Synergistic enhancement of CD8+ T cell-mediated tumor vaccine efficacy by an anti-transforming growth factor-beta monoclonal antibody

Abstract

Purpose: Transforming growth factor-beta (TGF-beta) is an immunosuppressive cytokine, having direct suppressive activity against conventional CD4(+) and CD8(+)T cells and natural killer cells, thereby inhibiting tumor immunosurveillance. Here, we investigated possible synergy between anti-TGF-beta (1D11) and a peptide vaccine on induction of antitumor immunity, and the mechanisms accounting for synergistic efficacy.

Experimental design: The effect of combination treatment with a peptide vaccine and anti-TGF-beta was examined in a subcutaneous TC1 tumor model, as well as the mechanisms of protection induced by this treatment.

Results: Anti-TGF-beta significantly and synergistically improved vaccine efficacy as measured by reduction in primary tumor growth, although anti-TGF-beta alone had no impact. The number of tumor antigen-specific CTL with high functional avidity as measured by IFN-gamma production and lytic activity was significantly increased in vaccinated mice by TGF-beta neutralization. Although TGF-beta is known to play a critical role in CD4(+)Foxp3(+) Treg cells, Treg depletion/suppression by an anti-CD25 monoclonal antibody (PC61) before tumor challenge did not enhance vaccine efficacy, and adding anti-TGF-beta did not affect Treg numbers in lymph nodes or tumors or their function. Also, TGF-beta neutralization had no effect on interleukin-17-producing T cells, which are induced by TGF-beta and interleukin-6. Absence of type II NKT cells, which induce myeloid cells to produce TGF-beta, was not sufficient to eliminate all sources of suppressive TGF-beta. Finally, the synergistic protection induced by anti-TGF-beta vaccine augmentation was mediated by CD8(+) T cells since anti-CD8 treatment completely abrogated the effect.

Conclusions: These results suggest that TGF-beta blockade may be useful for enhancing cancer vaccine efficacy.

Fig 1

Anti-TGF-β (1D11) synergistically enhanced the efficacy of a CTL-inducing peptide tumor vaccine. TC1 cells (2× 10⁴) were injected s.c. in the right flank of C57BL/6 mice on day 0. Some mice were treated with 100μg of 1D11 every other day from the time of tumor challenge (filled diamonds) or day 4 after tumor challenge (filled triangles) until the end of the experiment. Some mice were given a vaccine consisting of HPV16 E7_49-57 peptide (100 μg) emulsified in IFA together with 50 nmol of HBVc_128-140 and 1 μg of mouse GM-CSF s.c. 4 days after tumor injection (filled circle). Some mice given the vaccine were also treated with 1D11 (100 μg) i.p. every other day from the time of immunization until 30 days after tumor challenge (filled squares). Each data point represents mean ± SD. p=0.032 by Mann-Whitney test between vaccine alone group and vaccine+ TGF-β group on day 30. The synergy between the vaccine and anti-TGF-β was confirmed on day 30 by least squares regression test (p=0.0054). All groups had 5 mice each except for the control group. which had 4 mice. These experiments were repeated at least twice with comparable results.

Fig 2

Anti-TGF-β increased tumor antigen specific CD8⁺ T cell responses induced by the CTL-inducing peptide vaccine. TC1 cells (2 × 10⁴) were injected s.c. in the right flank of C57BL/6 mice. Four days after tumor challenge, some mice were immunized with the peptide vaccine as described in Fig 1. Some vaccinated mice were also treated i.p. with anti-TGF-β (1D11) every other day from the time of immunization. A and B. Eighteen days after tumor injection (14 days after vaccination), all mice were euthanized and spleen cells were prepared. A. The spleen cells were stained with anti-CD3, anti- CD8 and E7_49-57-loaded H-2D^b tetramer and analyzed by flow cytometry. The proportions of tetramer⁺ cells were determined among CD3⁺CD8⁺ cells. B. The spleen cells were stimulated with spleen cells of naïve C57BL/6 mice pulsed with either 0.1 μM or 0.1 nM of the E7_49-57 peptide. After overnight culture, the cells were stained with anti-CD3, anti-CD8 and anti-IFN-γ by the intracellular cytokine staining method described in the Materials and Methods. The proportion of IFN-γ⁺ cells was determined among CD3⁺CD8⁺ cells. C. Eighteen days after tumor injection (14 days after vaccination), all mice were injected i.v. with a mixture of naïve C57BL/6 spleen cells labeled with different concentrations of CFSE and pulsed with different concentrations of E7_49-57 peptides as described in Materials and Methods. Sixteen hours after the spleen cell injection, spleen cells were prepared, and residual CFSE⁺ cells were analyzed by flow cytometry. Each symbol represents one data point. Medians are shown as bars. p-values by Student’s t-test (A and right panel of B, where the data distribution is consistent with Gaussian) or Mann-Whitney (left panel of B and C) test are indicated. These experiments were repeated at least twice with comparable results.

