Blockade of TGF-beta enhances tumor vaccine efficacy mediated by CD8(+) T cells

Abstract

Though TGF-beta inhibition enhances antitumor immunity mediated by CD8(+) T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF-beta blockade, we tested the effect of anti-TGF-beta combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti-TGF-beta antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti-TGF-beta antibodies compared to vaccinated mice without anti-TGF-beta, suggesting that anti-TGF-beta synergistically enhanced irradiated tumor vaccine efficacy. CD8(+) T-cell depletion completely abrogated the vaccine efficacy, and so protection required CD8(+) T cells. Depletion of CD25(+) T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti-TGF-beta did not change the number of CD25(+) T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d-restricted NKT cells, which have been reported to induce TGF-beta production by MDSC through an IL-13-IL-4R-STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti-TGF-beta enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF-beta levels found in patients with cancer and that the effect is not dependent on TGF-beta solely from CD4(+)CD25(+) T regulatory cells or the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway.

Fig.1. Blockade of TGF-β synergistically enhances whole cell vaccine efficacy in mice and the protection is mediated by CD8⁺ T cells

1×10⁵ irradiated (25,000 rad) CT26 cells were injected subcutaneously (sc.) into BALB/c mice. Some vaccinated or unvaccinated mice were treated with anti-TGF-β monoclonal antibody (1D11.16) or control antibody (13C4) intraperitoneally (ip) first with 200 μg at the time of vaccination and of the CT26 challenge, and then with 100 μg three times a week from the time of vaccination to 2 weeks after CT26 challenge. The control group was untreated. Three weeks after vaccination, 1×10⁶ live CT26 cells were injected sc. into these mice. Two and 1 day before, and 4, 7, 10, and 14 days after CT26 challenge, some vaccinated mice treated with 1D11 were also treated with 0.5 mg of anti-CD8 monoclonal antibody (2.43). Tumors were measured by a caliper gauge, and tumor size was determined as the product of tumor length (mm) × tumor width (mm). Five female BALB/c mice were used for each group per each experiment. Mice were euthanized when the tumor area exceeded 1 cm², and data were represented as percent survival. (A) The results were pooled from six different experiments comparing vaccine with anti-TGF-β vs control antibody (n=30, each group). p<0.0001 by log-rank test between control and vaccine plus control antibody group. p<0.002 by log-rank test between vaccine plus control antibody group and vaccine plus anti-TGF-β monoclonal antibody group. (B) The results were pooled from two different experiments in which an anti-CD8-treated group was included. p<0.002 by log-rank test between control and vaccine plus control antibody group. p<0.005 by log-rank test between vaccine plus control antibody group and vaccine plus anti-TGF-β monoclonal antibody group.

Fig.2. Depletion of CD4⁺CD25⁺ T regulatory cells leads to tumor rejection in mice regardless of vaccination

3 and 1 day prior to vaccination, 1 mg anti-CD25 mAb (PC-61) or rat IgG mAb was injected ip. into BALB/c mice, and then 1×10⁵ irradiated (25,000 rad) CT26 cells as a vaccine were injected sc. into these mice. 3 weeks after vaccination, 1×10⁶ live CT26 cells were injected sc. into these mice. Tumors were measured by a caliper gauge, and tumor size was determined as the product of tumor length (mm) × tumor width (mm). Five female BALB/c mice were used for each group per each experiment. Mice were euthanized when the tumor area exceeded 1 cm², and the data were represented as percent survival. The results were pooled from two independent experiments. p<0.01 by log-rank test between control and vaccine alone group. p<0.0001 by log-rank test between control group and PC-61-treated groups with or without vaccination.

Fig.3. Enhancement of tumor vaccine efficacy by anti-TGF-β antibody is independent of CD4⁺CD25⁺ T regulatory cells

1×10⁵ irradiated (25,000 rad) CT26 cells were injected sc. into BALB/c mice. Some vaccinated or unvaccinated mice were treated with 200 μg (at the time of vaccination and CT26 challenge) or 100 μg (other time points) 1D11 or 13C4 i.p. three times a week. 3 weeks after vaccination, spleens were removed from these mice. These splenocytes were stained for CD4 and CD25 followed by anti-Foxp3 intracellular staining and analyzed by flow cytometry. 4∼5 female mice were used for each group. A. % of CD4⁺ CD25⁺ Foxp3⁺ cells per spleen. B. 10 days after tumor injection, tumor draining lymph node cells were recovered and stained with anti-CD3, anti-CD4, anti-CD25, and anti-Foxp3 in 1D11- and 13C4-treated mice without the tumor vaccine. The proportions of CD3⁺CD4⁺CD25⁺Foxp3⁺ T cells were determined by flow cytometry. 10 female mice were used for each group. Each symbol represents one data point. Median is shown as bars. C. 10 days after tumor challenge, tumor infiltrating Treg cells were examined by flow cytometry. Tumor infiltrating lymphocytes were recovered from 5 pooled tumors as described in the materials and methods section, and stained with anti-CD3, anti-CD4, anti-CD25, and anti-Foxp3 in 1D11- and 13C4-treated mice without the tumor vaccine. Presented density plots were gated on the CD3⁺CD4⁺ population. These experiments were repeated at least twice with comparable results.

Fig. 4. Abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway does not enhance vaccine efficacy

1×10⁵ irradiated (25,000 rad) CT26 cells were injected sc. into wt. BALB/c, BALB-CD1KO (A), BALB- IL-13 KO (B), BALB-IL-4-13KO (C), BALB-STAT-6 KO (D), BALB-IL-4R KO (E), or BALB-IL-4 KO (F) mice. 3 weeks after vaccination, 1×10⁶ live CT26 cells were injected sc. into these mice. Tumors were measured by a caliper gauge, and tumor size was determined as the product of tumor length (mm) × tumor width (mm). Five female BALB/c mice were used for each group per each experiment. Mice were euthanized when the tumor area exceeded 1 cm², and data were represented as percent survival. The results were pooled from two independent experiments. p<0.001 in A and B, p<0.002 in C, p<0.0005 in D, E, and F by log-rank test between un-vaccinated BALB/c mice and vaccinated BALB/c mice. p<0.01 in A by log-rank test between un-vaccinated BALB/c mice and CD1KO mice. p<0.02 in E by log-rank test between un-vaccinated IL-4R KO mice and vaccinated IL-4R KO mice.