Dying cells initiate adaptive immunity by providing both antigens and inflammatory stimuli for dendritic cells (DCs), which in turn activate CD8+ T cells through a process called antigen cross-priming. To define how different forms of programmed cell death influence immunity, we established models of necroptosis and apoptosis, where dying cells are generated by RIPK3 and CASP8 dimerization, respectively. We found that release of inflammatory mediators such as damage-associated molecular patterns (DAMPs) by dying cells was not sufficient for CD8+ T cell cross-priming. Instead, robust cross-priming required RIPK1 signaling and NF-κB-induced transcription within dying cells. Decoupling NF-κB signaling from necroptosis or inflammatory apoptosis reduced priming efficiency and tumor immunity. Our results reveal that coordinated inflammatory and cell death signaling pathways within dying cells orchestrate adaptive immunity.

Therapeutic intravesical instillation of bacillus Calmette-Guérin (BCG) is effective at triggering inflammation and eliciting successful tumor immunity in patients with non-muscle invasive bladder cancer, with 50 to 70% clinical response. Therapeutic success relies on repeated instillations of live BCG administered as adjuvant therapy shortly after tumor resection; however, the precise mechanisms remain unclear. Using an experimental model, we demonstrate that after a single instillation, BCG could disseminate to bladder draining lymph nodes and prime interferon-γ-producing T cells. Nonetheless, repeated instillations with live BCG were necessary for a robust T cell infiltration into the bladder. Parenteral exposure to BCG before instillation overcame this requirement; after the first intravesical instillation, BCG triggered a more robust acute inflammatory process and accelerated T cell entry into the bladder, as compared to the standard protocol. Moreover, parenteral exposure to BCG before intravesical treatment of an orthotopic tumor markedly improved response to therapy. Indeed, patients with sustained preexisting immunity to BCG showed a significant improvement in recurrence-free survival. Together, these data suggest that monitoring patients' response to purified protein derivative, and, in their absence, boosting BCG responses by parenteral exposure before intravesical treatment initiation, may be a safe and effective means of improving intravesical BCG-induced clinical responses.

Cross-presentation of cell-associated antigen is important in the priming of CD8 þ T-cell responses to proteins that are not expressed by antigen-presenting cells (APCs). In vivo, dendritic cells are the main cross-presenting APC, and much is known regarding their ability to capture and process cell-associated antigen. In contrast, little is known about the way death effector pathways influence the efficiency of cross-priming. Here, we compared two important mechanisms of programmed cell death: classical apoptosis, as it occurs in wild-type (WT) fibroblasts, and caspase-independent cell death, which occurs with increased features of autophagy in Bax/Bak À/À fibroblasts. We assessed virally infected WT and Bax/Bak À/À fibroblasts as a source of cell-associated antigen. We found that immunization with cells undergoing autophagy before cell death was superior in facilitating the cross-priming of antigen-specific CD8 þ T cells. Strikingly, silencing of Atg5 expression inhibited priming. We interpret this to be a novel form of 'immunogenic death' with the enhanced priming efficiency being a result of persistent MHC I cross-presentation and the induction of type I interferons. These results offer the first molecular evidence that catabolic pathways, including autophagy, influence the efficiency of cross-priming. We predict that targeting the autophagy cascade may provide a therapeutic strategy for achieving robust cross-priming of viral and tumor-specific CD8 þ T cells. There is now evidence to suggest that antigen-presenting cells (APCs) have evolved mechanisms to capture immunologically relevant information from internalized apoptotic cells, thus offering a mechanism whereby cell death may influence subsequent immune response(s). 1-3 Specifically, it has been shown that antigen derived from phagocytosed dying cells may be processed by dendritic cells (DCs) to generate MHC I/peptide, resulting in the activation of CD8 þ T cells. This pathway is called crosspriming due to the 'crossing' of the classically defined restriction of MHC I for presentation of endogenously synthesized protein. 4 In addition, dying cells are also capable of skewing cytokine production in phagocytes. 5 For example, macrophages and DCs engulf dying cells through specific phagocytic receptors (e.g., CD36, a v b 5 , CR3 and others), which may result in the inhibition of proinflammatory cytokine secretion (e.g., IL-1b, IL-12 and TNF-a). [6][7][8][9] These studies have all addressed the downstream effects of dying cells on the immune system with little attention to the complexity of cell death pathways. We hypothesized that the molecular mechanism of cell death influences the immunologic instruction given to the phagocyte and we focused our attention on two of the major classes of PCD that have been described: type I PCD (or classical apoptosis) and type II PCD (referred to as autophagic cell death or caspase-independent cell death). 10 Distinct, yet intertwined pathways are involved in executing cell death programs. Briefly, apoptosis is mediated ...