Neutrophil-activating therapy for the treatment of cancer.

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

Longitudinal study of 2 patients with cyclic thrombocytopenia, STAT3 and MPL mutations.

Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to 2 patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients, rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells.

Dendritic cells (DCs) are powerful antigen presenting cells, derived from bone marrow progenitors (cDCs) and monocytes (moDCs), that can shape the immune response by priming either proinflammatory or tolerogenic immune effector cells. The cellular mechanisms responsible for the generation of DCs that will prime a proinflammatory or tolerogenic response are poorly understood. Here we describe a novel mechanism by which tolerogenic DCs are formed from monocytes. When human monocytes were cultured with CD4+FoxP3+ natural regulatory T cells (Tregs) and T helper cells (Th) from healthy donor blood, they differentiated into regulatory DCs (DC Reg ), capable of generating induced Tregs from naïve T cells. DC Reg exhibited morphology, surface phenotype, cytokine secretion, and transcriptome that were distinct from other moDCs including those derived from monocytes cultured with Th or with GM-CSF/IL-4, as well as macrophages (MΦ). Direct cell contact between monocytes, Tregs and Th, along with Treg-derived CTLA-4, IL-10 and TGF-ß, was required for the phenotypic differentiation of DC Reg , although only IL-10 was required for imprinting the Treg-inducing capacity of DC Reg . High ratios of Treg:Th, along with monocytes and DC Reg similar in function and phenotype to those induced in vitro, were present in situ in human colorectal cancer specimens. Thus, through the combined actions of Tregs and Th, monocytes differentiate into DCs with regulatory properties, forming a positive feedback loop to reinforce Treg initiated immune regulation. This mechanism may contribute to immune tolerance in tissues such as tumors, which contain an abundance of Tregs, Th and monocytes.

An Immunosuppressive Dendritic Cell Subset Accumulates at Secondary Sites and Promotes Metastasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) after complete surgical resection is often followed by distant metastatic relapse for reasons that remain unclear. In this study, we investigated how the immune response at secondary sites affects tumor spread in murine models of metastatic PDAC. Early metastases were associated with dense networks of CD11b+CD11c+MHC-II+CD24+CD64lowF4/80low dendritic cells (DC), which developed from monocytes in response to tumor-released GM-CSF. These cells uniquely expressed MGL2 and PD-L2 in the metastatic microenvironment and preferentially induced the expansion of T regulatory cells (Treg) in vitro and in vivo Targeted depletion of this DC population in Mgl2DTR hosts activated cytotoxic lymphocytes, reduced Tregs, and inhibited metastasis development. Moreover, blocking PD-L2 selectively activated CD8 T cells at secondary sites and suppressed metastasis, suggesting that the DCs use this particular pathway to inhibit CD8 T-cell-mediated tumor immunity. Phenotypically similar DCs accumulated at primary and secondary sites in other models and in human PDAC. These studies suggest that a discrete DC subset both expands Tregs and suppresses CD8 T cells to establish an immunosuppressive microenvironment conducive to metastasis formation. Therapeutic strategies to block the accumulation and immunosuppressive activity of such cells may help prevent PDAC progression and metastatic relapse after surgical resection. Cancer Res; 77(15); 4158-70. ©2017 AACR.

A distinct subset of plasmacytoid dendritic cells induces activation and differentiation of B and T lymphocytes.

Plasmacytoid dendritic cells (pDCs) are known mainly for their secretion of type I IFN upon viral encounter. We describe a CD2hiCD5+CD81+ pDC subset, distinguished by prominent dendrites and a mature phenotype, in human blood, bone marrow, and tonsil, which can be generated from CD34+ progenitors. These CD2hiCD5+CD81+ cells express classical pDC markers, as well as the toll-like receptors that enable conventional pDCs to respond to viral infection. However, their gene expression profile is distinct, and they produce little or no type I IFN upon stimulation with CpG oligonucleotides, likely due to their diminished expression of IFN regulatory factor 7. A similar population of CD5+CD81+ pDCs is present in mice and also does not produce type I IFN after CpG stimulation. In contrast to conventional CD5-CD81- pDCs, human CD5+CD81+ pDCs are potent stimulators of B-cell activation and antibody production and strong inducers of T-cell proliferation and Treg formation. These findings reveal the presence of a discrete pDC population that does not produce type I IFN and yet mediates important immune functions previously attributed to all pDCs.

