Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine comprised of three isoforms, TGF-beta1, 2, and 3, that exhibits immunosuppressive functions. In the context of immunosuppression, TGF-beta dampens activation and maturation of both innate and adaptive immune cells, including natural killer cells, macrophages, and dendritic cells, as well as CD4+ and CD8+ T cells. Thus, TGF-beta inhibition, like blockade of other negative regulators of immunity, provides a potential modality of cancer treatment that can be used in combination therapy. Although we have previously demonstrated that blockade of all three isoforms of TGF-beta using a monoclonal antibody increases tumor vaccine efficacy, as measured by the reduction in tumor growth and increase in the number of antigen-specific CTLs, the function of individual isoforms of TGF-beta in the framework of tumor immunity is not well understood. In this study, we examined whether it is necessary to block TGF-beta3, in addition to the two other isoforms of TGF-beta, to improve vaccine-induced tumor immunity against the syngeneic TC1 tumor model, a C57BL/6 lung epithelial cell line transfected with HPV-16 E6 and E7 genes. When the subcutaneous tumors became approximately 5 mm in diameter, the mice were immunized with a peptide-based vaccine that targets the E7 oncogene with or without the simultaneous administration of anti-TGF-beta antibodies with unique specificities for the three isoforms. Mice immunized with the vaccine alone exhibited slightly delayed tumor growth relative to the untreated control. This effect could be enhanced by the combination of anti-TGF-beta antibodies that neutralize all three isoforms of TGF-beta. Interestingly, blockade of only TGF-beta1 & 2 also improved vaccine efficacy to a similar degree as the blockade of all three isoforms, though neutralization of TGF-beta alone could not suppress tumor growth. Therefore, it is not necessary to inhibit TGF-beta3 to generate significant vaccine-induced anti-tumor immunity. Systemic depletion of CD8+ T cells using anti-CD8-depleting antibodies completely abrogated the tumor protection observed in mice immunized with vaccine alone or vaccine in combination with TGF-beta blockade, suggesting that the mechanism of anti-tumor immunity in these conditions relies entirely on CD8+ T cells. In vivo CTL assays revealed that anti-TGF-beta antibodies tended to increase the quality of CTL activity induced by the vaccine. To better understand the effect of treatment on the tumor microenvironment, we used flow cytometric analysis to examine T cells in the tumor and tumor draining lymph nodes. While the vaccine significantly increased the number of tumor antigen-specific CD8+ T cells and IFN-gamma producing T cells in both lymph nodes and tumors, TGF-beta inhibition at two weeks, but not one week, after vaccination further augmented the increase in the number of T cells infiltrating tumors. Vaccine in combination with blockade of TGF-beta1 & 2, regardless of TGF-beta3 inhibition, also led to the greatest increase in the number of T-bet-expressing CD4+ and CD8+ T cells in tumors. This observation implies that blockade of TGF-beta1 & 2 alone may be sufficient to improve therapeutic tumor vaccines by facilitating the infiltration of more Th1/Tc1-type T cells into tumors. Because several studies suggest that TGF-beta3 actually plays a protective role against tumorigenesis in a range of tissues, developing TGF-beta blockade strategies that do not inhibit this isoform could be beneficial in certain settings.

Citation Format: Katharine Clark, Faith C. Robertson, Emma De Ravin, Shingo Kato, Anja Bloom, Amer Mirza, Jay A. Berzofsky, Masaki Terabe. Inhibition of TGF-beta isoforms 1 and 2 enhances therapeutic tumor vaccine efficacy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A054.