Immunologic and therapeutic synergy of IL-27 and IL-2: enhancement of T cell sensitization, tumor-specific CTL reactivity and complete regression of disseminated neuroblastoma metastases in the liver and bone marrow

Abstract

IL-27 exerts antitumor activity in murine orthotopic neuroblastoma, but only partial antitumor effect in disseminated disease. This study demonstrates that combined treatment with IL-2 and IL-27 induces potent antitumor activity in disseminated neuroblastoma metastasis. Complete durable tumor regression was achieved in 90% of mice bearing metastatic TBJ-IL-27 tumors treated with IL-2 compared with only 40% of mice bearing TBJ-IL-27 tumors alone and 0% of mice bearing TBJ-FLAG tumors with or without IL-2 treatment. Comparable antitumor effects were achieved by IL-27 protein produced upon hydrodynamic IL-27 plasmid DNA delivery when combined with IL-2. Although delivery of IL-27 alone, or in combination with IL-2, mediated pronounced regression of neuroblastoma metastases in the liver, combined delivery of IL-27 and IL-2 was far more effective than IL-27 alone against bone marrow metastases. Combined exposure to IL-27 produced by tumor and IL-2 synergistically enhances the generation of tumor-specific CTL reactivity. Potentiation of CTL reactivity by IL-27 occurs via mechanisms that appear to be engaged during both the initial sensitization and effector phase. Potent immunologic memory responses are generated in mice cured of their disseminated disease by combined delivery of IL-27 and IL-2, and depletion of CD8(+) ablates the antitumor efficacy of this combination. Moreover, IL-27 delivery can inhibit the expansion of CD4(+)CD25(+)Foxp3(+) regulatory and IL-17-expressing CD4(+) cells that are otherwise observed among tumor-infiltrating lymphocytes from mice treated with IL-2. These studies demonstrate that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing widely metastatic neuroblastoma tumors.

FIGURE 1

IL-27 and IL-2 synergistically enhance the proliferation and IFN-γ production by murine splenocytes. Normal murine splenocytes were cultured in the presence or absence of IL-27 (50 ng/ml) ± IL-2 (50 IU/ml) or medium alone for 48 h. Proliferative responses in the respective groups were assessed by [³H]thymidine (1 µCi/well) incorporation as described in Materials and Methods. Values shown represent the mean value ± SEM for triplicate samples (A). To investigate the impact of IL-27 and IL-2 combination on the production of IFN-γ, normal spleen cells were cultured in the presence of IL-27 ± IL-2 or medium alone for 72 h as described in Materials and Methods. Supernatants were then harvested and the concentration of IFN-γ in the respective culture supernatants was assayed using ELISA. The values shown represent the mean ± SEM for duplicate samples (B). *, p < 0.001; IL-2 plus IL-27 vs either IL-2 or IL-27 or medium alone.

FIGURE 2

Combined delivery of IL-27 and IL-2 mediates complete tumor regression and long term survival in mice bearing disseminated TBJ neuroblastoma metastases. Cohorts of 10 A/J mice per group were injected i.v. with 1 × 10⁵ TBJ FLAG or TBJ IL-27 neuroblastoma tumor cells on day 0. Mice were then treated with either IL-2 or medium alone on days 5–9, 12–16, 19–23, or 26–30 (depicted by bars) post tumor implantation (A). Cohorts of 10 A/J mice per group were injected i.v. with 1 × 10⁵ TBJ parental cells on day 0. Mice were treated with IL-27 hydrodynamic delivery administered on days 5 and 15 (depicted by arrows) post tumor injection. IL-2 or medium alone was injected i. p. on days 5–9, 12–16, 19–23, or 26–30, as described in Materials and Methods and monitored for survival (B).

FIGURE 3

Combined delivery of IL-27 and IL-2 mediates complete regression of disseminated TBJ neuroblastoma metastases in the liver. Cohorts of mice (10 mice/group) bearing established disseminated TBJ IL-27 or TBJ FLAG (control) tumors were treated with IL-2 (200,000 IU/injection) or vehicle alone on days 5–9 or 12–15 post tumor implantation as described in Materials and Methods. Mice were then euthanized at day 16 post tumor implantation and livers were resected for imaging of metastatic disease burden in the respective treatment groups. Light images of the respective organs were captured at ×1 magnification as described in Materials and Methods. Photographs of five liver/group are shown (A). The number of macrometastatic lesions at day 16 post tumor injection was also scored (B). *, p < 0.035; TBJ IL-27 plus IL-2 vs TBJ IL-27 plus vehicle.