Adipose tissue macrophages impair preadipocyte differentiation in humans.

AIM: The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation. METHODS: Abdominal subcutaneous(SAT) and visceral(VAT) adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified. RESULTS: Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p<0.001). With macrophage removal, SAT preadipocyte differentiation increased significantly as measured by immunofluorescence and gene expression, whereas VAT preadipocyte differentiation was unchanged. Adipocyte-secreted proinflammatory cytokines were higher and adiponectin lower in media from VAT vs SAT: macrophage removal reduced inflammatory cytokine and increased adiponectin secretion from both SAT and VAT adipocytes. Differentiation of preadipocytes from SAT but not VAT correlated inversely with systemic insulin resistance. CONCLUSIONS: The current results reveal that proinflammatory immune cells in human SAT are causally-related to impaired preadipocyte differentiation, which in turn is associated with systemic insulin resistance. In VAT, preadipocyte differentiation is poor even in the absence of tissue macrophages, pointing to inherent differences in fat storage potential between the two depots.

The receptor CD44 is associated with systemic insulin resistance and proinflammatory macrophages in human adipose tissue.

AIMS/HYPOTHESIS: Proinflammatory immune cell infiltration in human adipose tissue is associated with the development of insulin resistance. We previously identified, via a gene expression-based genome-wide association study, the cell-surface immune cell receptor CD44 as a functionally important gene associated with type 2 diabetes. We then showed that, compared with controls, Cd44 knockout mice were protected from insulin resistance and adipose tissue inflammation during diet-induced obesity. We thus sought to test whether CD44 is associated with adipose tissue inflammation and insulin resistance in humans. METHODS: Participants included 58 healthy, overweight/moderately obese white adults who met predetermined criteria for insulin resistance or insulin sensitivity based on the modified insulin-suppression test. Serum was collected from 43 participants to measure circulating concentrations of CD44. Subcutaneous adipose tissue was obtained from 17 participants to compare CD44, its ligand osteopontin (OPN, also known as SPP1) and pro-inflammatory gene expression. CD44 expression on adipose tissue macrophage (ATM) surfaces was determined by flow cytometry. RESULTS: Serum CD44 concentrations were significantly increased in insulin-resistant (IR) participants. CD44 gene expression in subcutaneous adipose tissue was threefold higher in the IR subgroup. The expression of OPN, CD68 and IL6 was also significantly elevated in IR individuals. CD44 gene expression correlated significantly with CD68 and IL6 expression. CD44 density on ATMs was associated with proinflammatory M1 polarisation. CONCLUSIONS/INTERPRETATION: CD44 and OPN in human adipose tissue are associated with localised inflammation and systemic insulin resistance. This receptor-ligand pair is worthy of further research as a potentially modifiable contributor to human insulin resistance and type 2 diabetes.

T(H)1, T(H)2, and T(H)17 cells instruct monocytes to differentiate into specialized dendritic cell subsets.

Monocytes and T helper (T(H)) cells rapidly infiltrate inflamed tissues where monocytes differentiate into inflammatory dendritic cells (DCs) through undefined mechanisms. Our studies indicate that T(H) cells frequently interact with monocytes in inflamed skin and elicit the differentiation of specialized DC subsets characteristic of these lesions. In psoriasis lesions, T(H)1 and T(H)17 cells interact with monocytes and instruct these cells to differentiate into T(H)1- and T(H)17-promoting DCs, respectively. Correspondingly, in acute atopic dermatitis, T(H)2 cells interact with monocytes and elicit the formation of T(H)2-promoting DCs. DC formation requires GM-CSF and cell contact, whereas T(H) subset specific cytokines dictate DC function and the expression of DC subset specific surface molecules. Moreover, the phenotypes of T cell-induced DC subsets are maintained after subsequent stimulation with a panel of TLR agonists, suggesting that T(H)-derived signals outweigh downstream TLR signals in their influence on DC function. These findings indicate that T(H) cells govern the formation and function of specialized DC subsets.