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 immunostimul atory properties, The majority of studies done so far on HBcAg induced resp onses 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 p roliferation of murine T cells. The advantage with this technique was that we could analyse the cytokine phenotype of proliferating cells of a particu lar cell type. We found that IL-10 expression was strongly induced in CD4 T cells after HBcAg immunization. Importantly, we found that IL-4 producin g HBcAg-specific CD4 + T cells are common after immunization although detec tion of IL-4 in culture supernatants indicates only low levels of IL-4. In contrast, IFN-gamma producing HBcAg-specific CD4 + T cells were found at lo wer numbers despite the detection of high levels of IFN-gamma in culture su pernatants. 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 cytokin e phenotype of the CD4 + T cells primed by HBcAg. (C) 2001 Elsevier Science B.V. All rights reserved.