Fig 3

The effect of anti-TGF-β on vaccine efficacy is not due to blocking Treg cells. TC1 cells (2 × 10⁴) were injected s.c. in the right flank of C57BL/6 mice. Four days after tumor challenge, some mice were immunized with the peptide vaccine as described in Fig 1. Some immunized mice were also treated i.p with 1D11 (100 μg) every other day from the time of immunization for two weeks. A. Four days or 13 days after tumor injection, tumor draining lymph node cells were recovered and stained with anti-CD3, anti-CD4, anti-CD25 and anti-Foxp3. The proportions of CD3⁺CD4⁺CD25⁺Foxp3⁺ cells were determined by flow cytometry. Each symbol represents one data point. Median are shown as bars. B. Sixteen days after tumor challenge, tumor infiltrating Tregs were examined by flow cytometry. Tumor infiltrating lymphocytes were recovered as described in the Materials and Methods section, stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3. Presented density plots were gated on the CD3⁺CD4⁺ population (upper row). Presented pseudo dot plots represented entire population (lower row). C. Some mice were treated with anti-CD25 (1 mg) five and three days before tumor injection. The experiment was terminated on day40 after tumor challenge. Each data point represents mean ± SD. All groups had 5 mice each. D. Eleven days after tumor challenge, tumor draining lymph node cells were used to examine Treg suppressive activity. Fifty thousand CD4⁺CD25⁻ cells with 5 × 10⁴ accessory cells with/without 1.25 × 10⁴ CD4⁺CD25⁺ Treg cells were stimulated with 0.5 μg/ml anti-CD3 for 72 hr. Cell proliferation was measured by ³H-thymidine incorporation, The % suppression was determined as described in the Materials and Methods section. These experiments were repeated at least twice with comparable results.

Fig. 4

Anti-TGF-β with the peptide vaccine does not affect IL-17 production by T cells. TC1 cells (2 × 10⁴) were injected s.c. in the right flank of C57BL/6 mice. Four days after tumor challenge, some mice were immunized with the peptide vaccine as described in Fig 1. Some immunized mice were also treated i.p. with 1D11 (100 μg) every other day from the time of immunization throughout the experiment. Eighteen days after tumor injection (14 days after vaccination), all mice were euthanized and tumor draining lymph node cells were harvested and stained for intracellular IL-17 together with surface CD3, CD4, CD8 staining. Median are shown as bars. These experiments were repeated at least twice with comparable results.

Fig. 5

The effect of anti-TGF-β on vaccine efficacy is not due solely to blocking TGF-β resulting from NKT cell-mediated immunosuppression. TC1 cells (2 × 10⁴) were injected s.c. in the right flank of C57BL/6 or CD1d KO mice. Four days after tumor challenge, some mice were immunized with the peptide vaccine as described in Fig 1. Some immunized mice were also treated i.p. with 1D11 (100 μg) every other day from the time of immunization for two weeks. A. Tumor size was followed for 28 days. Each data point was shown as mean ± SD. All groups had 5 mice each. p<0.03 by Mann-Whitney test between vaccine group and vaccine + anti-TGF-β group on day 26. B. Tumor infiltrating CD11b⁺Gr-1⁺ cells were examined in tumors from wild-type mice 16 days after tumor challenge. Tumor infiltrating lymphocytes prepared as in Fig 3B were stained with antibodies against CD11b and Gr-1. These experiments were repeated at least twice with comparable results.

Fig. 6

The protection induced by the combination of anti-TGF-β and the vaccine is mediated by CD8⁺ T cells. TC1 cells (2 × 10⁴) were injected s.c. in the right flank of C57BL/6 mice. Four days after tumor challenge, some mice were immunized with the peptide vaccine as described in Fig 1. Some immunized mice were also treated i.p. with 1D11 (100 μg) every other day from the time of immunization for two weeks. Some mice were also treated with anti-CD8 after immunization as indicated. p<0.03 by Mann-Whitney test between vaccine+cont mAb group and vaccine+anti-CD8 group. All groups had 5 mice each. p<0.03 by Mann-Whitney test between vaccine+anti-TGF-β+cont mAb group and vaccine+ TGF-β+anti-CD8 group on day 29. Each data point was shown as mean ± SD. These experiments were repeated at least twice with comparable results.