Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Introduction: Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. Methods: In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Results: Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Discussion: Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Altered Treg Infiltration after Discoidin Domain Receptor 1 (DDR1) Inhibition and Knockout Promotes Tumor Growth in Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer-related death worldwide. Discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, has been associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). However, its role in tumorigenesis remains poorly understood. This work aimed to explore the impact of DDR1 expression on immune cell infiltration in lung adenocarcinoma. Pharmacological inhibition and knockout of DDR1 were used in an immunocompetent mouse model of KRAS/p53-driven lung adenocarcinoma (LUAD). Tumor cells were engrafted subcutaneously, after which tumors were harvested for investigation of immune cell composition via flow cytometry. The Cancer Genome Atlas (TCGA) cohort was used to perform gene expression analysis of 509 patients with LUAD. Pharmacological inhibition and knockout of DDR1 increased the tumor burden, with DDR1 knockout tumors showing a decrease in CD8+ cytotoxic T cells and an increase in CD4+ helper T cells and regulatory T cells. TCGA analysis revealed that low-DDR1-expressing tumors showed higher FoxP3 (regulatory T-cell marker) expression than high-DDR1-expressing tumors. Our study showed that under certain conditions, the inhibition of DDR1, a potential therapeutic target in cancer treatment, might have negative effects, such as inducing a pro-tumorigenic tumor microenvironment. As such, further investigations are necessary.

Identification of Novel Low-Density Neutrophil Markers Through Unbiased High-Dimensional Flow Cytometry Screening in Non-Small Cell Lung Cancer Patients.

Neutrophils have been described as a phenotypically heterogeneous cell type that possess both pro- and anti-tumor properties. Recently, a subset of neutrophils isolated from the peripheral blood mononuclear cell (PBMC) fraction has been described in cancer patients. These low-density neutrophils (LDNs) show a heterogeneous maturation state and have been associated with pro-tumor properties in comparison to mature, high-density neutrophils (HDNs). However, additional studies are necessary to characterize this cell population. Here we show new surface markers that allow us to discriminate between LDNs and HDNs in non-small cell lung cancer (NSCLC) patients and assess their potential as diagnostic/prognostic tool. LDNs were highly enriched in NSCLC patients (median=20.4%, range 0.3-76.1%; n=26) but not in healthy individuals (median=0.3%, range 0.1-3.9%; n=14). Using a high-dimensional human cell surface marker screen, we identified 12 surface markers that were downregulated in LDNs when compared to HDNs, while 41 surface markers were upregulated in the LDN subset. Using flow cytometry, we confirmed overexpression of CD36, CD41, CD61 and CD226 in the LDN fraction. In summary, our data support the notion that LDNs are a unique neutrophil population and provide novel targets to clarify their role in tumor progression and their potential as diagnostic and therapeutic tool.

Monoacylglycerol lipase deficiency in the tumor microenvironment slows tumor growth in non-small cell lung cancer.

Monoacylglycerol lipase (MGL) expressed in cancer cells influences cancer pathogenesis but the role of MGL in the tumor microenvironment (TME) is less known. Using a syngeneic tumor model with KP cells (KrasLSL-G12D/p53fl/fl; from mouse lung adenocarcinoma), we investigated whether TME-expressed MGL plays a role in tumor growth of non-small cell lung cancer (NSCLC). In sections of human and experimental NSCLC, MGL was found in tumor cells and various cells of the TME including macrophages and stromal cells. Mice treated with the MGL inhibitor JZL184 as well as MGL knock-out (KO) mice exhibited a lower tumor burden than the controls. The reduction in tumor growth was accompanied by an increased number of CD8+ T cells and eosinophils. Naïve CD8+ T cells showed a shift toward more effector cells in MGL KOs and an increased expression of granzyme-B and interferon-γ, indicative of enhanced tumoricidal activity. 2-arachidonoyl glycerol (2-AG) was increased in tumors of MGL KO mice, and dose-dependently induced differentiation and migration of CD8+ T cells as well as migration and activation of eosinophils in vitro. Our results suggest that next to cancer cell-derived MGL, TME cells expressing MGL are responsible for maintaining a pro-tumorigenic environment in tumors of NSCLC.

Burst statistics in an early biofilm quorum sensing model: the role of spatial colony-growth heterogeneity.

Quorum-sensing bacteria in a growing colony of cells send out signalling molecules (so-called "autoinducers") and themselves sense the autoinducer concentration in their vicinity. Once-due to increased local cell density inside a "cluster" of the growing colony-the concentration of autoinducers exceeds a threshold value, cells in this clusters get "induced" into a communal, multi-cell biofilm-forming mode in a cluster-wide burst event. We analyse quantitatively the influence of spatial disorder, the local heterogeneity of the spatial distribution of cells in the colony, and additional physical parameters such as the autoinducer signal range on the induction dynamics of the cell colony. Spatial inhomogeneity with higher local cell concentrations in clusters leads to earlier but more localised induction events, while homogeneous distributions lead to comparatively delayed but more concerted induction of the cell colony, and, thus, a behaviour close to the mean-field dynamics. We quantify the induction dynamics with quantifiers such as the time series of induction events and burst sizes, the grouping into induction families, and the mean autoinducer concentration levels. Consequences for different scenarios of biofilm growth are discussed, providing possible cues for biofilm control in both health care and biotechnology.

The Iceberg Map of germline mutations in childhood cancer: focus on primary immunodeficiencies.

PURPOSE OF REVIEW: The risk of cancer is higher, and its outcome is worse in patients with primary immunodeficiency (PID) than in members of the general population. Thus, the inter-relationship of malignant diseases with PIDs requires more study. RECENT FINDINGS: Large genetic screens identified a vast number of germline mutations in childhood cancer patient samples. Although TP53 was the most frequent single gene identified as mutated, many PID disorders like DNA repair defects are among the inborn causes of childhood cancer. We provide a comprehensive analysis of compiled data from seven recent studies that focused on germline genetic landscapes and preexisting conditions in pediatric oncology. As potentially causal germline variants were identified in ≈8% of malignancies in children and adolescents, we visualized this proportion as the 'tips of the icebergs'. The results of additional network analyses showed the shared patterns of germline mutations in various malignancies and yielded a spatial distribution of the 'icebergs'. SUMMARY: The 'iceberg map of germline mutations in childhood cancers' was created to increase the awareness of the inborn genetic underpinnings of childhood malignancies and their relationships with immunodeficiencies. Needs and perspectives of clinical immunologists and pediatric oncologists to both improve patient care and guide research at this critical interface are discussed. VIDEO ABSTRACT.