Search

Supplementary Figures 1-11

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.

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

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

Antibodies for flow cytometry (S1); List of gene specific primer sequences for qRT-PCR (S2).

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-IIhi and MHC-IIlo tumor-associated macrophage (TAM) subpopulations that originate from Ly6Chi monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6Chi monocytic precursors. M-CSFR signaling blockade shifted the MHC-IIlo/MHC-IIhi TAM balance in favor of the latter as observed by the preferential differentiation of Ly6Chi monocytes into MHC-IIhi TAMs. In addition, the genetic and functional signatures of MHC-IIlo TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-IIlo TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-IIhi phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade. Cancer Res; 76(1); 35–42. ©2015 AACR.

Effects of α-M-CSFR Ab treatment on Ly6Chi monocyte function (S1); Effect of M-CSFR blockade on the survival of Ly6Chi monocytes and TAM (S2); Effect of M-CSFR blockade on Ly6Chi monocyte percentages in peripheral blood, spleen and bone marrow (S3); Absolute numbers of myeloid cell subsets in tumors from isotype or α-M-CSFR Ab-treated animals (S4); Production of M-CSF, IL-34 and GM-CSF by 3LL-R tumors (S5); Effect of M-CSFR blockade on monocyte differentiation in TS/A breast carcinoma tumors (S6); M-CSFR-mediated regulation of gene expression is IL-4Rα- independent (S7); Mixed Leukocyte Reaction (MLR) by TAM subsets from isotype or α-M-CSFR Ab treated animals (S8); M-CSFR blockade enhanced the M1-like MHC-IIhi TAM gene signature independently of GM-CSFR signaling (S9).

PDF - 410K, Molecular and functional heterogeneity of TAM and TADC subsets in 3LL-R tumors (S1); Characterization of the myeloid cell infiltrate in orthotopic 3LL-R tumors and effect of tumor growth on the lung macrophage subsets (S2); Intratumoral localization of the distinct myeloid cell subpopulations in 3LL-R tumors (S3); PHD2-haplodeficiency increases tumor vessel perfusion and vessel coverage in 3LL-R tumors (S4); Expression of hypoxia-regulated proteins in sorted TAM (S5).

PDF - 49K, Description of additional methods and procedures used in the study. Also includes supplementary references.

Description of additional methods and procedures used in the study.

PDf - 36K, List of antibodies (S1); List of gene specific primers (S2).

Junctional adhesion molecule-A (JAM-A), expressed on the surface of myeloid cells, is required for extravasation at sites of inflammation and may also modulate myeloid cell activation. Infiltration of myeloid cells is a common feature of tumors that drives disease progression, but the function of JAM-A in this phenomenon and its impact on tumor-infiltrating myeloid cells is little understood. Here we show that systemic cancer-associated inflammation in mice enhanced JAM-A expression selectively on circulating monocytes in an IL1β-dependent manner. Using myeloid-specific JAM-A-deficient mice, we found that JAM-A was dispensable for recruitment of monocytes and other myeloid cells to tumors, in contrast to its reported role in inflammation. Single-cell RNA sequencing revealed that loss of JAM-A did not influence the transcriptional reprogramming of myeloid cells in the tumor microenvironment. Overall, our results support the notion that cancer-associated inflammation can modulate the phenotype of circulating immune cells, and we demonstrate that tumors can bypass the requirement of JAM-A for myeloid cell recruitment and reprogramming. ispartof: FRONTIERS IN IMMUNOLOGY vol:13 ispartof: location:Switzerland status: published

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.

Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.

