RNA fluorescence in situ hybridization and flow cytometry (FISH-Flow) detects antigen-specific T cell responses associated with latent tuberculosis infection (TECH3P.940)

Abstract

Abstract We have previously described fluorescence in situ hybridization for mRNA detection combined with flow cytometry (FISH-Flow) as a novel method to detect gene expression at a single-cell level. Using mRNA as analyte in flow cytometry presents multiple, biological and technical advantages. Proof-of-principle experiments have also shown that FISH-Flow can detect rare events including antigen-specific T cell responses. Whether the method is applicable to a biomedical problem remains to be determined. Here we applied FISH-Flow to detection of latent Mycobacterium tuberculosis infection (LTBI), a condition affecting approximately a third of the world population. LTBI diagnosis is based on measuring IFN-γ release from peripheral blood mononuclear cells (PBMC) stimulated ex vivo with M. tuberculosis antigen. We obtained PBMC from 60 donors equally distributed between LTBI+ and LTBI- individuals, stimulated them for 6 hrs with M. tuberculosis purified protein derivative (PPD), and measured expression of IFNG and IL2 mRNA by FISH-Flow. The proportions of antigen-induced IFNG and IL2 expression in CD3+ cells were >2-fold higher in LTBI+ than in LTBI- donors (p<0.005). Detection of multiple surface protein markers in combination with mRNA probes determined that CD4+ T cells are the primary cytokine-expressing subset. These results show that the FISH-Flow assay can identify a biological response and demonstrates the usefulness of this technique for basic and clinical immunological studies.