Effector CD8 T cells fail to differentiate into memory cells in persisting antigen environment. (a) Purification and transfer of effector CD8 T cells into antigen-free versus antigen-persistent hosts. A schematic view of the adoptive transfer experiments. (b) CFSE-labelled naive HA-specific CD8 T cells were adoptively transferred into B10.D2 mice infected with rVV-HA (rVV-HA) or C3-HAhi mice. Four days later, cells were harvested from peripheral lymph nodes and spleen, and CD8+ Thy-1.1+ T cells were enriched by negative selection using magnetic beads before sorting gated on CD8, Thy-1.1 and at least three divisions by CFSE as shown in Fig. 1(a). Cells were stained with CD8 and KdHA tetramer. The percentage of clonotypic T cells before enrichment and sorting (Pre-sort) and the purity of clonotypic T cells after cell sorting (Post-sort) are indicated. (c) HA-specific effector CD8 T cells purified from rVV-HA infected B10.D2 mice (rVV-HA) were transferred into naive B10.D2 (rVV-HA (r) B10.D2), C3-HAhi (rVV-HA (r) C3-HAhi), or C3-HAlo (rVV-HA (r) C3-HAlo), and those effector cells from C3-HAhi mice were transferred into naive B10.D2 (C3-HAhi (r) B10.D2), C3-HAhi (C3-HAhi (r) C3-HAhi), or C3-HAlo (C3-HAhi (r) C3-HAlo). After 42 days, mice were challenged with Ad-HA or Ad-LacZ; 7 days after the challenge, cells were analysed for the expansion of clonotypic T cells. The percentages of clonotypic T cells (CD8+ Thy-1.1+ cells ± SD) in total CD8 T cells for all mice in each group are indicated. (d) Function of clonotypic CD8 T cells after challenge with Ad-HA were analysed by IFN-γ intracellular staining and the percentages of IFN-γ-producing clonotypic CD8 T cells in total CD8 T cells are indicated. Representative data of three independent experiments are shown.