A deep convolutional neural network for segmentation of whole-slide pathology images identifies novel tumour cell-perivascular niche interactions that are associated with poor survival in glioblastoma

Background: Glioblastoma is the most aggressive type of brain cancer with high-levels of intra- and inter-tumour heterogeneity that contribute to its rapid growth and invasion within the brain. However, a spatial characterisation of gene signatures and the cell types expressing these in different tumour locations is still lacking. Methods: We have used a deep convolutional neural network (DCNN) as a semantic segmentation model to segment seven different tumour regions including leading edge (LE), infiltrating tumour (IT), cellular tumour (CT), cellular tumour microvascular proliferation (CTmvp), cellular tumour pseudopalisading region around necrosis (CTpan), cellular tumour perinecrotic zones (CTpnz) and cellular tumour necrosis (CTne) in digitised glioblastoma histopathological slides from The Cancer Genome Atlas (TCGA). Correlation analysis between segmentation results from tumour images together with matched RNA expression data was performed to identify genetic signatures that are specific to different tumour regions. Results: We found that spatially resolved gene signatures were strongly correlated with survival in patients with defined genetic mutations. Further in silico cell ontology analysis along with single-cell RNA sequencing data from resected glioblastoma tissue samples showed that these tumour regions had different gene signatures, whose expression was driven by different cell types in the regional tumour microenvironment. Our results further pointed to a key role for interactions between microglia/pericytes/monocytes and tumour cells that occur in the IT and CTmvp regions, which may contribute to poor patient survival. Conclusions: This work identified key histopathological features that correlate with patient survival and detected spatially associated genetic signatures that contribute to tumour-stroma interactions and which should be investigated as new targets in glioblastoma. The source codes and datasets used are available in GitHub: https://github.com/amin20/GBM_WSSM.Amin Zadeh Shirazi, Mark D. McDonnell, Eric Fornaciari, Narjes Sadat Bagherian, Kaitlin G. Scheer, Michael S. Samuel, Mahdi Yaghoobi, Rebecca J. Ormsby, Santosh Poonnoose, Damon J. Tumes, and Guillermo A. Gome

The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota

Immune agonist antibodies (IAAs) are promising immunotherapies that target co-stimulatory receptors to induce potent anti-tumor immune responses, particularly when combined with checkpoint inhibitors. Unfortunately, their clinical translation is hampered by serious dose-limiting, immune-mediated toxicities, including high-grade and sometimes fatal liver damage, cytokine release syndrome (CRS), and colitis. We show that the immunotoxicity, induced by the IAAs anti-CD40 and anti-CD137, is dependent on the gut microbiota. Germ-free or antibiotic-treated mice have significantly reduced colitis, CRS, and liver damage following IAA treatment compared with conventional mice or germ-free mice recolonized via fecal microbiota transplant. MyD88 signaling is required for IAA-induced CRS and for anti-CD137-induced, but not anti-CD40-induced, liver damage. Importantly, antibiotic treatment does not impair IAA anti-tumor efficacy, alone or in combination with anti-PD1. Our results suggest that microbiota-targeted therapies could overcome the toxicity induced by IAAs without impairing their anti-tumor activity.Stephen J. Blake, Jane James, Feargal J. Ryan, Jose Caparros-Martin, Georgina L. Eden, Yee C. Tee, John R. Salamon, Saoirse C. Benson, Damon J. Tumes, Anastasia Sribnaia, Natalie E. Stevens, John W. Finnie, Hiroki Kobayashi, Deborah L. White, Steve L. Wesselingh, Fergal O’Gara, Miriam A. Lynn, and David J. Lyn

