Effective induction of antitumor immunity by immunization with plasmid DNA encoding TRP-2 plus neutralization of TGF-beta

Abstract

Plasmid DNA vaccine is an appealing cancer immunotherapy. However, it is a weak immunogen and immunization with plasmid DNA encoding self-antigens, such as melanoma-associated antigens, could not induce antitumor immunity because of tolerance. In this study, we investigated the feasibility of using a plasmid DNA encoding Xenopus laevis transforming growth factor-beta 5 (aTGF-beta5) as an immunogen to induce neutralizing antibodies against murine TGF-beta1 (mTGF-beta1) and thus enhance the efficacy of plasmid DNA vaccine encoding murine tyrosinase-related protein 2 (mTRP-2) through neutralization of TGF-beta. The results showed that immunization with aTGF-beta5 resulted in the generation of mTGF-beta1-neutralizing antibodies, and immunization with a combination of aTGF-beta5 and mTRP-2 induced specific cytotoxic T lymphocytes (CTLs). On the contrary, immunization with mTRP-2 alone could not elicit the CTL response. Moreover, immunization of C57BL/6 wild-type mice with a combination of aTGF-beta5 and mTRP-2 induced the protective and therapeutic antitumor immunity to B16F10 melanoma, whereas the antitumor activity was abrogated in both CD4-deficient mice and CD8-deficient mice on the C57BL/6 background. Our results indicate that immunization with aTGF-beta5 is capable of breaking immune tolerance and induces mTGF-beta1-neutralizing antibodies. Neutralization of TGF-beta can enhance the efficacy of DNA vaccine encoding mTRP-2 and the induction of antitumor immunity by this immunization strategy is associated with CD4+ and CD8+ T cells.

Fig. 1

Induction of TGF-β-specific antibodies. Mice were immunized by i.m. injection of pCI-aTGFβ5. Sera samples were obtained at day 7 after the last immunization and tested for the presence of TGF-β-specific antibodies by immunoblotting supernatants from CHO cells transfected with pCI-mTGFβ1 (lane 1) or pCI-aTGFβ5 (lane 2), which were separated by SDS-PAGE under nonreducing conditions and blotted onto PVDF membranes.

Fig. 2

Effect of the antibodies on the bioactivity of mTGF-β1. Supernatant from pCI-mTGFβ1-transfected CHO cells was collected, acidified and lyophilized as mTGF-β1. The growth inhibition of Mv 1 Lu cells mediated by mTGF-β1 was assessed by treatment with mTGF-β1 alone (a) or together with purified Ig derived from mice immunized with pCI-aTGFβ5 (b), pCI-mTGFβ1 (c), pCI-neo (d) or PBS (e) in the Mv 1 Lu bioassay system. *P<0.05 compared with the group treated with mTGF-β1 alone.

Fig. 3

Plasma levels of mTGF-β1 in untreated controls (a) and mice immunized with pCI-aTGFβ5 (b), pCI-mTGFβ1 (c), pCI-neo (d) or PBS (e). *P<0.0005 compared with controls.

Fig. 4

Induction of mTRP-2-specific CTLs. Mice were immunized with a combination of aTGF-β5 and mTRP-2. One or 2 weeks later, splenocytes were harvested and cultured for 5 days in the presence of 1 μg/ml mTRP-2_aa180-188 and 10 units/ml recombinant murine interleukin 2, and tested in 6-h ⁵¹Cr release assays. Curves represent the cytotoxicity against mTRP-2_aa180-188-pulsed EL-4 (filled circles), B16F10 (squares) and unpulsed EL-4 (open circles). Values are expressed as percentage (average of triplicates) of specific ⁵¹Cr release (percentage of specific lysis) at the indicated E/T ratios.

Fig. 5

Induction of the protective antitumor immunity. Mice were immunized with a combination of aTGF-β5 and mTRP-2 (filled circles), mTRP-2 alone (squares) or pVAX1 (triangles), or were injected with PBS as controls (open circles). One week after the last immunization, mice were challenged with 2.5×10³ B16F10 cells. Values are expressed as percentage of survival animals at the indicated time after tumor challenge.

Fig. 6

Roles of CD4⁺ and CD8⁺ T cells in the antitumor immunity. WT mice (filled circles), CD4-deficient mice (squares) and CD8-deficient mice ( triangles) were immunized with a combination of aTGF-β5 and mTRP-2, and additional WT mice were injected with PBS as controls (open circles). One week later, mice were challenged with 2.5×10³ B16F10 cells. Values are expressed as percentage of survival animals at the indicated time after tumor challenge.

Fig. 7

Induction of the therapeutic antitumor immunity. Mice were inoculated with 2.5×10³ B16F10 melanoma cells first and then treated with a combination of aTGF-β5 and mTRP-2 (filled circles), mTRP-2 alone (squares) or were injected with PBS as controls (open circles) at day 1 after tumor inoculation. Values are expressed as percentage of survival animals at the indicated time after tumor inoculation.