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. ispartof: CANCER IMMUNOLOGY RESEARCH vol:9 issue:3 pages:309-323 ispartof: location:United States status: published

The liver ischemia-reperfusion (IR) injury that occurs consequently to hepatic resection performed in patients with metastases can lead to tumor relapse for not fully understood reasons. We assessed the effects of liver IR on tumor growth and the innate immune response in a mouse model of colorectal (CR) liver metastasis. Mice subjected to liver ischemia 2 days after intrasplenic injection of CR carcinoma cells displayed a higher metastatic load in the liver, correlating with Kupffer cells (KC) death through the activation of receptor-interating protein 3 kinase (RIPK3) and caspase-1 and a recruitment of monocytes. Interestingly, the immunoregulatory mediators, tumor necrosis factor-α (TNF-α) and heme oxygenase-1 (HO-1) were strongly upregulated in recruited monocytes and were also expressed in the surviving KC following IR. Using TNFflox/flox LysMcre/wt mice, we showed that TNF deficiency in macrophages and monocytes favors tumor progression after IR. The antitumor effect of myeloid cell-derived TNF involved direct tumor cell apoptosis and a reduced expression of immunosuppressive molecules such as transforming growth factor-β, interleukin (IL)-10, inducible nitric oxyde synthase (iNOS), IL-33 and HO-1. Conversely, a monocyte/macrophage-specific deficiency in HO-1 (HO-1flox/flox LysMcre/wt ) or the blockade of HO-1 function led to the control of tumor progression post-liver IR. Importantly, host cell RIPK3 deficiency maintains the KC number upon IR, inhibits the IR-induced innate cell recruitment, increases the TNF level, decreases the HO-1 level and suppresses the tumor outgrowth. In conclusion, tumor recurrence in host undergoing liver IR is associated with the death of antitumoral KC and the recruitment of monocytes endowed with immunosuppressive properties. In both of which HO-1 inhibition would reinforce their antitumoral activity., info:eu-repo/semantics/published

BackgroundModulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker.MethodsWe employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models.ResultsWe were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs.ConclusionsCollectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.

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.

Leishmania major (L. major) parasites are intracellular parasites belong to the Trypanosomatidae family and are the causative agent of cutaneous leishmaniasis. This disease affects approximately 1.5 million per year worldwide and there is currently no prophylactic vaccine available. L. major is transmitted by the bite of an infected sandfly and has been considered for decades now as a mouse model of choice to identify the factors implicated in T helper (Th)1 and Th2 polarization due to the natural resistance and susceptibility to infection of C57BL/6 and BALB/c mice, respectively. In this study, we refine the role of IL-12p40 cytokine, which is implicated the development of a protective Th1 response, and STAT6, a transcription factor involved in the signaling via detrimental interleukin (IL)-4 and IL-13 associated Th2 cytokines during L. major infection in the BALB/c model. In the absence of STAT6 and IL-12p40 signaling, double knockout (DKO) susceptible BALB/c mice displayed reduced footpad swelling and ulcerative lesion compared to IL-12p40-/- mice upon L. major infection. Hence, they expressed slower upregulation of keratinocyte markers implicated in the inhibition of wound healing, such as keratin 6a (Krt6a) and Krt16. This coincides with the presence of neutrophils displaying an altered phenotype characterized by a lower expression of surface markers Ly6C, CD11b, and Ly6G. These neutrophils exhibited very lower levels of apoptosis similarly to neutrophils present in resistant STAT6-/- mice. Interestingly, the reduced footpad swelling in DKO mice is associated with a high footpad parasite level similar to susceptible IL-12p40-/- mice. In conclusion, this study demonstrate that in the absence of both STAT6 and IL-12p40 signaling, L. major-infected mice display smaller and less ulcerated lesions, which does, however, not correlate with reduced parasite load. In addition, the presence of neutrophils with an altered phenotype is associated with reduced apoptosis and delayed immunopathologies, demonstrating the detrimental role of STAT6 in infected susceptible BALB/c mice., SCOPUS: ar.j, info:eu-repo/semantics/published

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident MΦ types, designated MHC-II(lo) and MHC-II(hi) MΦs, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas MΦs differed at the molecular level, with MHC-II(lo) MΦs being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) MΦ peak and ultimately a restoration of the MHC-II(hi) MΦ-dominated steady-state equilibrium. These intricate MΦ dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local MΦ proliferation. Functionally, MHC-II(lo) MΦs were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue MΦs, rather than Ly6C(hi) monocyte-derived MΦs, contributed to β-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.

