Development of a High-Throughput Functional Screen Using Nanowell-Assisted Cell Patterning

Living-cell-based screens can facilitate lead discovery of functional therapeutics of interest. A versatile and scalable method is reported that uses dense arrays of nanowells for imparting defined patterns on monolayers of cells. It is shown that this approach can coordinate a multi-component biological assay by designing and implementing a high-throughput, functional nanoliter-scale neutralization assay to identify neutra­lizing antibodies against HIV.National Cancer Institute (U.S.) (P30-CA14051

Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris

Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, ‘hit-to-lead identification’ remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigenspecific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).Bill & Melinda Gates FoundationUnited States. Defense Advanced Research Projects AgencySpace and Naval Warfare Systems Center San Diego (U.S.) (Contract N66001-13-C-4025)W. M. Keck FoundationNational Institute of Allergy and Infectious Diseases (U.S.) (U19AI090970).National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Support (Core) Grant P30-CA14051

Diverse antiviral IgG effector activities are predicted by unique biophysical antibody features

Background: The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated. Results: In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach. Conclusions: In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies

TGF-β1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from the rupture of a blood vessel in the brain, leading to a mass of blood within the brain parenchyma. The injury causes a rapid inflammatory reaction that includes activation of the tissue-resident microglia and recruitment of blood-derived macrophages and other leukocytes. In this work, we investigated the specific responses of microglia following ICH with the aim of identifying pathways that may aid in recovery after brain injury. We used longitudinal transcriptional profiling of microglia in a murine model to determine the phenotype of microglia during the acute and resolution phases of ICH in vivo and found increases in TGF-β1 pathway activation during the resolution phase. We then confirmed that TGF-β1 treatment modulated inflammatory profiles of microglia in vitro. Moreover, TGF-β1 treatment following ICH decreased microglial Il6 gene expression in vivo and improved functional outcomes in the murine model. Finally, we observed that patients with early increases in plasma TGF-β1 concentrations had better outcomes 90 days after ICH, confirming the role of TGF-β1 in functional recovery from ICH. Taken together, our data show that TGF-β1 modulates microglia-mediated neuroinflammation after ICH and promotes functional recovery, suggesting that TGF-β1 may be a therapeutic target for acute brain injury

Molecular characterization of T cells across disease states in the central nervous system

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017Cataloged from PDF version of thesis. "February 2017."Includes bibliographical references (pages 157-170).The local cytokine milieu shapes the nature and function of immune cells and by extension the overall course of tissue-specific immune responses. In the context of cancer and autoimmunity, two opposing immune responses, the local immune environment can lead to dysfunctional T cell states. Understanding the mechanisms that distinguish these two states is key to identifying unique pathways that could be induced in autoimmunity and relieved without causing autoimmunity in cancer. We sought to characterize unique and shared T cell states in the context of multiple sclerosis (MS) and glioblastoma (GBM) using a combination of immunophenotyping and functional approaches, including ultra low-input transcriptional profiling. First we use a series of unbiased approaches to identify functional differences between auto-reactive T cells derived from MS or healthy donors.We found that MS-derived CCR6⁺ T cells produce pathogenic inflammatory cytokines IFN-[upsilon], IL-17, and GM-CSF, while healthy controls produce IL-10 in response to myelin antigen. We also identified a transcriptional signature that characterizes these cells from MS donors and highlights the role of CTLA-4 signaling in autoreactive T cells derived from healthy donors. Next, we sought to better understand the role of another co-inhibitory receptor, PD-1, specifically in the context of Treg and CD4⁺ T effector function in GBM, a central nervous system cancer. We identify unique signatures of dysfunction in both the CD4⁺ T cell and Treg compartment correlated with PD-1 expression. Finally, adverse development of autoimmunity in the context of treatment with blocking CTLA-4 antibodies suggests that studies directly comparing T cells in the context of autoimmunity and cancer can yield valuable insight into mechanisms of T cell dysfunction.Through a transcriptional comparison of isolated T cell subsets from the spinal fluid of MS patients, tumors of GBM patients, and matched blood we identify common biological mechanisms of CNS T cells and identify unique signatures of CNS exhaustion. Taken together, our data suggests that the CNS may be enriched for pathogenic cells in the context of both MS and GBM.by Brittany A. Goods.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Biological Engineerin