Understanding mast cell heterogeneity at single cell resolution

Mast cells (MC)s are evolutionarily conserved, tissue-resident immune cells with diverse roles in allergy, cancer, and protection from infection by helminths and microorganisms. The significant diversity in MC development and tissue-specific functional characteristics has recently begun to be understood. Exciting developments in single-cell-based RNA, protein, and chromatin profiling technologies offer new opportunities to characterize MC heterogeneity and to uncover novel MC functions and subtypes; these developments might lead to new and clinically effective therapies for certain pathologies. In this review, we provide an overview of the current understanding of MC development and heterogeneity and discuss new insights gained from single-cell-based studies that may lead to future research directions and therapeutic opportunities.Gökhan Cildir, Kwok Ho Yip, Harshita Pant, Vinay Tergaonkar, Angel F. Lopez, and Damon J. Tume

Translating the biology of B common receptor-engaging cytokines into clinical medicine

The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called β-common or βc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.Harshita Pant, Timothy R. Hercus, Damon J. Tumes, Kwok Ho Yip, Michael W. Parker, Catherine M. Owczarek, Angel F. Lopez, and David P. Husto

Immunisation with the BCG and DTPw vaccines induces different programs of trained immunity in mice

Available online 22 April 2021In addition to providing pathogen-specific immunity, vaccines can also confer nonspecific effects (NSEs) on mortality and morbidity unrelated to the targeted disease. Immunisation with live vaccines, such as the BCG vaccine, has generally been associated with significantly reduced all-cause infant mortality. In contrast, some inactivated vaccines, such as the diphtheria, tetanus, whole-cell pertussis (DTPw) vaccine, have been controversially associated with increased all-cause mortality especially in female infants in high-mortality settings. The NSEs associated with BCG have been attributed, in part, to the induction of trained immunity, an epigenetic and metabolic reprograming of innate immune cells, increasing their responsiveness to subsequent microbial encounters. Whether non-live vaccines such as DTPw induce trained immunity is currently poorly understood. Here, we report that immunisation of mice with DTPw induced a unique program of trained immunity in comparison to BCG immunised mice. Altered monocyte and DC cytokine responses were evident in DTPw immunised mice even months after vaccination. Furthermore, splenic cDCs from DTPw immunised mice had altered chromatin accessibility at loci involved in immunity and metabolism, suggesting that these changes were epigenetically mediated. Interestingly, changing the order in which the BCG and DTPw vaccines were co-administered to mice altered subsequent trained immune responses. Given these differences in trained immunity, we also assessed whether administration of these vaccines altered susceptibility to sepsis in two different mouse models. Immunisation with either BCG or a DTPw-containing vaccine prior to the induction of sepsis did not significantly alter survival. Further studies are now needed to more fully investigate the potential consequences of DTPw induced trained immunity in different contexts and to assess whether other non-live vaccines also induce similar changes.Natalie E. Stevens, Marjolein van Wolfswinkel, Winnie Bao, Feargal J. Ryan, Byron Brook, Nelly Amenyogbe … et al

IgE receptor of mast cells signals mediator release and inflammation via adaptor protein 14-3-3ζ

Background: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation–dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. Objective: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. Methods: Genetic manipulation of 14-3-3z expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3z knockout mice were used to assess 14-3-3z function in a MCdependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3z were assessed in human nasal polyp tissue MCs. Results: IgE-dependent mediator release from human MCs was decreased by 14-3-3z knockdown and increased by 14-3-3z overexpression. Deletion of the 14-3-3z gene decreased IgEdependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. Conclusion: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3z. We identify a critical role for this p-Ser/ Thr–binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.Kwok Ho Yip, Jessica Chao, Carl Coolen, Harshita Pant, Anita Kral, William Smith, Quenten Schwarz, Michele A. Grimbaldeston, Stuart Pitson, Angel F. Lopez, Joanna Woodcock and Damon J. Tume

Anti-beta c mAb CSL311 inhibits human nasal polyp pathophysiology in a humanized mouse xenograft model

Letter to the EditorAbstract not availableKwok Ho Yip, Nicholas J. Wilson, Harshita Pant, Christopher L. Brown Samantha Busfield, Milica Ng ... et al