ObjectivePostoperative ileus (POI) is assumed to result from myeloid cells infiltrating the intestinalmuscularis externa(ME) in patients undergoing abdominal surgery. In the current study, we investigated the role of infiltrating monocytes in a murine model of intestinal manipulation (IM)-induced POI in order to clarify whether monocytes mediate tissue damage and intestinal dysfunction or they are rather involved in the recovery of gastrointestinal (GI) motility.DesignIM was performed in mice with defective monocyte migration to tissues (C-C motif chemokine receptor 2,Ccr2−/−mice) and wild-type (WT) mice to study the role of monocytes and monocyte-derived macrophages (MΦs) during onset and resolution of ME inflammation.ResultsAt early time points, IM-induced GI transit delay and inflammation were equal in WT andCcr2−/−mice. However, GI transit recovery after IM was significantly delayed inCcr2−/−mice compared with WT mice, associated with increased neutrophil-mediated immunopathology and persistent impaired neuromuscular function. During recovery, monocyte-derived MΦs acquire pro-resolving features that aided in the resolution of inflammation. In line, bone marrow reconstitution and treatment with MΦ colony-stimulating factor 1 enhanced monocyte recruitment and MΦ differentiation and ameliorated GI transit inCcr2−/−mice.ConclusionOur study reveals a critical role for monocyte-derived MΦs in restoring intestinal homeostasis after surgical trauma. From a therapeutic point of view, our data indicate that inappropriate targeting of monocytes may increase neutrophil-mediated immunopathology and prolong the clinical outcome of POI, while future therapies should be aimed at enhancing MΦ physiological repair functions.

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.

Various steady state and inflamed tissues have been shown to contain a heterogeneous DC population consisting of developmentally distinct subsets, including cDC1s, cDC2s and monocyte-derived DCs, displaying differential functional specializations. The identification of functionally distinct tumour-associated DC (TADC) subpopulations could prove essential for the understanding of basic TADC biology and for envisaging targeted immunotherapies. We demonstrate that multiple mouse tumours as well as human tumours harbour ontogenically discrete TADC subsets. Monocyte-derived TADCs are prominent in tumour antigen uptake, but lack strong T-cell stimulatory capacity due to NO-mediated immunosuppression. Pre-cDC-derived TADCs have lymph node migratory potential, whereby cDC1s efficiently activate CD8+ T cells and cDC2s induce Th17 cells. Mice vaccinated with cDC2s displayed a reduced tumour growth accompanied by a reprogramming of pro-tumoural TAMs and a reduction of MDSCs, while cDC1 vaccination strongly induces anti-tumour CTLs. Our data might prove important for therapeutic interventions targeted at specific TADC subsets or their precursors., Dendritic cells are antigen-presenting cells consisting of distinct subsets originating from different lineages. Here, the authors identify the subsets of dendritic cells populating the tumour tissue in both mice and humans and find they have opposing functions in regulating the anti-tumour immune response.

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-IIhi and MHC-IIlo tumor-associated macrophage (TAM) subpopulations that originate from Ly6Chi monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6Chi monocytic precursors. M-CSFR signaling blockade shifted the MHC-IIlo/MHC-IIhi TAM balance in favor of the latter as observed by the preferential differentiation of Ly6Chi monocytes into MHC-IIhi TAMs. In addition, the genetic and functional signatures of MHC-IIlo TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-IIlo TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-IIhi phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade. Cancer Res; 76(1); 35–42. ©2015 AACR.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident MΦ types, designated MHC-II(lo) and MHC-II(hi) MΦs, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas MΦs differed at the molecular level, with MHC-II(lo) MΦs being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) MΦ peak and ultimately a restoration of the MHC-II(hi) MΦ-dominated steady-state equilibrium. These intricate MΦ dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local MΦ proliferation. Functionally, MHC-II(lo) MΦs were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue MΦs, rather than Ly6C(hi) monocyte-derived MΦs, contributed to β-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.

