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.

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.

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.

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.

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.

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.

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.