One unlabelled mAb was adsorbed
to the microtitre plate well and used as a capture antibody, and the other labelled mAb served as the probe. For the multicytokine assay, we have used Multiflex Biomarker Immunoassay (Millipore, Billerica, MA). To determine T-cell proliferation, TCR-Tg spleen T cells were labelled in vitro with the intracellular dye carboxyfluorescein EPZ-6438 in vivo succinimidyl ester (CFSE) by using the Vybrant CFSE SE tracer kit (Molecular Probes, Eugene, OR.). Carboxyfluorescein succinimidyl ester (CFSE; 0·5 mm) was added to the cell suspensions, and the mixture was incubated for 10 min at 37°. The labelling reaction was stopped by repetitive washing with ice-cold RPMI-1640 medium containing 10% fetal calf serum. Labelled cells (4 × 106/ml) were cultured with various concentrations of antigen for 2, 3 or 4 days. Cultured
cells were harvested, washed and labelled with anti-TCR Vβ11 antibodies. The number of cell divisions was determined by selleck compound dilution of the intracellular dye CFSE in TCR Vβ11+ gated T cells by flow cytometry. In some experiments, Bodipy-FL (Invitrogen, Carlsbad, CA) was used for the cell proliferation assay instead of CFSE. To measure the effect of HBeAg on effector T-cell activation in vitro, we cultured splenocytes from naive 7/16-5 TCR-Tg and 7/16-5 × HBeAg dbl-Tg mice in the presence of 1 μg/ml of the HBeAg-derived peptide p120–140 and analysed CD69 expression by FACS. There was a dramatic difference between T-cell activation in 7/16-5 TCR-Tg mice versus 7/16-5 × HBeAg dbl-Tg mice. A high percentage (59·30%) of T cells derived from 7/16-5 TCR-Tg mice expressed CD69 after 3 days in culture, which was sustained after 6 days. In contrast, only 18·56% of T cells derived from HBeAg × 7/16-5 dbl-Tg mice expressed CD69 at 3 days of culture and the expression of CD69 remained low (11·17%) at day 6 (Fig. 1a). Similarly, in vitro IL-2 production by 7/16-5 × HBeAg dbl-Tg splenic T cells cultured with either HBeAg or HBcAg was significantly reduced
compared with T cells from 7/16-5 single TCR-Tg mice (Fig. 1b). It is notable that the tolerance exhibited in 7/16-5 × HBeAg dbl-Tg mice is not the result of clonal deletion of 7/16-5 TCR-Tg T cells either Phospholipase D1 in the thymus or in the spleen (data not shown,30). CFSE-labelled splenocytes from 7/16-5 TCR-Tg and 7/16-5 × HBeAg dbl-Tg mice and 7/16-5 × HBcAg dbl-Tg mice were cultured in vitro in the presence of HBeAg peptide p120–140 to examine CD4+ and CD8+ effector cell proliferation. As shown in Fig. 2, after 4 days in culture both CD4+ and CD8+ Vβ11+ TCR-Tg T cells from 7/16-5 mice and 7/16-5 × HBc dbl-Tg mice proliferated. In contrast, the vast majority of the proliferating Vβ11+ TCR-Tg T cells from 7/16-5 × HBeAg dbl-Tg mice were neither CD4+ cells nor CD8+ cells (Fig. 2, middle column) and represented a DN, Vβ11+ population.