OPTIMIZATION OF MURINE CD8(-LYMPHOCYTE RESPONSES TO PSEUDORABIES VIRUS() CYTOTOXIC T)

The aim of this work was to optimize the procedures used to elicit a c ellular immune response to pseudorabies virus (PrV) in mice, using var ious immunization schedules and routes. An Eu-labeling-based cytotoxic T-lymphocyte (CTL) test was developed to measure the response. This n ecessitated optimization of numerous steps. In suspension, Eu labeling required high concentrations of dextran-sulfate (DXS) and Eu with a 3 0-min labeling time at room temperature. For anchored cells, the label ing time was 1 to 48 h, and the labeling efficiency depended strongly on the Eu concentration, but only marginally on the DXS concentration. In vivo and in vitro stimulation protocols were also optimized for th e CTL test. For in vitro stimulation, spleen cells were cultured in T- 25 flasks at a multiplicity of infection (m.o.i.) of 2. The CTL test w as validated by specific depletion of CD8(+) CTL, FACS analysis, and b y comparing Eu and Cr-51 labeling. Then groups of mice were vaccinated once or twice by various routes (intraperitoneal (i.p.), intravenous (i.v.), subcutaneous (s.c.) and in the rear footpads (FP)) and accordi ng to different time schedules. CTL activity was detected only in boos ted animals immunized FP, i.p. or i.v. That the cellular immune respon se contributes to protection was further suggested by the observation that i.p. immunization conferred better protection against challenge t han s.c. immunization.