title: Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status.
abstract: RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. We tested whether antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI), a condition affecting one-third of the world population. Peripheral-blood mononuclear cells from donors, identified as positive or negative for LTBI by current medical practice, were stimulated ex vivo with mycobacterial antigen. IFNG and IL2 mRNA production was assayed by FISH-Flow. Concurrently, immunophenotypes of the cytokine mRNA-positive cells were characterized by conventional, antibody-based staining of cell-surface markers. An association was found between donor LTBI status and antigen-specific induction of IFNG and IL2 transcripts. Induction of these cytokine genes, which was detected by FISH-Flow in a quarter the time required to see release of the corresponding proteins by ELISA, occurred primarily in activated CD4+ T cells via T-cell receptor engagement. Moreover, NK cells contributed to IFNG gene induction. These results show that antigen-driven induction of T-cell cytokine mRNA is a measurable single-cell parameter of the host responses associated with latent tuberculosis. FISH-Flow read-outs contribute a multi-scale dimension to the immunophenotyping afforded by antibody-based flow cytometry. Multi-scale, single-cell analyses may satisfy the need to determine disease stage and therapy response for tuberculosis and other infectious pathologies.
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Pooja
authors.initials: P
authors.lastname: Vir
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Arrigucci
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Karim
authors.initials: K
authors.lastname: Lakehal
authors.affiliation: Department of Biostatistics, School of Public Health, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Davidow
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Richard
authors.initials: R
authors.lastname: Pine
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Tyagi
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Bushkin
authors.affiliation: Global Tuberculosis Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Alfred
authors.initials: A
authors.lastname: Lardizabal
authors.affiliation: Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Gennaro
mesh_terms: D016022:Case-Control Studies; D016207:Cytokines / Q000096:biosynthesis / Q000097:blood*; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D007371:Interferon-gamma / Q000097:blood; D007376:Interleukin-2 / Q000097:blood; D055985:Latent Tuberculosis / Q000097:blood* / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008213:Lymphocyte Activation; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D016207:Cytokines; D007376:Interleukin-2; D007371:Interferon-gamma