Deterministic reprogramming of neutrophils within tumors.

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.

IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases.

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Targeted inducible delivery of immunoactivating cytokines reprograms glioblastoma microenvironment and inhibits growth in mouse models.

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor characterized by a strongly immunosuppressive tumor microenvironment (TME) that represents a barrier also for the development of effective immunotherapies. The possibility to revert this hostile TME by immunoactivating cytokines is hampered by the severe toxicity associated with their systemic administration. Here, we exploited a lentiviral vector-based platform to engineer hematopoietic stem cells ex vivo with the aim of releasing, via their tumor-infiltrating monocyte/macrophage progeny, interferon-α (IFN-α) or interleukin-12 (IL-12) at the tumor site with spatial and temporal selectivity. Taking advantage of a syngeneic GBM mouse model, we showed that inducible release of IFN-α within the TME achieved robust tumor inhibition up to eradication and outperformed systemic treatment with the recombinant protein in terms of efficacy, tolerability, and specificity. Single-cell RNA sequencing of the tumor immune infiltrate revealed reprogramming of the immune microenvironment toward a proinflammatory and antitumoral state associated with loss of a macrophage subpopulation shown to be associated with poor prognosis in human GBM. The spatial and temporal control of IL-12 release was critical to overcome an otherwise lethal hematopoietic toxicity while allowing to fully exploit its antitumor activity. Overall, our findings demonstrate a potential therapeutic approach for GBM and set the bases for a recently launched first-in-human clinical trial in patients with GBM.

CRISPR-based gene disruption and integration of high-avidity, WT1-specific T cell receptors improve antitumor T cell function.

T cell receptor (TCR)-based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01-restricted TCRs, three that were specific to the less explored immunodominant WT137-45 and two that were specific to the noncanonical WT1-78-64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR ß constant (TRBC) knockout, thus avoiding TCRαß mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137-45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.

A PGE2-MEF2A axis enables context-dependent control of inflammatory gene expression.

Tight control of inflammatory gene expression by antagonistic environmental cues is key to ensure immune protection while preventing tissue damage. Prostaglandin E2 (PGE2) modulates macrophage activation during homeostasis and disease, but the underlying mechanisms remain incompletely characterized. Here we dissected the genomic properties of lipopolysaccharide (LPS)-induced genes whose expression is antagonized by PGE2. The latter molecule targeted a set of inflammatory gene enhancers that, already in unstimulated macrophages, displayed poorly permissive chromatin organization and were marked by the transcription factor myocyte enhancer factor 2A (MEF2A). Deletion of MEF2A phenocopied PGE2 treatment and abolished type I interferon (IFN I) induction upon exposure to innate immune stimuli. Mechanistically, PGE2 interfered with LPS-mediated activation of ERK5, a known transcriptional partner of MEF2. This study highlights principles of plasticity and adaptation in cells exposed to a complex environment and uncovers a transcriptional circuit for IFN I induction with relevance for infectious diseases or cancer.

TIM4 expression by dendritic cells mediates uptake of tumor-associated antigens and anti-tumor responses.

Acquisition of cell-associated tumor antigens by type 1 dendritic cells (cDC1) is essential to induce and sustain tumor specific CD8+ T cells via cross-presentation. Here we show that capture and engulfment of cell associated antigens by tissue resident lung cDC1 is inhibited during progression of mouse lung tumors. Mechanistically, loss of phagocytosis is linked to tumor-mediated downregulation of the phosphatidylserine receptor TIM4, that is highly expressed in normal lung resident cDC1. TIM4 receptor blockade and conditional cDC1 deletion impair activation of tumor specific CD8+ T cells and promote tumor progression. In human lung adenocarcinomas, TIM4 transcripts increase the prognostic value of a cDC1 signature and predict responses to PD-1 treatment. Thus, TIM4 on lung resident cDC1 contributes to immune surveillance and its expression is suppressed in advanced tumors.

Determinants, mechanisms, and functional outcomes of myeloid cell diversity in cancer.

