Flow cytometric quantification of T cell proliferation and division kinetics in woodchuck model of hepatitis B.
Woodchucks infected with woodchuck hepatitis virus (WHV) represent the closest natural animal model to study the immunopathogenesis of liver injury caused by essentially noncytopathic, highly human specific hepatitis B virus (HBV). The importance of antiviral T cell response in induction of hepatitis and in control of HBV replication has been demonstrated. However, the understanding of how these responses contribute to the development of different immunomorphological forms of liver disease and their outcomes remain elusive. In this study, we established and standardized a flow cytometry assay using peripheral blood mononuclear cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to assess WHV-specific and mitogen-driven T lymphocyte proliferative responses in woodchucks. The assay is of significantly greater sensitivity than the adenine incorporation assay currently used when applied to measure either WHV-specific T cell responses in acute (P < 0.001) and chronic (P < 0.03) viral hepatitis or those induced by mitogens in both healthy and WHV-infected animals. It also provides a new type of information, not previously available, characterizing the strength of woodchuck T cell proliferative reactivity by measuring cell division rates. The study shows that woodchuck PBMC labeled with CFSE exhibit light scatter and fluorescence profiles compatible to those of human PBMC, allowing quantitation and deconvolution of the flow cytometric data by applying the existing analytical softwares. The availability of this novel assay should facilitate a more precise and comprehensive evaluation of hepadnavirus-specific and generalized T cell responses in experimental WHV hepatitis.
Gujar
Shashi A
SA
Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.
Michalak
Tomasz I
TI
D000818:Animals; D049109:Cell Proliferation; D004195:Disease Models, Animal; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D006509:Hepatitis B / Q000276:immunology*; D006515:Hepatitis B virus / Q000502:physiology; D008213:Lymphocyte Activation; D008392:Marmota; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013194:Staining and Labeling; D013388:Succinimides; D013601:T-Lymphocytes / Q000276:immunology*
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D013388:Succinimides