Determination of cytokine co-expression in individual splenic CD4(+) and CD8(+) T cells from influenza virus-immune mice

We have studied the patterns of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) co-expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet-inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4 and IFN-gamma were determined by three-colour flow cytometric analysis of fixed and saponin-permeabilized cells fluorescent-stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL-2/IL-4, IL-2/IFN-gamma and IL-4/IFN-gamma. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus-immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co-expressing combinations of them (i.e. IL-4/IL-2, IL-4/IFN-gamma and IL-2/IFN-gamma), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.