Complete tumour regressions induced by vaccination with IL-12 gene-transduced tumour cells in combination with IL-15 in a melanoma model in mice

Abstract

In the present study, IL-12 gene-transduced B78-H1 melanoma cells (B78/IL-12) were used in combination with IL-15 to treat melanoma-bearing mice. Genetically modified B78/IL-12 cells, when injected subcutaneously, induced strong activation of antitumour mechanisms resulting in complete loss of tumourigenicity. In a therapeutic model, intratumoural injection of irradiated B78/IL-12 cells significantly delayed tumour growth and led to the regression of melanoma in one case. Similarly, consecutive daily injections of IL-15 markedly inhibited tumour progression with occasional curative effects. When used in combination, vaccination with B78/IL-12 cells and treatment with IL-15 caused eradication of established tumours in all treated mice. The combined treatment with B78/IL-12 cells and IL-15 activated not only a local response against tumour, but also induced systemic antitumour immunity that led to a delay or inhibition of tumour development at a distant site. In vitro studies demonstrated that when used together, B78/IL-12 cells and IL-15 induced a shift from a type Th2 to a type Th1 response. Activation of the antitumour immune response in double-treated mice resulted, in part, from stimulation of IFN-gamma production and was accompanied by the development of cytotoxic effectors in the spleen. As shown in a macrophage tumouricidal assay, macrophages could also play a role in the antitumour effects. The results confirmed that vaccination with IL-12 gene-modified tumour cells is superior to the treatment with unmodified tumour cell vaccine and, additionally, showed that IL-15 is an excellent candidate for adjuvant therapy, inducing synergistically not only a delay of tumour growth but also its complete eradication.

Fig. 1

In vivo growth of IL-12 gene-transduced B78-H1 melanoma cells (B78/IL-12). 5×10⁵ B78/IL-12 melanoma cells or non-transduced parental B78-H1 cells were injected into the footpad of the right hind limb of B6D2F1 mice

Fig. 2

Therapeutic effects of B78/IL-12 vaccine in combination with IL-15. Mice were injected into the footpad of the right hind limb with 2×10⁵ B78-H1 melanoma cells. On day 3, mice were treated intratumourally (i.t.) with 1×10⁶ irradiated live B78/IL-12 cells (or control B78/e.v. cells) followed by seven consecutive daily i.t. injections of IL-15 (2 μg per dose, from day 6 to 12). (A) Growth kinetics and (B) percentage of tumour-free mice vaccinated and/or treated with IL-15. Groups contained seven mice

Fig. 3

Systemic therapeutic effect of B78/IL-12 vaccine in combination with IL-15 against B78-H1 melanoma. Mice were injected into the footpad of the left hind limb with 1×10⁵ B78-H1 melanoma cells. On day 3, mice were injected into the contralateral footpad with a vaccine containing 1×10⁶ irradiated live B78/IL-12 cells and from days 6 to 12 with 2 μg IL-15. Horizontal bars indicate the median value for each group

Fig. 4

FACS analysis of lymph node cells from mice treated with B78/IL-12 cells in combination with IL-15. Mice inoculated with 2×10⁵ B78-H1 melanoma cells (day 0) were treated with 1×10⁶ live irradiated B78/IL-12 cells on day 3 and/or 2 μg IL-15, injected daily for 7 consecutive days (starting from day 6). On day 13 lymph nodes were isolated, lymphocytes were stained with anti-CD3, anti-CD4, anti-CD8, anti-NK or anti-CD19 mAbs and analyzed using FACSCalibur flow microfluorocytometer. Groups contained four to six mice

Fig. 5

Cytotoxic effect of splenocytes from mice treated with B78/IL-12 vaccine and/or IL-15 against B78-H1 cells. Details of treatment (see Materials and methods). Splenocytes were incubated for 3 days in the presence of irradiated stimulatory B78-H1 cells with or without IL-2 (10 units/ml). Cytotoxic effect was measured against ⁵¹Cr-labelled B78-H1 melanoma cells (E:T =50:1); 6 h incubation time. *P<0.001, in comparison with all other groups (IL-15 alone, vaccine alone and control)

Fig. 6

Cytotoxic effect of splenocytes from mice treated with B78/IL-12 vaccine and/or IL-15 during stimulation with irradiated B78-H1 melanoma cells. Photographs show 2-day cultures of irradiated B78-H1 cells admixed with splenocytes from mice treated with B78/IL-12 cells + IL-15 (A), B78/IL-12 cells alone (B), IL-15 alone (C) or diluent (D). Details of treatment (see Materials and methods). Note the strong cytotoxic effect against stimulatory cells in culture of splenocytes from double-treated mice—almost no live tumour cells are present in this culture, in comparison with other cultures (arrows). Large dark aggregations indicate accumulation of effector cells and apoptotic tumour cells

Fig. 7

IFN-γ production in cultures of splenocytes from mice treated with B78/IL-12 vaccine and/or IL-15. Details of treatment (see Materials and methods). Splenocytes were stimulated with either irradiated B78-H1 cells or IL-12 (1 ng/ml), and the amount of IFN-γ in supernatants from 1- or 3-day cultures was measured by ELISA

Fig. 8

Treatment with B78/IL-12 vaccine and IL-15 stimulates cells secreting IFN-γ. Mice were injected with B78-H1 melanoma cells and treated with B78/IL-12 vaccine and/or IL-15. Details of treatment (see Materials and methods). One day after the last injection of IL-15, lymphocytes from tumour-draining lymph nodes were isolated and restimulated with B78-H1 cells for 24 or 48 h. Frequencies of cells secreting IFN-γ (A) or IL-4 (B) are expressed as dot numbers per well. *P<0.05, in comparison with all other groups (IL-15 alone, vaccine alone and control)