Cytosolic bacterial pathogens activate TLR pathways in tumors that synergistically enhance STING agonist cancer therapies.

Bacterial pathogens that invade the eukaryotic cytosol are distinctive tools for fighting cancer, as they preferentially target tumors and can deliver cancer antigens to MHC-I. Cytosolic bacterial pathogens have undergone extensive preclinical development and human clinical trials, yet the molecular mechanisms by which they are detected by innate immunity in tumors is unclear. We report that intratumoral delivery of phylogenetically distinct cytosolic pathogens, including Listeria, Rickettsia, and Burkholderia species, elicited anti-tumor responses in established, poorly immunogenic melanoma and lymphoma in mice. We were surprised to observe that although the bacteria required entry to the cytosol, the anti-tumor responses were largely independent of the cytosolic sensors cGAS/STING and instead required TLR signaling. Combining pathogens with TLR agonists did not enhance anti-tumor efficacy, while combinations with STING agonists elicited profound, synergistic anti-tumor effects with complete responses in >80% of mice after a single dose. Small molecule TLR agonists also synergistically enhanced the anti-tumor activity of STING agonists. The anti-tumor effects were diminished in Rag2-deficient mice and upon CD8 T cell depletion. Mice cured from combination therapy developed immunity to cancer rechallenge that was superior to STING agonist monotherapy. Together, these data provide a framework for enhancing the efficacy of microbial cancer therapies and small molecule innate immune agonists, via the co-activation of STING and TLRs.

Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1 blockade.

Checkpoint blockade immunotherapy targeting the PD-1/PD-L1 inhibitory axis has produced remarkable results in the treatment of several types of cancer. Whereas cytotoxic T cells are known to provide important antitumor effects during checkpoint blockade, certain cancers with low MHC expression are responsive to therapy, suggesting that other immune cell types may also play a role. Here, we employed several mouse models of cancer to investigate the effect of PD-1/PD-L1 blockade on NK cells, a population of cytotoxic innate lymphocytes that also mediate antitumor immunity. We discovered that PD-1 and PD-L1 blockade elicited a strong NK cell response that was indispensable for the full therapeutic effect of immunotherapy. PD-1 was expressed on NK cells within transplantable, spontaneous, and genetically induced mouse tumor models, and PD-L1 expression in cancer cells resulted in reduced NK cell responses and generation of more aggressive tumors in vivo. PD-1 expression was more abundant on NK cells with an activated and more responsive phenotype and did not mark NK cells with an exhausted phenotype. These results demonstrate the importance of the PD-1/PD-L1 axis in inhibiting NK cell responses in vivo and reveal that NK cells, in addition to T cells, mediate the effect of PD-1/PD-L1 blockade immunotherapy.