Flow cytometric determination of cytokine production and proliferation in Hepatitis B core antigen specific murine CD4 cells: Lack of correlation between number of cytokine producing cells and cytokine levels in supernatant

Hepatitis B virus (HBV) core antigen (HBcAg) has extraordinary immunostimulatory properties. The majority of studies done so far on HBcAg induced responses have used ELISA or bioassay for cytokine determination and the ³[H]thymidine incorporation assay to measure proliferation. Here multiparameter flow cytometry was used to measure HBcAg induced cytokine production and proliferation of murine T cells. The advantage with this technique was that we could analyse the cytokine phenotype of proliferating cells of a particular cell type. We found that IL-10 expression was strongly induced in CD4 + T cells after HBcAg immunization. Importantly, we found that IL-4 producing HBcAg-specific CD4 + T cells are common after immunization although detection of IL-4 in culture supernatants indicates only low levels of IL-4. In contrast, IFN-γ producing HBcAg-specific CD4 + T cells were found at lower numbers despite the detection of high levels of IFN-γ in culture supernatants. Thus, the frequency of these cells is not accurately reflected by the detectability of the respective cytokine in culture supernatants. A low number of specific CD4 + T cells may effectively produce high levels of cytokine. We therefore suggest that different types of cytokine assays are used in order to obtain the most accurate picture of the intrinsic cytokine phenotype of the CD4 + T cells primed by HBcAg.