Most, if not all, aspects of carcinogenesis are influenced by the tumor microenvironment (TME), a complex architecture of cells, matrix components, soluble signals, and their dynamic interactions in the context of physical traits of the tissue. Expanding application of technologies for high-dimensional analyses with single-cell resolution has begun to decipher the contributions of the immune system to cancer progression and its implications for therapy. In this review, we will discuss the multifaceted roles of tumor-associated macrophages and neutrophils, focusing on factors that subvert tissue immune homeostasis and offer therapeutic opportunities for TME reprogramming. By performing a critical analysis of available datasets, we elaborate on diversification mechanisms and unifying principles of myeloid cell heterogeneity in human tumors.

Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

Tumor-Derived Prostaglandin E2 Promotes p50 NF-κB-Dependent Differentiation of Monocytic MDSCs.

Myeloid-derived suppressor cells (MDSC) include immature monocytic (M-MDSC) and granulocytic (PMN-MDSC) cells that share the ability to suppress adaptive immunity and to hinder the effectiveness of anticancer treatments. Of note, in response to IFNγ, M-MDSCs release the tumor-promoting and immunosuppressive molecule nitric oxide (NO), whereas macrophages largely express antitumor properties. Investigating these opposing activities, we found that tumor-derived prostaglandin E2 (PGE2) induces nuclear accumulation of p50 NF-κB in M-MDSCs, diverting their response to IFNγ toward NO-mediated immunosuppression and reducing TNFα expression. At the genome level, p50 NF-κB promoted binding of STAT1 to regulatory regions of selected IFNγ-dependent genes, including inducible nitric oxide synthase (Nos2). In agreement, ablation of p50 as well as pharmacologic inhibition of either the PGE2 receptor EP2 or NO production reprogrammed M-MDSCs toward a NOS2low/TNFαhigh phenotype, restoring the in vivo antitumor activity of IFNγ. Our results indicate that inhibition of the PGE2/p50/NO axis prevents MDSC-suppressive functions and restores the efficacy of anticancer immunotherapy. SIGNIFICANCE: Tumor-derived PGE2-mediated induction of nuclear p50 NF-κB epigenetically reprograms the response of monocytic cells to IFNγ toward an immunosuppressive phenotype, thus retrieving the anticancer properties of IFNγ. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2874/F1.large.jpg.

Heterogeneity of neutrophils.

Structured models of ontogenic, phenotypic and functional diversity have been instrumental for a renewed understanding of the biology of immune cells, such as macrophages and lymphoid cells. However, there are no established models that can be used to define the diversity of neutrophils, the most abundant myeloid cells. This lack of an established model is largely due to the uniquely short lives of neutrophils, a consequence of their inability to divide once terminally differentiated, which has been perceived as a roadblock to functional diversity. This perception is rapidly evolving as multiple phenotypic and functional variants of neutrophils have been found, both in homeostatic and disease conditions. In this Opinion article, we present an overview of neutrophil heterogeneity and discuss possible mechanisms of diversification, including genomic regulation. We suggest that neutrophil heterogeneity is an important feature of immune pathophysiology, such that co-option of the mechanisms of diversification by cancer or other disorders contributes to disease progression.

Immune signature drives leukemia escape and relapse after hematopoietic cell transplantation.

Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-γ or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.

Interferon gene therapy reprograms the leukemia microenvironment inducing protective immunity to multiple tumor antigens.

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing.

Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells.

In the clinic, chimeric antigen receptor-modified T (CAR T) cell therapy is frequently associated with life-threatening cytokine-release syndrome (CRS) and neurotoxicity. Understanding the nature of these pathologies and developing treatments for them are hampered by the lack of appropriate animal models. Herein, we describe a mouse model recapitulating key features of CRS and neurotoxicity. In humanized mice with high leukemia burden, CAR T cell-mediated clearance of cancer triggered high fever and elevated IL-6 levels, which are hallmarks of CRS. Human monocytes were the major source of IL-1 and IL-6 during CRS. Accordingly, the syndrome was prevented by monocyte depletion or by blocking IL-6 receptor with tocilizumab. Nonetheless, tocilizumab failed to protect mice from delayed lethal neurotoxicity, characterized by meningeal inflammation. Instead, the IL-1 receptor antagonist anakinra abolished both CRS and neurotoxicity, resulting in substantially extended leukemia-free survival. These findings offer a therapeutic strategy to tackle neurotoxicity and open new avenues to safer CAR T cell therapies.

