Objective To investigate the composition of Th 0/Th 1/Th 2 in chronic Hepatitis B Virus (HBV) infected individuals by determining the expression of IL 4/IFN γ in single CD4 + T cell from peripheral blood, and to analyze the role of Th 1/Th 2 cytokines in chronic HBV infection Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 3?ml fresh heparinized blood by Ficoll density gradient centrifugation The interphase PBMCs were suspended in RPMI 1640 culture medium supplemented with 10% heat inactivated fetal calf serum (FCS), glutamine and antibiotics Furthermore, in order to isolate CD4 + cell rich population, anti CD8 monoclone antibody (mAb) was added to the cell suspension at a final dilution of 1/120 After 60 minutes incubation at 37℃, fresh rabbit sera as a source of complement were added Freshly isolated CD8 + free PBMCs were enriched for lymphocytes by removal of adherent cells after 1 5 hours adherence to plastic culture flasks Cells were stimulated with 1?ng/ml PMA, 1?μmol/L Ionomycin and 1?μmol/L monensin for 4 hours at 37℃ Cells were washed with PBS containing 0 5% (w/v) BSA Then washing with PBS, cells were fixed with PBS containing 4% (v/v) paraformaldehyde for 10 minutes on ice Cells were washed twice with PBS and permeabilized with PBS containing 0 1% (w/v) saponin, 0 5% BSA and 10% pooled human serum for 10 minutes on ice Cells were then incubated with PE labled antibodies against IFN γ and FITC labled antibodies against IL 4 for 45 minutes at 4℃ Before, between and after incubations with mAb, cells were washed with PBS containing 0 5% BSA and 0 1% saponin Cell samples were analyzed on a fluorescence activated Department of Infectious Diseases, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China (Jiang RL, Lu QS and Luo KX) Department of Immunology, First Military Medical University, Guangzhou 510515, China (Fu N) cell sorter (FACS) The percentage of IL 4/IFN γ cytokines producing cells was expressed In each experiment, cryopreserved PBMC from the same healthy donor were analyzed in parallel to serve as an internal control Results We developed an assay for intracellular staining of cytokines Cells were able to produce IFN γ (but not IL 4) which were defined as Th 1; cells were able to produce IL 4 (but not IFN γ) which were defined as Th 2; Cells were able to produce IFN γplus IL 4 which were defined as Th 0 Cells cultured in medium alone do not give a signal with any of the Abs used; whereas upon stimulation with PMA and ionomycin , part of the cells were positive for IL 4 and IFN γ, but not the control Ab Monensin was added to the cultures, which caused intracellular accumulation of newly synthesized proteins by arresting their transport principally in the Golgi complex These results in a higher intracellular concentration for both cytokines, while in non stimulated cells the cytokines remained undetectable in the presence of monensin The percentage of IFN γ producing T cells and IL 4 producing T cells ranged from 2 3% to 18 6% and from 1 1% to 8 7% respectively in CD4 + cells in non infected individuals The majority of CD4 + T cells in peripheral blood from chronic HBV infected individuals were Th 0 cells The percentage of Th 1 cells increased significantly with the hepatic inflammation activity The percentage of Th 1 cells in advanced period of chronic hepatitis B was higher than in stable period of chronic hepatitis B The percentage of Th 2 cells in CD4 + T cells from HBV infected individuals did not differ significantly, but was higher than controls Conclusions Th 1 cells are associated with hepatic inflammation activity of chronic hepatitis B, and Th 2 cells may be associated with the chronicity of HBV infection Objective To investigate the composition of Th 0 / Th 1 / Th 2 in chronic Hepatitis B Virus (HBV) infected individuals by determining the expression of IL 4 / IFN γ in single CD4 + T cell from peripheral blood, and to analyze the role of Th1 / Th2 cytokines in chronic HBV infection Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 3 ml of fresh heparinized blood by Ficoll density gradient centrifugation The interphase PBMCs were suspended in RPMI 1640 culture medium supplemented with 10% heat inactivated fetal calf serum (FCS), glutamine and antibiotics Furthermore, in order to isolate CD4 + cell rich population, anti CD8 monoclone antibody (mAb) was added to the cell suspension at a final dilution of 1/120 after 60 minutes incubation at 37 ° C, fresh rabbit sera as a source of complement were added Freshly isolated CD8 + free PBMCs were enriched for lymphocytes by removal of adherent cells after 1 5 hours adherence to plastic culture flasks Cells were stimulated with 1 ng / ml PMA, 1 μmol / L Ionomycin and 1 μmol / L monensin for 4 hours at 37 ° C. Cells were washed with PBS containing 0.5% (w / v) cells were fixed with PBS containing 4% (v / v) paraformaldehyde for 10 minutes on ice cells were washed twice with PBS and permeabilized with PBS containing 0 1% (w / v) saponin, 0 5% BSA and 10% pooled human serum for 10 minutes on ice Cells were then incubated with PE labled antibodies against IFN γ and FITC labled antibodies against IL 4 for 45 minutes at 4 ° C. Before, between and after incubations with mAb, cells were washed with PBS containing 0 5% BSA and 0 1% saponin Cell samples were analyzed on a fluorescence activated Department of Infectious Diseases, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China (Jiang RL, Lu QS and Luo KX) Department of Immunology, First Military Medical University, Guangzhou 510515, China (Fu N) cell sorter (FACS) The percent age of IL4 / IFNγ cytokines producing cells was expressed in each experiment, cryopreserved PBMC from the same healthy donor were analyzed in parallel to serve as an internal control Results We developed an assay for intracellular staining of cytokines Cells were able to produce IFNγ (but not IL 4) which were defined as Th 1; cells were able to produce IL 4 (but not IFN γ) which were defined as Th 2; Cells were able to produce IFN γplus IL 4 which were defined as Th 0 Cells cultured in medium alone do not give a signal with any of the Abs used; and prior upon stimulation with PMA and ionomycin, part of the cells were positive for IL 4 and IFN γ, but not the control Ab Monensin was added to the cultures, which caused intracellular accumulation of newly synthesized proteins by arresting their transport principally in the Golgi complex These results in a higher intracellular concentration for both cytokines, while in non stimulated cells the cytokines remained u ndetectable in the presence of monensin The percentage of IFN γ producing T cells and IL 4 producing T cells ranged from 2 3% to 18 6% and from 1 1% to 8 7% respectively in CD4 + cells in non-infected individuals The majority of CD4% T cells in peripheral blood from chronic HBV infected individuals were Th0 cells increased significantly with the hepatic inflammation activity The percentage of Th1 cells in advanced period of chronic hepatitis B was higher than in stable period of chronic hepatitis B The percentage of Th 2 cells in CD4 + T cells from HBV infected individuals did not differ significantly, but was higher than controls Conclusions Th 1 cells are associated with hepatic inflammation activity of chronic hepatitis B, and Th 2 cells may be associated with the chronicity of HBV infection