Analyzing the distribution of cells expressing mRNA for T cell receptor γ and δ chains in a virus-induced inflammatory process

Abstract

Acute inflammatory processes are extremely complex, containing sets of activated cells that may be difficult to categorize. The interface between two methodologies for characterizing the involvement of γδ T cells, in situ hybridization to detect T cell receptor (TCR) mRNA and flow cytometric analysis of surface TCR expression, is utilized here to study the pneumonia caused by intranasal (i.n.) infection of mice with influenza A viruses. Substantial numbers of cells expressing mRNA for the γ and δ TCR chains are present in bronchoalveolar lavage (BAL) populations obtained either late in the course of primary infection with an H3N2 virus or within a few days of secondary challenge with an H1N1 virus. The majority of the γδ TCR mRNA + cells detected in FACS-separated BAL populations partition to the Thy1 + γδ TCR + subset, while relatively few (<10%) Cδ mRNA transcripts are found in cells that phagocytose latex particles. However, an additional set of γδ TCR mRNA + cells is also located in a high side scatter (H-SSC) population, which stains nonspecifically with monoclonal antibodies (mAbs) and is normally gated out in the process of flow cytometric analysis. This H-SSC population tends to be enriched for cells expressing Cγ 1 2 rather than Cγ4 mRNA. While some γδ TCR lymphocytes can be demonstrated by in vitro stimulation of the CD3ϵ + subset within this H-SSC population, the majority of the γδ T cell precursors that can be expanded in culture demonstrate a low side scatter (L-SSC) profile more characteristic of normal T lymphocytes. The possibility that subsets of activated, granular (H-SSC) αβ TCR + and Cγ 1 2 mRNA + γδ T cells are being missed when conventional FACS analysis is used to study this viral pneumonia is discussed.