Opposing macrophage polarization programs show extensive epigenomic and transcriptional cross-talk.

Stimulation of macrophages with interferon-γ (IFN-γ) and interleukin 4 (IL-4) triggers distinct and opposing activation programs. During mixed infections or cancer, macrophages are often exposed to both cytokines, but how these two programs influence each other remains unclear. We found that IFN-γ and IL-4 mutually inhibited the epigenomic and transcriptional changes induced by each cytokine alone. Computational and functional analyses revealed the genomic bases for gene-specific cross-repression. For instance, while binding motifs for the transcription factors STAT1 and IRF1 were associated with robust and IL-4-resistant responses to IFN-γ, their coexistence with binding sites for auxiliary transcription factors such as AP-1 generated vulnerability to IL-4-mediated inhibition. These data provide a core mechanistic framework for the integration of signals that control macrophage activation in complex environmental conditions.

Mutual epithelium-macrophage dependency in liver carcinogenesis mediated by ST18.

The ST18 gene has been proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis has been lacking thus far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 messenger RNA (mRNA) and protein were detectable neither in normal liver nor in cultured hepatoblasts, but were readily expressed after subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as coculture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre-established tumors caused rapid tumor involution associated with pervasive morphological changes, proliferative arrest, and apoptosis in tumor cells, as well as depletion of tumor-associated macrophages, vascular ectasia, and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in hepatoblasts. Finally, RNA sequencing of ST18-depleted tumors before involution revealed down-regulation of inflammatory response genes, pointing to the suppression of nuclear factor kappa B-dependent transcription. CONCLUSION: ST18 expression in epithelial cells is induced by tumor-associated macrophages, contributing to the reciprocal feed-forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18-dependent epithelium-macrophage interactions in a therapeutic setting. (Hepatology 2017;65:1708-1719).

TNF-Mediated Restriction of Arginase 1 Expression in Myeloid Cells Triggers Type 2 NO Synthase Activity at the Site of Infection.

Neutralization or deletion of tumor necrosis factor (TNF) causes loss of control of intracellular pathogens in mice and humans, but the underlying mechanisms are incompletely understood. Here, we found that TNF antagonized alternative activation of macrophages and dendritic cells by IL-4. TNF inhibited IL-4-induced arginase 1 (Arg1) expression by decreasing histone acetylation, without affecting STAT6 phosphorylation and nuclear translocation. In Leishmania major-infected C57BL/6 wild-type mice, type 2 nitric oxide (NO) synthase (NOS2) was detected in inflammatory dendritic cells or macrophages, some of which co-expressed Arg1. In TNF-deficient mice, Arg1 was hyperexpressed, causing an impaired production of NO in situ. A similar phenotype was seen in L. major-infected BALB/c mice. Arg1 deletion in hematopoietic cells protected these mice from an otherwise lethal disease, although their disease-mediating T cell response (Th2, Treg) was maintained. Thus, deletion or TNF-mediated restriction of Arg1 unleashes the production of NO by NOS2, which is critical for pathogen control.

Macrophages and cancer: from mechanisms to therapeutic implications.

Infiltration by immune cells is a hallmark of most forms of malignancy. In this context, tumor-associated macrophages (TAMs) represent key regulators of the complex interplay between the immune system and cancer. We discuss evidence indicating that in many settings TAMs fuel, rather than limit, tumor progression, and negatively impact on responses to therapy. We discuss how the unique functional properties of TAMs are shaped by tumor-derived signals, placing TAM development in the context of the broader understanding of the cellular and molecular mechanisms controlling macrophage origin, differentiation, and maintenance in tissues. Finally, we provide examples of how a molecular understanding of the relationships between TAMs and the tumor microenvironment may lead to improved cancer therapies.

A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages.

The transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8-PU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRF-AP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites.