8-enriched pmel-1 splenocytes, pmel^IL2, and pmel^IL15 cells were cocultured with EL-4 targets pulsed with titrated doses of hgp100_25–33 peptide. Both pmel^IL2 and pmel^IL15 cells recognized only targets pulsed with hgp100_25–33 peptide and not EL-4 alone or EL-4 pulsed with an irrelevant H2-D^b-restricted peptide derived from nucleoprotein (data not shown). Compared with naïve controls, pmel^IL2 and pmel^IL15 cells had a similar increase in functional avidity and secreted comparable, dose-dependent amounts of IFN-γ (Fig. 4A) and TNF-α (Fig. 4B). Thus, Ag-primed pmel-1 splenocytes cultured in the presence of either IL-2 or IL-15 were activated relative to naïve CD8⁺ T cells.

Fig. 4.

Tumor/self-reactive CD8⁺ T cells cultured in IL-15 or IL-2 polarize to two functionally distinct populations with unique in vitro and in vivo functions. IFN-γ (A), TNF-α (B), IL-10 (C), and IL-2 (D) production by CD8-enriched pmel-1 splenocytes (▴), pmel^IL2 (○), and pmel^IL15 (▪) cells cocultured for 24 h with EL-4 pulsed with titrated doses of hgp100_25–33 peptide. Data shown are representative of at least two independent coculture experiments. (E) Quantitative RT-PCR for IL-2 mRNA in CD8-enriched pmel-1 splenocytes (gray bar), pmel^IL2 (open bar), and pmel^IL15 cells (black bar) after a 6-h coculture with EL-4 targets pulsed with 1 × 10^–7 M hgp100_25–33. Data shown are representative of three independent experiments. (F) pmel^IL15 cells at d 8 are less cytolytic than pmel^IL2 cells directed against hgp100_25–33 pulsed MCA-205 targets. Data shown are representative of three independent cytolytic assays. (G) Absolute number of adoptively transferred cells on d 6 in the blood of tumor-bearing, sublethally irradiated WT mice from groups treated with rFPhgp100, IL-2 (36 μg per dose), and 1 × 10⁶ pmel^IL2 cells (open bar) or pmel^IL15 cells (filled bar). Data shown are mean ± SEM of three mice per group and represent the global maximum in cell number for groups receiving pmel^IL2 and pmel^IL15 cells. Similar results were also observed in the spleens of treated animals (data not shown). This experiment was performed twice with similar results.

Significantly, only pmel^IL2 cells secreted the immunosuppressive cytokine IL-10, whereas pmel^IL15 cells produced no detectable IL-10 across all peptide concentrations tested (Fig. 4C). Conversely, only pmel^IL15 cells produced dose-dependent amounts of IL-2 (Fig. 4D). No IL-2 was detected in supernatants taken from cocultures that used pmel^IL2 cells. To confirm that this difference in detectable IL-2 was due to differential production by pmel^IL15 cells, and not differential consumption by pmel^IL2 cells, we performed quantitative RT-PCR for IL-2 mRNA. IL-2 message levels were at least 10-fold higher in pmel^IL15 cells compared with naïve pmel-1 and pmel^IL2 cells in three independent experiments (Fig. 4E). Both naïve CD8⁺ pmel-1 and pmel^IL2 cells expressed similar levels of IL-2 mRNA. Thus, the difference in detectable levels of IL-2 was due to differential production by pmel^IL15 cells, and not enhanced consumption by pmel^IL2 cells.

In addition to cytokine secretion, we evaluated the cytolytic activity of pmel^IL2 and pmel^IL15 cells against MCA-205 targets pulsed with hgp100_25–33 or nucleoprotein-derived peptides. In three independent assays, pmel^IL15 cells were significantly less lytic than pmel^IL2 cells against targets pulsed with relevant peptide (Fig. 4F).

Last, we compared the in vivo proliferative capacity of pmel^IL2 and pmel^IL15 cells. Ag-primed pmel-GFP splenocytes were cultured in the presence of IL-2 or IL-15 and adoptively transferred into sublethally irradiated, tumor-bearing WT mice in combination with rFPhgp100 vaccination and exogenous IL-2 (36 μg per dose). In two independent experiments, the number of CD8⁺GFP⁺ cells per μL of blood reached a peak in both treatment groups on d 6, and was significantly higher in groups that received IL-15-cultured cells compared with groups that received IL-2-cultured cells (Fig. 4G). A similar pattern of proliferation was also observed in the spleens of treated mice (data not shown). Therefore, in addition to being phenotypically distinct, pmel^IL2 and pmel^IL15 cells represent two functionally distinct populations of tumor/self-reactive CD8⁺ T cells.

