Enumeration of lymphokine mRNA-containing cells in vivo in a murine graft-versus-host reaction using the PCR

Abstract

A method of enumerating lymphokine mRNA-containing cells in vivo was developed by combining limiting dilution analysis with PCR amplification of cDNA. Single-hit kinetics revealed that the PCR-limiting dilution analysis could detect a single positive cell among greater than 40,000 negative cells. With this method, spleens and lymph nodes of mice undergoing an acute allogeneic graft-versus-host reaction were found to contain lymphokine mRNA-expressing cells at frequencies of 3% for interferon gamma, 0.05% for granulocyte/macrophage colony-stimulating factor, 0.002% for interleukin 3, and 0.03% for interleukin 4; these frequencies were 20- to 175-fold higher than in lymphoid tissues of normal mice. In contrast to their low frequencies of lymphokine mRNA-containing cells in vivo, graft-versus-host reaction populations restimulated in vitro for 24 hr with anti-CD3 antibody yielded frequencies ranging from 3% for interleukin 4 to nearly 70% for interferon gamma. Furthermore, lymphokine transcripts were also detected in single micromanipulated cells from these populations. Because frequencies of anti-CD3-inducible lymphokine mRNA-containing cells in normal mice were only 0.03-1%, it was concluded that lymphoid tissues of graft-versus-host reaction mice contained high frequencies of cells that had been primed for lymphokine synthesis. Only a small fraction of these cells, however, expressed lymphokine mRNAs at a given time point in vivo.