Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation

Abstract

We examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade, alone or in combination with a granulocyte/macrophage colony-stimulating factor (GM-CSF)-expressing tumor cell vaccine, on rejection of the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6. Recently established tumors could be eradicated in 80% (68/85) of the cases using combination treatment, whereas each treatment by itself showed little or no effect. Tumor rejection was dependent on CD8(+) and NK1.1(+) cells but occurred irrespective of the presence of CD4(+) T cells. Mice surviving a primary challenge rejected a secondary challenge with B16-BL6 or the parental B16-F0 line. The same treatment regimen was found to be therapeutically effective against outgrowth of preestablished B16-F10 lung metastases, inducing long-term survival. Of all mice surviving B16-BL6 or B16-F10 tumors after combination treatment, 56% (38/68) developed depigmentation, starting at the site of vaccination or challenge and in most cases progressing to distant locations. Depigmentation was found to occur in CD4-depleted mice, strongly suggesting that the effect was mediated by CTLs. This study shows that CTLA-4 blockade provides a powerful tool to enhance T cell activation and memory against a poorly immunogenic spontaneous murine tumor and that this may involve recruitment of autoreactive T cells.

Figure 1

Successful treatment of preestablished B16-BL6 using anti–CTLA-4 and GM-CSF–producing BL6 vaccine. C57BL/6 female mice (five per group) were injected with 10⁴ B16-BL6 cells subcutaneously on the back, on the same day (A) or 4, 8, or 12 d (B–D) before treatment was started. Treatment consisted of three consecutive injections (in a 6-d time frame as indicated in Materials and Methods) of anti–CTLA-4 antibody 9H10 intraperitoneally (•), control hamster IgG (100, 50, 50 μg; ○), or 10⁶ irradiated BL6/g cells subcutaneously, in combination with 9H10 (▪) or hamster IgG (□). Tumor growth (mm²) was scored by measuring perpendicular diameters and was averaged for all mice within each group. In some treatment groups, only a fraction of the mice (indicated between brackets) developed a tumor.

Figure 2

A single dose of GM-CSF–producing vaccine cooperates with CTLA-4 blockade to induce 100% cure of B16-BL6. Mice were inoculated subcutaneously with 10⁴ B16-BL6 cells. On the same day, combination treatment was initiated using triple BL6/g vaccine (days 0, 3, and 6) combined with either hamster IgG (100, 50, 50 μg on days 3, 6, and 9; □) or anti–CTLA-4 (▪). Control treatments consisted of antibody injections alone: hamster IgG (○) or anti–CTLA-4 (•). Also, anti–CTLA-4 treatment was combined with a single (▴) or double injection (♦) of the BL6/g vaccine. Average tumor size was calculated for all mice within a treatment group (mm²). The fraction of mice developing tumors is shown between brackets.

Figure 3

Anti–CTLA-4 enhances IFN-γ production by B16-specific T cells induced in vivo. Mice (four per group) were vaccinated with irradiated BL6/g (10⁶ per mouse) and cotreated with control hamster IgG (A) or anti–CTLA-4 (B). After 4 wk, mice were challenged with 2 × 10⁴ B16-BL6, and 10 d later, splenocytes were pooled and restimulated in vitro using B16-BL6/B7.1 (open bars) or a mixture of B16-F10 and DC2.4 dendritic cells (filled bars). On day 8, cultures were tested for tumor-specific IFN-γ release as described in Materials and Methods. Targets included B16 sublines -F0, -F10, and -BL6, as well as unrelated H-2^b tumors EL4 and MC38.

Figure 3

Anti–CTLA-4 enhances IFN-γ production by B16-specific T cells induced in vivo. Mice (four per group) were vaccinated with irradiated BL6/g (10⁶ per mouse) and cotreated with control hamster IgG (A) or anti–CTLA-4 (B). After 4 wk, mice were challenged with 2 × 10⁴ B16-BL6, and 10 d later, splenocytes were pooled and restimulated in vitro using B16-BL6/B7.1 (open bars) or a mixture of B16-F10 and DC2.4 dendritic cells (filled bars). On day 8, cultures were tested for tumor-specific IFN-γ release as described in Materials and Methods. Targets included B16 sublines -F0, -F10, and -BL6, as well as unrelated H-2^b tumors EL4 and MC38.

Figure 4

Mice bearing B16-F10 lung metastases show enhanced survival when treated with anti–CTLA-4 and F10/g vaccine. B16-F10 cells (5 × 10⁴ per mouse) were injected into the tail vein and 24 h later, treatment was started using control hamster IgG (10 mice, ○), anti–CTLA-4 antibody 9H10 (9 mice; •), irradiated F10/g (10⁶ subcutaneously) in combination with hamster IgG (10 mice; □) or 9H10 (13 mice; ▪) on days 1, 4, and 7, according to the dosing schedule used for subcutaneous tumors (see Fig. 1 legend). Mice were followed for survival, and in some subjects death due to extensive pulmonary metastasis was confirmed by harvesting lungs postmortem.

Figure 5

B16-F10 metastases demonstrate lymphocytic infiltration after treatment with anti–CTLA-4 and F10/g vaccine. Mice injected with 10⁵ B16-F10 intravenously and treated with control hamster IgG (A), 9H10 (B), or F10/g vaccine in combination with either hamster IgG (C) or 9H10 (D) on days 1, 4, and 7, as outlined in the Fig. 4 legend. On day 25, lungs were harvested, fixed in 10% neutral-buffered formalin, and processed for hematoxylin–eosin staining.

Figure 6

Rejection of B16-BL6 or B16-F10 as a result of treatment with anti–CTLA-4 and GM-CSF–producing vaccines causes autoimmune skin and hair depigmentation. After successful treatment for B16-BL6 subcutaneously or B16-F10 intravenously, C57Bl/6 mice developed skin and hair depigmentation. (A) Depigmentation of both sites of vaccination and challenge, after rejection of a day 0 tumor. (B) Progressive depigmentation found in a mouse rejecting a B16-BL6 subcutaneous tumor, established 8 d before treatment started. (C) Depigmentation at the site of vaccination of a mouse cured from preestablished B16-F10 lung metastases.

Figure 6

Rejection of B16-BL6 or B16-F10 as a result of treatment with anti–CTLA-4 and GM-CSF–producing vaccines causes autoimmune skin and hair depigmentation. After successful treatment for B16-BL6 subcutaneously or B16-F10 intravenously, C57Bl/6 mice developed skin and hair depigmentation. (A) Depigmentation of both sites of vaccination and challenge, after rejection of a day 0 tumor. (B) Progressive depigmentation found in a mouse rejecting a B16-BL6 subcutaneous tumor, established 8 d before treatment started. (C) Depigmentation at the site of vaccination of a mouse cured from preestablished B16-F10 lung metastases.

Figure 6

Rejection of B16-BL6 or B16-F10 as a result of treatment with anti–CTLA-4 and GM-CSF–producing vaccines causes autoimmune skin and hair depigmentation. After successful treatment for B16-BL6 subcutaneously or B16-F10 intravenously, C57Bl/6 mice developed skin and hair depigmentation. (A) Depigmentation of both sites of vaccination and challenge, after rejection of a day 0 tumor. (B) Progressive depigmentation found in a mouse rejecting a B16-BL6 subcutaneous tumor, established 8 d before treatment started. (C) Depigmentation at the site of vaccination of a mouse cured from preestablished B16-F10 lung metastases.