Adoptive Transfer Using Cells Cultured in IL-15 vs. Cells Cultured in IL-2. To explore differential antitumor function in vivo, we adoptively transferred pmel^IL2 or pmel^IL15 cells (1 × 10⁶) into sublethally irradiated WT mice bearing 14-d s.c. B16 tumors. These mice also received rFPhgp100 vaccination and exogenous IL-2 (36 μg per dose). In three independent experiments, treatment with pmel^IL15 cells caused a more pronounced delay in tumor growth compared with groups that received pmel^IL2 cells (Fig. 5A). In addition to tumor size, groups that received pmel^IL15 cells had a statistically significant (P = 0.006) improvement in survival compared with groups that received pmel^IL2 cells (Fig. 5B). Similar results were observed when nonirradiated tumor-bearing hosts were used. These data demonstrated that adoptive transfer of a T_CM-like population of cells may be superior to that of a T_EM-like population in an adoptive tumor immmunotherapy treatment regimen that employs a systemically administered, Ag-specific vaccination.

Fig. 5.

IL-15-cultured tumor-reactive CD8⁺ T cells treat established B16 melanoma with enhanced efficacy compared with IL-2-cultured cells. WT mice bearing 14-d established s.c. B16 tumors were sublethally irradiated and either were left untreated as controls (⋄) or received adoptive transfer of 1 × 10⁶ pmel-1 cells cultured in 10 ng/ml rhIL-2 (•) or 10 ng/ml rhIL-15 (▪), rFPhgp100 vaccination, and exogenous IL-2 (36 μg per dose). Tumor area (A) and survival (B) in mice receiving pmel^IL2 or pmel^IL15 cells, rFPhgp100 vaccination, and exogenous IL-2 compared with untreated controls. Data shown are representative of three independent experiments.

Forced Expression of IL-15 by Tumor-Reactive T Cells Enhances Antitumor Function. Genetic modification, including the insertion of autocrine growth factors such as IL-2, has been proposed as a means of enhancing the survival and function of adoptively transferred T cells (23). To assess whether the expression of IL-15 by adoptively transferred, tumor-reactive CD8⁺ T cells could improve their in vivo antitumor function, we crossed the pmel-1 Tg mouse with a previously described human IL-15 Tg mouse (20). CD8⁺ T cells from this cross (henceforth described as pmel-IL-15) spontaneously assumed the phenotype (CD8⁺CD44^hiCD122^hi) of a recently described IL-15-dependent memory CD8⁺ T cell population (Fig. 6A) (13). Pmel-IL-15 double Tg CD8⁺ T cells did not become spontaneously activated in vivo; they did not express the activation markers CD25 and CD69, nor did the cells exhibit enhanced reactivity against hgp100_25–33 pulsed targets compared with cells from a pmel-1 single Tg littermate control (data not shown). In five independent experiments, pmel-IL-15 double Tg splenocytes treated established s.c. B16 tumors superiorly to those of pmel-1 splenocytes when combined with rFPhpg100 vaccination and exogenous IL-2 (36 μg per dose) (Fig. 6B). In some experiments, treatment with pmel-IL-15 cells plus rFPhgp100 vaccine treated better than pmel-1 cells plus rFPhgp100, suggesting that the secretion of IL-15 might reduce the dependency of a T cell on an exogenously administered cytokine to induce antitumor activity. Treatment groups that received vaccination, IL-2, and pmel-IL-15 splenocytes survived statistically longer (P = 0.0016) than groups that received pmel-1 splenocytes (data not shown). Together, these data revealed that the forced expression of IL-15 by adoptively transferred CD8⁺ T cells could substantially enhance their in vivo antitumor function.

Fig. 6.

