title: Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry.
abstract: Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from pre-existing in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103; and.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Bushkin
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103; and.
authors.forename: Felix
authors.initials: F
authors.lastname: Radford
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103; and.
authors.forename: Richard
authors.initials: R
authors.lastname: Pine
authors.affiliation: Global Tuberculosis Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103.
authors.forename: Alfred
authors.initials: A
authors.lastname: Lardizabal
authors.affiliation: Global Tuberculosis Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103.
authors.forename: Bonita T
authors.initials: BT
authors.lastname: Mangura
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103; and.
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Gennaro
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers University, Newark, NJ 07103; and bushkiyu@njms.rutgers.edu sanjay.tyagi@rutgers.edu.
authors.forename: Sanjay
authors.identifier: http://orcid.org/0000-0001-7303-5827
authors.initials: S
authors.lastname: Tyagi
mesh_terms: D000328:Adult; D000368:Aged; D000942:Antigens, Bacterial / Q000276:immunology; D016207:Cytokines / Q000097:blood / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000097:blood / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology* / Q000473:pathology; D014397:Tuberculosis, Pulmonary / Q000097:blood / Q000276:immunology* / Q000473:pathology
chemical_list: D000942:Antigens, Bacterial; D016207:Cytokines; D012333:RNA, Messenger