Combination of in situ hybridization and quantitative, competitive rt-pcr for the determination of cytokine mRNA copy number in activated T cells

Academic Article

Abstract

  • The elucidation of cytokine expressing cells during T cell activation is critical for understanding the formation and progression of the immune response. An important aspect of this understanding is determining cytokine expression at the single cell level. We have used a combination of in situ hybridization (ISH) and quantitative, competitive RT-PCR (QC-RTPCR) for measuring cytokine mRNA in activated T cells at the single cell level. Our previous results with ISH have demonstrated that only a subpopulation of clonotype transgenic T cells produce cytokine mRNA when stimulated both in vitro and m vivo. Utilizing limiting dilution QCRT-PCR, the frequency of mRNA expressing cells by PCR was equivalent to that found with ISH. Furthermore, analysis of T cell populations by QCRT-PCR has allowed the determination of the copy number of cytokine mRNAs in ThO, Thl, and Th2 clonal populations. Remarkably, the copy number for IL-2 (2,500-3,300 copies/cell), IL-4 (12,000-18,000 copies/cell), IL-10 (1,000-1,500 copies/cell) and IFNy (200-800 copies/cell) remains relatively constant over a broad antigen range, suggesting that when activated to produce cytokines, T cells are either on or off. This data has important implications for control of cvtokine expression and the nature of T cell activation.
  • Authors

    Published In

  • The FASEB Journal  Journal
  • Author List

  • Sweenev SD; Bucy RP; Hockett RD
  • Volume

  • 10
  • Issue

  • 6