Tumor-associated myeloid cells, including dendritic cells (DCs) and macrophages, are immune suppressive. This study demonstrates a novel mechanism involving FOXO3 and NF-κB RelA that controls myeloid cell signaling and impacts their immune-suppressive nature. We find that FOXO3 binds NF-κB RelA in the cytosol, impacting both proteins by preventing FOXO3 degradation and preventing NF-κB RelA nuclear translocation. The location of protein–protein interaction was determined to be near the FOXO3 transactivation domain. In turn, NF-κB RelA activation was restored upon deletion of the same sequence in FOXO3 containing the DNA binding domain. We have identified for the first time, to our knowledge, a direct protein–protein interaction between FOXO3 and NF-κB RelA in tumor-associated DCs. These detailed biochemical interactions provide the foundation for future studies to use the FOXO3–NF-κB RelA interaction as a target to enhance tumor-associated DC function to support or enhance antitumor immunity.

Background The development of memory responses is an evolutionary function of the adaptive immune system. We propose that for the immune system to populate the memory compartment with the best-suited CD8 T cells it utilizes a process of certification or molecular accreditation mediated through Natural Killer Group 2D (NKG2D). This process of certification assures that the memory compartment is filled with CD8 T cells that have demonstrated their ability to kill their cognate targets through a two-step process that utilizes T cell receptor (TCR) and NKG2D signaling. Methods One week after immunization with peptide-pulsed dendritic cells, NKG2D signaling was transiently blocked in vivo with a single injection of neutralizing antibodies. Under such conditions, we determined the importance of NKG2D signaling during the effector phase for memory formation without compromising NKG2D signaling at the memory phase. Both open (polyclonal) and closed (monoclonal) CD8 T cell repertoires were studied. Results We show that signaling through NKG2D mediated this certification. Temporary blockade of NKG2D signaling during the effector phase resulted in the formation of highly defective memory CD8 T cells characterized by altered expression of the ribosomal protein S6 and epigenetic modifiers, suggesting modifications in the T cell translational machinery and epigenetic programming. Finally, these uncertified memory cells were not protective against a B16 tumor challenge. Conclusion Signaling through NKG2D during the effector phase (certification) favors the development of functional memory CD8 T cells, a previously undescribed role for NKG2D. Temporary blockade of NKG2D signaling during the effector phase results in the formation of highly defective memory CD8 T cells potentially by affecting the expression of the ribosomal protein S6 and epigenetic modifiers, suggesting alterations in T cell translational machinery and epigenetic programming.

Cancers exploit coinhibitory receptors on T cells to escape tumor immunity, and targeting such mechanisms has shown remarkable clinical benefit, but in a limited subset of patients. We hypothesized that cancer cells mimic noncanonical mechanisms of early development such as axon guidance pathways to evade T cell immunity. Using gain-of-function genetic screens, we profiled axon guidance proteins on human T cells and their cognate ligands and identified fibronectin leucine-rich transmembrane protein 3 (FLRT3) as a ligand that inhibits T cell activity. We demonstrated that FLRT3 inhibits T cells through UNC5B, an axon guidance receptor that is up-regulated on activated human T cells. FLRT3 expressed in human cancers favored tumor growth and inhibited CAR-T and BiTE + T cell killing and infiltration in humanized cancer models. An FLRT3 monoclonal antibody that blocked FLRT3-UNC5B interactions reversed these effects in an immune-dependent manner. This study supports the concept that axon guidance proteins mimic T cell checkpoints and can be targeted for cancer immunotherapy.