Abstract 4986: Regulation of radiation-induced in situ tumor vaccination by TGFβ superfamily members
Authors: Vanpouille-Box Claire I. , Formenti Silvia C. , Demaria Sandra 
Published: July 2016

Abstract
Transforming Growth Factor-beta (TGFβ) and activin A (actA) are members of the TGFβ superfamily with overlapping as well as distinct functions in many processes including regulation of inflammation and immunity. Similar to TGFβ, actA has been shown to promote the conversion of CD4+CD25- T cells into induced regulatory T cells (iTregs), and to potentiate iTregs generation by TGFβ.
Many cancer cells produce actA, and we have recently found that levels of actA released in vitro by breast cancer cells are enhanced by radiotherapy (RT). Interestingly, prolonged exposure to TGFβ inhibitors also resulted in enhanced actA release, consistent with a compensatory mechanism described in development (Carvalho et al., J Cell Sci, 2007). Here we tested the hypothesis that actA plays a role in regulating anti-tumor immunity induced by local tumor RT and TGFβ blockade in vivo (Vanpouille-Box et al., Cancer Res 2015).
4T1 derivatives with conditional actA knockdown (4T1shActA) or non-silencing control (4T1shNS) were engineered using inducible doxycycline (DOX) plasmids and injected s.c. in BALB/c mice (day 0). ActA gene knockdown (KD) was induced by DOX at day 8. TGFβ neutralizing 1D11 or isotype control antibodies were given i.p. every other day starting on day 12. RT was delivered to the primary tumor in 6Gy fractions on five consecutive days starting at day 13. Mice were followed for tumor growth and survival or euthanized at day 22 or 28 for analysis.
Consistent with in vitro data, ActA gene expression was upregulated at day 28 in 4T1 tumors of mice after prolonged TGFβ blockade by 1D11. Neither 1D11 nor ActA gene KD by themselves inhibited tumor growth. However, each intervention significantly improved tumor control achieved by RT. ActA KD in mice treated with RT+1D11 prevented tumor recurrence and improved survival (RT+1D11 vs RT+1D11+shActA p = 0.026; RT+shActA vs RT+1D11+shActA p = 0.0008). Interestingly, blockade of TGFβ or actA resulted in increased intratumoral Tregs (Control: 11.6%; 1D11: 26.2%, shActA: 21%) and markedly enhanced the increase in Tregs seen with RT alone (RT: 15.7%; RT+1D11: 27.5%; RT+shActA: 30.3%). In marked contrast, when both TGFβ and actA were inhibited Tregs significantly decreased in both non-irradiated (1D11+shActA: 13.6%) and irradiated tumors (RT+1D11+shActA: 7.9% of Tregs). IFNγ production by CD8+ T cells in response to the tumor-specific AH1 peptide was significantly higher in RT+1D11+shActA treated mice compared to RT+1D11 (p = 0.045) and RT+shActA (p = 0.0147). Additionally, in RT+1D11+shActA mice expression of the activation marker CD69 was markedly increased in intra-tumoral CD8+ PD-1+ T cells (MFI Control: 187; 1D11: 139.5; shActA: 162; 1D11+shActA: 111; RT: 138.5; RT+1D11: 379; RT+shActA: 182; RT+1D11+shActA: 498.5).
Overall, data indicate that TGFβ and actA regulate Tregs numbers in tumors and show a complex interaction with RT. Combined blockade of TGFβ and actA during RT may be required to improve in situ vaccination by RT.
Citation Format: Claire I. Vanpouille-Box, Silvia C. Formenti, Sandra Demaria. Regulation of radiation-induced in situ tumor vaccination by TGFβ superfamily members. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4986.