Biology subjects

Kimball, A. K.

Publications and source records attributed to Kimball, A. K..

4 recordsLinked to original sources

bioRxiv2020

Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

Virus infection is frequently characterized using bulk cell populations. How these findings correspond to infection in individual cells remains unclear. Here, we integrate high-dimensional single-cell approaches to quantify viral and host RNA and protein expression signatures using de novo infection with a well-characterized model gammaherpesvirus. While infected cells demonstrated genome-wide transcription, individual cells revealed pronounced variation in gene expression, with only 9 of 80 annotated viral open reading frames uniformly expressed in all cells, and a 1000-fold variation in viral RNA expression between cells. Single-cell analysis further revealed positive and negative gene correlations, many uniquely present in a subset of cells. Beyond variation in viral gene expression, individual cells demonstrated a pronounced, dichotomous signature in host gene expression, revealed by measuring host RNA abundance and post-translational protein modifications. These studies provide a resource for the high-dimensional analysis of virus infection, and a conceptual framework to define virus infection as the sum of virus and host responses at the single-cell level. HIGHLIGHTSO_LICyTOF and scRNA-seq identify wide variation in gene expression between infected cells. C_LIO_LIHost RNA expression and post-translational modifications stratify virus infection. C_LIO_LISingle cell RNA analysis reveals new relationships in viral gene expression. C_LIO_LISimultaneous measurement of virus and host defines distinct infection states. C_LI

microbiology↗

bioRxiv2019

Tumor-Intrinsic Response to IFNgamma Shapes the Tumor Microenvironment and Anti-PD-1 Response in NSCLC

Targeting PD-1/ PD-L1 is only effective in ~20% of lung cancer patients, but determinants of this response are poorly defined. We previously observed differential responses of two murine K-Ras lung cancer cell lines to anti-PD-1 therapy: CMT167 tumors were eliminated while LLC tumors were resistant. The goal of this study was to define mechanism(s) mediating this difference. RNA-Seq analysis of cancer cells recovered from lung tumors revealed that CMT167 cells induced an IFN{gamma} signature that was absent in LLC cells. Silencing Ifngr1 in CMT167 resulted in tumors resistant to IFN{gamma} and anti-PD-1 therapy. Conversely, LLC cells had high basal expression of Socs1, an inhibitor of IFN{gamma}. Silencing Socs1 increased response to IFN{gamma} in vitro and sensitized tumors to anti-PD-1. This was associated with a reshaped TME, characterized by enhanced T cell infiltration and enrichment of PD-L1 high myeloid cells. These studies demonstrate that targeted enhancement of tumor-intrinsic IFN{gamma} signaling can induce of cascade of changes associated with increased therapeutic vulnerability. SummaryMechanisms regulating response to anti-PD-1 therapy in lung cancer are not well defined. This study, using orthotopic immunocompetent mouse models of lung cancer, demonstrates that intrinsic sensitivity of cancer cells to IFN{gamma} determines anti-PD-1 responsiveness through alterations in the tumor microenvironment.

cancer biology↗

bioRxiv2018

High-dimensional characterization of IL-10 production and IL-10 dependent regulation during primary gammaherpesvirus infection

Interleukin (IL)-10 is a potent immunomodulatory cytokine produced by multiple cell types to restrain immune activation. Many herpesviruses use the IL-10 pathway to facilitate infection, but how endogenous IL-10 is regulated during primary infection in vivo remains poorly characterized. Here, we infected mice with murine gammaherpesvirus 68 ({gamma}HV68) and analyzed the production, and genetic contribution, of IL-10 by mass cytometry (cytometry by time-of-flight, CyTOF) analysis.{gamma} HV68 infection elicited a breadth of effector CD4 T cells in the lungs of acutely infected mice, including a highly activated effector subset that co-expressed IFN{gamma}, TNF, and IL-10. By using IL-10 green fluorescent protein (gfp) transcriptional reporter mice, we identified that IL-10 was primarily expressed within CD4 T cells during acute infection in the lungs. IL10gfp expressing CD4 T cells were highly proliferative and characterized by the expression of multiple co-inhibitory receptors including PD-1 and LAG-3. When we analyzed acute{gamma} HV68 infection of IL-10 deficient mice, we found that IL-10 limits the frequency of both myeloid and effector CD4 T cell subsets in the infected lung, with minimal changes at a distant mucosal site. These data emphasize the unique insights that high-dimensional analysis can afford in investigating antiviral immunity, and provide new insights into the breadth, phenotype and function of IL-10 expressing effector CD4 T cells during acute virus infection.\n\nThis work was funded by National Institutes of Health Grants R01 AI121300 and R01 CA168558 (to L.F.v.D.), an American Heart Association National Scientist Development grant (#13SDG14510023), the Crohns and Colitis Foundation of America (#311295), a pilot grant from the Lung, Head and Neck Cancer program within the University of Colorado Cancer Center, and a Career Enhancement Award from the University of Colorado Lung Cancer Specialized Program of Research Excellence (P50CA58187) (all to E.T.C.).\n\nThe Lung, Head and Neck program within the University of Colorado Cancer Center, and the Flow Cytometry Shared Resource, are directly funded through support from the National Cancer Institute Cancer Center Support Grant P30CA046934.\n\nAbbreviations used in this article

immunology↗

bioRxiv2018

Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 ({gamma}HV68), Kaposis sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during {gamma}HV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with {gamma}HV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive {gamma}HV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level.\n\nAUTHOR SUMMARY\n\nThe gammaherpesviruses are a group of DNA tumor viruses that establish lifelong infection. How these viruses infect and manipulate cells has frequently been studied in bulk populations of cells. While these studies have been incredibly insightful, there is limited understanding of how virus infection proceeds within a single cell. Here we present a new approach to quantify gammaherpesvirus gene expression at the single-cell level. This method allows us to detect cell-to-cell variation in the expression of virus non-coding RNAs, an important and understudied class of RNAs which do not encode for proteins. By examining multiple features of virus gene expression, this method further reveals significant variation in infection between cells across multiple stages of infection. These studies emphasize that gammaherpesvirus infection can be surprisingly heterogeneous when viewed at the level of the individual cell. Because this approach can be broadly applied across diverse viruses, this study affords new opportunities to understand the complexity of virus infection within single cells.

microbiology↗