Siew Cheng Wong

The three human monocyte subsets: implications for health and disease

Human blood monocytes are heterogeneous and conventionally subdivided into two subsets based on CD16 expression. Recently, the official nomenclature subdivides monocytes into three subsets, the additional subset arising from the segregation of the CD16+ monocytes into two based on relative expression of CD14. Recent whole genome analysis reveal that specialized functions and phenotypes can be attributed to these newly defined monocyte subsets. In this review, we discuss these recent results, and also the description and utility of this new segregation in several disease conditions. We also discuss alternative markers for segregating the monocyte subsets, for example using Tie-2 and slan, which do not necessarily follow the official method of segregating monocyte subsets based on relative CD14 and CD16 expressions.

Increased Myeloid-Derived Suppressor Cells in Gastric Cancer Correlate with Cancer Stage and Plasma S100A8/A9 Proinflammatory Proteins

Immune dysfunction may contribute to tumor progression in gastric cancer (GC) patients. One mechanism of immune dysfunction is the suppression of T cell activation and impairment of the efficacy of cancer immunotherapy by myeloid-derived suppressor cells (MDSCs). We assessed the phenotype and immunosuppressive function of MDSCs in GC patients. We further investigated the role of S100A8/A9 in GC and the relationship between S100A8/A9 and MDSC function. Lastly, the effect of MDSCs on survival rates and its potential as a prognostic factor in GC patients were investigated. MDSCs from PBMCs of GC patients were identified by comparing the expression of specific surface markers with PBMCs from healthy individuals. The ability of MDSCs to suppress T lymphocyte response and the effect of S100A8/A9 and RAGE blocking were tested in vitro by (autologous) MLR. GC patients had significantly more MDSCs than healthy individuals. These MDSCs suppressed both T lymphocyte proliferation and IFN-γ production and had high arginase-I expression. Levels of S100A8/A9 in plasma were higher in GC patients compared with healthy individuals, and they correlated with MDSC levels in the blood. Blocking of S100A8/A9 itself and the S100A8/A9 receptor RAGE on MDSCs from GC patients abrogated T cell effector function. We found that high levels of MDSCs correlated with more advanced cancer stage and with reduced survival (p = 0.006). S100A8/A9 has been identified as a potential target to modulate antitumor immunity by reversing MDSC-mediated immunosuppression.

Susceptibility and Response of Human Blood Monocyte Subsets to Primary Dengue Virus Infection

Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16− and CD16+ subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16− and CD16+ blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16+ monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1β, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16+ monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease.

Warburg metabolism in tumor-conditioned macrophages promotes metastasis in human pancreatic ductal adenocarcinoma

ABSTRACT Patients with pancreatic ductal adenocarcinoma (PDAC) face a clinically intractable disease with poor survival rates, attributed to exceptionally high levels of metastasis. Epithelial-to-mesenchymal transition (EMT) is pronounced at inflammatory foci within the tumor; however, the immunological mechanisms promoting tumor dissemination remain unclear. It is well established that tumors exhibit the Warburg effect, a preferential use of glycolysis for energy production, even in the presence of oxygen, to support rapid growth. We hypothesized that the metabolic pathways utilized by tumor-infiltrating macrophages are altered in PDAC, conferring a pro-metastatic phenotype. We generated tumor-conditioned macrophages in vitro, in which human peripheral blood monocytes were cultured with conditioned media generated from normal pancreatic or PDAC cell lines to obtain steady-state and tumor-associated macrophages (TAMs), respectively. Compared with steady-state macrophages, TAMs promoted vascular network formation, augmented extravasation of tumor cells out of blood vessels, and induced higher levels of EMT. TAMs exhibited a pronounced glycolytic signature in a metabolic flux assay, corresponding with elevated glycolytic gene transcript levels. Inhibiting glycolysis in TAMs with a competitive inhibitor to Hexokinase II (HK2), 2-deoxyglucose (2DG), was sufficient to disrupt this pro-metastatic phenotype, reversing the observed increases in TAM-supported angiogenesis, extravasation, and EMT. Our results indicate a key role for metabolic reprogramming of tumor-infiltrating macrophages in PDAC metastasis, and highlight the therapeutic potential of using pharmacologics to modulate these metabolic pathways.

Contact-dependent carcinoma aggregate dispersion by M2a macrophages via ICAM-1 and β2 integrin interactions

Tumor-associated macrophages (TAMs) can constitute up to 50% of the tumor mass and have strong implications in tumor progression and metastasis. Macrophages are plastic and can polarize to various subtypes that differ in terms of surface receptor expression as well as cytokine and chemokine production and effector function. Conventionally, macrophages are grouped into two major subtypes: the classically activated M1 macrophages and the alternatively activated M2 macrophages. M1 macrophages are pro-inflammatory, promote T helper (Th) 1 responses, and show tumoricidal activity, whereas M2 macrophages contribute to tissue repair and promote Th2 responses. Herein, we present a microfluidic system integrating tumor cell aggregates and subtypes of human monocyte-derived macrophages in a three-dimensional hydrogel scaffold, in close co-culture with an endothelial monolayer to create an in vitro tumor microenvironment. This platform was utilized to study the role of individual subtypes of macrophages (M0, M1, M2a, M2b and M2c) in human lung adenocarcinoma (A549) aggregate dispersion, as a representation of epithelial-mesenchymal transition (EMT). A significant difference was observed when M2a macrophages were in direct contact with or separated from A549 aggregates, suggesting a possible mechanism for proximity-induced, contact-dependent dissemination via ICAM-1 and integrin β2 interactions. Indeed, M2a macrophages tended to infiltrate and release cells from carcinoma cell aggregates. These findings may help in the development of immunotherapies based on enhancing the tumor-suppressive properties of TAMs.

