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IL1β is a central mediator of inflammation. Secretion of IL1β typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL1β in promoting cancer progression in patients, but the underlying mechanisms are ill-defined. Here, we have shown a key role for IL1β in driving tumor progression in two distinct mouse tumor models. Notably, activation of the inflammasome, caspase-8, as well as the pore-forming proteins GSDMD and mixed lineage kinase domain–like protein in the host were dispensable for the release of intratumoral bioactive IL1β. Inflammasome-independent IL1β release promoted systemic neutrophil expansion and fostered accumulation of T-cell–suppressive neutrophils in the tumor. Moreover, IL1β was essential for neutrophil infiltration triggered by antiangiogenic therapy, thereby contributing to treatment-induced immunosuppression. Deletion of IL1β allowed intratumoral accumulation of CD8+ effector T cells that subsequently activated tumor-associated macrophages. Depletion of either CD8+ T cells or macrophages abolished tumor growth inhibition in IL1β-deficient mice, demonstrating a crucial role for CD8+ T-cell–macrophage cross-talk in the antitumor immune response. Overall, these results support a tumor-promoting role for IL1β through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for release of this cytokine in tumors.

Supplementary Data from Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages

Agonistic αCD40 therapy has been shown to inhibit cancer progression in only a fraction of patients. Understanding the cancer cell–intrinsic and microenvironmental determinants of αCD40 therapy response is therefore crucial to identify responsive patient populations and to design efficient combinatorial treatments. Here, we show that the therapeutic efficacy of αCD40 in subcutaneous melanoma relies on preexisting, type 1 classical dendritic cell (cDC1)–primed CD8+ T cells. However, after administration of αCD40, cDC1s were dispensable for antitumor efficacy. Instead, the abundance of activated cDCs, potentially derived from cDC2 cells, increased and further activated antitumor CD8+ T cells. Hence, distinct cDC subsets contributed to the induction of αCD40 responses. In contrast, lung carcinomas, characterized by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumor growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell death–inducing chemotherapy sensitized lung tumors to αCD40 therapy in subcutaneous and orthotopic settings. These insights into the microenvironmental regulators of response to αCD40 suggest that different tumor types would benefit from different combinations of therapies to optimize the clinical application of CD40 agonists.Significance:This work highlights the temporal roles of different dendritic cell subsets in promoting CD8+ T-cell–driven responses to CD40 agonist therapy in cancer.

Supplementary Figure from Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages

Radioimmunotherapy (RIT) aims to deliver a high radiation dose to cancer cells, while minimizing the exposure of normal cells. Typically, monoclonal antibodies are used to target the radionuclides to cancer cell surface antigens. However, antibodies face limitations due to their poor tumor penetration and suboptimal pharmacokinetics, while the expression of their target on the cancer cell surface may be gradually lost. In addition, most antigens are expressed in a limited number of tumor types. To circumvent these problems, we developed a Nanobody (Nb)-based RIT against a prominent stromal cell (stromal-targeting radioimmunotherapy or STRIT) present in nearly all tumors, the tumor-associated macrophage (TAM). Macrophage Mannose Receptor (MMR) functions as a stable molecular target on TAM residing in hypoxic areas, further allowing the delivery of a high radiation dose to the more radioresistant hypoxic tumor regions. Since MMR expression is not restricted to TAM, we first optimized a strategy to block extra-tumoral MMR to prevent therapy-induced toxicity. A 100-fold molar excess of unlabeled bivalent Nb largely blocks extra-tumoral binding of Lu-177-labeled anti-MMR Nb and prevents toxicity, while still allowing the intra-tumoral binding of the monovalent Nb. Interestingly, three doses of Lu-177-labeled anti-MMR Nb resulted in a significantly retarded tumor growth, thereby outcompeting the effects of antiPD1, anti-VEGFR2, doxorubicin and paclitaxel in the TS/A mammary carcinoma model. Together, these data propose anti-MMR STRIT as a valid new approach for cancer treatment.

Interleukin-1β (IL-1β) is a central mediator of inflammation whose secretion typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL-1β in promoting cancer progression in patients, but the underlying mechanisms are little understood. Here, we show a key role for IL-1β in driving tumor progression in two distinct mouse tumor models. Notably, inflammasome activation and GSDMD were dispensable for the production of intratumoral bioactive IL-1β, which promoted systemic mobilization and infiltration of neutrophils into tumors. Neutrophils recruited via IL-1β suppressed the acquisition of an effector T-cell phenotype and subsequent antitumor immune response. Moreover, IL-1β was essential for neutrophil accumulation upon antiangiogenic therapy, thereby contributing to therapy-induced immunosuppression. Antitumor immunity in the absence of IL-1β-dependent neutrophil recruitment relied on immunostimulatory macrophages which promoted the infiltration and activation of cytotoxic T-cells. Overall, these results support a tumor-promoting role for IL-1β through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for its release in tumors.