Forced expression of IL-15 by adoptively transferred CD8⁺ T cells enhances their in vivo antitumor function. (A) FACS analysis for surface expression of CD122 (IL-2 and IL-15Rβ) and CD44 on CD8⁺ T cells derived from a pmel-IL-15 double Tg mouse or an age-matched pmel-1 single Tg littermate. Percentages shown represent the percentage of gated cells within the respective quadrant. Results from one representative experiment are shown after gating on propidium iodide-negative and CD8⁺ T cells. (B) WT mice were inoculated with s.c. B16 melanoma, and 14 d later they received adoptive transfer of 5 × 10⁶ pmel-1 or pmel-IL-15 splenocytes, rFPhgp100 vaccination, and exogenous IL-2 (36 μg per dose) or were left untreated as controls. In addition to tumor size, groups that received pmel-IL-15 splenocytes, rFPhgp100, and IL-2 survived significantly longer (P = 0.0016) compared with groups that received pmel-1 splenocytes, rFPhgp100, and IL-2 (data not shown). Data shown are representative of four independent experiments.

Discussion

In the experiments described in this manuscript, we found that IL-15 was a versatile cytokine capable of enhancing the in vivo antitumor activity of adoptively transferred, tumor/self-reactive CD8⁺ T cells. Although use of IL-15 as a vaccine adjuvant has been described (16, 18), these reports did not directly evaluate the ability of systemic IL-15 to enhance CD8⁺ T cell function against tumor or pathogens. By comparing treatment responses in groups that received titrated doses of either exogenous IL-15 or IL-2, we determined that the two cytokines possess a similar dose-response relationship in the range of 12–108 μg per dose. Based on preclinical data in mice (24), IL-15 may possess a greater therapeutic index in humans compared with IL-2. Thus, the use of exogenous IL-15 in patients may be preferable to that of IL-2 to support the function of adoptively transferred CD8⁺ T cells.

One of the most significant recent improvements in immunotherapy has been the use of a lymphodepletive conditioning regimen before adoptive cell transfer (25). The mechanisms underlying this augmented immune function, however, remain obscure. We found that the endogenous production of IL-15, but not IL-2, by a tumor-bearing host enhanced the in vivo antitumor efficacy of adoptively transferred CD8⁺ T cells. In infectious disease models, host-derived IL-15 augments protective immunity by maintaining the survival and function of natural killer and memory CD8⁺ T cells (20). Thus, depletion of these lymphoid subsets before adoptive transfer may augment the ability of transferred tumor-specific CD8⁺ T cells to perceive endogenous IL-15.

Currently, IL-2 is used to activate and expand T cells before adoptive transfer into patients. Using this approach, transferred cells assume a highly differentiated, effector phenotype (25). We show that, in vitro, IL-15 expands tumor/self-reactive CD8⁺ T cells with the phenotype and function of naturally arising T_CM cells (26, 27). Specifically, we found that Ag-primed CD8⁺ T cells grown in the presence of either IL-2 or IL-15 produced comparable amounts of IFN-γ and TNF-α on restimulation, but only cells grown in IL-15 produced their own IL-2 and retained the expression of CD62L. Further, we discovered a previously undescribed functional difference between T_EM- and T_CM-like CD8⁺ cells: the ability of T_EM-like cells, and not T_CM-like cells, to produce IL-10. It remains to be determined whether this functional distinction will hold true for naturally arising T_EM and T_CM populations. Most importantly, we found that a T_CM-like population of tumor/self-reactive CD8⁺ T cells, like virus-specific T_CM CD8⁺ cells (27), proliferate more in vivo and treat established tumors better than a T_EM-like population when used in an adoptive immunotherapy treatment regimen.

Finally, we discovered that the forced expression of IL-15 by adoptively transferred, tumor-reactive CD8⁺ T cells treats established s.c. tumors better than tumor-reactive CD8⁺ T cells that do not secrete IL-15, a finding that may be surprising given the ability of IL-15 to be presented in trans by IL-15Rα (28). These experiments model the clinical possibility of transducing an exogenous IL-15 gene into human tumor-infiltrating lymphocytes, a procedure that has been accomplished by using an exogenous IL-2 gene (23).

In summary, we determined that IL-15 could enhance the in vivo function of tumor/self-reactive CD8⁺ T cells by using the pmel-1 model. By removing endogenous IL-15 by using tumor-bearing IL-15 knockout hosts or supplementing IL-15 by means of exogenous administration, as a component of culture media, or as a transgene expressed by adoptively transferred T cells, we found that IL-15 improves the antitumor function of adoptively transferred T cells. Current efforts are focused on the translation of these observations in mice to use in improving adoptive immunotherapies in humans.