Abstract A187: RIG-I agonists reinforce antitumor adaptive immunity and decrease Treg activity in breast cancer

Abstract

Abstract RIG-I like receptors, RNA helicases that sense viral oligonucleotide motifs and activate innate immunity, are gaining interest in cancer therapy, given their ability to redirect immune responses within the tumor microenvironment (TME), and increase efficacy of experimental cancer vaccines. RIG-I agonists are not well studied in breast cancers, a type of cancer that is often considered immunologically “silent.” We recently reported that therapeutic delivery of RIG-I agonists increase tumor-infiltrating leukocytes (TILs) and expression of proinflammatory Th1 cytokines in the 4T1 mouse model of aggressive, metastatic breast cancer through tumor cell-intrinsic mechanisms. However, these studies do not rule out the importance of myeloid immune responders (e.g., macrophages and dendritic cells) in propagating the effects of RIG-I agonists against tumor cells in vivo, nor do they rule out the impact of RIG-I agonists on adaptive antitumor immunity, a subject that is relatively understudied. We assessed the effects of the RIG-I agonist SLR20 on the the activity of effector T-lymphocytes (TEff) and regulatory T-lymphocytes (TReg) in the TME. Interestingly, SLR20 treatment of mouse and human breast tumor cells increased expression of FAS and MHC-I on tumor cells, and caused tumor cells to express T-cell chemoattractants (e.g., CXCL10, RANTES), potentially increasing T-cells recruitment to tumors, and increasing tumor cell susceptibility to TEff recognition and killing. Using an ex vivo co-culture assay in which 4T1 mouse mammary tumor cells were co-cultured with CD8+ T-cells harvested from mice pre-inoculated with SLR20-treated 4T1 tumor cells, we measured the rate of CD8+-mediated tumor cell killing. This approach revealed that T-cells harvested from mice inoculated with SLR20-treated cells caused greater tumor cell killing than what was seen by CD8+ T-cells harvested from untreated mice. We also found that conditioned media harvested from 4T1 cells treated with SLR20 increased clonal expansion of CD3/CD28-activated T-cells above what was seen with conditioned media harvested from 4T1 cells treated with a control oligonucleotide or from untreated 4T1 cells. TGFβ-mediated differentiation of CD4+ T-cells into tolerogenic and immunosuppressive TRegs was measured in cultures of CD4+ T-cells treated with cultured media derived from SLR20-treated 4T1 cells. These studies showed that cultured media harvested from 4T1 cells treated with SLR20, but not from untreated 4T1 cells or 4T1 cells treated with a control ligand, diminished TReg differentiation, and decreased CD4+ T-cells surface expression of PD-1, CTLA4, and CCR8. Importantly, in vivo experiments assessing therapeutic treatment of 4T1 tumors with SLR20 revealed greater tumor growth inhibition when SLR20 was combined with PD-L1 targeting antibodies. Taken together, these findings indicate that therapeutic activation of RIG-I signaling operates at the interface of innate and adaptive immunity within breast tumors to redirect the TME from an immunosuppressed state to one that is immunogenic and receptive to clinically relevant checkpoint inhibitors. Citation Format: David L. Elion, Max E. Jacobson, Donna J. Hicks, Bushra Rahman, Violeta Sanchez, Paula I Gonzales-Ericsson, Olga Fedorova, Anna M. Pyle, John T. Wilson, Rebecca S. Cook. RIG-I agonists reinforce antitumor adaptive immunity and decrease Treg activity in breast cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A187.