Macrophages are often prominently present in the tumor microenvironment, where distinct macrophage populations can differentially affect tumor progression. Although the metabolism of macrophages influences their function, little is known about the metabolic characteristics of tumor-associated macrophage (TAM) subsets. Using transcriptomic and metabolomic analyses, we now reveal that pro-inflammatory MHC-IIhi TAMs display a hampered TCA cycle, while reparative MHC-IIlo TAMs show a higher oxidative and glycolytic metabolism. Both TAM populations preferred lactate over glucose as a carbon source to fuel the TCA cycle. However, while lactate supported the oxidative metabolism in MHC-IIlo TAMs, it decreased the metabolic activity of MHC-IIhi TAMs. Lactate subtly affected the transcriptome of MHC-IIlo TAMs, increased L-arginine metabolism and enhanced T-cell suppressive capacity of these TAMs. Overall, our data uncover the metabolic intricacies of distinct TAM subsets and identify lactate as an important carbon source, and metabolic and functional regulator of TAMs.

Background: Chronic inflammation in the tumor microenvironment (TME) sustained by immune cells has a crucial role both in tumor initiation and progression. One of the central cytokines of inflammation, IL-1β, is produced as a biologically inactive precursor that requires proteolytic processing by caspase-1. Activation of caspase-1 is triggered by the formation of inflammasomes, multiprotein complexes that detect microbial and endogenous danger signals primarily via NOD-like receptors, such as NLRP3 and NLRC4. Biologically active IL-1β is believed to be released through membrane pores formed by gasdermin D during a lytic form of cell death called pyroptosis. Although IL-1β-mediated inflammation has been shown to have a detrimental role in tumor progression, the signaling pathway controlling IL-1β release in the TME and the exact effect of the cytokine on antitumor T-cell responses have not been fully elucidated. A better understanding of how IL-1β release is controlled in tumors will also pave the way towards the therapeutic utilization of small-molecule inhibitors available to target NOD-like receptors and caspase-1. Methods: First, we characterized the impact of IL-1β in the TME by assessing the immune cell composition and vasculature of Lewis lung carcinomas (LLC) and E0771 breast carcinomas in IL-1β-deficient mice using flow cytometry and histologic analysis. Next, we used mice deficient in different inflammasome components, including NLRP3, NLRC4 and caspase-1, to investigate the involvement of these proteins in controlling IL-1β release in LLC and E0771 tumors. Using immunoblots and small-molecule inhibitors, we further characterized the activation of alternative enzymatic pathways and their involvement in IL-1β release by tumor-associated myeloid cells. Finally, we examined the role of pyroptosis and necroptosis in IL-1β release using gasdermin D- and MLKL-deficient mice, respectively. Release of IL-1β was assessed using ELISA and immunoblots. Results: We found that IL-1β secretion was restricted to myeloid cells and promoted tumor progression in mouse models of lung and breast carcinoma. IL-1β deletion abrogated the tumor-induced mobilization of immunosuppressive neutrophils and normalized the tumor vasculature, thereby alleviating hypoxia. Consequently, proliferation of effector T-cells in the TME was enhanced, leading to higher CD4+ and CD8+ T-cell abundance in the absence of IL-1β. We observed that, although the NLRP3 inflammasome was active in tumor-infiltrating myeloid cells, NLRP3 and caspase-1 were not essential for the proteolytic maturation of pro-IL-1β and secretion of biologically active IL-1β in the TME. Inhibition or genetic deletion of caspase-8 reduced inflammasome-independent IL-1β release, indicating that caspase-8 provides an alternative pathway for proteolytic activation and secretion of IL-1β in tumor-infiltrating myeloid cells. Moreover, IL-1β release by tumor-infiltrating myeloid cells was independent of lytic cell death modalities including gasdermin D-mediated pyroptosis and MLKL-mediated necroptosis, suggesting an alternative release mechanism for the cytokine in the TME. Conclusions: Overall, our results demonstrate that tumor-infiltrating myeloid cells are able to release IL-1β independently of inflammasomes. We show that proteolytic maturation of IL-1β via caspase-8 in myeloid cells acts as an important driver of immune suppression in the TME through vascular destabilization, recruitment of immunosuppressive neutrophils and consequential inhibition of antitumor T-cell responses. We also show, that, unlike in autoinflammation, gasdermin D-mediated pyroptosis is not essential for the release of IL-1β in tumors. These results suggest that therapeutic inhibition of inflammasomes or pyroptosis will likely not be beneficial in certain tumor types due to the presence of an alternative caspase-8-mediated IL-1β release pathway in tumor-associated myeloid cells. Citation Format: Máté Kiss, Lieselotte Vande Walle, Helena Van Damme, Aleksandar Murgaski, Evangelia Bolli, Jiri Keirsse, Maria Solange Martins, Yvon Elkrim, Amelie Fossoul, Jens Serneels, Massimiliano Mazzone, Mohamed Lamkanfi, Jo A. Van Ginderachter, Damya Laoui. Inflammasome-independent IL-1β release by myeloid cells promotes vessel destabilization and immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A083.