Engineering a 3D microfluidic culture platform for tumor-treating field application

The limitations of current cancer therapies highlight the urgent need for a more effective therapeutic strategy. One promising approach uses an alternating electric field; however, the mechanisms involved in the disruption of the cancer cell cycle as well as the potential adverse effects on non-cancerous cells must be clarified. In this study, we present a novel microfluidic device with embedded electrodes that enables the application of an alternating electric field therapy to cancer cells in a 3D extracellular matrix. To demonstrate the potential of our system to aid in designing and testing new therapeutic approaches, cancer cells and cancer cell aggregates were cultured individually or co-cultured with endothelial cells. The metastatic potential of the cancer cells was reduced after electric field treatment. Moreover, the proliferation rate of the treated cancer cells was lower compared with that of the untreated cells, whereas the morphologies and proliferative capacities of the endothelial cells were not significantly affected. These results demonstrate that our novel system can be used to rapidly screen the effect of an alternating electric field on cancer and normal cells within an in vivo-like microenvironment with the potential to optimize treatment protocols and evaluate synergies between tumor-treating field treatment and chemotherapy.

Human Regulatory B Cells Combine Phenotypic and Genetic Hallmarks with a Distinct Differentiation Fate

Regulatory B cells (B-reg) produce IL-10 and suppress inflammation in both mice and humans, but limited data on the phenotype and function of these cells have precluded detailed assessment of their contribution to host immunity. In this article, we report that human B-reg cannot be defined based on a phenotype composed of conventional B cell markers, and that IL-10 production can be elicited in both the CD27+ memory population and naive B cell subset after only a brief stimulation in vitro. We therefore sought to obtain a better definition of IL-10–producing human B-regs using a multiparameter analysis of B cell phenotype, function, and gene expression profile. Exposure to CpG and anti-Ig are the most potent stimuli for IL-10 secretion in human B cells, but microarray analysis revealed that human B cells cotreated with these reagents resulted in only ∼0.7% of genes being differentially expressed between IL-10+ and IL-10− cells. Instead, connectivity map analysis revealed that IL-10–secreting B cells are those undergoing specific differentiation toward a germinal center fate, and we identified a CD11c+ B cell subset that was not capable of producing IL-10 even under optimal conditions. Our findings will assist in the identification of a broader range of human pro–B-reg populations that may represent novel targets for immunotherapy.

CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes

Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16− expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated β2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases.

The need to identify myeloid dendritic cell progenitors in human blood

Dendritic cells (DC) are professional phagocytes possessing a unique ability to sense perturbations in the tissue microenvironment and promote adaptive immune responses, whilst maintaining immunological tolerance. Mouse myeloid DC progenitors with the ability to migrate through the blood and replenish the DC pool have been identified in bone marrow but the ontogeny of human DC is poorly understood. Access to lymphoid tissues for human DC isolation is severely limited and researchers have resorted to the use of in vitro derivation systems in attempts to understand DC development, which may result in misleading conclusions. The identification of a human DC progenitor in blood would greatly enhance the understanding of DC homeostasis and their role in pathogenesis.

Checkpoint blockade immunotherapy reshapes the high-dimensional phenotypic heterogeneity of murine intratumoural neoantigen-specific CD8 + T cells

The analysis of neoantigen-specific CD8+ T cells in tumour-bearing individuals is challenging due to the small pool of tumour antigen-specific T cells. Here we show that mass cytometry with multiplex combinatorial tetramer staining can identify and characterize neoantigen-specific CD8+ T cells in mice bearing T3 methylcholanthrene-induced sarcomas that are susceptible to checkpoint blockade immunotherapy. Among 81 candidate antigens tested, we identify T cells restricted to two known neoantigens simultaneously in tumours, spleens and lymph nodes in tumour-bearing mice. High-dimensional phenotypic profiling reveals that antigen-specific, tumour-infiltrating T cells are highly heterogeneous. We further show that neoantigen-specific T cells display a different phenotypic profile in mice treated with anti-CTLA-4 or anti-PD-1 immunotherapy, whereas their peripheral counterparts are not affected by the treatments. Our results provide insights into the nature of neoantigen-specific T cells and the effects of checkpoint blockade immunotherapy.Immune checkpoint blockade (ICB) therapies can unleash anti-tumour T-cell responses. Here the authors show, by integrating MHC tetramer multiplexing, mass cytometry and high-dimensional analyses, that neoantigen-specific, tumour-infiltrating T cells are highly heterogeneous and are subjected to ICB modulations.