TLR5 signaling causes dendritic cell dysfunction and orchestrates failure of immune checkpoint therapy against ovarian cancer
Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system. Patients bearing ovarian tumors infiltrated with high frequencies of T cells are associated with a greater survival probability. However, therapies to revitalize tumor-associated T cells, such as PD-L1/PD-1 or CTLA4 blockade , are ineffective for the treatment of ovarian cancer. We demonstrate that for ovarian cancer, Toll-Like Receptor 5 (TLR5) signaling, the only known ligand for which is bacterial flagellin, governs failure of PD-L1 and CTLA4 blockade. Mechanistically, chronic TLR5 signaling on CD11c+ cells in vivo and in vitro impairs the differentiation of functional IL-12-producing XCR1+, CD103+ cDC1 subsets, biasing CD11c+ precursor cells towards myeloid subsets expressing high levels of PD-L1. This culminates in impaired activation of CD8 T cells, reducing CD8 T cell function and ability to persist within the ovarian tumor microenvironment. Expansion of cDC1s in situ using FLT3L in combination with PD-L1 blockade achieved significant survival benefit, but only in TLR5 KO mice, whereas no benefit was observed in the presence of TLR5 signaling. Thus, we identify a host-intrinsic mechanism leading to the failure of PD-L1 blockade for ovarian cancer, demonstrating that chronic TLR5 signaling on CD11c+ cells is a barrier limiting the efficacy of checkpoint therapy. Overall design: Bone marrow cells were collected from femurs and tibias a wild-type mouse and cultured with 400ng/ml of FLT3L (BioXCell, cat# BE0342) in 3ml of RPMIc in a 6-well plate (Thermo Scientific™ BioLite™ Microwell Plates, cat #12-556-004) at 3e6 cells per well on day one 10ng/ml of ultra-purified Flagellin derived from S. typhimurium (InvivoGen, cat# tlrl-epstfla-5) and incubated at 37°C in a humidified incubator containing 5% CO2. Additional RPMIc + FLT3L with or without flagellin was added at 3ml on day four. Samples were collected and stained with TotalSeq™-B Mouse Myeloid Cocktail, V1.0 (Biolegend, cat# 199904) separately after 8 days of culture. RNA library preparation and sequencing was performed by the UVA Genome Analysis and Technology Core using a 10X Chromium X.