Adaptive immune responses rely on differentiation of CD4 T helper cells into subsets with distinct effector functions best suited for host defence against the invading pathogen. Interleukin (IL)-17-producing T helper cells (T(H)17) are a recently identified subset, separate from the T helper type 1 (T(H)1) and T helper type 2 (T(H)2) subsets. Synergy between the cytokines transforming growth factor-beta and IL-6 in vitro induces development of T(H)17 cells in mouse and human systems, whereas IL-23 supports expansion of these cells. However, it is not known which conditions in vivo would induce this combination of cytokines. Furthermore, it is enigmatic that a combination of pro-inflammatory and anti-inflammatory cytokines would be required to generate an effector T(H)17 response. Here we show that the relevant physiological stimulus triggering this combination of cytokines is the recognition and phagocytosis of infected apoptotic cells by dendritic cells. Phagocytosis of infected apoptotic cells uniquely triggers the combination of IL-6 and transforming growth factor-beta through recognition of pathogen-associated molecular patterns and phosphatidylserine exposed on apoptotic cells, respectively. Conversely, phagocytosis of apoptotic cells in the absence of microbial signals induces differentiation of the closely related regulatory T cells, which are important for controlling autoimmunity. Blocking apoptosis during infection of the mouse intestinal epithelium with the rodent pathogen Citrobacter rodentium, which models human infections with the attaching and effacing enteropathogenic and enterohaemorrhagic Escherichia coli, impairs the characteristic T(H)17 response in the lamina propria. Our results demonstrate that infected apoptotic cells are a critical component of the innate immune signals instructing T(H)17 differentiation, and point to pathogens particularly adept at triggering apoptosis that might preferentially induce T(H)17-mediated immunity. Because T(H)17 cells have been correlated with autoimmune diseases, investigation of the pathways of innate recognition of infected apoptotic cells might lead to improved understanding of the causative defects in autoimmunity.

Macrophages tightly scale their core metabolism after being activated, but the precise regulation of the mitochondrial electron-transport chain (ETC) and its functional implications are currently unknown. Here we found that recognition of live bacteria by macrophages transiently decreased assembly of the ETC complex I (CI) and CI-containing super-complexes and switched the relative contributions of CI and CII to mitochondrial respiration. This was mediated by phagosomal NADPH oxidase and the reactive oxygen species (ROS)-dependent tyrosine kinase Fgr. It required Toll-like receptor signaling and the NLRP3 inflammasome, which were both connected to bacterial viability-specific immune responses. Inhibition of CII during infection with Escherichia coli normalized serum concentrations of interleukin 1β (IL-1β) and IL-10 to those in mice treated with dead bacteria and impaired control of bacteria. We have thus identified ETC adaptations as an early immunological-metabolic checkpoint that adjusts innate immune responses to bacterial infection.

Interaction of T cell with antigen-bearing dendritic cells (DC) results in T cell activation, but whether this interaction has physiological consequences on DC function is largely unexplored. Here we show that when antigen-bearing DCs contact T cells, DCs initiate anti-pathogenic programs. Signals of this interaction are transmitted from the T cell to the DC, through extracellular vesicles (EV) that contain genomic and mitochondrial DNA, to induce antiviral responses via the cGAS/STING cytosolic DNA-sensing pathway and expression of IRF3-dependent interferon regulated genes. Moreover, EV-treated DCs are more resistant to subsequent viral infections. In summary, our results show that T cells prime DCs through the transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection. The reciprocal communication between innate and adaptive immune cells thus allow efficacious responses to unknown threats.

Toll-like receptor (TLR) ligands are increasingly being used as adjuvants in cancer vaccine trials to harness innate immunity and prime effective antitumor immune responses. Despite some success, enhancing tumor antigen presentation, promoting a protective antitumor response, and overcoming the immunosuppressive tumor microenvironment pose considerable challenges that necessitate further improvements in vaccine design. Here, we show that expression of the TLR ligand flagellin within tumor cells constitutes an effective antitumor vaccination strategy that relies on simultaneous engagement of TLR5 and the Nod-like receptors (NLRs) NLRC4/NAIP5 (neuronal apoptosis inhibitory protein 5) by flagellin along with associative recognition of tumor antigen for optimal antigen presentation to T cells. Although TLR5 signaling was critical for mediating rapid macrophage-dependent clearance of flagellin-expressing tumor cells in vivo, TLR5 and NLRC4/NAIP5 were equally important for priming antitumor CD4(+) and CD8(+) T cells and suppressing tumor growth. Vaccination with irradiated flagellin-expressing tumor cells prevented tumor development, and disrupting flagellin recognition by TLR5 or NLRC4/NAIP5 impaired protective immunization against an existing or subsequent tumor. Our findings delineate a new strategy to induce anticancer immune responses consisting of introducing microbial structures with dual TLR and NLR stimulatory activity into tumor cells. This ensures recognition of tumor-derived antigen within the inflammatory context of microbial recognition and additionally activates both the phagocytic and the cytosolic pathways of innate immune defense against the tumor.