Adenosine 5′-monophosphate (AMP) and methacholine are commonly used to assess airway hyperreactivity. However, it is not fully known whether the site of airway constriction primarily involved during challenges with either agent is similar. Using a ventilation distribution test, we investigated whether the constriction induced by each agent involves the lung periphery in a similar fashion. Ventilation distribution was evaluated by the phase III slope (S) of the single-breath washout, using gases with different diffusivities like helium (He) and hexafluorosulfur (SF6). A greater postchallenge increase in SHe reflects alterations at the level of terminal and respiratory bronchioles, while a greater increase in SSF6 reflects alterations in alveolar ducts, increases to an equal extent reflecting alterations in more proximal airways where gas transport is still convective for both gases. SSF6 and SHe were measured in 15 asthma patients before and after airway challenges (20% forced expired volume in 1-s fall) with AMP and methacholine. SHe increased to a greater extent than SSF6 after AMP challenge (5.7 vs. 3.7%/l; P = 0.002), with both slopes increasing to an equal extent after methacholine challenge (3.1%/l; P = 0.959). The larger increase in SHe following AMP challenge suggests distal ventilation impairment up to the level of terminal and respiratory bronchioles. With methacholine, the similar increases in SHe and SSF6 suggest a less distal impairment. AMP, therefore, seems to affect more extensively the very peripheral airways, whereas methacholine seems to have an effect on less distal airways.

In homeostasis, the liver is critical for the metabolism of nutrients including sugars, lipids, proteins and iron, for the clearance of toxins, and to induce immune tolerance to gut-derived antigens. These functions predispose the liver to infection by blood-borne pathogens, and to a variety of diseases ranging from toxin and medication-induced disorders (CCl4, acetaminophen) to metabolic disorders (steatohepatitis, alcoholic liver disease, biliary obstruction, cholestasis) or autoimmunity. Chronic liver injury often progresses to life threatening fibrosis and can end in liver cirrhosis and hepatocellular carcinoma (Pellicoro et al., 2014). The liver contains parenchymal cells or hepatocytes that make up the majority of hepatic cells. It also contains non-parenchymal structural cells such as sinusoidal endothelial cells and a large number of non-parenchymal innate immune cells, mainly monocytes, neutrophils, macrophages, DCs, NK and NKT cells that can trigger an adaptive immune response in the case of infections or other pathogenic insults (Jenne and Kubes, 2013). How this immune response is regulated determines the extent of acute and chronic liver injury (Stijlemans et al., 2014). In this context, liver macrophages have been demonstrated to play central but divergent (from initiating to resolving) functions in liver injury (Sica et al., 2014). It has become clear in the last years that hepatic macrophages consist of two classes, tissue-resident macrophages, the Kupffer cells (KCs) originating from yolk sac/fetal liver progenitors and tissue-infiltrating macrophages originating from bone marrow-derived Ly6CHi monocytes (Jinhoux and Jung, 2014; Tacke and Zimmerman, 2014). Distinguishing the activities of KCs from those of monocyte-derived macrophages during liver injury or repair is currently a frontline research topic in the macrophage field. Indeed, considering that clinical management of liver failure remains problematic, a better understanding of the immune mechanisms regulating liver injury is expected to allow the development of new therapeutic modalities. Here, we describe an isolation technique for liver non-parenchymal polymorphonuclear (PMN) and mononuclear myeloid cells permitting their molecular and functional characterization.

Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11bhiF4/80hiLy6Clo TAM subsets, designated as MHC-IIlo and MHC-IIhi TAM, both of which were derived from tumor-infiltrating Ly6Chi monocytes. MHC-IIlo TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-IIlo TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-IIhi counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (PHD2)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-IIlo TAM subset. The same observation in PHD2+/+→ PHD2+/− bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-IIlo TAM. Cancer Res; 74(1); 24–30. ©2013 AACR.