Cyclone: an accessible pipeline to analyze, evaluate and optimize multiparametric cytometry data.

In the past decade, high-dimensional single cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation which are computationally intense and difficult to evaluate and optimize. Here we present Cyclone, an analysis pipeline integrating dimensionality reduction, clustering, evaluation and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification, but also enables the unsupervised identification of lymphocytes and mononuclear phagocytes subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on variety of cytometry datasets which will further power immunology research and provide a scaffold for biological discovery.

Cyclone: an accessible pipeline to analyze, evaluate, and optimize multiparametric cytometry data.

In the past decade, high-dimensional single-cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation, which are computationally intense and difficult to evaluate and optimize. Here, we present Cytometry Clustering Optimization and Evaluation (Cyclone), an analysis pipeline integrating dimensionality reduction, clustering, evaluation, and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full-spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification but also enables the unsupervised identification of lymphocytes and mononuclear phagocyte subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on a variety of cytometry datasets, which will further power immunology research and provide a scaffold for biological discovery.

A tumor-specific mechanism of Treg enrichment mediated by the integrin αvß8.

Regulatory T cells (Tregs) that promote tumor immune evasion are enriched in certain tumors and correlate with poor prognosis. However, mechanisms for Treg enrichment remain incompletely understood. We described a mechanism for Treg enrichment in mouse and human tumors mediated by the αvß8 integrin. Tumor cell αvß8 bound to latent transforming growth factor-ß (L-TGF-ß) presented on the surface of T cells, resulting in TGF-ß activation and immunosuppressive Treg differentiation in vitro. In vivo, tumor cell αvß8 expression correlated with Treg enrichment, immunosuppressive Treg gene expression, and increased tumor growth, which was reduced in mice by αvß8 inhibition or Treg depletion. Structural modeling and cell-based studies suggested a highly geometrically constrained complex forming between αvß8-expressing tumor cells and L-TGF-ß-expressing T cells, facilitating TGF-ß activation, independent of release and diffusion, and providing limited access to TGF-ß inhibitors. These findings suggest a highly localized tumor-specific mechanism for Treg enrichment.

Integrin αvß8-expressing tumor cells evade host immunity by regulating TGF-ß activation in immune cells.

TGF-ß is a promising immunotherapeutic target. It is expressed ubiquitously in a latent form that must be activated to function. Determination of where and how latent TGF-ß (L-TGF-ß) is activated in the tumor microenvironment could facilitate cell- and mechanism-specific approaches to immunotherapeutically target TGF-ß. Binding of L-TGF-ß to integrin αvß8 results in activation of TGF-ß. We engineered and used αvß8 antibodies optimized for blocking or detection, which - respectively - inhibit tumor growth in syngeneic tumor models or sensitively and specifically detect ß8 in human tumors. Inhibition of αvß8 potentiates cytotoxic T cell responses and recruitment of immune cells to tumor centers - effects that are independent of PD-1/PD-L1. ß8 is expressed on the cell surface at high levels by tumor cells, not immune cells, while the reverse is true of L-TGF-ß, suggesting that tumor cell αvß8 serves as a platform for activating cell-surface L-TGF-ß presented by immune cells. Transcriptome analysis of tumor-associated lymphoid cells reveals macrophages as a key cell type responsive to ß8 inhibition with major increases in chemokine and tumor-eliminating genes. High ß8 expression in tumor cells is seen in 20%-80% of various cancers, which rarely coincides with high PD-L1 expression. These data suggest tumor cell αvß8 is a PD-1/PD-L1-independent immunotherapeutic target.

An OX40/OX40L interaction directs successful immunity to hepatitis B virus.

Depending on age of acquisition, hepatitis B virus (HBV) can induce a cell-mediated immune response that results in either cure or progressive liver injury. In adult-acquired infection, HBV antigens are usually cleared, whereas in infancy-acquired infection, they persist. Individuals infected during infancy therefore represent the majority of patients chronically infected with HBV (CHB). A therapy that can promote viral antigen clearance in most CHB patients has not been developed and would represent a major health care advance and cost mitigator. Using an age-dependent mouse model of HBV clearance and persistence in conjunction with human blood and liver tissue, we studied mechanisms of viral clearance to identify new therapeutic targets. We demonstrate that age-dependent expression of the costimulatory molecule OX40 ligand (OX40L) by hepatic innate immune cells is pivotal in determining HBV immunity, and that treatment with OX40 agonists leads to improved HBV antigen clearance in young mice, as well as increased strength of T cell responses in young mice and adult mice that were exposed to HBV when they were young and developed a CHB serological profile. Similarly, in humans, we show that hepatic OX40L transcript expression is age-dependent and that increased OX40 expression on peripheral CD4+ T cells in adults is associated with HBV clearance. These findings provide new mechanistic understanding of the immune pathways and cells necessary for HBV immunity and identify potential therapeutic targets for resolving CHB.

FIV diversity: FIV Ple subtype composition may influence disease outcome in African lions.

Feline immunodeficiency virus (FIV) infects domestic cats and at least 20 additional species of non-domestic felids throughout the world. Strains specific to domestic cat (FIV(Fca)) produce AIDS-like disease progression, sequelae and pathology providing an informative model for HIV infection in humans. Less is known about the immunological and pathological influence of FIV in other felid species although multiple distinct strains of FIV circulate in natural populations. As in HIV-1 and HIV-2, multiple diverse cross-species infections may have occurred. In the Serengeti National Park, Tanzania, three divergent subtypes of lion FIV (FIV(Ple)) are endemic, whereby 100% of adult lions are infected with one or more of these strains. Herein, the relative distribution of these subtypes in the population are surveyed and, combined with observed differences in lion mortality due to secondary infections based on FIV(Ple) subtypes, the data suggest that FIV(Ple) subtypes may have different patterns of pathogenicity and transmissibility among wild lion populations.