Single-cell transcriptomic atlas of the human retina identifies cell types associated with age-related macular degeneration

Genome-wide association studies (GWAS) have identified genetic variants associated with age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. However, it has been challenging to identify the cell types associated with AMD given the genetic complexity of the disease. Here we perform massively parallel single-cell RNA sequencing (scRNA-seq) of human retinas using two independent platforms, and report the first single-cell transcriptomic atlas of the human retina. Using a multi-resolution network-based analysis, we identify all major retinal cell types, and their corresponding gene expression signatures. Heterogeneity is observed within macroglia, suggesting that human retinal glia are more diverse than previously thought. Finally, GWAS-based enrichment analysis identifies glia, vascular cells, and cone photoreceptors to be associated with the risk of AMD. These data provide a detailed analysis of the human retina, and show how scRNA-seq can provide insight into cell types involved in complex, inflammatory genetic diseases.National Institutes of Health (U.S.) (Grant F32-AI136459)National Institutes of Health (U.S.) (Grant U01-MH119509)National Institutes of Health (U.S.) (Grant R01-AG062335)National Institutes of Health (U.S.) (Grant U01-NS110453

Functional inflammatory profiles distinguish myelin-reactive T cells from patients with multiple sclerosis

Myelin-reactive T cells have been identified in patients with multiple sclerosis (MS) and healthy subjects with comparable frequencies, but the contribution of these autoreactive T cells to disease pathology remains unknown. A total of 13,324 T cell libraries generated from blood of 23 patients and 22 healthy controls were interrogated for reactivity to myelin antigens. Libraries derived from CCR6+ myelin-reactive T cells from patients with MS exhibited significantly enhanced production of interferon-γ (IFN-γ), interleukin-17 (IL-17), and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared to healthy controls. Single-cell clones isolated by major histocompatibility complex/peptide tetramers from CCR6+ T cell libraries also secreted more proinflammatory cytokines, whereas clones isolated from controls secreted more IL-10. The transcriptomes of myelin-specific CCR6+ T cells from patients with MS were distinct from those derived from healthy controls and, notably, were enriched in T helper cell 17 (TH17)–induced experimental autoimmune encephalitis gene signatures, and gene signatures derived from TH17 cells isolated other human autoimmune diseases. These data, although not causal, imply that functional differences between antigen-specific T cells from MS and healthy controls are fundamental to disease development and support the notion that IL-10 production from myelin-reactive T cells may act to limit disease progression or even pathogenesis

Blood handling and leukocyte isolation methods impact the global transcriptome of immune cells

Background Transcriptional profiling with ultra-low input methods can yield valuable insights into disease, particularly when applied to the study of immune cells using RNA-sequencing. The advent of these methods has allowed for their use in profiling cells collected in clinical trials and other studies that involve the coordination of human-derived material. To date, few studies have sought to quantify what effects that collection and handling of this material can have on resulting data. Results We characterized the global effects of blood handling, methods for leukocyte isolation, and preservation media on low numbers of immune cells isolated from blood. We found overall that storage/shipping temperature of blood prior to leukocyte isolation and sorting led to global changes in both CD8+ T cells and monocytes, including alterations in immune-related gene sets. We found that the use of a leukocyte filtration system minimized these alterations and we applied this method to generate high-quality transcriptional data from sorted immune cells isolated from the blood of intracerebral hemorrhage patients and matched healthy controls. Conclusions Our data underscore the necessity of processing samples with comparably defined protocols prior to transcriptional profiling and demonstrate that a filtration method can be applied to quickly isolate immune cells of interest while minimizing transcriptional bias. Keywords: Immune profiling; Peripheral blood mononuclear cells; Transcriptome; RNA-seqNational Institute of Neurological Diseases and Stroke (U.S.) (Grant 5R01NS097728–02)National Cancer Institute (U.S.) (Grant P30-CA14051