FIGURE 4

Combined delivery of IL-27 and IL-2 mediates complete regression of induced neuroblastoma metastases in the bone marrow. Cohorts of 10 A/J mice/group were injected i.v. with 1 × 10⁵ TBJ IL-27 or TBJ FLAG (control) neuroblastoma tumor cells on day 0 as described in Materials and Methods. Mice were then treated with either IL-2 or vehicle alone on days 5–9, 12–16, 19–23, or 26–30 post tumor cell implantation. Mice were harvested individually as they became sick, bone marrow was extracted, and tumor burden in each marrow specimen was assessed by colony assay in the presence of G418 (1 mg/ml) as described in Materials and Methods. The data shown represent the proportion of mice developing colonies in each of the respective treatment groups, *, p = 0.0198; TBJ IL-27 plus IL-2 vs TBJ IL-27 plus vehicle (A). In complementary studies, cohorts of mice (10 mice/group) were injected with 1 × 10⁵ TBJ FLAG or TBJ IL-27 cells on day 0, followed by therapy with IL-2 or vehicle on days 5–9 or 12–16 post tumor cell injection. Nineteen days post tumor injection, mice were euthanized and bone marrow cells were isolated as described in Materials and Methods. Single cell suspensions containing both marrow cells and metastatic neuroblastoma tumor cells were prepared and injected s.c. (1 × 10⁶ cells/injection) into naive mice to assess the tumorigenicity of contaminating neuroblastoma metastases in the bone marrow. Tumor size was monitored twice a week. The proportion of mice that developed a tumor at any time post injection of bone marrow cells from the respective groups is shown in B. **, p = 0.011, TBJ IL-27 plus IL-2 vs TBJ IL-27 plus vehicle.

FIGURE 5

Role of T vs NK cell subsets in the antitumor activity of IL-27/IL-2 therapy. Cohorts of 10 wild-type A/J were injected i.v. with either TBJ-IL-27 or TBJ-FLAG tumor cells (1 × 10⁵ cells/animal) on day 0. Mice were concurrently depleted of NK cells, CD4⁺ T cells or CD8⁺ T cells as described in Materials and Methods. Mice were treated with either IL-2 or medium alone on days 5–9, 12–16, 19–23, or 26–30 post tumor implantation as described in Materials and Methods and monitored for survival. Mice surviving at last follow-up were tumor free.

FIGURE 6

IL-27 inhibits IL-2-induced increases in the proportion of CD4⁺CD25⁺FoxP3⁺ T cells within TIL from TBJ neuroblastoma tumors. Mice bearing primary orthotopic TBJ Flag or TBJ-IL-27 tumors treated with vehicle control or IL-2 were euthanized as described in the Materials and Methods section. Tumors were dissected and TIL were isolated and stained with PECy7 conjugated anti-mouse CD4, allophycocyanin conjugated anti-mouse CD25, PE conjugated anti-mouse IL17A, and FITC-conjugated antimouse Foxp3 as described in Materials and Methods. CD45 was used as marker for leukocytes. The percentage of CD4⁺ CD25⁺ Foxp3⁺ T cells within the total CD4⁺ T cell population is shown A and C. The percentage of CD4⁺ IL-17⁺ cells within the CD45⁺ gate (B) and the percentage of IL-17⁺ expression within CD4⁺ TILs is shown for tumor-derived samples from different treatment groups (D).

FIGURE 7

Combined exposure to IL-27 and IL-2 synergistically enhances immune responsiveness: IFN-γ production. Spleens were resected under sterile conditions from mice bearing disseminated TBJ IL-27 or TBJ FLAG (control) tumors at day 15 post tumor implantation. Single cell suspensions were prepared and splenocytes were then restimulated with corresponding irradiated TBJ FLAG or TBJ IL-27 tumors cells ± IL-2 (10 IU/ml) for 72 h as described in Materials and Methods. IFN-γ concentration in the respective culture supernatants were then assayed by ELISA. Values shown represent the mean ± SEM of triplicate samples.

FIGURE 8

Combined exposure to IL-27 and IL-2 synergistically enhances tumor-specific immune responsiveness: generation of neuroblastoma- specific CTL activity. Spleens were resected under sterile conditions from mice bearing disseminated TBJ IL-27 or TBJ FLAG (control) tumors at day 15 post tumor implantation. Single cell suspensions were prepared and splenocytes were restimulated with irradiated TBJ IL-27 or TBJ-FLAG tumor cells ± IL-2 for 6 days as described in Materials and Methods. The cytolytic activity directed against TBJ FLAG or irrelevant syngeneic SA-1 tumor cells was assayed by ¹¹¹Indium release assay. Combined exposure to IL-27 and IL-2 can synergistically enhance the generation of CTL reactivity (A). Additionally, IL-27 also can mediate effects during both the priming and effector phases of the generation of CTL reactivity and this effect was further enhanced by IL-2 (B). This CTL reactivity is tumor specific, because enhancement of reactivity against syngeneic irrelevant SA-1 tumor cells was not observed (data not shown). Values shown represent the mean percentage of specific lysis for each of the respective groups assessed in triplicate at the indicated E:T ratios.