Caspase-1 signaling in myeloid suppressor cells can promote T-cell independent cancer progression, but the regulation of inflammasome signaling within the highly heterogeneous myeloid population in the tumor milieu remains elusive. To resolve this complexity, single cell transcriptomic profile of Head and Neck Squamous Cell Carcinoma (HNSCC) identified distinct inflammasome-associated genes within specific clusters of tumor-infiltrating myeloid cells. Among these myeloid cells, the sensor protein, NLRP3, and downstream effector IL-1β transcripts were enriched in discreet monocytic and macrophage subtypes in the TME. We showed that deletion of NLRP3, but not AIM2, phenocopied caspase-1/IL-1β dependent tumor progression in vivo. Paradoxically, we found myeloid-intrinsic caspase-1 signaling increased myeloid survival contrary to what would be predicted from the canonical pyroptotic function of caspase-1. This myeloid NLRP3/IL-1β signaling axis promotion of tumor growth was found to be gasdermin D independent. Mechanistically, we found that phagocyte-mediated efferocytosis of dying tumor cells in the TME directly activated NLRP3-dependent inflammasome signaling to drive IL-1β secretion. Subsequently we showed that NLRP3-mediated IL-1β production drives tumor growth in vivo. Dynamic RNA velocity analysis showed a robust directional flow from efferocytosis gene-set high macrophages to an inflammasome gene-set high macrophage population. We provide a novel efferocytosis-dependent inflammasome signaling pathway which mediates homeostatic tumor cell apoptosis that characterizes chronic inflammation-induced malignancy.

With the clinical approval of T-cell–dependent immune checkpoint inhibitors for many cancers, therapeutic cancer vaccines have re-emerged as a promising immunotherapy. Cancer vaccines require the addition of immunostimulatory adjuvants to increase vaccine immunogenicity, and increasingly multiple adjuvants are used in combination to bolster further and shape cellular immunity to tumor antigens. However, rigorous quantification of adjuvants’ synergistic interactions is challenging due to partial redundancy in costimulatory molecules and cytokine production, leading to the common assumption that combining both adjuvants at the maximum tolerated dose results in optimal efficacy. Herein, we examine this maximum dose assumption and find combinations of these doses are suboptimal. Instead, we optimized dendritic cell activation by extending the Multidimensional Synergy of Combinations (MuSyC) framework that measures the synergy of efficacy and potency between two vaccine adjuvants. Initially, we performed a preliminary in vitro screening of clinically translatable adjuvant receptor targets (TLR, STING, NLL, and RIG-I). We determined that STING agonist (CDN) plus TLR4 agonist (MPL-A) or TLR7/8 agonist (R848) as the best pairwise combinations for dendritic cell activation. In addition, we found that the combination of R848 and CDN is synergistically efficacious and potent in activating both murine and human antigen-presenting cells (APCs) in vitro. These two selected adjuvants were then used to estimate a MuSyC-dose optimized for in vivo T-cell priming using ovalbumin-based peptide vaccines. Finally, using B16 melanoma and MOC1 head and neck cancer models, MuSyC-dose–based adjuvating of cancer vaccines improved the antitumor response, increased tumor-infiltrating lymphocytes, and induced novel myeloid tumor infiltration changes. Further, the MuSyC-dose–based adjuvants approach did not cause additional weight changes or increased plasma cytokine levels compared to CDN alone. Collectively, our findings offer a proof of principle that our MuSyC-extended approach can be used to optimize cancer vaccine formulations for immunotherapy.

BackgroundThe inflammasome is a multi-protein signaling pathway in immune and epithelial cells that is important for activation of the innate immune system and protection from pathogens. This pathway is well characterized in myeloid cell populations, however the T cell intrinsic effects of the inflammasome are not well understood.MethodsIn this study we utilize an inflammasome null mouse model to investigate the functional and phenotypic differences in inflammasome null and wildtype T cells. We utilize a whole cell vaccine against B16 mouse tumors to generate B16 tumor antigen specific T cells. In addition, we utilize clinically relevant PD-1 inhibitory antibodies to model checkpoint inhibition with inflammasome null T cells.ResultsHere we show that the inflammasome is expressed and activated in tumor infiltrating T cells in both humans and mice. We find that inflammasome null T cells have an altered phenotype causing them to become more proliferative and increase killing capacity. In addition, caspase 1 null T cells are present in the TME at a greater frequency than wildtype T cells. We also show that caspase 1 knockout T cells have higher checkpoint expression, most notably an increase in PD-1 expression, and combination caspase 1 and PD-1 blockade results in a significant reduction in tumor burden.ConclusionsTherefore, we propose that T cell intrinsic inflammasome signaling acts as a negative regulator to inhibit T cell activation and cytotoxicity. Together our findings reveal the inflammasome as an attractive pathway that can be targeted in combination with checkpoint blockade therapies to improve anti-tumor T cell responses.

BackgroundInflammation has long been associated with different stages of tumorigenesis as well as response to therapy. A key signaling pathway in this context is the casp-1 inflammasome. However, to date, its role in cancer has been contradictory and context dependent. We previously reported myeloid casp-1 can promote tumor growth in T cell independent manner. However, the regulatory mechanism that drives the myeloid intrinsic inflammasome signaling in the context of tumor growth remains largely unknown.MethodsIn order to gain finer details about the inflammasome pathway components in the different myeloid clusters, we analyzed tumor and blood samples from head and neck cancer patients using bulk as well as 10X single cell sequencing platforms. For in vivo tumor studies, genetically engineered preclinical mice models were used. For in vitro functional studies, cells were isolated from mice or human tumors/blood and differentiated to either MDSC or macrophages and subjected to various assays.ResultsOur bulk sequencing of myeloid cells isolated from treatment naïve head and neck tumors revealed an enrichment for inflammasome genes. Unbiased pathway analysis of tumor infiltrating myeloid cells compared to matched peripheral blood monocytes revealed IL-1β signaling to be significantly altered in the tumor myeloids. In our single cell transcriptomic sequencing dataset on human head & neck carcinoma with matched peripheral blood monocytes, we observed similar elevated inflammasome transcriptomic activity within specific clusters of tumor-infiltrating macrophages and myeloid derived suppressor cells. Interestingly, distinct inflammasome sensor genes, specifically NLRP3, had distinct co-expressions with IL-1β in specific myeloid subsets within the TME. Our data also indicates that myeloid-intrinsic caspase-1 signaling paradoxically increased tumor infiltrating myeloid cell survival without significant intratumoral trafficking into the tumor. When we explored the TME regulatory factors that regulate intratumoral myeloid inflammasome signaling, we found that NLRP3 dependent inflammasome signaling and IL-1β production promotes tumor growth in a Gasdermin D independent mechanism. Mechanistically, we show that efferocytosis of dying tumor cells by myeloid cells in the TME directly activates NLRP3 dependent inflammasome signaling and IL-1 β production in myeloid cells to promote tumor growth rate.ConclusionsTo our knowledge, we are the first to attribute the tumor supporting role of myeloid inflammasome signaling to efferocytic clearance of apoptotic debris in the tumor microenvironment. Our study thus opens an enticing option of novel therapeutic modality for treatment of solid tumors in future.Ethics ApprovalAll experimental procedures were approved by the Institutional Review Board of Vanderbilt University Medical Center (IRB: 170172).