Determination of peripheral blood mononuclear cell responses to mitogens and woodchuck hepatitis virus core antigen in woodchucks by 5-bromo-2′-deoxyuridine or 2[³H]adenine incorporation

Summary

Characterization of cellular immune response to antigens of woodchuck hepatitis virus (WHV) can contribute to the understanding of acute resolving and chronic outcome of hepadnavirus infection. Studies were limited because peripheral blood mononuclear cells (PBMCs) of woodchucks failed to incorporate [³H]thymidine sufficiently. Therefore, we established a non-radioactive proliferation assay for woodchuck PBMCs using 5-bromo-2′deoxyuridine (BrdU) as thymidine analogue. Mitogen- and WHV core protein(WHcAg) induced PBMC proliferation was detected by BrdU incorporation and compared to an assay using 2[³H]adenine. After stimulation with concanavalin A (ConA) and phytohaemagglutinin (PHA) we observed significant PBMC proliferation with both assays. Mitogen-induced nucleoside uptake of PBMCs into cellular DNA was confirmed by detection of 1′,2′[³H]BrdU and 2[³H]adenine in extracted DNA. PBMCs obtained during the acute phase of WHV infection could be stimulated by WHcAg, whereas no WHcAg-induced proliferation of PBMCs was found in WHV-negative animals. PBMCs of chronic WHV carriers showed only a weak response to WHcAg. The established assays will be useful in determining the kinetics of cellular immune responses to different WHV antigens in the course of WHV infection and may provide an insight into mechanisms responsible for chronic outcome of hepadnavirus infection.