Response of intrahepatic lymphocytes (IHLs) from chimpanzees 251 and 325 to HCV after challenge with different doses. IHLs prepared as described under Materials and methods, from chimpanzee 251 (A) or chimpanzee 325 (B), were tested for anti-HCV cellular response using direct ex vivo IFN-γ ELISPOT assay. Autologous PBLs from time 0 (before challenge) were infected with recombinant HCV-C-NS3-vaccinia (filled column), HCV-NS3-NS5-vaccinia (unfilled column), at an m.o.i. of 10:1 and used as stimulator cells. IHLs at 1, 6, and 12 months postchallenge were incubated with stimulator cells at 37°C and 5% CO2. The numbers of direct ex vivo IFN-γ-secreting cells (ISCs) were measured 18 h after in vitro stimulation. The numbers of HCV-specific ISCs/106 cells were plotted after subtracting the background (parental vaccinia virus alone). The cutoff value of direct ex vivo ISCs (horizontal line) was calculated as described under Materials and methods as approximately 185.5 ISCs/106. The data represent the average of two experiments.